Lipophilicity of Bacteriochlorin-Based Photosensitizers as a Determinant for PDT Optimization through the Modulation of the Inflammatory Mediators

Photodynamic therapy (PDT) augments the host antitumor immune response, but the role of the PDT effect on the tumor microenvironment in dependence on the type of photosensitizer and/or therapeutic protocols has not been clearly elucidated. We employed three bacteriochlorins (F2BOH, F2BMet and Cl2BHep) of different polarity that absorb near-infrared light (NIR) and generated a large amount of reactive oxygen species (ROS) to compare the PDT efficacy after various drug-to-light intervals: 15 min. (V-PDT), 3h (E-PDT) and 72h (C-PDT). We also performed the analysis of the molecular mechanisms of PDT crucial for the generation of the long-lasting antitumor immune response. PDT-induced damage affected the integrity of the host tissue and developed acute (protocol-dependent) local inflammation, which in turn led to the infiltration of neutrophils and macrophages. In order to further confirm this hypothesis, a number of proteins in the plasma of PDT-treated mice were identified. Among a wide range of cytokines (IL-6, IL-10, IL-13, IL-15, TNF-α, GM-CSF), chemokines (KC, MCP-1, MIP1α, MIP1β, MIP2) and growth factors (VEGF) released after PDT, an important role was assigned to IL-6. PDT protocols optimized for studied bacteriochlorins led to a significant increase in the survival rate of BALB/c mice bearing CT26 tumors, but each photosensitizer (PS) was more or less potent, depending on the applied DLI (15 min, 3 h or 72 h). Hydrophilic (F2BOH) and amphiphilic (F2BMet) PSs were equally effective in V-PDT (>80 cure rate). F2BMet was the most efficient in E-PDT (DLI = 3h), leading to a cure of 65 % of the animals. Finally, the most powerful PS in the C-PDT (DLI = 72 h) regimen turned out to be the most hydrophobic compound (Cl2BHep), allowing 100 % of treated animals to be cured at a light dose of only 45 J/cm2.


Introduction
Photodynamic therapy (PDT) is a promising therapeutic approach to cancer, which in recent years entered the mainstream therapeutic options in oncology [1][2][3]. PDT aims at the destruction of the primary tumor without damaging surrounding, healthy tissues, and at the stimulation of anti-tumor immunity. There are three main mechanisms of PDT: the direct killing of cancer cells by photogenerated reactive oxygen species (ROS), tumor-associated vascular damage and activation of anti-tumor immune responses [2,4]. These mechanisms suggest that intricate relations between the nature of the photosensitizer (PS), PS dose, light dose, drug-to-light interval (DLI), the oxygen concentration in the targeted tissue and the mechanism of cell death [2,[5][6][7] are likely to affect the outcome of PDT efficacy.
Changes in the peritumoral microenvironment induced by PDT seem to influence the activity of non-specific mechanisms of the innate immune system. The cells involved in non-specific mechanisms include phagocytic cells-macrophages, granulocytes, monocytes and several mediators (chemokines and cytokines) [43,44]. PDT-induced inflammation may also activate mechanisms of adaptive (or specific) immune response generated when antigens are recognized by the lymphocyte population, capable of inducing immunological memory and of causing the regression of distant established tumors that received no treatment [17]. Cells dying after PDT can be a source of antigens and stimulate cells of the immune system that infiltrate the tumor, eventually leading to anti-tumor immunity [45][46][47]. The development of effective, adaptive immune responses requires the prior engagement of innate immunity [39].
The main purpose of this work is to investigate PDT regimes with three bacteriochlorin-type photosensitizers of different polarity and to relate the outcomes of these regimes to the properties of the PSs, namely their physicochemical properties, subcellular localization, pharmacokinetics, ROS generation and immune response. This work offers a basis for the optimization of immune responses with PDT and the rational design of combinations between PDT and immunotherapies. The bacteriochlorins selected for this study are presented in Scheme 1. The synthesis and some photophysical properties of F2BOH, F2BMet (named redaporfin) and Cl2BHep have been reported [21][22][23]48], as well as the pharmacokinetics and biodistribution of F2BOH and F2BMet [14,15,24]. Although some in vitro studies and the PDT of tumor-bearing mice have been published for F2BOH and F2BMet [21][22][23][24], they were repeated and extended in this work to afford a more direct comparison with the corresponding studies with Cl2BHep. Scheme 1. Chemical structure of investigated halogenated bacteriochlorin derivatives.

Spectroscopic Studies
Electronic absorption spectra were recorded in a Hewlett Packard HP8453 spectrophotometer. Solutions containing samples of photosensitizers were dissolved in the selected solvents in quartz cuvettes. Using measured absorbance for various concentrations of bacteriochlorins, either in dimethyl sulfoxide (DMSO) or in ethanol, the molar absorption coefficients were determined from Beer's law. Fluorescence emission spectra were recorded from 700 nm to 800 nm with excitation at λ ≈ 505 nm. The excitation and emission slits were both set to 8 nm and scanning speed to 50 nm/min. Scheme 1. Chemical structure of investigated halogenated bacteriochlorin derivatives.

Spectroscopic Studies
Electronic absorption spectra were recorded in a Hewlett Packard HP8453 spectrophotometer. Solutions containing samples of photosensitizers were dissolved in the selected solvents in quartz cuvettes. Using measured absorbance for various concentrations of bacteriochlorins, either in dimethyl sulfoxide (DMSO) or in ethanol, the molar absorption coefficients were determined from Beer's law. Fluorescence emission spectra were recorded from 700 nm to 800 nm with excitation at λ ≈ 505 nm. The excitation and emission slits were both set to 8 nm and scanning speed to 50 nm/min. Fluorescence spectra were recorded with a Perkin Elmer Fluorescence Spectrometer LS 55 (Perkin Elmer, Waltham, MA, USA). Fluorescence lifetimes were determined using a Time-Correlated Single Photon Counting (TCSPC) mode using the FluoroLog-3 Spectrophotometer (Horiba Jobin Yvon, Glasgow, UK).
The instrument was equipped with 340 nm picoseconds-pulsed light-emitting diodes (LEDs) as the excitation source in the MCS mode. During measurements, the Instrument Response Function (IRF) was obtained from a non-fluorescence suspension of colloidal silica (LUDOX 30%, Sigma Aldrich, Schnelldorf, Germany), held in a 10 mm path length quartz cell, and was considered to be wavelength independent. All lifetimes were fit to a χ 2 value of less than 1.1, and with residuals trace symmetrically distributed around the zero axes. Fluorescence quantum yield for F 2 BOH and Cl 2 BHep in DMSO was determined using the comparative method according to the Equation (1): where F and F Std are the areas under the fluorescence emission curves of the sample and the standard, A and A Std are the absorbance of the sample and the standard, and η/ηStd are the refractive indices of the solvent used for the sample and the standard, respectively. Redaporfin (Φ FStd = 0.138) was employed as the standard. The absorbance of the solutions at the excitation wavelength was in the range of 0.02.

ROS Detection with Fluorescent Probes
3'-p-(aminophenyl)fluorescein (APF) and 3'-p-(hydroxyphenyl)fluorescein (HPF) were used to detect hydroxyl radicals. Singlet Oxygen Sensor Green®(SOSG) was applied as a probe for singlet oxygen. Dihydroethidinum (DHE) was used to identify the superoxide ion. All probes were employed for the detection of various ROSs after the illumination of the photosensitizer. PS solutions were diluted to a final concentration of 5 µM per well. A tested fluorescent probe was added at a final concentration of 10 µM. PS solutions were irradiated for various time intervals with a red light from the LED-based device (735 ± 20 nm). A microplate reader (Tecan, Infinite M200 Reader, Tecan, Männedorf, Switzerland) was used for the acquisition of fluorescence signal immediately before and after illumination. APF fluorescence emission at 515 nm was determined upon excitation at 490 nm. For SOSG, the corresponding values were 525 and 505 nm, and 480 nm and 580 nm for DHE.

Photodecomposition Experiments
The photostability of bacteriochlorins was investigated following the measurement of their optical absorption in 5 µM solutions prepared in DMSO and diluted in phosphate-buffered saline (PBS) (the DMSO content did not exceed 0.5%). Continuous irradiation of an aerated solution was carried out using a LED at 735 ± 20 nm. The reaction progress was monitored by electronic spectroscopy using a Hewlett Packard HP8453 spectrophotometer.

LogP Determination
The n-octanol/PBS partition coefficients (logP) were determined following the shake-flask method. The photosensitizers were dissolved in the mixture of n-octanol previously saturated with a solution of PBS and the same volume of PBS saturated with n-octanol. After the addition of the samples of each bacteriochlorin, the solutions were mixed on a vortex device (horizontally) and then the phases were separated by centrifugation (15 min, 8000 rpm, RT). Next, the PBS/n-octanol phase was taken and diluted to obtain 0.5% of PBS/n-octanol content in the final solution. This solution was left into the ultrasonic bath. The fluorescence of each solution was measured using a Fluorescence Spectrometer LS 55 (Perkin Elmer, Waltham, MA, USA) and compared with the calibration curve to obtain the concentration of the photosensitizer. Partition coefficients were calculated from the ratio c oct /c PBS , where c oct and c PBS are the concentrations of the porphyrin derivatives in the n-octanol and in the PBS.

Photosensitizer Formulation
For the biological studies, due to the reduced solubility of Cl 2 BHep in water, the Pluronic-based micelles were applied to deliver it either to cells in vitro or to tumors in vivo. The formulation was prepared by a thin-film hydration method. Dynamic Light Scattering (DLS) using a Malvern Zetaziser Nano ZS system was employed to measure the hydrodynamic diameters (D H ), size distributions and zeta potentials (ζ) of polymeric micelles. Cl 2 BHep concentrations in the formulations were determined and controlled by UV−vis spectrophotometry. Results are reported as the mean of three independent measurements with three different samples (n = 3). The weight percentage of Pluronic-based formulations was determined spectrophotometrically using the absorption calibration curves generated from the Cl 2 BHep standard solutions at known concentrations, as well as DLS measurements. Drug loading content (DL) and drug encapsulating efficiency (EE) were calculated based on the Equations (2) and (3)  Electronic absorption spectra were recorded by HP8453 spectrophotometer using a 1 cm path-length quartz cell.

Cell Cultures
Murine colon carcinoma (CT26) cells were grown in full-strength Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose, L-glutamine, sodium pyruvate and 3.7 g/L NaHCO 3 (BioTech) with the addition of 10% fetal bovine serum (FBS, PAN-Biotech, Aidenbach, Germany) and supplemented by antibiotics (penicillin/streptomycin, PAN-Biotech, Aidenbach, Germany). The cells were cultured in incubators maintained at 37 • C with 5% CO 2 under fully humidified conditions. All experiments were performed on cells in the logarithmic phase of growth. Media were replaced every two days and cells were subcultured using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA).

Cellular Uptake
CT26 cells were seeded on 96-plate microplate (10 4 per well). After 24 h, the cells were incubated with various PS concentrations for time intervals from 2 h up to 24 h. Bacteriochlorin solutions were prepared by diluting stock solutions in DMSO or in micellar formulations (for Cl 2 BHep) with the culture medium to the desired final concentration (5 µM). The highest concentration of DMSO in the medium did not exceed 0.5%. After incubation, the cells were washed twice with PBS and solubilized in 30 µL of Triton X-100 and 70 µL of DMSO/ethanol solution (1:3). The retention of cell-associated PSs was detected by fluorescence (λ exc = 505, λ em = 750 nm) with the microplate reader (Tecan Infinite M200 Reader, Tecan, Männedorf, Switzerland).

