Photodynamic Therapeutic Effect during 5-Aminolevulinic Acid-Mediated Photodynamic Diagnosis-Assisted Transurethral Resection of Bladder Tumors

Background Photodynamic diagnosis-assisted transurethral resection of bladder tumors (PDD-TURBT) enhances detection of elusive lesions compared to standard white light-transurethral resection of bladder tumors (WL-TURBT). If minimal light exposure during PDD-TURBT induces the accumulation of reactive oxygen species (ROS), potentially resulting in phototoxicity in small lesions, apoptosis may be triggered in residual small tumors, allowing them to escape resection. We investigated the hypothesis of a potential photodynamic therapeutic effect during PDD-TURBT. Methods and Materials Our study, conducted between January 2016 and December 2020 at Nara Medical University Hospital, focused on a specific emphasis on ROS production. Immunohistochemical analysis for thymidine glycol and Nε-hexanoyl-lysine was performed on 69 patients who underwent 5-aminolevulinic acid-mediated PDD-TURBT and 28 patients who underwent WL-TURBT. Additionally, we incrementally applied the minimal irradiation energy to T24 and UM-UC-3 cells treated with 5-aminolevulinic acid using instruments similar to those used in PDD-TURBT and evaluated intracellular ROS production and phototoxicity. Results Immunohistochemical analysis revealed a significant increase in production of thymidine glycol and Nε-hexanoyl-lysine within the PDD-TURBT group. In T24 and UM-UC-3 cells treated with 5-aminolevulinic acid and light exposure, immunofluorescent staining demonstrated a dose-dependent increase in intracellular ROS production. In addition, higher irradiation energy levels were associated with a greater increase in ROS production and phototoxicity, as well as more significant decrease in mitochondrial membrane potential. Conclusion Although the irradiation energy used in PDD-TURBT did not reach the levels commonly used in photodynamic therapy, our findings support the presence of a potential cytotoxic effect on bladder lesions during PDD-TURBT.


Introduction
Transurethral resection of bladder tumors (TURBT) is the primary diagnostic and therapeutic approach for nonmuscle invasive bladder cancer (NMIBC).Recently, photodynamic diagnosis (PDD)-assisted TURBT has garnered attention owing to its ability to enhance tumor detection rates and reduce intravesical recurrence [1][2][3].PDD is a medical imaging technique used to detect cancerous tissues, particularly in the bladder during TURBT procedures.Te term "photodynamic" comes from the Greek words "photo," meaning light, and "dynamics," meaning force or power.In PDD, a photosensitizing agent, such as 5-aminolevulinic acid (5-ALA) or hexaminolevulinate (HAL), is administered to the patient.Tese agents preferentially accumulate in cancerous tissues.When the bladder is illuminated with blue light during cystoscopy, the cancerous tissues fuoresce (emit a red or pink light), making them more visible compared to normal tissues.Tis enhanced visibility aids surgeons in identifying and removing cancerous tissues more accurately [4,5].Te efectiveness of PDD-TURBT in preventing intravesical recurrence is primarily attributed to its superior ability to detect microlesions that are challenging to identify with conventional white light-transurethral resection of bladder tumors (WL-TURBT) [6][7][8][9].
In cancer cells, 5-aminolevulinic acid (5-ALA) is not metabolized into heme in the mitochondria, leading to the intracellular accumulation of the intermediate metabolite protoporphyrin IX (PpIX) [9].PpIX is a photosensitizer that is activated and emits red fuorescence (around 635 nm) when exposed to blue light (400−410 nm) provided by a laser.Additionally, in photodynamic therapy (PDT), red light with wavelengths ranging from 600 to 800 nm is commonly used, leading to the production of reactive oxygen species (ROS) by PpIX [10][11][12].Red light is frequently used in PDT due to its deeper tissue penetration capabilities and its efectiveness in activating photosensitizers that generate cytotoxic ROS, which are crucial for the treatment's efcacy.On the other hand, in PDD-TURBT, the purpose is the detection of tumors.Tumors with high accumulation of PpIX are exposed to blue light, causing them to emit red fuorescent light.Tis is why blue light is used during PDD-TURBT.Tumor observation typically begins under blue light, but after circumferential marking around tumor, the procedure usually switches to white light mode for tumor resection.White light has a broad spectrum, encompassing wavelengths around 600−800 nm commonly used in PDT.We believe that the signifcant decrease in intravesical recurrence rate of PDD-TURBT is attributed to the efcient mechanical resection of tumors facilitated by the visualization of tumors through red fuorescence and this approach helps prevent any residual tumor during PDD-TURBT.Additionally, we hypothesized that PDD-TURBT could potentially induce PDD efect in very small and fat lesions that may be difcult to detect even with PDD-TURBT.Many clinical trials of PDT for bladder cancer have utilized a relatively high energy dose of 25-100 J/cm 2 when applying PDT with 5-ALA [13][14][15].Tis energy dose was considerably higher than that emitted by the light source used for TURBT.However, we consider the possibility that during PDD-TURBT, minimal light exposure induces the accumulation of ROS similar to PDT, resulting in phototoxicity in small tumors that are difcult to detect even with PDD-TURBT.Compared to PDT, the energy levels of PDD-TURBT are obviously lower.Tey might be too low to induce apoptosis in bladder cancer.However, what if a PDT-like efect occurs even at the low energy levels of PDD-TURBT?Te residual tumors from resection might be caused to die because of this PDT-like efect, which could potentially contribute to a decrease in intravesical recurrence.Here, we conducted a series of experiments to investigate whether these efects similar to PDT are achieved in tumor tissue and tumor cells with the minimal energy generated during PDD-TURBT.

