Quantitative analysis of vascular changes during photoimmunotherapy using speckle variance optical coherence tomography (SV-OCT)

: Near-infrared (NIR) photoimmunotherapy (NIR-PIT) is an emerging cancer therapy based on a monoclonal antibody and phthalocyanine dye conjugate. Direct tumor necrosis and immunogenic cell death occur during NIR irradiation. However, the alteration of tumor blood vessels and blood volume inside the blood vessels induced by the NIR-PIT process is still unknown. In our study, a speckle variance (SV) algorithm combined with optical coherence tomography (OCT) technology was applied to monitor the change of blood vessels and the alterations of the blood volume inside the blood vessels during and after NIR-PIT treatment. Vascular density and the measurable diameter of the lumen in the blood vessel (the diameter of the region filled with blood) were extracted for quantitively uncovering the alterations of blood vessels and blood volume induced by NIR-PIT treatment. The results indicate that both the density and the diameter of the lumen in the blood vessels decrease during the NIR-PIT process, while histological results indicated the blood vessels were dilated. The increase of permeability of blood vessels could lead to the increase of the blood pool volume within the tumor (shown in histology) and results in the decrease of free-moving red blood cells inside the blood vessels (shown in SV-OCT).

Abstract: Near-infrared (NIR) photoimmunotherapy (NIR-PIT) is an emerging cancer therapy based on a monoclonal antibody and phthalocyanine dye conjugate. Direct tumor necrosis and immunogenic cell death occur during NIR irradiation. However, the alteration of tumor blood vessels and blood volume inside the blood vessels induced by the NIR-PIT process is still unknown. In our study, a speckle variance (SV) algorithm combined with optical coherence tomography (OCT) technology was applied to monitor the change of blood vessels and the alterations of the blood volume inside the blood vessels during and after NIR-PIT treatment. Vascular density and the measurable diameter of the lumen in the blood vessel (the diameter of the region filled with blood) were extracted for quantitively uncovering the alterations of blood vessels and blood volume induced by NIR-PIT treatment. The results indicate that both the density and the diameter of the lumen in the blood vessels decrease during the NIR-PIT process, while histological results indicated the blood vessels were dilated. The increase of permeability of blood vessels could lead to the increase of the blood pool volume within the tumor (shown in histology) and results in the decrease of free-moving red blood cells inside the blood vessels (shown in SV-OCT).