Cytotoxicity and Cell Survival Assay
The MTT (3-(4,5dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay was used to quantify cell survival and PS-mediated cytotoxicity. After the cells were attached to the microplates, PS solutations at concentrations of 0 to 100 µM were added to the cell medium. Cells were incubated for the appropriate time in the dark. Next, the PS solution of each well was removed, cells were washed in PBS and fresh culture medium supplemented with FBS and antibiotics was added to each well, and cells were returned to the incubator for 24 h. MTT dissolved in PBS at content 10% of final solution was added to each well, and the microplates were further incubated for ca. 3 h. Then, the medium was re-extracted four more times using the procedure described above to ensure complete recovery of the drug. The extracts were pooled and the fluorescence analysis of the extracts was done. The samples were excited at 505 nm, and the fluorescence spectra were recorded in a range of 600−800 nm. The amount of Cl 2 BHep in the tissues was reported as the average from four animals (with the standard error of the mean (SEM)). The concentration of Cl 2 BHep was estimated from the fluorescence intensity of this bacteriochlorin in samples with known concentrations.

Spectroscopic Characterization of Photosensitizers
All investigated photosensitizers exhibit the characteristic bacteriochlorin absorption spectrum with a broad near-UV Soret, a narrow band in the green (≈ 510 nm) and a near-infrared Qy band (≈ 745 nm) of high intensity that is crucial for PDT. Representative absorption and fluorescence spectra recorded in DMSO are shown in Figure 1. Table 2 summarizes the important photophysical properties of these photosensitizers. The high molar absorption coefficients of these photosensitizers enable their use at much lower concentrations than their porphyrin and chlorin analogs [5]. Moreover, their absorption of NIR light allows for the use of light that penetrates more deeply into human tissues than red light. Scheme 2. Scheme for carrying out immunological analyses using the MAGPIX system.

Statistical Analysis
In both physicochemical and photochemical studies, as well as in vitro biological activity evaluation, the results are expressed as an average ± the standard error of the mean (SEM). The sample size in biological tests in vitro was n = 6-12 in each experimental group. All experiments were repeated independently at least three times. The Student's t-test or Welch's Mann-Whitney U-test (Luminex analysis) was used for the determination of statistical significance. Results were considered as statistically significant with a confidence level of 95% (p < 0.05). Statistical analysis was performed with the STATISTICA 12.5 (StatSoft, Kraków, Poland).

Spectroscopic Characterization of Photosensitizers
All investigated photosensitizers exhibit the characteristic bacteriochlorin absorption spectrum with a broad near-UV Soret, a narrow band in the green (≈ 510 nm) and a near-infrared Q y band (≈ 745 nm) of high intensity that is crucial for PDT. Representative absorption and fluorescence spectra recorded in DMSO are shown in Figure 1. Table 2 summarizes the important photophysical properties of these photosensitizers. The high molar absorption coefficients of these photosensitizers enable their use at much lower concentrations than their porphyrin and chlorin analogs [5]. Moreover, their absorption of NIR light allows for the use of light that penetrates more deeply into human tissues than red light.  The fluorescence lifetimes of F2BOH and F2BMet are comparable. This is due to their structural similarity and the presence of eight fluorine atoms in the phenyl rings located in the meso positions of the tetrapyrrole macrocycle. Cl2BHep is characterized by a much shorter fluorescence lifetime, as expected from the presence of eight chlorine atoms and their higher spin-orbital coupling constants (ζ) than fluorine atoms. The difference in fluorescence quantum yields can be assigned to the same effect. Cl2BHep indicates the lowest ΦF value among other tested compounds (ΦF = 0.008). The relatively high value of ΦF determined for F2MBet suggests the use of this PS in photodiagnosis.

ROS Detection by Fluorescent Probes
The ability of bacteriochlorins to produce different ROSs after illumination was monitored using APF, HPF, DHE and SOSG fluorescent probes. Figure 2 shows that all investigated bacteriochlorins activate probes specific for singlet oxygen as well as for oxygen-centered radicals. F2BOH and F2BMet tend to give lower SOSG fluorescence intensities than Cl2BHep (Figure 2d), which is consistent with the higher singlet oxygen quantum yield of Cl2BHep presented in Table 2. Cl2BHep also tends to give higher APF and HPF fluorescences than other bacteriochlorins, suggesting that this derivative is the most effective in producing oxygen-centered radicals (Figures 2a-b). This is consistent with determinations of the triplet quantum yields of chlorinated and fluorinated tetraphenylbacteriochlorin derivatives which show that the triplet quantum yields of chlorinated derivatives reach unity, whereas those of the fluorinated compounds do not seem to exceed 0.8 [21][22][23]. All bacteriochlorins studied here activate dihydroethidium (DHE), thereby indicating the presence of a superoxide anion, as shown in Figure 2c. F2BOH led to higher activations than F2BMet and Cl2BHep. It will be shown below that, under the same condition, F2BOH is substantially decomposed. It is possible that this decomposition affects the probe. The balance between type I (hydroxyl radicals) and type II (singlet oxygen) photochemical mechanisms is thought to play an important role in the PDT activity because the hydroxyl radicals are the most cytotoxic species, and are expected to be a major contributor to tumor damage in PDT. Tookad Soluble [50], which is also a bacteriochlorin derivative, acts mainly via type I photochemistry in aqueous media [51,52]. This PS forms both  The fluorescence lifetimes of F 2 BOH and F 2 BMet are comparable. This is due to their structural similarity and the presence of eight fluorine atoms in the phenyl rings located in the meso positions of the tetrapyrrole macrocycle. Cl 2 BHep is characterized by a much shorter fluorescence lifetime, as expected from the presence of eight chlorine atoms and their higher spin-orbital coupling constants (ζ) than fluorine atoms. The difference in fluorescence quantum yields can be assigned to the same effect. Cl 2 BHep indicates the lowest Φ F value among other tested compounds (Φ F = 0.008). The relatively high value of Φ F determined for F 2 MBet suggests the use of this PS in photodiagnosis.

ROS Detection by Fluorescent Probes
The ability of bacteriochlorins to produce different ROSs after illumination was monitored using APF, HPF, DHE and SOSG fluorescent probes. Figure 2 shows that all investigated bacteriochlorins activate probes specific for singlet oxygen as well as for oxygen-centered radicals. F 2 BOH and F 2 BMet tend to give lower SOSG fluorescence intensities than Cl 2 BHep (Figure 2d), which is consistent with the higher singlet oxygen quantum yield of Cl 2 BHep presented in Table 2. Cl 2 BHep also tends to give higher APF and HPF fluorescences than other bacteriochlorins, suggesting that this derivative is the most effective in producing oxygen-centered radicals (Figure 2a,b). This is consistent with determinations of the triplet quantum yields of chlorinated and fluorinated tetraphenyl-bacteriochlorin derivatives which show that the triplet quantum yields of chlorinated derivatives reach unity, whereas those of the fluorinated compounds do not seem to exceed 0.8 [21][22][23]. All bacteriochlorins studied here activate dihydroethidium (DHE), thereby indicating the presence of a superoxide anion, as shown in Figure 2c. F 2 BOH led to higher activations than F 2 BMet and Cl 2 BHep. It will be shown below that, under the same condition, F 2 BOH is substantially decomposed. It is possible that this decomposition affects the probe. The balance between type I (hydroxyl radicals) and type II (singlet oxygen) photochemical mechanisms is thought to play an important role in the PDT activity because the hydroxyl radicals are the most cytotoxic species, and are expected to be a major contributor to tumor damage in PDT. Tookad Soluble [50], which is also a bacteriochlorin derivative, acts mainly via type I photochemistry in aqueous media [51,52]. This PS forms both superoxide and hydroxyl radicals upon illumination and the reaction of superoxide with nitric oxide in tumor blood vessels was proposed as the major factor related to the antivascular effect induced by Tookad-V-PDT [51,53]. The propensity of halogenated bacteriochlorins to produce ROS via a type I mechanism was attributed to the formation of a charge-transfer complex with molecular oxygen [3].
J. Clin. Med. 2020, 9,8 10 of 25 superoxide and hydroxyl radicals upon illumination and the reaction of superoxide with nitric oxide in tumor blood vessels was proposed as the major factor related to the antivascular effect induced by Tookad-V-PDT [51,53]. The propensity of halogenated bacteriochlorins to produce ROS via a type I mechanism was attributed to the formation of a charge-transfer complex with molecular oxygen [3].

Photostability
The photodecomposition quantum yield Φpb is a measure of photostability. According to Table  2, F2BOH is the least stable bacteriochlorin of this series. This can also be appreciated in Figure 3. It was shown that bacteriochlorins with phenylsulfonic groups have lower oxidation potentials than bacteriochlorins with phenylsulfonamide groups, and this is associated with more facile oxidation [21][22][23]. Figure 3b shows that the disappearance of the bacteriochorin bands at 375, 510 and 743 nm is not accompanied by corresponding changes in the shoulder at 410 nm or in the small band at 660 nm, both assigned to small chlorin contamination in this sample. This suggests that the photodecomposition of F2BOH will eventually lead to the opening of the macrocycle. This photodecomposition is likely triggered by the ROS produced by F2BOH, and may lead to other radical intermediates that react with fluorescent probes, namely DHE. In contrast, the sulfonamide bacteriochlorins are remarkably stable. Generation of reactive oxygen species (ROSs) in bacteriochlorin solution (in phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO) did not exceed 0.5%) assessed by the fluorescence probes selective towards: (a) overall radical species (APF), (b) hydroxyl radicals (HPF), (c) superoxide radical anion (DHE) or (d) SOSG specific for singlet oxygen. The concentrations of the probes were initially 10 µM, and bacteriochlorin was 5 µM in each sample. Samples were irradiated with a 735 ± 20 nm LED light. Data are presented as mean ± the standard error of the mean (SEM) (N = 12).

Photostability
The photodecomposition quantum yield Φ pb is a measure of photostability. According to Table 2, F 2 BOH is the least stable bacteriochlorin of this series. This can also be appreciated in Figure 3. It was shown that bacteriochlorins with phenylsulfonic groups have lower oxidation potentials than bacteriochlorins with phenylsulfonamide groups, and this is associated with more facile oxidation [21][22][23]. Figure 3b shows that the disappearance of the bacteriochorin bands at 375, 510 and 743 nm is not accompanied by corresponding changes in the shoulder at 410 nm or in the small band at 660 nm, both assigned to small chlorin contamination in this sample. This suggests that the photodecomposition of F 2 BOH will eventually lead to the opening of the macrocycle. This photodecomposition is likely triggered by the ROS produced by F 2 BOH, and may lead to other radical intermediates that react with fluorescent probes, namely DHE. In contrast, the sulfonamide bacteriochlorins are remarkably stable.

Lipophilicity as Assessed by logP Determinations
n-Octanol:PBS partition coefficients are a convenient measure of polarity and are related to solubility in biocompatible solvents. The POW values determined using the shake-flask method are presented in Table 2. The obtained logP values are similar to those previously reported [48]. The logP values range from -1.4 to 4.5, meaning that F2BOH is soluble in water, but Cl2BHep is insoluble in water. Fluorine and chlorine atoms present in the ortho-position of the phenyl group have a similar effect on the photostability of the studied compounds. On the other hand, substituents in the metaposition determine their hydrophilicity. Therefore, the most hydrophobic compound is a derivative containing n-heptyl chain-Cl2BHep. Moreover, such large differences in polarity are expected to lead to very different biological effects [54].