Cell Culture.
In order to translate this experiment to actual clinical settings, we used two bladder urothelial cancer cell lines, namely T24 and University of Michigan-Urinary Carcinoma-3 (UM-UC-3).Tese cells are derived from human bladder cancer.For example, T24 is derived from muscle-invasive bladder cancer, and UM-UC-3 is derived from NMIBC.Te cell lines were maintained in RPMI1640 medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Nichirei Biosciences Inc., Tokyo, Japan), 100 U/mL penicillin and 100 µg/mL streptomycin (Nacalai Tesque, Kyoto, Japan) in a standard humidifed incubator at 37 °C in an atmosphere containing 5% CO 2 .

Tumor Specimens Obtained by TURBT.
All procedures involving human participants performed in this study were in accordance with the ethical standards of the Nara Medical University Ethical Committee (Ethical Approval Number: 1256) and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Immunohistochemical staining was performed to evaluate ROS production in tumor tissues during transurethral surgery.A total of 239 patients with primary NMIBC underwent initial WL or PDD TURBT between January 2016 and December 2020 at Nara Medical University Hospital.Of these, 109 patients who had multiple tumors, 28 patients who had carcinoma in situ, and fve patients with a lack of clinicopathological data were excluded from this study, leaving 69 patients who underwent PDD-TURBT and 28 patients who underwent WL-TURBT (Figure 1).To establish the control group for the TURBT group, tumor areas and normal mucosa areas were extracted from the specimens of both groups.Patients with sufcient tumor area for evaluation were included in this analysis.Te following section describes how we evaluated tumor and normal bladder mucosa areas for immunohistochemical analysis.For the immunohistochemical analysis, tumor areas from all 69 patients in the PDD-TURBT group and 28 patients in the WL-TURBT group were used.However, for the immunohistochemical analysis of the bladder mucosa area, 29 patients in the PDD-TURBT group and 10 patients in the WL-TURBT group were excluded from this analysis because their specimens did not have sufcient volume of bladder mucosa.Finally, 69 tumor areas and 40 normal bladder mucosa areas in the PDD-TURBT group were included in this study.Additionally, 28 tumor areas and 18 normal bladder mucosa areas in the WL-TURBT group were included in this study.