Introduction
Targeted cancer therapies offer the promise of more effective tumor treatment with lower side-effects than conventional cancer therapies [1]. Near infrared (NIR) photoimmunotherapy (NIR-PIT) is an emerging cancer therapy with low side effects, which is based on a targeted monoclonal antibody-photon absorber conjugate (APC) [mAb conjugated with a near-infrared phthalocyanine dye (IR700)] that induces highly selective and rapid cellular necrosis after exposure to NIR light [2,3]. The death of cancer cells will further lead to immunogenic cell death that initiates local immune reactions to released cancer antigens from dying cancer cells [4,5]. U.S. Food and Drug Administration (FDA) has initiated a clinical trial of EGFR targeting NIR-PIT in patients with inoperable head and neck cancer in April 2015, and entered into the third clinical efficacy trial in February 2020 [6]. The first EGFR targeting NIR-PIT agent (Akalux) has recently been conditionally approved with a laser system (Bioblade) for clinical use in September 2020 in Japan [7]. Since NIR-PIT has been proposed, numbers of studies have been performed to investigate its treatment effects. Results show PIT is a well effective treatment in treating tumors based on histopathological analysis and bioluminescence imaging [1,[8][9][10][11][12][13]. However, real-time vascular changes during PIT treatment is still unknown. In this study, we focus on evaluating the alterations of blood vessels and the blood volume inside the blood vessels during and after NIR-PIT treatment.
Quantitative analysis of blood cells or vasculatures in clinic are mainly based on immunohistochemical staining [14,15] or fluorescent proteins [16,17], which are excellent endogenous fluorescence emitters to be used for depicting various biological processes in cells both in vitro and in vivo. However, histological method is invasive and time-consuming. By labeling circulating cells with fluorescent proteins, blood flow in blood vessels can be clearly monitored [18]. Fluorescent proteins labeling is a powerful tools in aiding development of anti-tumor drugs, but the use of fluorescence proteins requires virus-mediated in vivo gene transfection, which is unlikely to be permitted in humans. Thus they are unsuitable for monitoring therapy process in clinical studies. Imaging techniques such as Doppler ultrasound and Magnetic Resonance Imaging (MRI) have been applied to detect blood vessel information in clinics [19,20]. However, Doppler ultrasound as a nondestructive imaging method is mainly applied in imaging the blood vessels with large diameter (diameter > 200 µm) [19]. For blood vessels with small diameter (< 100 µm), the detection sensitivity of Doppler ultrasound is limited. MRI is able to image small blood vessels only with contrast agent [20]. It is necessary to develop a real-time label-free imaging technology to monitor the blood vessels and the blood inside the blood vessels in vivo, and could be potentially used to evaluate the cancer therapy efficacy.
Optical coherence tomography (OCT) is an established biomedical imaging technology for subsurface imaging of tissue samples with high image resolution of less than ten micrometers and a penetration depth of several millimeters, comparable to the size of a standard punch biopsy [21,22]. OCT is based on optical scattering characteristics of tissues with a certain contrast ratio with biological structure and phase information [23]. It has been widely used in biomedical research because of its high imaging speed, noninvasiveness, and high signal-to-noise ratio [24]. OCT imaging is analogous to ultrasound B-mode imaging, with the exception of measuring the echo time delay and intensity of backscattered light rather than sound [24]. In OCT, the optical beam is scanned across the tissue and the backscattered light is measured as a function of axial depth and transverse location. In this way, OCT can generate cross-sectional, tomographic images of subsurface tissue microstructures and obtain 3D tissue morphology reconstruction by stacking a series of 2D OCT tomograms [24]. OCT has been explored and extended not only for structural imaging but also for functional imaging, such as OCT angiography. OCT angiography is becoming a useful and important imaging technique due to its ability to provide volumetric microvascular networks in vivo without the need of exogenous contrast agent [25]. In clinical study, OCT angiography has been shown to help clinicians and physicians monitor the progression of ophthalmology diseases, such as retinopathy [26][27][28][29][30][31][32][33][34][35][36], vascular dysfunction between normal and glaucomatous eyes [37,38]. In dermatology, OCT angiography allows the visualization of in vivo blood vessels and their distribution with lesions and offers promising functional information within dermis for up to a depth of 1-2 mm [25]. OCT angiography has been demonstrated to image the vascular networks of two distinct horizontal plexuses in the dermis in normal skin [39], lesion skin [40], and skin cancer [41]. OCT angiography has the potential to improve the diagnostic accuracy of skin diseases by distinguishing the tumor risk, and aiding in the early diagnosis of skin cancer [25]. Besides ophthalmology and dermatology, OCT angiography also showed its feasibility in neuroscience and brain imaging [42]. In addition, OCT angiography has recently been incorporated into endoscopic setup to image neoplastic progression of Barrett's esophagus (BE) [43] and the microvasculature of the human palm [44], which will further broaden the potential clinical applications of the OCT angiography.
Since OCT signal contains both magnitude and phase information, there are three main types of angiographic methods classified based on utilized OCT signals [25,45]: the first type is based on complex OCT signal (both the magnitude and phase signal), such as optical microangiography [46], the second type is based on intensity signal, such as speckle variance (SV) [47], correlation mapping [25,48], and split-spectrum amplitude decorrelation angiography (SSADA) [49]; the third type is based on phase signal, such as phase variance [50]. Each type of these three OCT based angiographic methods has its own advantage due to its unique physics and mathematics behind the blood flow contrast mechanism [45]. Since both complex-signal-based and phase-signal-based methods are phase-sensitive, therefore a phase-stable system is required for high-contrast images [51]. On the other hand, intensity-signal-based OCT angiography does not suffer from phase noise artifacts and is relatively less sensitive to phase noise, making it particularly helpful in situations where the phase stability of light source is an issue [25,45,49]. In our study, since a swept-source laser is used, thus we selected the intensity-signal-based method. In a previous quantitative comparison study, SV OCT angiography has demonstrated better performance in terms of vessel connectivity, image contrast, and signal-to-noise ratio (SNR) than other intensity-signal-based methods, such as correlation mapping and SSADA [45]. Besides, less computation time is needed for SV OCT angiography due to the simple subtraction operation [45]. Taking all the factors into consideration, SV OCT angiography was selected for monitoring PIT process in this study.
The first application of using speckle analysis in OCT images to acquire a depth-resolved flow signals is reported in 2005 [52]. And then, SV algorithm has been used to image blood vessels based on the amplitude information in OCT structural images [53][54][55][56]. In a previous study, the hemodynamic alterations in a single large vessel with diameter over 150 µm were monitored during PIT by OCT, including SV analysis and Doppler flow measurement, the results show that blood velocity in peripheral tumor vessels quickly drops below the detection limit during PIT [57].
In this study, we employed conventional structural OCT together with SV-OCT to monitor the lumen in tumor blood vessels with various sizes in real time during and after NIR-PIT in situ and in vivo. Quantitative parameters of tumor blood vessels, such as vascular density and diameters of lumen in the blood vessels, were analyzed to further evaluate the NIR-PIT therapy. This technology could potentially be used to optimize the effectiveness of treatment.