Biological Studies -Optimization of PS Formulation
Formulations of F2BMet in Pluronic poloxamers were recently described [12] and motivated the development of similar formulations fo Cl2BHep. Absorption spectra of Cl2BHep in PBS with < 0.5% DMSO and encapsulated in polymer micelles are shown in Figure 4. The encapsulation of this bacteriochlorin derivative significantly increases the sharpness of the bands, suggesting disaggregation in P123 micelles. Particle size distribution estimated by DLS (Figure 4b) shows that the hydrodynamic diameter of Cl2BHep-P123 micelles prepared in PBS at pH 7.4 is ca. 27 nm with a relatively narrow and homogeneous size distribution. Similarly to P123 micelles prepared for F2BMet described previously [12], the calculated (see equations above) encapsulation efficacy (EE) reached ca. 87% with 12.12 (wt %) drug loading content (DL). The zeta potential for Cl2BHep was slightly negative and reached ζ = −1.07. These data indicate that P123 gave the stable structures, with the ability to form micelles and to provide a slightly hydrophobic environment to maintain the photosensitizer in the monomeric form [12]. P123 micelles may increase cellular uptake, efficient photogeneration of hydroxyl radicals, as well as preferable tissue distribution and pharmacokinetics [12].

Lipophilicity as Assessed by logP Determinations
n-Octanol:PBS partition coefficients are a convenient measure of polarity and are related to solubility in biocompatible solvents. The P OW values determined using the shake-flask method are presented in Table 2. The obtained logP values are similar to those previously reported [48]. The logP values range from −1.4 to 4.5, meaning that F 2 BOH is soluble in water, but Cl 2 BHep is insoluble in water. Fluorine and chlorine atoms present in the ortho-position of the phenyl group have a similar effect on the photostability of the studied compounds. On the other hand, substituents in the meta-position determine their hydrophilicity. Therefore, the most hydrophobic compound is a derivative containing n-heptyl chain-Cl 2 BHep. Moreover, such large differences in polarity are expected to lead to very different biological effects [54].

Biological Studies-Optimization of PS Formulation
Formulations of F 2 BMet in Pluronic poloxamers were recently described [12] and motivated the development of similar formulations fo Cl 2 BHep. Absorption spectra of Cl 2 BHep in PBS with < 0.5% DMSO and encapsulated in polymer micelles are shown in Figure 4. The encapsulation of this bacteriochlorin derivative significantly increases the sharpness of the bands, suggesting disaggregation in P123 micelles. Particle size distribution estimated by DLS (Figure 4b) shows that the hydrodynamic diameter of Cl 2 BHep-P123 micelles prepared in PBS at pH 7.4 is ca. 27 nm with a relatively narrow and homogeneous size distribution. Similarly to P123 micelles prepared for F 2 BMet described previously [12], the calculated (see equations above) encapsulation efficacy (EE) reached ca. 87% with 12.12 (wt %) drug loading content (DL). The zeta potential for Cl 2 BHep was slightly negative and reached ζ = −1.07. These data indicate that P123 gave the stable structures, with the ability to form micelles and to provide a slightly hydrophobic environment to maintain the photosensitizer in the monomeric form [12]. P123 micelles may increase cellular uptake, efficient photogeneration of hydroxyl radicals, as well as preferable tissue distribution and pharmacokinetics [12].

Cellular Uptake of Bacteriochlorins
The time-dependent accumulation of F2BOH, F2BMet and Cl2BHep (in PBS and in P123 micelles) in CT26 cells exposed to a 5 μM photosensitizer solution are shown in Figure 5a. In vitro activity of investigated photosensitizers: (a) time-dependent uptake of halogenated bacteriochlorins at 5 μM by CT26 cells determined based on fluorescence measurements of cellular extracts; (b) cytotoxicity in the dark toward CT26 cells determined by MTT assay after optimal incubation time estimated in uptake studies; (c) photodynamic efficacy of F2BOH, F2BMet and Cl2BHep after their incubation at 5 μM with CT26 for optimal time and irradiation with 735 ± 20 nm LED light; (d) phototoxicity of F2BOH at 5 μM without washing the medium before illumination, and of Cl2BHep encapsulated in P123 micelles at 5 μM. Data are presented as mean ± SEM (N = 12), * p < 0.05.

Cellular Uptake of Bacteriochlorins
The time-dependent accumulation of F 2 BOH, F 2 BMet and Cl 2 BHep (in PBS and in P123 micelles) in CT26 cells exposed to a 5 µM photosensitizer solution are shown in Figure 5a.

Cellular Uptake of Bacteriochlorins
The time-dependent accumulation of F2BOH, F2BMet and Cl2BHep (in PBS and in P123 micelles) in CT26 cells exposed to a 5 μM photosensitizer solution are shown in Figure 5a. In vitro activity of investigated photosensitizers: (a) time-dependent uptake of halogenated bacteriochlorins at 5 μM by CT26 cells determined based on fluorescence measurements of cellular extracts; (b) cytotoxicity in the dark toward CT26 cells determined by MTT assay after optimal incubation time estimated in uptake studies; (c) photodynamic efficacy of F2BOH, F2BMet and Cl2BHep after their incubation at 5 μM with CT26 for optimal time and irradiation with 735 ± 20 nm LED light; (d) phototoxicity of F2BOH at 5 μM without washing the medium before illumination, and of Cl2BHep encapsulated in P123 micelles at 5 μM. Data are presented as mean ± SEM (N = 12), * p < 0.05. In vitro activity of investigated photosensitizers: (a) time-dependent uptake of halogenated bacteriochlorins at 5 µM by CT26 cells determined based on fluorescence measurements of cellular extracts; (b) cytotoxicity in the dark toward CT26 cells determined by MTT assay after optimal incubation time estimated in uptake studies; (c) photodynamic efficacy of F 2 BOH, F 2 BMet and Cl 2 BHep after their incubation at 5 µM with CT26 for optimal time and irradiation with 735 ± 20 nm LED light; (d) phototoxicity of F 2 BOH at 5 µM without washing the medium before illumination, and of Cl 2 BHep encapsulated in P123 micelles at 5 µM. Data are presented as mean ± SEM (N = 12), * p < 0.05.
The results show that for all tested compounds, a gradual, time-dependent increase in PS accumulation was observed. The optimal accumulation for each bacteriochlorin ranged from 18 to 24 h, and then the uptake decreases, or a plateau phase is observed. F 2 BMet (log P = 1.9) showed the most effective accumulation in cells at a concentration of 24 pM per cell after 20 h of incubation. F 2 BOH is deprotonated in the culture medium (log P = −1.4), and its negative charges limit its ability to penetrate cell membranes, leading to a relatively low accumulation in cells (3.88 pM per cell). For this type of compound, penetration into cells is mainly through the endocytosis. Cl 2 BHep (log P ≈ 4.5) is likely to aggregate in the medium which limits its bioavailability and justifies its low concentration in cells (ca. 7.5 pM). The encapsulation of Cl 2 BHep in P123 micelles improves the cellular uptake (22 pM), especially at shorter incubation times (12 h).
The results also indicate very low cytotoxic behavior in the dark in the whole wide range of PS concentrations investigated ( Figure 5b) and pronounced phototoxicity especially for F 2 BMet (Figure 5c). Based on these data, it can be concluded that without the use of micelles, the largest photodynamic effect was noted for F 2 BMet, followed by Cl 2 BHep and finally by F 2 BOH. This is also the order of their uptake (Figure 5a). The lower uptake and lower phototoxicity of F 2 BOH can be overcome by illuminating the cells after the incubation with this photosensitizer, but without washing the photosensitizer molecules that remain in the culture medium. This procedure has a close analogy with V-PDT, where the PS is present in the blood, but not inside the cells. The results obtained in these conditions are presented in Figure 5d as F 2 BOH V-PDT. The possible aggregation of Cl 2 BHep can be avoided using Pluronic formulations. Figure 5d also shows that a P123 formulation of Cl 2 BHep significantly increases its phototoxicity without increasing the cytotoxicity in the dark (Figure 5b) The lethal doses of light causing the killing of 50% of the cancer cells population (LLD 50 ) of the three photosensitizers are presented in Table 3.

Intracellular Localization-CLSM Imaging
The intracellular localization in CT26 cells of F 2 BOH, F 2 BMet and Cl 2 BHep was investigated by confocal laser scanning microscopy (CLSM). Each of these photosensitizers was incubated with a specific probe for mitochondria (MitoTracker), lysosomes (LysoTracker) and endoplasmic reticulum (ER-Tracker). The intracellular localization of F 2 BOH and F 2 BMet in different cell lines has been published before [21][22][23]41]. The more hydrophilic bacteriochlorin is accumulated in lysosomes, whereas the amphiphilic bacteriochlorin is mostly found in the ER (and Golgi), with a smaller localization in the mitochondria. The overlapped images corresponding to Cl 2 BHep are shown in Figure 6, in which the fluorescence of the bacteriochlorin is shown in red, and the fluorescence of the organelle-specific probe is shown in green (ER, mitochondria) or in yellow for lysosomes, respectively. The nuclei were also stained with Hoechst33342 and indicate the characteristic blue fluorescence. The lipophilic Cl 2 BHep possesses a high degree of localization in mitochondria and lysosomes, and a slightly lower one in the ER. The higher subcellular localization of sulfonamide bacteriochlorins in the ER and mitochondria is consistent with their higher photoxicities than F 2 BOH. It is worth to notice that the localization of a photosensitizer is important to determine the initial targets of PDT [18,55,56]. Furthermore, to confirm the colocalization of Cl2BHep (red signal) with each organelle-specific probe (green signal) more clearly, the spectral profiles for these components were defined. The obtained profiles are presented in Figure 7. Localization of hydrophobic PSs in mitochondria may result from the influence of mitochondrial membrane potential and lipid bilayer membrane. Moreover, P123 strongly attenuated the uptake and photocytotoxicity of hydrophobic PSs and redirected the cellular uptake to endocytosis. For example, thiamine pyrophosphate (ThPP) solubilized with P123 was found to accumulate in endocytic vesicles, indicating endocytosis as the main mechanism for the cellular uptake of P123-solubilized Furthermore, to confirm the colocalization of Cl 2 BHep (red signal) with each organelle-specific probe (green signal) more clearly, the spectral profiles for these components were defined. The obtained profiles are presented in Figure 7. Furthermore, to confirm the colocalization of Cl2BHep (red signal) with each organelle-specific probe (green signal) more clearly, the spectral profiles for these components were defined. The obtained profiles are presented in Figure 7. Localization of hydrophobic PSs in mitochondria may result from the influence of mitochondrial membrane potential and lipid bilayer membrane. Moreover, P123 strongly attenuated the uptake and photocytotoxicity of hydrophobic PSs and redirected the cellular uptake to endocytosis. For example, thiamine pyrophosphate (ThPP) solubilized with P123 was found to accumulate in endocytic vesicles, indicating endocytosis as the main mechanism for the cellular uptake of P123-solubilized Localization of hydrophobic PSs in mitochondria may result from the influence of mitochondrial membrane potential and lipid bilayer membrane. Moreover, P123 strongly attenuated the uptake and photocytotoxicity of hydrophobic PSs and redirected the cellular uptake to endocytosis. For example, thiamine pyrophosphate (ThPP) solubilized with P123 was found to accumulate in endocytic vesicles, indicating endocytosis as the main mechanism for the cellular uptake of P123-solubilized THPP. Berg and co-workers found that the hydrophobic 5,10,15,20-tetrakis(4-hydroxyohenyl)porphine solubilized with P123 accumulated in endocytic vesicles, and suggested that in this case, the endocytosis is the main mechanism for cellular uptake [57]. The accumulation of the photosensitizer in certain specific organelles (mitochondria, endoplasmic reticulum or Golgi) is thought to lead to the more efficient triggering of cell death upon illumination [17,18,56].