2.3.
Immunohistochemical Staining Analysis.Immunohistochemical staining was performed as previously described [16].Briefy, antibodies against thymidine glycol (TG) and N ε -hexanoyl-lysine (N ε -HEL) were used as primary antibodies (dilution ratio of 1 : 100 for overnight incubation at 4 °C) and were purchased from the Japan Institute for the Control of Aging, NIKKEN SEIL Co, Ltd (Shizuoka, Japan).Te secondary antibody, peroxidase-2 Advances in Urology conjugated anti-mouse immunoglobulin G (H + L) (Nichirei Biosciences Inc., Tokyo, Japan), was used at a dilution ratio of 1 : 1,000.ImageJ (National Institute of Health, Rockville, MD, USA, freeware) with Java ™ version 1.8.0_112 (64-bit) under Windows 10 Pro edition was used to quantify stain-positive cells [17].Figure 2 shows representative images of immunohistochemical staining analysis using anti-TG and N ε -HEL primary antibodies, as well as images reconstructed using ImageJ.For immunohistochemical staining analysis, fve sites were randomly selected from each specimen and observed under a high-power feld, and the percentage of stained areas in one feld of view was measured using ImageJ.Furthermore, we evaluated ROS production in the normal bladder mucosa area as well as in tumors from PDD-TURBT.Te percentage of the area stained with TG and N ε -HEL was measured by dividing the staining area of TG and N ε -HEL by the total tumor area and total bladder mucosa area in one feld of view, respectively.For each specimen, the mean stained area was calculated from fve felds of view and the mean values were compared between the PDD and WL-TURBT groups.

PDT Using 5-ALA and WL Exposure to Urothelial Cancer
Cells.Experimental PDT procedures were performed in dark conditions as much as possible.Te cells were initially seeded at a density of 0.5 × 10 5 cells per well in 24-well plates.Subsequently, the cells were treated with and without 1 mM 5-ALA.Te previous study reported that higher ALA concentrations were associated with increased PpIX accumulation in urothelial cell lines, with a defned concentration of 1 mM for 5-ALA [18].Te cells treated with 5-ALA were kept in the dark for 3 hours within a standard humidifed incubator at 37 °C with 5% CO 2 , followed by WL exposure at increasing intensities of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 mJ/cm 2 using a TURBT light source (KARL STORZ GmbH & Co. KG; Tuttlingen, Germany).During WL exposure, the cumulative energy was measured using an irradiance meter (Delta OHM, Padova, Italy).Supplementary Figure S1 shows a photograph taken during WL exposure.Te cells treated with PDT were incubated for an additional 1 hr within a standard humidifed incubator at 37 °C with 5% CO 2 .

Immunofuorescent Staining
Analysis.Immunofuorescent staining of the cultured cells was performed as previously described [19].Cells treated with PDT were fxed with 4% paraformaldehyde, permeabilized with phosphate-bufered saline (PBS) containing 0.1% Triton X-100 (PBS-T) (Nacalai Tesque, Kyoto, Japan), and blocked with PBS-Tcontaining 1% bovine serum albumin (BSA).Te primary anti-TG and N ε -HEL antibodies were added to the samples at a dilution ratio of 1 : 100 in PBS-T containing 1% BSA overnight at 4 °C in a humidifed chamber.Subsequently, a goat anti-mouse IgG (H + L) highly crossadsorbed secondary antibody (Termo Fischer Scientifc, Massachusetts, US) at a 1 : 500 dilution in PBS-T containing 1% BSA was added to the samples at room temperature for 1 h in the dark.Nuclear counterstaining and mounting were performed simultaneously using Ibidi Mounting Medium with DAPI (NIPPON Genetics Co., Ltd.Japan).
For immunofuorescent staining, the mean percentage of primary antibody-positive cells among the DAPI-stained cells was compared for each irradiation energy level.Cell Advances in Urology 3 counting was performed automatically using ImageJ software (National Institute of Health, Rockville, MD, USA, freeware), similar to the immunohistochemical staining analysis.Te phototoxicity induced by PDT was evaluated by the WST-8 assay using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan).100 μL of WST-8 solution was added to each well, and the cells were further incubated at 37 °C for 1 h.Absorbance was measured at 450 nm using a microplate reader (TECAN, Männedorf, Switzerland).For statistical comparisons, three wells were prepared for each condition.