OCT system and SV-OCT
A swept-source OCT system (VEG220C1, Thorlabs, Newton, New Jersey, USA) was employed in our experiments [58]. The swept-source laser is centered at 1300 nm with a spectrum bandwidth of 100 nm. The axis resolution in the air can reach 16 µm, the lateral resolution is 13 µm [58]. The imaging depth (coherence length) can reach 8 mm in air. The laser source operates at a sweep rate of 100 kHz with 12 mW output power. A three-dimensional data set was acquired in monitoring PIT process ( . The effective imaging frame rate of this system was 66 frames/s and the acquisition time for one 3D dataset took less than 10 seconds. To characterize the tumor structure and the lumen of blood vessels in tumor, speckle variance (SV) and conventional structural OCT were applied in our experiments. SV-OCT images microvasculature by calculating the interline or interframe speckle variance of the intensity-based structural OCT images [47,51]. The SV analysis was performed as follow [51,59,60]: where I is the OCT signal intensity at each pixel, and i, j, k are indices for the B scan (frame, transverse, and axial pixels). N is the number of frames used in variance calculation and 4 frames were used in our experiment.

Phantom design
To demonstrate the accuracy of SV-OCT system for characterizing the blood inside the blood vessels, three silicone capillaries with an inner diameter of 200 ± 20 µm (ID: 0.2 mm, OD: 2.4 mm, VitroCom Inc.) were fabricated as vessel phantom [61]. These three silicone tubes were filled with intralipid solution of 1% volume concentration to mimic the blood. Two sides of each silicone tube were sealed by glue. The filled capillaries were fixed side by side on an empty petri dish. Then, 1.5-g agar powder (Now Inc.), 0.5-mL Intralipid solution (Hospira Inc.), and 49.5-mL PBS buffer were mixed and stirred well. After heating in a microwave for ∼2 minutes, the mixed liquid gel was poured into the petri dish on which the glass capillaries were fixed until the capillaries were submerged in the liquid gel. After the liquid gel cooled and solidified, the vessel phantom was ready to be imaged.