In Vivo Studies: PDT Protocol Optimization-Biodistribution
Biodistribution and pharmacokinetic studies of is done in [24] and amphiphilic F 2 BMet [15,22] were reported recently, and reveal the highest tumor-to-muscle or -to-skin ratio immediately after PS intravenous administration [15,22,24]. F 2 BOH is characterized by high blood concentration after intravenous administration, relatively low tissue accumulation and rapid elimination time (44 h). Its i.v. injection does not require the use of any carriers and is done in PBS [24]. On the other hand, F 2 BMet is effectively administered in micellar form based on the low content of the solubilizer CrEL (CrEL/EtOH/0.9% NaClaq 0.2:1:98.8 v/v/v), indicating appropriate tissue distribution in many types of tumors [12,15,22,24]. 15 min after administration it shows high blood concentration, which decreases with time, and good affinity to cancer tissues. It is characterized by a slightly longer stay in the body (about 65 h) [15,24] The tissue distribution of Cl 2 BHep is described for the first time and is presented in Figure 8. The tissue distribution of Cl 2 BHep was determined using the extraction of tissue and biological fluids followed by homogenization and fluorescence measurements. THPP. Berg and co-workers found that the hydrophobic 5,10,15,20-tetrakis(4hydroxyohenyl)porphine solubilized with P123 accumulated in endocytic vesicles, and suggested that in this case, the endocytosis is the main mechanism for cellular uptake [57]. The accumulation of the photosensitizer in certain specific organelles (mitochondria, endoplasmic reticulum or Golgi) is thought to lead to the more efficient triggering of cell death upon illumination [17,18,56].

In Vivo Studies: PDT Protocol Optimization -Biodistribution
Biodistribution and pharmacokinetic studies of is done in [24] and amphiphilic F2BMet [15,22] were reported recently, and reveal the highest tumor-to-muscle or -to-skin ratio immediately after PS intravenous administration [15,22,24]. F2BOH is characterized by high blood concentration after intravenous administration, relatively low tissue accumulation and rapid elimination time (44 h). Its i.v. injection does not require the use of any carriers and is done in PBS [24]. On the other hand, F2BMet is effectively administered in micellar form based on the low content of the solubilizer CrEL (CrEL/EtOH/0.9% NaClaq 0.2:1:98.8 v/v/v), indicating appropriate tissue distribution in many types of tumors [12,15,22,24]. 15 minutes after administration it shows high blood concentration, which decreases with time, and good affinity to cancer tissues. It is characterized by a slightly longer stay in the body (about 65 h) [15,24] The tissue distribution of Cl2BHep is described for the first time and is presented in Figure 8. The tissue distribution of Cl2BHep was determined using the extraction of tissue and biological fluids followed by homogenization and fluorescence measurements. The results clearly indicate that this hydrophobic PS can be favorable in C-PDT due to the significantly increased tumor accumulation in the prolonged incubation time (especially 72 h after PS injection). At this point, the PS concentration in tumors is four times higher than that obtained for 15 min, and the double of that for 3 h. The calculated tumor to muscle (T:M) ratios at 15 min., 3 h and 72 h are 3.6, 12.5 and 20.9, respectively. The tumor to skin (T:S) ratios are equal to 1.3, 1.38 and 2.11 at the same time. The highest T:M and T:S ratios at 72 h suggest a superior selectivity for Cl2BHep and a remarkable potential for cellular-targeted protocols with DLI = 72 h (C-PDT).
The summary of the photosensitizers' properties crucial for optimization and appropriate choice of PDT protocols, as well as their systemic administration, are listed in Table 4. Table 4. Properties of investigated bacteriochlorin-based photosensitizers and those used in clinical practice, crucial for determining the photodynamic therapy (PDT) protocols in vivo. The results clearly indicate that this hydrophobic PS can be favorable in C-PDT due to the significantly increased tumor accumulation in the prolonged incubation time (especially 72 h after PS injection). At this point, the PS concentration in tumors is four times higher than that obtained for 15 min, and the double of that for 3 h. The calculated tumor to muscle (T:M) ratios at 15 min., 3 h and 72 h are 3.6, 12.5 and 20.9, respectively. The tumor to skin (T:S) ratios are equal to 1.3, 1.38 and 2.11 at the same time. The highest T:M and T:S ratios at 72 h suggest a superior selectivity for Cl 2 BHep and a remarkable potential for cellular-targeted protocols with DLI = 72 h (C-PDT).
The summary of the photosensitizers' properties crucial for optimization and appropriate choice of PDT protocols, as well as their systemic administration, are listed in Table 4.

PDT Efficacy
We investigated the three basic PDT protocols, involving drug-to-light intervals of 15 min (V-PDT), 3 h (E-PDT) and 72 h (C-PDT) between the injection of the photosensitizer and the illumination. V-PDT aims at the destruction of tumor vessels, limiting the tumor nutrition and controlling tumor growth. In V-PDT, light is applied very soon after PS administration, while it is still within the vascular compartment. This protocol was tested for all the photosensitizers because all of them are mostly in the vascular compartment 15 min after i.v. administration. Alternatively, in tumor endothelial cells-targeted PDT (E-PDT), the drug-to-light interval is increased up to 3 h to take advantage of the accumulation of the photosensitizer both in the tumor microenvironment and tumor tissue. C-PDT with DLI = 72 h aims at the selective destruction of the tumor tissue while sparing the normal tissues. The latter two protocols were not tested for F2BOH because of its faster elimination from the organism. Figure 9 shows the Kaplan-Meier survival plots for all protocols with an indication of the percentage of animals with local tumor control.

PDT Efficacy
We investigated the three basic PDT protocols, involving drug-to-light intervals of 15 min (V-PDT), 3 h (E-PDT) and 72 h (C-PDT) between the injection of the photosensitizer and the illumination. V-PDT aims at the destruction of tumor vessels, limiting the tumor nutrition and controlling tumor growth. In V-PDT, light is applied very soon after PS administration, while it is still within the vascular compartment. This protocol was tested for all the photosensitizers because all of them are mostly in the vascular compartment 15 min after i.v. administration. Alternatively, in tumor endothelial cells-targeted PDT (E-PDT), the drug-to-light interval is increased up to 3 h to take advantage of the accumulation of the photosensitizer both in the tumor microenvironment and tumor tissue. C-PDT with DLI = 72 h aims at the selective destruction of the tumor tissue while sparing the normal tissues. The latter two protocols were not tested for F2BOH because of its faster elimination from the organism. Figure 9 shows the Kaplan-Meier survival plots for all protocols with an indication of the percentage of animals with local tumor control.   Figure 9 shows that tumor growth control was observed in all groups. For F2BOH-V-PDT (Figure 9a), complete long-term remission was obtained for 85% of the treated animals. In the case of F 2 BMet (Figure 9b), the V-PDT protocol was also the most effective, resulting in about 80% of total cures. The F 2 BMet-E-PDT was also highly effective, resulting in 65% long-term cures. F 2 BMet C-PDT was less successful, with only 20% of cured mice. Interestingly, results obtained for the most hydrophobic photosensitizer Cl 2 BHep encapsulated in Pluronic P123 micelles indicate the highest percentage of cures, especially after C-PDT (Figure 9c,d). Cl 2 BHep C-PDT with 74 J/cm 2 led to a very strong systemic inflammatory response that led to the death of some treated mice (35%) in the first 24 h after treatment (thus, survival analysis in Figure 8c starts at 65%). Subsequently, the light dose was de-escalated to obtain a safe protocol. C-PDT with a light dose of 60 J/cm2 remained lethal for 40% of the mice. A light dose of 45 J/cm2 no longer caused strong inflammatory reactions, and long-term cures were obtained for all of the animals. This is one of the most remarkable results with C-PDT protocols at such low light doses. The efficiency of Cl2BHep-C-PDT may be the result of the synergistic interaction of the following factors: (1) the formulation based on the PluronicP123 polymer; (2) appropriate accumulation of the photosensitizer in tumor tissue, which was demonstrated in a tissue distribution study; (3) efficient generation of ROSs, including type I mechanism.

The Inflammatory Changes Observed after PDT
The PDT efficacy presented in the Kaplan-Meier plots indicates that the hydrophilic PS is most effective in V-PDT, the amphiphilic compound is suitable for both V-and E-PDT and the hydrophobic one shows superior activity in C-PDT. It is interesting to further explore the biological responses of these protocols and their possible connection with the very diverse therapeutic outcomes.
We observed differences in acute biological responses to PDT, such as edema and erythema, in vascular and cellular protocols ( Figure 10). Edema usually appeared immediately after V-PDT and E-PDT, attained its maximum 24 h post-treatment and lasted until day 3-4. However, strong erythema and hyperemia were only present in animals subjected to F2BOH-V-PDT. For F2BMet-V-PDT the illumination area is characterized by large swelling, whereas after Cl2BHep-C-PDT, any strong edema is not observed. In the case of the F2BMet-V-PDT edema of the leg with the tumor, which was maximal at 3 h then decreased up to day 4, and then the progressive darkening of the tumor and scab formation are observed. For C-PDT, the inflammatory changes appear milder without significant edema, and tumor involution occurs over a prolonged period with the increase in necrotic area, as opposed to the acute effects observed with V-PDT. The different therapeutic protocols seem to generate a different immune response, which is likely to influence the therapeutic outcome. Figure 9 shows that tumor growth control was observed in all groups. For F2BOH-V-PDT ( Figure  9a), complete long-term remission was obtained for 85% of the treated animals. In the case of F2BMet (Figure 9b), the V-PDT protocol was also the most effective, resulting in about 80% of total cures. The F2BMet-E-PDT was also highly effective, resulting in 65% long-term cures. F2BMet C-PDT was less successful, with only 20% of cured mice. Interestingly, results obtained for the most hydrophobic photosensitizer Cl2BHep encapsulated in Pluronic P123 micelles indicate the highest percentage of cures, especially after C-PDT (Figures 9c-d). Cl2BHep C-PDT with 74 J/cm 2 led to a very strong systemic inflammatory response that led to the death of some treated mice (35%) in the first 24 hours after treatment (thus, survival analysis in Figure 8c starts at 65%). Subsequently, the light dose was de-escalated to obtain a safe protocol. C-PDT with a light dose of 60 J/cm 2 remained lethal for 40% of the mice. A light dose of 45 J/cm 2 no longer caused strong inflammatory reactions, and long-term cures were obtained for all of the animals. This is one of the most remarkable results with C-PDT protocols at such low light doses. The efficiency of Cl2BHep-C-PDT may be the result of the synergistic interaction of the following factors: (1) the formulation based on the PluronicP123 polymer; (2) appropriate accumulation of the photosensitizer in tumor tissue, which was demonstrated in a tissue distribution study; (3) efficient generation of ROSs, including type I mechanism.