Measurement of ROS Production.
Te accumulation of ROS from the experimental PDT procedures was assessed using the Highly Sensitive DCFH-DA-ROS assay kit (Dojindo Molecular Technologies, Kumamoto, Japan).DCFH-DA is a dye used to detect ROS with high sensitivity.Briefy, the cells were washed twice with hypo-and normo-VICs using Hanks' Balanced Salt Solution (HBSS) (FUJI-FILM Wako Chemicals Co., Japan) and then treated with the highly sensitive DCFH-DA dye working solution for 30 minutes.Fluorescence intensity was measured using a microplate reader (TECAN, Männedorf, Switzerland).Fluorescence intensity measurements were conducted using an excitation wavelength of 490 nm and an emission wavelength of 540 nm.For statistical comparisons, three wells were prepared for each condition.

Measurement of Mitochondrial Membrane Potential.
Mitochondrial membrane potential (MMP) was evaluated using the JC-1 MitoMP Detection Kit (Dojindo Molecular Technologies, Kumamoto, Japan).Mitochondria are also the primary site of ROS production within cells.In the ATP production pathway via oxidative phosphorylation, leaked electrons react with oxygen, producing superoxide anions, which are highly reactive ROS.Additionally, mitochondrial DNA and the electron transport chain are susceptible to damage by ROS.With aging, mitochondrial dysfunction occurs, leading to a decrease in mitochondrial membrane potential.Tis kit allows for the evaluation of mitochondrial membrane potential and assessment of the potential for cell apoptosis.JC-1 emits red fuorescence when it aggregates in mitochondria with normal membrane potential.When the membrane potential decreases, JC-1 exists as a monomer and emits green fuorescence.Changes in the intensity of red and green fuorescence can be used to assess the state of the mitochondria.Normally, JC-1 emits both green and red fuorescence, but as the membrane potential decreases, the red fuorescence diminishes and the green fuorescence becomes more intense.After the experimental PDT procedures, cells were exposed to JC-1 solution in the culture medium at a fnal concentration of 4 μM and then incubated at 37 °C for 30 minutes.After two washes with culture medium, fuorescence intensity was measured using a microplate reader (TECAN, Männedorf, Switzerland).Te fuorescence ratio (Red/Green) was calculated and compared between two groups treated with and without PDT.
For green fuorescence intensity, measurements were conducted with an excitation wavelength of 485 nm and an emission wavelength of 530 nm.For red fuorescence intensity, measurements were conducted with an excitation wavelength of 535 nm and an emission wavelength of 600 nm.For statistical comparisons, three wells were prepared for each condition.4 Advances in Urology signifcance was set at a P value <0.05.Tese analyses were performed using EZR software (Saitama Medical Center, Jichi Medical University, Saitama, Japan) [20].

Results
3.1.Production of TG and N ε -HEL during PDD-TURBT and WL-TURBT.) for the PDD-TURBT group and 20.9 minutes (SD, ±7.5) for the WL-TURBT group.Figure 3 shows a comparison of area positive for TG and N ε -HEL between the PDD-and WL-TURBT groups.TG is expressed in the nucleus when thymidine is damaged by hydroxyl radicals, and it is used to evaluate DNA damage [21].N ε -HEL is a marker of lipid peroxidation-induced oxidative stress [22].Te production of TG and N ε -HEL in cancer cells was signifcantly higher in the PDD-TURBT group compared to the WL-TURBT group.Additionally, ROS production in the PDD-TURBT-treated tumor group was signifcantly higher than in the normal bladder mucosa group.

PDT-Induced Intracellular Production of TG and N ε -HEL in T24
and UM-UC-3.Figure 4 shows representative images of immunofuorescent staining analysis of T24 and UM-UC-3 cells treated with PDT and 1.0 mJ/cm 2 of WL exposure.In both T24 and UM-UC-3 cells, more than half of the cells were positive for the production of TG and N ε -HEL.Figure 5 shows a comparison of cells positive for TG and N ε -HEL at each irradiation energy level that were treated with and without PDT.Approximately 0.6 seconds of exposure resulted in an irradiation energy level of 0.01 mJ/cm 2 , and with 1 minute of exposure, it reached exactly 1.0 mJ/cm 2 .In both T24 and UM-UC-3 cells, only a few cells were positive for TG and N ε -HEL at an irradiation energy level of 0.01 and 0.02 mJ/cm 2 .Immunofuorescent staining demonstrated that the production of intracellular ROS increased in a dosedependent manner with increasing light exposure level.Furthermore, at 0.5 and 1.0 mJ/cm 2 , the percentage of stained cells for TG and N ε -HEL were signifcantly higher in the PDT-treated cells.