Synthesis of mAb and IR700
Conjugation of IR700 dyes with mAbs was performed as described previously [10]. In brief, panitumumab (1.0 mg, 6.8 nmol) was incubated with IRDye 700DX NHS ester (66.8 µg, 34.2 nmol) in 0.1 M Na2HPO4 (pH 8.6) at room temperature for 1 h. The mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare Life Sciences, Pittsburgh, PA, USA). Coomassie Plus protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA) was applied to determine protein concentration by measuring the absorption at 595 nm with UV-Vis (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). The concentration of IR700 was measured by absorption at 689 nm to confirm the number of fluorophore molecules per mAb. This synthesis was controlled so that there are about two IR700 molecules bound to a single antibody on average. IR700 conjugated to EGFR-targeting panitumumab were abbreviate as pan-IR700. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as a quality control for the conjugate. Conjugate was separated by SDS-PAGE with a 4-20% gradient polyacrylamide gel (Life Technologies, Gaithersburg, MD, USA). A standard marker (Crystalgen Inc., Commack, NY, USA) was used as a protein molecular weight marker. After electrophoresis at 80 V for 2.5 h, the gel was imaged with a Pearl Imager (LI-COR Biosciences, Lincoln, Nebraska, USA) using a 700 nm fluorescence channel. Diluted panitumumab was used as a control. The gel was stained with Colloidal Blue staining to determine the molecular weight of conjugate.

Animal and tumor models
Six 6-week-old female nude mice were purchased from Charles River (NCI-Frederick, Frederick, Maryland). All in vivo animals' procedures were performed in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the Institutional Animal Care and Use Committees (IACUCs) at the University of Maryland and NIH. Two million A431 cells were inoculated to two sides of dorsum of each nude mouse. Tumor volumes were measured and calculated using the following formula: tumor volume = ½(length ×width 2) [3,57]. Mice were monitored for their general health and tumor sizes every day. Tumor volumes reached 100 mm 3 were selected for experiment. 100 µg of pan-IR700 was injected into mice via the lateral tail vein. 24 h after injection, mice were prepared for experiment. Before imaging, the skin above the tumor was removed and a window chamber was used to fix the tumor for minimizing the imaging artifact from animal movement as previous reported [56,62]. Near infrared LED used in our experiments has a wavelength spectrum of 680 -700 nm (Tech-LED, Marubeni America Corp., Santa Clara, CA, USA) [63,64]. LED was turned on for ∼30 min with a dose of ∼50 J/cm 2 to irradiate the tumor [64]. To record the alterations of tumor vasculature during and after NIR-PIT caused by LED, the data acquisition protocol is as follows: An image of tumor was taken every 10 min for the first 30 min before LED was turned on. This 30 min period of recording without LED irradiation was considered as control group. For the following 30 min, the LED was turned on and images were captured every 5 min, the NIR-PIT treatment was completed after 30 min of LED exposure. To record the tumor vasculature's post-treatment alterations, the response after NIR-PIT were continued to be observed. Images were taken every 5 min, which also lasted 30 min. Tumor xenografts were excised from mice after experiments. They were fixed with 10% formalin, embedded in paraffin, sectioned (10 µm thick) and stained with hematoxylin and eosin (H & E) for histological analysis. To clarify the shapes of blood vessels, anti-CD31 (clone D8V9E, 1:200 dilution; Cell Signaling Technology, Danvers, MA, USA) immunohistochemical staining was performed with Bond RXm auto stainer (Leica Biosystems, Wetzlar, Germany). Light microscopy with an Olympus BX61 (Olympus Corporation) was applied to evaluate histological changes.

Vessel characterization
To quantitatively analyze the change of tumor vasculatures in the NIR-PIT treatment, two parameters were extracted in this study, namely, the vascular density (VD) and the diameter of lumen in blood vessels (DLBV). The signal obtained from SV-OCT indicated the region inside the blood vessels that was filled with blood. The vascular density (VD) is defined as VD = A vessel A total , where A vessel is the area of vessels filled with blood, A total is the total area of image. In general, the DLBV value at the beginning of the experiment was normalized to 1, and the values at other time points were compared with the results at the beginning. The measurable diameter of lumen in blood vessel (MDLBV, the diameter of the region filled with blood) was calculated based on the centerline algorithm [65]. First, the vessel image was segmented and turned into binary image. Then the centerline and boundary in each vessel were determined by 'bwmorph' and 'bwboundaries' functions in MATLAB toolbox, respectively. After that, the shortest distance from each centerline point to the boundary was calculated as radius at the corresponding position.