The Inflammatory Changes Observed after PDT
The PDT efficacy presented in the Kaplan-Meier plots indicates that the hydrophilic PS is most effective in V-PDT, the amphiphilic compound is suitable for both V-and E-PDT and the hydrophobic one shows superior activity in C-PDT. It is interesting to further explore the biological responses of these protocols and their possible connection with the very diverse therapeutic outcomes.
We observed differences in acute biological responses to PDT, such as edema and erythema, in vascular and cellular protocols ( Figure 10). Edema usually appeared immediately after V-PDT and E-PDT, attained its maximum 24 h post-treatment and lasted until day 3-4. However, strong erythema and hyperemia were only present in animals subjected to F2BOH-V-PDT. For F2BMet-V-PDT the illumination area is characterized by large swelling, whereas after Cl2BHep-C-PDT, any strong edema is not observed. In the case of the F2BMet-V-PDT edema of the leg with the tumor, which was maximal at 3 h then decreased up to day 4, and then the progressive darkening of the tumor and scab formation are observed. For C-PDT, the inflammatory changes appear milder without significant edema, and tumor involution occurs over a prolonged period with the increase in necrotic area, as opposed to the acute effects observed with V-PDT. The different therapeutic protocols seem to generate a different immune response, which is likely to influence the therapeutic outcome. Figure 10. The differences in the inflammatory reaction occurring in BALB/c mice bearing CT26 tumors before and after PDT using optimal protocols with F2BOH, F2BMet, and Cl2BHep as the photosensitizer. Figure 10. The differences in the inflammatory reaction occurring in BALB/c mice bearing CT26 tumors before and after PDT using optimal protocols with F2BOH, F2BMet, and Cl2BHep as the photosensitizer.

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1β or TNFα. However, there are also differences between the protocols. Table 5.
The trend in changes of selected cytokines and chemokines determined after bacteriochlorin-based PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases ( Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and prot evaluating changes in selected cytokines and proinflammatory mediators present in the blood b and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in t of observed trends in all tested biomarkers changes are presented in Table 5. These biomarker inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as as the destruction of the vasculature [61]. A first overview identifies common trends su significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or T However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorin based PDT. The analysis was performed using Luminex multiplexing assay in the serum of individua mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration o biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A signi increase in the concentration of IL-6 was observed in all cases ( Figure 11a). The multidirect nature of IL-6 interactions includes the participation in the activation of antigen-recognizi lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early rea in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-tr mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pro or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as and sensitive, although nonspecific, indicators of various inflammations, which in this case sh be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PS evaluating changes in selected cytokines and proinflammatory mediators present in and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The of observed trends in all tested biomarkers changes are presented in Table 5. These inform on the degree and mode of inflammation, infiltration of neutrophils, tumor ce as the destruction of the vasculature [61]. A first overview identifies common significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, M However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bac based PDT. The analysis was performed using Luminex multiplexing assay in the serum mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each b and  are symbols for the non-significant (higher or lower) tendency in the conc biomarker. The tendencies were estimated in comparison to values obtained in the serum individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukin increase in the concentration of IL-6 was observed in all cases ( Figure 11a). The nature of IL-6 interactions includes the participation in the activation of antige lymphocytes (also LIF) as well as the production of acute-phase proteins indicating t in response to tissue damage. IL-6 expression was strongly and consistently enhanced mice, although the role of IL-6 in PDT-induced inflammation may vary depending on or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be con and sensitive, although nonspecific, indicators of various inflammations, which in be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induc
We further explored the inflammatory responses obse evaluating changes in selected cytokines and proinflammator and 24 h after PDT using the Luminex xMAP technology, as s of observed trends in all tested biomarkers changes are pres inform on the degree and mode of inflammation, infiltration o as the destruction of the vasculature [61]. A first overvie significant increases in IL-6 or KC, and tendencies to inc However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemok based PDT. The analysis was performed using Luminex multip mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or dec and  are symbols for the non-significant (higher or lowe biomarker. The tendencies were estimated in comparison to va individual animals before PDT. Figure 11 focuses on the changes observed in the bloo increase in the concentration of IL-6 was observed in all ca nature of IL-6 interactions includes the participation in th lymphocytes (also LIF) as well as the production of acute-pha in response to tissue damage. IL-6 expression was strongly an mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentratio and sensitive, although nonspecific, indicators of various inf be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases ( Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and pro evaluating changes in selected cytokines and proinflammatory mediators present in the blood and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in of observed trends in all tested biomarkers changes are presented in Table 5. These biomarke inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, a as the destruction of the vasculature [61]. A first overview identifies common trends su significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlori based PDT. The analysis was performed using Luminex multiplexing assay in the serum of individu mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker, and  are symbols for the non-significant (higher or lower) tendency in the concentration biomarker. The tendencies were estimated in comparison to values obtained in the serum of the sam individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A sign increase in the concentration of IL-6 was observed in all cases ( Figure 11a). The multidire nature of IL-6 interactions includes the participation in the activation of antigen-recogniz lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early re in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-t mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pr or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered a and sensitive, although nonspecific, indicators of various inflammations, which in this case s be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different P evaluating changes in selected cytokines and proinflammatory mediators present in and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. Th of observed trends in all tested biomarkers changes are presented in Table 5. Thes inform on the degree and mode of inflammation, infiltration of neutrophils, tumor as the destruction of the vasculature [61]. A first overview identifies common significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, M However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after ba based PDT. The analysis was performed using Luminex multiplexing assay in the serum mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each and  are symbols for the non-significant (higher or lower) tendency in the con biomarker. The tendencies were estimated in comparison to values obtained in the seru individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleuk increase in the concentration of IL-6 was observed in all cases ( Figure 11a). The nature of IL-6 interactions includes the participation in the activation of antig lymphocytes (also LIF) as well as the production of acute-phase proteins indicating in response to tissue damage. IL-6 expression was strongly and consistently enhance mice, although the role of IL-6 in PDT-induced inflammation may vary depending o or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be co and sensitive, although nonspecific, indicators of various inflammations, which in be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with evaluating changes in selected cytokines and proinflammatory mediator and 24 h after PDT using the Luminex xMAP technology, as shown in Sc of observed trends in all tested biomarkers changes are presented in Ta inform on the degree and mode of inflammation, infiltration of neutroph as the destruction of the vasculature [61]. A first overview identifi significant increases in IL-6 or KC, and tendencies to increase IL-10 However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determ based PDT. The analysis was performed using Luminex multiplexing assay mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the l and  are symbols for the non-significant (higher or lower) tendency biomarker. The tendencies were estimated in comparison to values obtaine individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels o increase in the concentration of IL-6 was observed in all cases (Figur nature of IL-6 interactions includes the participation in the activatio lymphocytes (also LIF) as well as the production of acute-phase proteins in response to tissue damage. IL-6 expression was strongly and consisten mice, although the role of IL-6 in PDT-induced inflammation may vary d or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasm and sensitive, although nonspecific, indicators of various inflammation be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PS evaluating changes in selected cytokines and proinflammatory mediators present in and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The of observed trends in all tested biomarkers changes are presented in Table 5. These inform on the degree and mode of inflammation, infiltration of neutrophils, tumor ce as the destruction of the vasculature [61]. A first overview identifies common significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, M However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bac based PDT. The analysis was performed using Luminex multiplexing assay in the serum mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each b and  are symbols for the non-significant (higher or lower) tendency in the conc biomarker. The tendencies were estimated in comparison to values obtained in the serum individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukin increase in the concentration of IL-6 was observed in all cases (Figure 11a). The nature of IL-6 interactions includes the participation in the activation of antige lymphocytes (also LIF) as well as the production of acute-phase proteins indicating t in response to tissue damage. IL-6 expression was strongly and consistently enhanced mice, although the role of IL-6 in PDT-induced inflammation may vary depending on or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be con and sensitive, although nonspecific, indicators of various inflammations, which in be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with d evaluating changes in selected cytokines and proinflammatory mediators and 24 h after PDT using the Luminex xMAP technology, as shown in Sch of observed trends in all tested biomarkers changes are presented in Tab inform on the degree and mode of inflammation, infiltration of neutrophi as the destruction of the vasculature [61]. A first overview identifies significant increases in IL-6 or KC, and tendencies to increase IL-10, However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determin based PDT. The analysis was performed using Luminex multiplexing assay i mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the lev and  are symbols for the non-significant (higher or lower) tendency biomarker. The tendencies were estimated in comparison to values obtained individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of increase in the concentration of IL-6 was observed in all cases (Figure nature of IL-6 interactions includes the participation in the activation lymphocytes (also LIF) as well as the production of acute-phase proteins i in response to tissue damage. IL-6 expression was strongly and consistent mice, although the role of IL-6 in PDT-induced inflammation may vary de or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma and sensitive, although nonspecific, indicators of various inflammations be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Cha
We further explored the inflammatory resp evaluating changes in selected cytokines and proin and 24 h after PDT using the Luminex xMAP techn of observed trends in all tested biomarkers chang inform on the degree and mode of inflammation, i as the destruction of the vasculature [61]. A fi significant increases in IL-6 or KC, and tenden However, there are also differences between the p Table 5. The trend in changes of selected cytokines based PDT. The analysis was performed using Lum mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) incre and  are symbols for the non-significant (hig biomarker. The tendencies were estimated in comp individual animals before PDT. Figure 11 focuses on the changes observed increase in the concentration of IL-6 was observ nature of IL-6 interactions includes the particip lymphocytes (also LIF) as well as the production o in response to tissue damage. IL-6 expression was s mice, although the role of IL-6 in PDT-induced infl or type of tumor treated [61][62][63]. Increased IL-6 co and sensitive, although nonspecific, indicators of be identified with the destruction of tumor tissue [

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different P evaluating changes in selected cytokines and proinflammatory mediators present in and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. Th of observed trends in all tested biomarkers changes are presented in Table 5. Thes inform on the degree and mode of inflammation, infiltration of neutrophils, tumor as the destruction of the vasculature [61]. A first overview identifies common significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, M However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after ba based PDT. The analysis was performed using Luminex multiplexing assay in the serum mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each and  are symbols for the non-significant (higher or lower) tendency in the con biomarker. The tendencies were estimated in comparison to values obtained in the seru individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleuk increase in the concentration of IL-6 was observed in all cases (Figure 11a). The nature of IL-6 interactions includes the participation in the activation of antig lymphocytes (also LIF) as well as the production of acute-phase proteins indicating in response to tissue damage. IL-6 expression was strongly and consistently enhance mice, although the role of IL-6 in PDT-induced inflammation may vary depending o or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be co and sensitive, although nonspecific, indicators of various inflammations, which in be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PS evaluating changes in selected cytokines and proinflammatory mediators present in and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The of observed trends in all tested biomarkers changes are presented in Table 5. These inform on the degree and mode of inflammation, infiltration of neutrophils, tumor ce as the destruction of the vasculature [61]. A first overview identifies common significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, M However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bac based PDT. The analysis was performed using Luminex multiplexing assay in the serum mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each b and  are symbols for the non-significant (higher or lower) tendency in the conc biomarker. The tendencies were estimated in comparison to values obtained in the serum individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukin increase in the concentration of IL-6 was observed in all cases (Figure 11a). The nature of IL-6 interactions includes the participation in the activation of antige lymphocytes (also LIF) as well as the production of acute-phase proteins indicating t in response to tissue damage. IL-6 expression was strongly and consistently enhanced mice, although the role of IL-6 in PDT-induced inflammation may vary depending on or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be con and sensitive, although nonspecific, indicators of various inflammations, which in be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with d evaluating changes in selected cytokines and proinflammatory mediators and 24 h after PDT using the Luminex xMAP technology, as shown in Sch of observed trends in all tested biomarkers changes are presented in Tab inform on the degree and mode of inflammation, infiltration of neutrophi as the destruction of the vasculature [61]. A first overview identifies significant increases in IL-6 or KC, and tendencies to increase IL-10, However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determin based PDT. The analysis was performed using Luminex multiplexing assay i mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the lev and  are symbols for the non-significant (higher or lower) tendency biomarker. The tendencies were estimated in comparison to values obtained individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of increase in the concentration of IL-6 was observed in all cases (Figure nature of IL-6 interactions includes the participation in the activation lymphocytes (also LIF) as well as the production of acute-phase proteins i in response to tissue damage. IL-6 expression was strongly and consistent mice, although the role of IL-6 in PDT-induced inflammation may vary de or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma and sensitive, although nonspecific, indicators of various inflammations be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9, 8 18 3.12.