PDT-Induced Phototoxicity.
Figure 6 shows a comparison between T24 and UM-UC-3 cells treated with and without PDTfor phototoxicity at each irradiation energy level.PDT-induced phototoxicity increased in a dose-dependent manner with increasing light exposure level.Signifcant PDTinduced toxicity was observed in T24 and UM-UC-3 cells treated with PDT using 0.05 mJ/cm 2 or more.
3.4.PDT-Induced ROS Production. Figure 7 shows representative images of fuorescence micrographs of ROS production in T24 and UM-UC-3 cells treated with and without PDT, and 0.2, 0.5, and 1.0 mJ/cm 2 of WL exposure.In both T24 and UMUC3 cells, we observed minimal intracellular ROS production in the cells without PDT at any irradiation energy level.However, in the cells treated with PDT, increasing fuorescence intensity correlating with higher energy levels was observed.Figure 8 shows a comparison of fuorescence intensity between the cells treated with and without PDT at each irradiation energy level.In both T24 and UMUC3 cells, a signifcant increase in ROS production was observed in cells treated with PDT compared to those without PDT, starting from an energy level of 0.2 mJ/cm 2 , which corresponds to an irradiation time exceeding 12 seconds.

Changes in Mitochondrial Membrane
Potential Induced by PDT. Figure 9 shows representative images of fuorescence micrographs of mitochondrial membrane potential in T24 and UM-UC-3 cells treated with PDT and 0.2, 0.5, and 1.0 mJ/cm 2 of WL exposure.Mitochondrial membrane potential is indicated by red fuorescence.Tere is no reduction in red fuorescence in cells without PDT at any irradiation energy level.However, in cells treated with PDT, a decrease in red fuorescence is observed with increasing irradiation energy.Figure 10 shows a comparison of fuorescence intensity ratio (Red/Green) between cells treated with and without PDT at each irradiation energy level.In T24 cells, a decrease in mitochondrial membrane potential was observed when the irradiation energy exceeded 0.5 mJ/cm 2 .Similarly, in UM-UC-3 cells, a decrease in mitochondrial membrane potential was observed when the irradiation energy exceeded 0.1 mJ/cm 2 .

Discussion
Several studies have investigated the in vivo dynamics of ROS production.Generally, ROS can induce cell death in tumor cells, making them potential candidates for disease treatment [23].5-ALA-PDT relies on a combination of visible light exposure and PpIX generation within cells.Exposure to light in the presence of PpIX and molecular oxygen leads to the formation of cytotoxic intermediates that induce tumor cell death [23].Although this efect has been utilized in applications such as brain tumors, esophageal cancer, and skin cancer, 5-ALA-PDT has not been widely adopted for bladder cancer.Szliszka et al. reported the potential of combined therapy using tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and PDT to induce cytotoxicity in bladder cancer cells [24].TRAIL can selectively induce apoptosis in cancer cells; however, T24 cells are resistant to TRAIL and showed enhanced cytotoxicity in combination with 5-ALA-PDT.In this report, visible light with a wavelength of 400-750 nm was used and an irradiation energy level was 7.5 J/cm 2 .Ekroll et al. reported on the cell destruction mechanism induced by PDT using the rat bladder cancer cell