Data analysis
Data are expressed as mean ± standard deviation. A two-sample t-test with unequal means was completed to determine whether the difference was significant in the statistical parameters between any two sample groups. Differences were regarded as statistically significant when p<0.05.

Phantom study
Vessel phantom diameters obtained from conventional structural OCT mode and SV-OCT mode are represented in Fig. 1. Figure 1(a) shows the cross-sectional image of three capillary tubes with inner diameter ∼200 µm in conventional structural OCT mode, which were embedded at different depth in the agar gel. Tubes 1-3 shown in Fig. 1(a) are located from shallow to deep. The embedded glass capillaries are located at different depths from surface with ∼25 µm, 50 µm, and 112 µm for tubes 1-3, respectively. Figure 1(b) displays the projection view of three capillaries tubes in SV-OCT mode. Since agar phantom is solid, there is no particle motion, and it generates limited signal in SV mode. While the intralipid particles can freely move inside the capillaries, they can be well exhibited through the SV calculation. An en face image in conventional structural OCT mode of phantom is displayed in Fig. 1(c). Both glass capillaries and agar phantom can be observed in conventional structural OCT mode. On the other hand, SV-OCT can reflect the space with free moving particles within vessels (inner diameter or lumen of the vessels), but not the vessels wall. Figure 1(d) shows the binary image and centerline processed from the SV-OCT image. Figure 1(e) compares the diameters of lumen in vessel phantom measured by conventional structural OCT and SV-OCT mode in three glass capillaries. For obtaining the accurate diameter of the tubes, tube 1 was measured from en face image at the depth of 165 µm, tube 2 was measured at about 190 µm, and tube 3 was measured at about 250 µm in conventional structural OCT mode. While in SV-OCT mode, the diameters were obtained from projection view. Since Intralipid has filled the entire glass capillaries, the diameter of intralipid obtained by SV-OCT can represent the size of the inner diameter of capillaries. Ten measurements from ten different locations in the same en face image of each capillary were averaged for statistical analysis. From Fig. 1(d), the diameter of tube 3 is smaller than that of tube 1 or 2. Tube 3 also seems uneven. It may be due to the fabrication variance while still within the 10% variance range the factory claimed. Results show the value is consistent from two imaging modes (conventional structural OCT and SV-OCT mode). Therefore, it is feasible to measure the diameter of blood vessels using conventional structural OCT and the diameter of the region filled with blood (lumen) using SV-OCT mode.

Vessel measurement in normal tissues
Vessel images of normal skin tissue from SV-OCT and conventional structural OCT are displayed in Fig. 2. Figure 2(a) is projection image of lumen in blood vessels (the region filled with blood), which is reconstructed from SV-OCT images from depth 0 µm-480 µm. The image contains different sizes of vessels from different depths of tissue.
From Fig. 2(a), SV-OCT can clearly distinguish the micro-vessels as small as 28 µm. Blood vessels distributed at different depths can also be clearly resolved. To further analyze the distribution of vessels at different depths, blood vessels are pseudo-colored with depth information ranging from 0 µm to 480 µm, as shown in Fig. 2(b). The surface (0 µm) is indicated by red color and the deepest position (480 µm) is indicated by blue color. Figure 2(c) shows the image from conventional structural OCT. Large vessels can be easily distinguished, while the microvasculature is indistinguishable compared to Fig. 2(a) using SV-OCT mode. The 3D overlay image of SV-OCT and conventional OCT image volume is displayed in Fig. 2(d). Figure 2(e), (f), and (g) display the cross-sectional image of SV-OCT mode, conventional structural OCT mode, and the fused image from the cross-section indicated in Fig. 2 (d), respectively. The location of blood vessels on tissue can be directly visualized on the fused SV-OCT and conventional structural OCT image.