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and prot evaluating changes in selected cytokines and proinflammatory mediators present in the blood b and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in t of observed trends in all tested biomarkers changes are presented in Table 5. These biomarker inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as as the destruction of the vasculature [61]. A first overview identifies common trends su significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or T However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorin based PDT. The analysis was performed using Luminex multiplexing assay in the serum of individua mice before and after PDT (N = 6-7).  Figure 11 focuses on the changes observed in the blood levels of interleukins. A signi increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirect nature of IL-6 interactions includes the participation in the activation of antigen-recognizi lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early rea in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-tr mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pro or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as and sensitive, although nonspecific, indicators of various inflammations, which in this case sh be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PS evaluating changes in selected cytokines and proinflammatory mediators present in and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The of observed trends in all tested biomarkers changes are presented in Table 5. These inform on the degree and mode of inflammation, infiltration of neutrophils, tumor ce as the destruction of the vasculature [61]. A first overview identifies common significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, M However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bac based PDT. The analysis was performed using Luminex multiplexing assay in the serum mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each b and  are symbols for the non-significant (higher or lower) tendency in the conc biomarker. The tendencies were estimated in comparison to values obtained in the serum individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukin increase in the concentration of IL-6 was observed in all cases (Figure 11a). The nature of IL-6 interactions includes the participation in the activation of antige lymphocytes (also LIF) as well as the production of acute-phase proteins indicating t in response to tissue damage. IL-6 expression was strongly and consistently enhanced mice, although the role of IL-6 in PDT-induced inflammation may vary depending on or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be con and sensitive, although nonspecific, indicators of various inflammations, which in be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induc
We further explored the inflammatory responses obse evaluating changes in selected cytokines and proinflammator and 24 h after PDT using the Luminex xMAP technology, as s of observed trends in all tested biomarkers changes are pres inform on the degree and mode of inflammation, infiltration o as the destruction of the vasculature [61]. A first overvie significant increases in IL-6 or KC, and tendencies to inc However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemok based PDT. The analysis was performed using Luminex multip mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or dec and  are symbols for the non-significant (higher or lowe biomarker. The tendencies were estimated in comparison to va individual animals before PDT. Figure 11 focuses on the changes observed in the bloo increase in the concentration of IL-6 was observed in all ca nature of IL-6 interactions includes the participation in th lymphocytes (also LIF) as well as the production of acute-pha in response to tissue damage. IL-6 expression was strongly an mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentratio and sensitive, although nonspecific, indicators of various inf be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9, 8 18

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and prot evaluating changes in selected cytokines and proinflammatory mediators present in the blood b and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in t of observed trends in all tested biomarkers changes are presented in Table 5. These biomarker inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as as the destruction of the vasculature [61]. A first overview identifies common trends su significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or T However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorin based PDT. The analysis was performed using Luminex multiplexing assay in the serum of individua mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration o biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A signi increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirect nature of IL-6 interactions includes the participation in the activation of antigen-recognizi lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early rea in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-tr mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pro or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as and sensitive, although nonspecific, indicators of various inflammations, which in this case sh be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with d evaluating changes in selected cytokines and proinflammatory mediators and 24 h after PDT using the Luminex xMAP technology, as shown in Sch of observed trends in all tested biomarkers changes are presented in Tab inform on the degree and mode of inflammation, infiltration of neutrophi as the destruction of the vasculature [61]. A first overview identifies significant increases in IL-6 or KC, and tendencies to increase IL-10, However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determin based PDT. The analysis was performed using Luminex multiplexing assay i mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the lev and  are symbols for the non-significant (higher or lower) tendency biomarker. The tendencies were estimated in comparison to values obtained individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of increase in the concentration of IL-6 was observed in all cases (Figure nature of IL-6 interactions includes the participation in the activation lymphocytes (also LIF) as well as the production of acute-phase proteins i in response to tissue damage. IL-6 expression was strongly and consistent mice, although the role of IL-6 in PDT-induced inflammation may vary de or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma and sensitive, although nonspecific, indicators of various inflammations be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8

Examination of Inflammatory Cytokines Cha
We further explored the inflammatory resp evaluating changes in selected cytokines and proin and 24 h after PDT using the Luminex xMAP techn of observed trends in all tested biomarkers chang inform on the degree and mode of inflammation, i as the destruction of the vasculature [61]. A fi significant increases in IL-6 or KC, and tenden However, there are also differences between the p Table 5. The trend in changes of selected cytokines based PDT. The analysis was performed using Lum mice before and after PDT (N = 6-7).

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9,8 18 of 25

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with d evaluating changes in selected cytokines and proinflammatory mediators and 24 h after PDT using the Luminex xMAP technology, as shown in Sch of observed trends in all tested biomarkers changes are presented in Tab inform on the degree and mode of inflammation, infiltration of neutrophi as the destruction of the vasculature [61]. A first overview identifies significant increases in IL-6 or KC, and tendencies to increase IL-10, However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determin based PDT. The analysis was performed using Luminex multiplexing assay i mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the lev and  are symbols for the non-significant (higher or lower) tendency biomarker. The tendencies were estimated in comparison to values obtained individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of increase in the concentration of IL-6 was observed in all cases (Figure nature of IL-6 interactions includes the participation in the activation lymphocytes (also LIF) as well as the production of acute-phase proteins i in response to tissue damage. IL-6 expression was strongly and consistent mice, although the role of IL-6 in PDT-induced inflammation may vary de or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma and sensitive, although nonspecific, indicators of various inflammations be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9, 8

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).  Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).  Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and pro evaluating changes in selected cytokines and proinflammatory mediators present in the blood and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in of observed trends in all tested biomarkers changes are presented in Table 5. These biomarke inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, a as the destruction of the vasculature [61]. A first overview identifies common trends su significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlori based PDT. The analysis was performed using Luminex multiplexing assay in the serum of individu mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker, and  are symbols for the non-significant (higher or lower) tendency in the concentration biomarker. The tendencies were estimated in comparison to values obtained in the serum of the sam individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A sign increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidire nature of IL-6 interactions includes the participation in the activation of antigen-recogniz lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early re in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-t mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pr or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered a and sensitive, although nonspecific, indicators of various inflammations, which in this case s be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with d evaluating changes in selected cytokines and proinflammatory mediators and 24 h after PDT using the Luminex xMAP technology, as shown in Sch of observed trends in all tested biomarkers changes are presented in Tab inform on the degree and mode of inflammation, infiltration of neutrophil as the destruction of the vasculature [61]. A first overview identifies significant increases in IL-6 or KC, and tendencies to increase IL-10, However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determin based PDT. The analysis was performed using Luminex multiplexing assay in mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the lev and  are symbols for the non-significant (higher or lower) tendency biomarker. The tendencies were estimated in comparison to values obtained individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of increase in the concentration of IL-6 was observed in all cases (Figure nature of IL-6 interactions includes the participation in the activation lymphocytes (also LIF) as well as the production of acute-phase proteins in in response to tissue damage. IL-6 expression was strongly and consistentl mice, although the role of IL-6 in PDT-induced inflammation may vary dep or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma and sensitive, although nonspecific, indicators of various inflammations, be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and prot evaluating changes in selected cytokines and proinflammatory mediators present in the blood b and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in t of observed trends in all tested biomarkers changes are presented in Table 5. These biomarker inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as as the destruction of the vasculature [61]. A first overview identifies common trends su significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or T However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorin based PDT. The analysis was performed using Luminex multiplexing assay in the serum of individua mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration o biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A signi increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirect nature of IL-6 interactions includes the participation in the activation of antigen-recognizi lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early rea in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-tr mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pro or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as and sensitive, although nonspecific, indicators of various inflammations, which in this case sh be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PS evaluating changes in selected cytokines and proinflammatory mediators present in and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The of observed trends in all tested biomarkers changes are presented in Table 5. These inform on the degree and mode of inflammation, infiltration of neutrophils, tumor ce as the destruction of the vasculature [61]. A first overview identifies common significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, M However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bac based PDT. The analysis was performed using Luminex multiplexing assay in the serum mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each b and  are symbols for the non-significant (higher or lower) tendency in the conc biomarker. The tendencies were estimated in comparison to values obtained in the serum individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukin increase in the concentration of IL-6 was observed in all cases (Figure 11a). The nature of IL-6 interactions includes the participation in the activation of antige lymphocytes (also LIF) as well as the production of acute-phase proteins indicating t in response to tissue damage. IL-6 expression was strongly and consistently enhanced mice, although the role of IL-6 in PDT-induced inflammation may vary depending on or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be con and sensitive, although nonspecific, indicators of various inflammations, which in be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induc
We further explored the inflammatory responses obse evaluating changes in selected cytokines and proinflammator and 24 h after PDT using the Luminex xMAP technology, as s of observed trends in all tested biomarkers changes are pres inform on the degree and mode of inflammation, infiltration o as the destruction of the vasculature [61]. A first overvie significant increases in IL-6 or KC, and tendencies to inc However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemok based PDT. The analysis was performed using Luminex multip mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or dec and  are symbols for the non-significant (higher or lowe biomarker. The tendencies were estimated in comparison to va individual animals before PDT. Figure 11 focuses on the changes observed in the bloo increase in the concentration of IL-6 was observed in all ca nature of IL-6 interactions includes the participation in th lymphocytes (also LIF) as well as the production of acute-pha in response to tissue damage. IL-6 expression was strongly an mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentratio and sensitive, although nonspecific, indicators of various inf be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induc
We further explored the inflammatory responses obse evaluating changes in selected cytokines and proinflammator and 24 h after PDT using the Luminex xMAP technology, as s of observed trends in all tested biomarkers changes are pres inform on the degree and mode of inflammation, infiltration o as the destruction of the vasculature [61]. A first overvie significant increases in IL-6 or KC, and tendencies to inc However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemok based PDT. The analysis was performed using Luminex multip mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or dec and  are symbols for the non-significant (higher or lowe biomarker. The tendencies were estimated in comparison to va individual animals before PDT. Figure 11 focuses on the changes observed in the bloo increase in the concentration of IL-6 was observed in all ca nature of IL-6 interactions includes the participation in th lymphocytes (also LIF) as well as the production of acute-pha in response to tissue damage. IL-6 expression was strongly an mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentratio and sensitive, although nonspecific, indicators of various inf be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9, 8

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induc
We further explored the inflammatory responses obse evaluating changes in selected cytokines and proinflammator and 24 h after PDT using the Luminex xMAP technology, as s of observed trends in all tested biomarkers changes are pres inform on the degree and mode of inflammation, infiltration o as the destruction of the vasculature [61]. A first overvie significant increases in IL-6 or KC, and tendencies to inc However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemok based PDT. The analysis was performed using Luminex multip mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or dec and  are symbols for the non-significant (higher or lowe biomarker. The tendencies were estimated in comparison to va individual animals before PDT. Figure 11 focuses on the changes observed in the bloo increase in the concentration of IL-6 was observed in all ca nature of IL-6 interactions includes the participation in th lymphocytes (also LIF) as well as the production of acute-pha in response to tissue damage. IL-6 expression was strongly an mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentratio and sensitive, although nonspecific, indicators of various inf be identified with the destruction of tumor tissue [64]. J. Clin. Med. 2020, 9, 8

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS F2BOH F2BMet
Cl2BHep ,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and proto evaluating changes in selected cytokines and proinflammatory mediators present in the blood be and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in t of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as as the destruction of the vasculature [61]. A first overview identifies common trends suc significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or T However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A signif increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirecti nature of IL-6 interactions includes the participation in the activation of antigen-recognizin lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early rea in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-tre mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pro or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as e and sensitive, although nonspecific, indicators of various inflammations, which in this case sh be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should J. Clin. Med. 2020, 9,8 18 of 25

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should J. Clin. Med. 2020, 9,8 18 of 25

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PS evaluating changes in selected cytokines and proinflammatory mediators present in and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The of observed trends in all tested biomarkers changes are presented in Table 5. These inform on the degree and mode of inflammation, infiltration of neutrophils, tumor ce as the destruction of the vasculature [61]. A first overview identifies common significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, M However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bac based PDT. The analysis was performed using Luminex multiplexing assay in the serum mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each b and  are symbols for the non-significant (higher or lower) tendency in the conc biomarker. The tendencies were estimated in comparison to values obtained in the serum individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukin increase in the concentration of IL-6 was observed in all cases (Figure 11a). The nature of IL-6 interactions includes the participation in the activation of antige lymphocytes (also LIF) as well as the production of acute-phase proteins indicating t in response to tissue damage. IL-6 expression was strongly and consistently enhanced mice, although the role of IL-6 in PDT-induced inflammation may vary depending on or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be con and sensitive, although nonspecific, indicators of various inflammations, which in

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64]. .