Advances in Urology
line AY-27, with a dose of 1.6 J/cm 2 [25].Numerous studies have been conducted on this topic.Te irradiation energy used in PDD-TURBT does not reach the levels used in PDT, which requires 20-100 J/cm 2 to achieve adequate cytotoxic efects on bladder cancer [26,27].Moreover, the wavelengths used in PDD-TURBT and PDT are diferent.PDD-TURBT uses blue light (410-420 nm), whereas PDT uses higher wavelengths (650-660 nm).Te depth of laser penetration difers as well.Although we cannot directly compare the energy levels between PDD-TURBT and PDT, it is clear that the energy level of PDD-TURBT is signifcantly lower than that of PDT.Tis likely results in diferent levels of ROS production.In PDT, the high level of ROS production may contribute to a more efcient cancer cell death.Te ROS    Advances in Urology induced by PDT might also afect other lesions in combination with systemic therapies such as chemotherapy and immune checkpoint inhibitors [28].In contrast, PDD-TURBT likely does not have such efects due to its lower ROS production.
In PDT, we cannot ignore the adverse events, including bladder spasms, hematuria, and urinary urgency [29,30].Tese adverse events are caused by the high level of ROS production.However, in the potential PDT efect introduced by PDD-TURBT, these adverse events do not occur.In fact, symptoms such as urinary urgency and hematuria after PDD-TURBT are caused by the resection of the bladder mucosa, not the PDT efect.Terefore, we can suggest that the potential PDT efect by PDD-TURBT might be obviously safer.
Te phototoxicity of PDD-TURBT was confrmed in our study.Furthermore, using the TURBT device, we observed accumulation of ROS within the bladder cancer cells and confrmed a decrease in mitochondrial membrane potential when the irradiation time exceeded approximately 10 seconds.Tis result suggests that during actual PDD-TURBT procedures, even with minimal irradiation time, there is a possibility of inducing phototoxicity in tumor cells located on the bladder mucosal surface.However, all experiments in this study were conducted in vitro, lacking structures such as microvasculature and stroma present in actual tumor tissues, which could not be taken into consideration.Drawing conclusions solely from these results can be challenging; however, using the PDD-TURBT device, we were able to confrm the accumulation of ROS within mitochondria and phototoxicity with very low doses of WL exposure.
We compared oxidative stress markers, including TG and N ε -HEL, using specimens obtained from TURBT.When evaluating ROS production, we must consider the infuence not only of PDT-like efects but also of the surgical procedure itself, which can induce ROS production through physical stimuli.To address this, we divided patients into two groups: PDD-TURBT and WL-TURBT.By ensuring both groups experienced similar levels of surgical invasion from physical stimuli, we conducted statistically appropriate analyses to mitigate concerns about the surgical procedure's infuence.Ultimately, our fndings suggest that PDD-TURBT may introduce higher ROS production compared to WL-TURBT.In an actual bladder cancer scenario, cancer cells are densely layered, and it is unlikely that the light from Tese images were taken using the JC-1 MitoMP detection kit (Dojindo Molecular Technologies, Kumamoto, Japan) immediately after the experimental PDT procedures.Tis kit allows the evaluation of mitochondrial membrane potential and the assessment of the potential for cell apoptosis.JC-1 emits red fuorescence when it aggregates in mitochondria with normal membrane potential.When the membrane potential decreases, JC-1 exists as a monomer and emits green fuorescence.Te changes in the intensity of red and green fuorescence can be used to assess the state of the mitochondria.Normally, JC-1 emits both green and red fuorescence, but when the membrane potential decreases, the red fuorescence diminishes and the green fuorescence becomes more intense.Eventually, the red fuorescence represents mitochondrial membrane potential.A decrease in red fuorescence indicates a reduction in mitochondrial membrane potential, which could induce apoptosis.5-ALA, 5-aminolevulinic acid.10 Advances in Urology PDD-TURBT with PpIX would reach the deep cancer cells.Similarly, red light commonly used in PDT with PpIX is mostly at 630 nm that is believed to penetrate to a depth of approximately 5 mm [31].Under white light exposure, tissue penetration depth is lower.Furthermore, laser light sources are specifcally manufactured for PDT, primarily using slender fbers to efciently deliver light to the tumor, and their energy efciency is not comparable to the light irradiation used in TURBT [32].Te radiance meter for TURBT light source showed an irradiation energy level of 1.0 mJ/cm 2 per 60 seconds.Even if it were to be applied continuously for one hour, it would only reach a cumulative value of 60 mJ/cm 2 .In reality, even with continuous irradiation for one hour, such a simple calculation may not apply because, during PDD-TURBT, the endoscope is constantly moved within the bladder, so it never remains in one place for long.However, it may be efective against residual tumors and very small lesions after TURBT.If there is a photodynamic therapeutic efect with PDD-TURBT, it may be benefcial to take the time during surgery to slowly and carefully observe while applying light to the entire bladder mucosa.For example, during PDD-TURBT, the operation period is sufciently long and the entire bladder mucosa is exposed to light from the TURBT instrument, potentially reducing the intravesical recurrence rate.Tis optimistic efect is attributed not only to the high detection rate resulting from thorough observation of the bladder mucosa but also to the potential PDT efect.Tis study has several limitations.First, experiments on ROS production and phototoxicity following light irradiation were conducted exclusively using cultured cells.Tis study was conducted entirely in vitro, which limits reproducibility.Actual tumors are thicker, posing challenges due to the limited tissue penetration depth of white light used in TURBT.Unfortunately, this aspect was not investigated in our study.Ideally, future research would involve a 3D cell model or in vivo studies using nude mice to address these limitations.Second, the evaluation of ROS production and phototoxicity was only based on immunostaining using oxidative stress markers, the WST-8 assay, the Highly Sensitive DCFH-DA-ROS assay kit, and the JC-1 MitoMP Detection Kit.Singlet oxygen used in PDT can induce apoptosis through both the extrinsic and intrinsic pathways.Methods allowing a more direct measurement of ROS levels are available, including electron spin resonance spectroscopy and the direct measurement of mitochondrial ROS [33].Terefore, alternative approaches should have been explored in addition to this study.Specifcally, multiplex staining would be suitable for assessing multiple indices simultaneously.Unfortunately, we are currently unable to utilize such a technique.Tird, there was a signifcant delay between sample collection by TURBT and evaluation of immunohistochemical staining analysis.Tis duration is approximately 2 years for the PDD-TURBT group and 4 years for the WL-TURBT group.ROS have a short half-life and are not expected to persist in the samples.We relied on oxidative stress markers in the cells resulting from ROS, which are known to remain for a certain period even after formalin fxation.However, the exact duration of retention is not defnitive, and in cases where samples have aged for several years, conducting an accurate evaluation may not be feasible.Fourth, the number of TURBT samples used for immunohistochemistry was limited.Excluding more than half of the initially included patients who underwent TURBT  for primary NMIBC between 2016 and 2020 introduced signifcant selection bias.Consequently, the cohort decreased from 239 to 97, leading to decreased reproducibility.Terefore, further accumulation of bladder cancer cases is necessary.