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols uating changes in selected cytokines and proinflammatory mediators present in the blood before 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms bserved trends in all tested biomarkers changes are presented in Table 5. These biomarkers can rm on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well he destruction of the vasculature [61]. A first overview identifies common trends such as ificant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. ever, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS F2BOH F2BMet
Cl2BHep ,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant ase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional re of IL-6 interactions includes the participation in the activation of antigen-recognizing T phocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction sponse to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated , although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol pe of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early sensitive, although nonspecific, indicators of various inflammations, which in this case should entified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and proto evaluating changes in selected cytokines and proinflammatory mediators present in the blood be and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in t of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as as the destruction of the vasculature [61]. A first overview identifies common trends suc significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or T However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).
,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A signif increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirecti nature of IL-6 interactions includes the participation in the activation of antigen-recognizin lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early rea in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-tre mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pro or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as e and sensitive, although nonspecific, indicators of various inflammations, which in this case sh be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m or type of tumor treated [61][62][63]. Increased IL-6 concentration and sensitive, although nonspecific, indicators of various infl be identified with the destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS F2BOH F2BMet
Cl2BHep  ,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and protocols evaluating changes in selected cytokines and proinflammatory mediators present in the blood before and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms of observed trends in all tested biomarkers changes are presented in Table 5. These biomarkers can inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well as the destruction of the vasculature [61]. A first overview identifies common trends such as significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlorinbased PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual mice before and after PDT (N = 6-7).

PS F2BOH F2BMet
Cl2BHep  ,  indicated significantly (min two-times) increased or decreased the level of each biomarker,  and  are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol J. Clin. Med. 2020, 9, 8 1

Examination of Inflammatory Cytokines Changes Induced by PDT
We further explored the inflammatory responses observed with different PSs and pro evaluating changes in selected cytokines and proinflammatory mediators present in the blood and 24 h after PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in of observed trends in all tested biomarkers changes are presented in Table 5. These biomarke inform on the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, a as the destruction of the vasculature [61]. A first overview identifies common trends su significant increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemokines determined after bacteriochlori based PDT. The analysis was performed using Luminex multiplexing assay in the serum of individu mice before and after PDT (N = 6-7).

PS F2BOH F2BMet
Cl2BHep ,  indicated significantly (min two-times) increased or decreased the level of each biomarker, and  are symbols for the non-significant (higher or lower) tendency in the concentration biomarker. The tendencies were estimated in comparison to values obtained in the serum of the sam individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A sign increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidire nature of IL-6 interactions includes the participation in the activation of antigen-recogniz lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early re in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-t mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, pr or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered a

Examination of Inflammatory Cytokines Changes Induce
We further explored the inflammatory responses observ evaluating changes in selected cytokines and proinflammatory and 24 h after PDT using the Luminex xMAP technology, as sh of observed trends in all tested biomarkers changes are presen inform on the degree and mode of inflammation, infiltration of as the destruction of the vasculature [61]. A first overview significant increases in IL-6 or KC, and tendencies to incr However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemoki based PDT. The analysis was performed using Luminex multiple mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or decr and  are symbols for the non-significant (higher or lower biomarker. The tendencies were estimated in comparison to valu individual animals before PDT. Figure 11 focuses on the changes observed in the blood increase in the concentration of IL-6 was observed in all cas nature of IL-6 interactions includes the participation in the lymphocytes (also LIF) as well as the production of acute-phas in response to tissue damage. IL-6 expression was strongly and mice, although the role of IL-6 in PDT-induced inflammation m 020, 9,8 18 of 25

ination of Inflammatory Cytokines Changes Induced by PDT
rther explored the inflammatory responses observed with different PSs and protocols changes in selected cytokines and proinflammatory mediators present in the blood before ter PDT using the Luminex xMAP technology, as shown in Scheme 2. The results in terms d trends in all tested biomarkers changes are presented in Table 5. These biomarkers can the degree and mode of inflammation, infiltration of neutrophils, tumor cell death, as well truction of the vasculature [61]. A first overview identifies common trends such as increases in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. there are also differences between the protocols.

5.
The trend in changes of selected cytokines and chemokines determined after bacteriochlorin-PDT. The analysis was performed using Luminex multiplexing assay in the serum of individual efore and after PDT (N = 6-7).

F2BOH
F2BMet  ndicated significantly (min two-times) increased or decreased the level of each biomarker,  are symbols for the non-significant (higher or lower) tendency in the concentration of rker. The tendencies were estimated in comparison to values obtained in the serum of the same ual animals before PDT.
11 focuses on the changes observed in the blood levels of interleukins. A significant the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional IL-6 interactions includes the participation in the activation of antigen-recognizing T es (also LIF) as well as the production of acute-phase proteins indicating the early reaction to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated ,

of 25 f Inflammatory Cytokines Changes Induced by PDT
plored the inflammatory responses observed with different PSs and protocols in selected cytokines and proinflammatory mediators present in the blood before using the Luminex xMAP technology, as shown in Scheme 2. The results in terms in all tested biomarkers changes are presented in Table 5. These biomarkers can e and mode of inflammation, infiltration of neutrophils, tumor cell death, as well of the vasculature [61]. A first overview identifies common trends such as s in IL-6 or KC, and tendencies to increase IL-10, MIP-2, MIP-1b or TNFa. also differences between the protocols. d in changes of selected cytokines and chemokines determined after bacteriochlorinanalysis was performed using Luminex multiplexing assay in the serum of individual after PDT (N = 6-7).

BOH
F2BMet  significantly (min two-times) increased or decreased the level of each biomarker,  bols for the non-significant (higher or lower) tendency in the concentration of endencies were estimated in comparison to values obtained in the serum of the same als before PDT.
ses on the changes observed in the blood levels of interleukins. A significant centration of IL-6 was observed in all cases (Figure 11a). The multidirectional ractions includes the participation in the activation of antigen-recognizing T IF) as well as the production of acute-phase proteins indicating the early reaction damage. IL-6 expression was strongly and consistently enhanced in PDT-treated ole of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol eated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early ugh nonspecific, indicators of various inflammations, which in this case should he destruction of tumor tissue [64].

Examination of Inflammatory Cytokines Chang
We further explored the inflammatory respons evaluating changes in selected cytokines and proinfla and 24 h after PDT using the Luminex xMAP technolo of observed trends in all tested biomarkers changes a inform on the degree and mode of inflammation, infil as the destruction of the vasculature [61]. A first significant increases in IL-6 or KC, and tendencie However, there are also differences between the proto Table 5. The trend in changes of selected cytokines and based PDT. The analysis was performed using Lumine mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increase and  are symbols for the non-significant (higher biomarker. The tendencies were estimated in comparis individual animals before PDT. Figure 11 focuses on the changes observed in increase in the concentration of IL-6 was observed nature of IL-6 interactions includes the participatio lymphocytes (also LIF) as well as the production of ac in response to tissue damage. IL-6 expression was stro mice, although the role of IL-6 in PDT-induced inflamm and J. Clin. Med. 2020, 9, 8

Examination of Inflammatory Cytokines Changes Ind
We further explored the inflammatory responses obs evaluating changes in selected cytokines and proinflammato and 24 h after PDT using the Luminex xMAP technology, as of observed trends in all tested biomarkers changes are pre inform on the degree and mode of inflammation, infiltration as the destruction of the vasculature [61]. A first overv significant increases in IL-6 or KC, and tendencies to in However, there are also differences between the protocols. Table 5. The trend in changes of selected cytokines and chemo based PDT. The analysis was performed using Luminex multi mice before and after PDT (N = 6-7).

PS
,  indicated significantly (min two-times) increased or de and  are symbols for the non-significant (higher or low biomarker. The tendencies were estimated in comparison to v individual animals before PDT. Figure 11 focuses on the changes observed in the blo increase in the concentration of IL-6 was observed in all nature of IL-6 interactions includes the participation in lymphocytes (also LIF) as well as the production of acute-ph in response to tissue damage. IL-6 expression was strongly a mice, although the role of IL-6 in PDT-induced inflammation or type of tumor treated [61][62][63]. Increased IL-6 concentrati and sensitive, although nonspecific, indicators of various in be identified with the destruction of tumor tissue [64]. are symbols for the non-significant (higher or lower) tendency in the concentration of biomarker. The tendencies were estimated in comparison to values obtained in the serum of the same individual animals before PDT. Figure 11 focuses on the changes observed in the blood levels of interleukins. A significant increase in the concentration of IL-6 was observed in all cases (Figure 11a). The multidirectional nature of IL-6 interactions includes the participation in the activation of antigen-recognizing T lymphocytes (also LIF) as well as the production of acute-phase proteins indicating the early reaction in response to tissue damage. IL-6 expression was strongly and consistently enhanced in PDT-treated mice, although the role of IL-6 in PDT-induced inflammation may vary depending on the PS, protocol or type of tumor treated [61][62][63]. Increased IL-6 concentration in plasma can be considered as early and sensitive, although nonspecific, indicators of various inflammations, which in this case should be identified with the destruction of tumor tissue [64]. In the experimental groups that underwent PDT, these also exhibited a significant increase in IL-15 level, Figure 11d. In cancer immunotherapy, this cytokine is considered as a strong adjuvant, stimulating cytotoxicity and the proliferation of lymphocytes (cytotoxic, NKT) as well as the proliferation and secretion of antibodies activated by B cells [65]. IL15 is also an important growth factor for memory T lymphocytes, especially CD8+ [66]. The lack of unambiguous changes in IL-13 and especially in IL-10 (with the possible exception of (F2BOH), the V-PDT concentration after PDT ( Figure 10b) using most protocols can be considered a good prognostic factor because it can ultimately lead to inhibition of the cell-type immune response and inflammatory processes [67]. In this respect, F2BOH V-PDT may not provide the best control of the inflammatory process. An increase in the concentration of CC-type chemokines (MCP-1, MIP1α, MIP1β) in the plasma after PDT can be seen (except for the F2BMet V-PDT and C-PDT protocol). A significant increase in the concentration of the inflammatory protein MCP-1 is evidence of increased inflammatory processes within the tumor microenvironment and the dominance of the necrotic pathway of cancer cell death and the infiltration of macrophages and monocytes into the tumor surrounding tissue [68,69].
Several preclinical studies have suggested that suppression of long-term tumor growth following Photofrin-PDT is dependent upon the presence of neutrophils [70]. Neutrophil migration into the treated tumor is associated with an increase in the expression of the chemokines macrophage inflammatory protein MIP-2 and KC. Figure 12 shows that MIP-2 and KC increase significantly after PDT. In particular, V-PDT with both F2BOH and F2BMet causes a large increase in KC that is constitutively expressed and is believed to be involved in basal neutrophil migration. In contrast, MIP-2, the major chemokine released after F2BMet-V-PDT, is inducible and thought to mediate stressor injury-induced neutrophil migration. In the experimental groups that underwent PDT, these also exhibited a significant increase in IL-15 level, Figure 11d. In cancer immunotherapy, this cytokine is considered as a strong adjuvant, stimulating cytotoxicity and the proliferation of lymphocytes (cytotoxic, NKT) as well as the proliferation and secretion of antibodies activated by B cells [65]. IL15 is also an important growth factor for memory T lymphocytes, especially CD8+ [66]. The lack of unambiguous changes in IL-13 and especially in IL-10 (with the possible exception of (F 2 BOH), the V-PDT concentration after PDT (Figure 10b) using most protocols can be considered a good prognostic factor because it can ultimately lead to inhibition of the cell-type immune response and inflammatory processes [67]. In this respect, F 2 BOH V-PDT may not provide the best control of the inflammatory process. An increase in the concentration of CC-type chemokines (MCP-1, MIP1α, MIP1β) in the plasma after PDT can be seen (except for the F 2 BMet V-PDT and C-PDT protocol). A significant increase in the concentration of the inflammatory protein MCP-1 is evidence of increased inflammatory processes within the tumor microenvironment and the dominance of the necrotic pathway of cancer cell death and the infiltration of macrophages and monocytes into the tumor surrounding tissue [68,69].
Several preclinical studies have suggested that suppression of long-term tumor growth following Photofrin-PDT is dependent upon the presence of neutrophils [70]. Neutrophil migration into the treated tumor is associated with an increase in the expression of the chemokines macrophage inflammatory protein MIP-2 and KC. Figure 12 shows that MIP-2 and KC increase significantly after PDT. In particular, V-PDT with both F 2 BOH and F 2 BMet causes a large increase in KC that is constitutively expressed and is believed to be involved in basal neutrophil migration. In contrast, MIP-2, the major chemokine released after F 2 BMet-V-PDT, is inducible and thought to mediate stressor injury-induced neutrophil migration.
The levels of KC and MIP-2 were unchanged in non-treated CT26 tumor-bearing mice and remained the comparable levels before PDT, which additionally indicates that the induction of MIP-2 and KC was not the result of a systemic mediator. Thus, bacteriochlorin-mediated V-PDT induced a time-dependent increase in two neutrophil attractant chemokines, MIP-2 and KC. The optimal protocols with sulfonamide bacteriochlorin (F2BMet-V-PDT and Cl2BHep-C-PDT) are characterized by increased secretion of MIP-1, which is also involved in neutrophilic inflammation. Only in the case of F2BOH-V-PDT can the higher level of MIG (CXCL9) and IP-10 (CXCL10) be detected [71]. Importantly, these chemokines are known to exhibit antitumor activity through strong T-cell chemotaxis and anti-angiogenic (angiostatic) properties. Thus, due to the fact that their production is induced by interferons (IFNγ, inducible protein 10, IP-10, IFNγ-induced chemokine monokine 9, MIG), their increased plasma concentration after PDT indirectly indicates the activity of IFNγ [72]. Figure 13 shows how TNFα had increased in the blood of all groups that were subjected to the optimal scheme of PDT. It is widely known that TNFα upregulation may induce changes in the tumor vasculature and stimulate the antitumor immune response [73]. Moreover, these data can also suggest the activity of macrophages triggering in these cells Toll-like receptor (TLR)-based signal transduction activity resulting in the production of TNFα [61,62].  The levels of KC and MIP-2 were unchanged in non-treated CT26 tumor-bearing mice and remained the comparable levels before PDT, which additionally indicates that the induction of MIP-2 and KC was not the result of a systemic mediator. Thus, bacteriochlorin-mediated V-PDT induced a time-dependent increase in two neutrophil attractant chemokines, MIP-2 and KC.
The optimal protocols with sulfonamide bacteriochlorin (F 2 BMet-V-PDT and Cl 2 BHep-C-PDT) are characterized by increased secretion of MIP-1, which is also involved in neutrophilic inflammation. Only in the case of F 2 BOH-V-PDT can the higher level of MIG (CXCL9) and IP-10 (CXCL10) be detected [71]. Importantly, these chemokines are known to exhibit antitumor activity through strong T-cell chemotaxis and anti-angiogenic (angiostatic) properties. Thus, due to the fact that their production is induced by interferons (IFNγ, inducible protein 10, IP-10, IFNγ-induced chemokine monokine 9, MIG), their increased plasma concentration after PDT indirectly indicates the activity of IFNγ [72]. Figure 13 shows how TNFα had increased in the blood of all groups that were subjected to the optimal scheme of PDT. It is widely known that TNFα upregulation may induce changes in the tumor vasculature and stimulate the antitumor immune response [73]. Moreover, these data can also suggest the activity of macrophages triggering in these cells Toll-like receptor (TLR)-based signal transduction activity resulting in the production of TNFα [61,62]. J. Clin. Med. 2020, 9, 8 20 of 25 The levels of KC and MIP-2 were unchanged in non-treated CT26 tumor-bearing mice and remained the comparable levels before PDT, which additionally indicates that the induction of MIP-2 and KC was not the result of a systemic mediator. Thus, bacteriochlorin-mediated V-PDT induced a time-dependent increase in two neutrophil attractant chemokines, MIP-2 and KC. The optimal protocols with sulfonamide bacteriochlorin (F2BMet-V-PDT and Cl2BHep-C-PDT) are characterized by increased secretion of MIP-1, which is also involved in neutrophilic inflammation. Only in the case of F2BOH-V-PDT can the higher level of MIG (CXCL9) and IP-10 (CXCL10) be detected [71]. Importantly, these chemokines are known to exhibit antitumor activity through strong T-cell chemotaxis and anti-angiogenic (angiostatic) properties. Thus, due to the fact that their production is induced by interferons (IFNγ, inducible protein 10, IP-10, IFNγ-induced chemokine monokine 9, MIG), their increased plasma concentration after PDT indirectly indicates the activity of IFNγ [72]. Figure 13 shows how TNFα had increased in the blood of all groups that were subjected to the optimal scheme of PDT. It is widely known that TNFα upregulation may induce changes in the tumor vasculature and stimulate the antitumor immune response [73]. Moreover, these data can also suggest the activity of macrophages triggering in these cells Toll-like receptor (TLR)-based signal transduction activity resulting in the production of TNFα [61,62]. Figure 13. Concentrations of (a) TNFα; (b) VEGF in the serum of mice before and after optimal PDT protocol with F2BOH (V-PDT), F2BMet (V-PDT) and Cl2BHep (C-PDT) as well as healthy mice and Figure 13. Concentrations of (a) TNFα; (b) VEGF in the serum of mice before and after optimal PDT protocol with F 2 BOH (V-PDT), F 2 BMet (V-PDT) and Cl 2 BHep (C-PDT) as well as healthy mice and mice bearing CT26 tumor controls. Luminex, N = 6-7; each dot represents one individual, line represents a mean; * p < 0.05.
Last, but not least, we examined the vascular endothelial growth factor (VEGF). Figure 12b reveals a more significant decrease of VEGF after vascular-targeted treatment than after C-PDT. This observation suggests that the formation of new vessels within the tumor is inhibited, thereby stopping its further local progression and preventing distant metastases [74].
Taken together, the inflammatory changes observed after PDT and the patterns of inflammatory cytokines changes reveal that water-soluble photosensitizers such as F 2 BOH, generally lead to an increase in the serum level of these cytokines in V-PDT, when compared with less polar photosensitizers. This is consistent with the observation of strong erythema and hyperemia in animals subject to F 2 BOH-V-PDT. This may be related to the preferential association of hydrophilic photosensitizers with albumin, as opposed to the association of the less polar photosensitizers with lipoproteins. The tumor localization of albumin-bound and lipoprotein-bound photosensitizers is likely the cause of the difference in inflammatory reactions, because in the conditions of this study (same drug and light doses, comparable tumor concentration in V-PDT), all the photosensitizers produce similar oxidative stresses (similar light absorption and similar singlet oxygen quantum yields). Interestingly, the cytokine patterns revealed that V-PDT, E-PDT and C-PDT with F 2 BMet are surprisingly similar, although the photosensitizer concentration in the target tissue decreases appreciably with time. This suggests that the re-localization of the photosensitizer in the tumor may compensate for its lower concentration to yields-comparable inflammatory responses.

Summary and Conclusions
We determined physicochemical, spectroscopic and photochemical properties of three bacteriochlorins with widely different polarities (F2BOH, F2BMet, Cl2BHep), and related them with phototoxicity in vitro and PDT efficacy in prototypical PDT regimes. The most important difference between the studied compounds is their hydrophilicity/lipophilicity (logP) caused by a change from sulfonic group, to methylsulfonamide and to heptylsulfonamide ones, respectively. For the reasons discussed above, the chlorinated substituent has a higher singlet oxygen quantum yield (Φ ∆ = 0.63) than the fluorinated compounds (Φ ∆ ranged from 0.43 to 0.44), but this does not make Cl2BHep more phototoxic in vitro. The advantage of having chlorine atoms in the least soluble compound is to help avoid aggregation. The bulky chlorine atoms prevent the bacteriochlorins from sitting on the top of each other and facilitating dissolution.
PDT in vitro is influenced by differences in cellular uptake and subcellular localization. The pharmacokinetics and biodistribution of the photosensitizers determine the PDT regime (V-PDT, E-PDT, C-PDT) most adequate for each one of them. The most effective photosensitizers in V-PDT (DLI = 15 min) have hydrophilic or amphiphilic character, i.e. sulfonic and selected sulfonamide derivatives of bacteriochlorins (F2BOH, logP = −1.4; F2BMet logP = 1.9). The PDT protocol optimized for these photosensitizers led to >80% cure rates. More lipophilic photosensitizers (Cl2BHep, logP ≈ 4.5) proved to be more effective in protocols characterized by a longer DLI of 72 h. Such compounds, due to their limited solubility in the aqueous medium, need specialized formulations, such as encapsulation in Pluronic poloxamers micelles. This may be an additional challenge, but it comes with the advantages of increased stability, selectivity and overall efficacy.
The different PDT regimes lead to different biological responses that can have distinct immunological components and contribute to the overall PDT efficacy. Various degree of local inflammation is observed after different PDT regimes. Multiplex analysis of selected cytokines present in the blood before and 24 h after the PDT showed increased IL-6 concentrations in plasma after PDT, as expected from the inflammations observed. The enhanced concentration of KC and MIP-2 chemokines after PDT suggest that tumor response may involve an influx of neutrophils to the tumor. The optimal protocols with sulfonamide bacteriochlorin (F2BMet-V-PDT and Cl2BHep-C-PDT) are characterized by increased secretion of MIP-1, which is also involved in neutrophilic inflammation.
The selected optimal protocols indicate the increased level of TNFα activity and decreased VEGF, especially after anti-vascular treatment. Further studies are necessary to fully understand how the changes in these biomarkers after PDT contribute to mounting an adaptive immune response capable of inducing immunological memory and recognition of tumor cells surviving the local PDT treatment.