Figure 1 :
Figure 1: Flowchart of patient selection for immunohistochemical staining analysis.Of the 239 patients who underwent TURBTfor primary NMIBC between 2016 and 2020, 69 who underwent PDD-TURBT and 28 who underwent WL-TURBT were included in this study.NMIBC, nonmuscle invasive bladder cancer; PDD, photodynamic diagnosis; TURBT, transurethral resection of bladder cancer; WL, white light.

Figure 2 :
Figure2: Micrographs before and after using ImageJ to reconstruct immunohistochemical staining analysis.Te areas stained through immunohistochemical staining analysis were reconstructed as red using ImageJ, allowing the calculation of the percentage of stained area per feld of view.

Figure 3 :Figure 4 :
Figure 3: A comparison of the percentage of area stained with TG and N ε -HEL in immunohistochemical staining analysis.Box plots show the comparison of area stained with TG (a) and N ε -HEL (b) between PDD-and WL-TURBT groups that, respectively, included patients who underwent initial PDD-and WL-TURBT for primary NMIBC.Te percentage of area stained with TG and N ε -HEL was measured by dividing the staining area of TG and N ε -HEL by the total tumor area and total bladder mucosa area in one feld of view, respectively.Te asterisk ( * ) indicates a P value <0.05, and double asterisks ( * * ) indicate a P value <0.01.PDD, photodynamic diagnosis; TURBT, transurethral resection of bladder cancer; WL, white light.

Figure 5 :
Figure 5: Evaluation of percentage of cells stained for TG and N ε -HEL at each irradiation energy level.T24 (a, b) and UM-UC-3 (c, d) cellswere treated with or without 5-ALA and WL exposure at increasing intensity of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 mJ/cm 2 .Te exposure time for each energy level is also indicated.Te percentage of cells stained for TG and N ε -HEL is compared between the cells with and without the experimental PDT procedures at each irradiation energy level.Since the staining process takes two days, this analysis was immediately conducted on the day following WL exposure.For statistical comparisons, three wells were prepared for each condition.An asterisk ( * ) indicates a P value <0.05, and double asterisks ( * * ) indicate a P value <0.01.5-ALA, 5-aminolevulinic acid; WL, white light.

Figure 6 :Figure 7 :
Figure 6: Evaluation of phototoxicity at each irradiation energy level.Te phototoxicity induced by PDT was evaluated by the WST-8 assay using a Cell Counting Kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan).Te lower absorbance at 450 nm indicates higher phototoxicity.Tis analysis was conducted immediately following WL exposure.T24 (a) and UM-UC-3 (b) cells were treated with or without 5-ALA and exposed to WL at increasing intensities of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 mJ/cm 2 .Te absorbance is compared between the cells with and without the experimental PDTprocedures at each irradiation energy level.Te exposure time for each energy level is also indicated.For statistical comparisons, three wells were prepared for each condition.An asterisk ( * ) indicates a P value <0.05, and double asterisks ( * * ) indicate a P value <0.01.5-ALA, 5-aminolevulinic acid; WL, white light.

Figure 8 :
Figure8: Evaluation of intracellular ROS production at each irradiation energy level.PDT-induced ROS production was evaluated using the highly sensitive DCFH-DA-ROS assay kit (Dojindo Molecular Technologies, Kumamoto, Japan).Tis analysis was conducted immediately following WL exposure.T24 (a) and UM-UC-3 (b) cells were treated with or without 5-ALA and exposed to WL at increasing intensities of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 mJ/cm 2 .Te fuorescence intensity was compared between cells with and without the experimental PDT procedures at each irradiation energy level.Te higher fuorescence intensity indicates an increase in ROS production.For statistical comparisons, three wells were prepared for each condition.An asterisk ( * ) indicates a P value <0.05, and double asterisks ( * * ) indicate a P value <0.01.5-ALA, 5-aminolevulinic acid.

T24UM-UC- 3 Figure 9 :
Figure9: Micrographs showing changes of mitochondrial membrane potential induced by PDT.Micrographs show changes of mitochondrial membrane potential induced by PDT in T24 and UM-UC-3 cells treated with or without 5-ALA at 0.2, 0.5, and 1.0 mJ/cm 2 WL exposure.Tese images were taken using the JC-1 MitoMP detection kit (Dojindo Molecular Technologies, Kumamoto, Japan) immediately after the experimental PDT procedures.Tis kit allows the evaluation of mitochondrial membrane potential and the assessment of the potential for cell apoptosis.JC-1 emits red fuorescence when it aggregates in mitochondria with normal membrane potential.When the membrane potential decreases, JC-1 exists as a monomer and emits green fuorescence.Te changes in the intensity of red and green fuorescence can be used to assess the state of the mitochondria.Normally, JC-1 emits both green and red fuorescence, but when the membrane potential decreases, the red fuorescence diminishes and the green fuorescence becomes more intense.Eventually, the red fuorescence represents mitochondrial membrane potential.A decrease in red fuorescence indicates a reduction in mitochondrial membrane potential, which could induce apoptosis.5-ALA, 5-aminolevulinic acid.

Figure 10 :
Figure 10: Evaluation of changes of mitochondrial membrane potential induced by PDT.Changes in mitochondrial membrane potential induced by PDT were evaluated using the JC-1 MitoMP Detection Kit (Dojindo Molecular Technologies, Kumamoto, Japan) immediately following WL exposure.T24 (a) and UM-UC-3 (b) cells were treated with or without 5-ALA and exposed to WL at increasing intensities of 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, and 1.0 mJ/cm 2 .Te fuorescence intensity ratio (red/green) was compared between cells with and without the experimental PDT procedures at each irradiation energy level.Te lower fuorescence intensity ratio indicates a reduction of mitochondrial membrane potential.For statistical comparisons, three wells were prepared for each condition.An asterisk ( * ) indicates a P value <0.05, and double asterisks ( * * ) indicate a P value <0.01.