Vascular changes in tumor without LED illumination
Before exposed to LED, tumors with pan-IR700 were imaged by OCT. The group that without LED exposure was taken as the control data (the baseline data of the same group of animals). The results of no-LED-exposure from SV-OCT mode are shown in Fig. 3. Figure 3(a) shows the vessels filled with blood pseudo-colored with depth from 0 µm to 480 µm. Figure 3(b) is the corresponding 3D images. The changes are negligible in the vessels. To further depict the changes, the VD and MDLBV from 0 to 30 min without LED exposure were calculated. Changes of VD over time are displayed in Fig. 3(c). Results show the density almost experience no change from 0 min to 30 min. Changes of MDLBV over time are shown in Fig. 3(d). Four blood vessels  were selected which are labeled by the white arrow 1, 2, 3, and 4 in Fig. 3(b). Results demonstrate that there are negligible changes in both VD and MDLBV without LED exposure.

Vascular changes in tumor with LED exposure
After obtaining the images of tumor vasculatures without LED exposure, the LED was turned on for PIT treatment. The results of tumor vascular changes with LED exposure are displayed in Fig. 4. Figure 4(a) shows the pseudo-colored SV-OCT image with depth from 0 µm to 480 µm at 0 min, 10 min, 20 min, and 30 min during LED exposure, respectively. Figure 4(b) is the corresponding 3D images. After 10 minutes of LED exposure, there was a slight decrease in the vascular density compared to that at 0 min (when the LED was turned on), especially in the position indicated by the 400 µm * 400 µm white square. After that, the vascular density decreased over time, and some small vessels vanished as labeled by the white arrows in Fig. 4(a). After 30 minutes of irradiation, the LED was turned off. The changes of vasculatures were continued to be monitored for another 30 minutes. The results of tumor vasculatures alteration after turning off the LED are shown in Fig. 4(c) and (d). Results show the density of vessels continues to decrease after turning off the LED, and some small vessels continue to vanish (indicated in the white square from 0 min to 60 min). Cross-sectional OCT images can clearly display the location of tumor vessels. Structure information from cross-sectional images were also analyzed in this study. Cross-sectional images of tumor from both conventional structural OCT mode and SV mode during and after NIR-PIT treatment are displayed in Fig. 5. Three time points were demonstrated: right before LED irradiation (0 min), right after LED irradiation (30 min), and 30 min after LED was turned off. The first row shows the cross-sectional images from conventional structural OCT mode, the second row shows the corresponding lumen in blood vessels (the region filled with blood) from SV mode, and the third row is the fused image of the two modes. The conventional structural OCT mode reflects the blood vessel position (labeled by white arrows). In the SV-OCT mode, both diameter and density of lumen in blood vessels decrease can be clearly visualized at 30 min and 60 min. The fused image can give better visualization about the change of lumen in blood vessels and the location of the whole blood vessels during and after PIT treatment (the third row).

Fig. 5.
Change of tumor vasculatures during and after NIR-PIT process in cross-sectional view. The first row is tumor structure obtained by conventional structural OCT mode, the second row is lumen blood vessel structure (the region filled with blood) obtained by SV mode, and the third row is the overlay of two modes. First column is at time point right before LED irradiation; the second column is right after 30 min LED irradiation. The third column is 30 min after LED was turned off. Scale bar: 200 µm. To quantify the changes of the blood volume inside the blood vessels, we calculated the VD (the density of area filled with blood) and MDLBV (the measurable diameter of the region filled with blood) during and after NIR-PIT treatment as shown in Fig. 6. Normalized VD was displayed in Fig. 6(b). In our study, density value at 0 min was normalized as 1. Density values at other time points were compared with the density value at 0 min. Results show that the density changed slightly at the first 5 min of LED irradiation, then gradually decreased after 10 min. The same trend continued after the LED was turned off. At 60 min, the density value decreased to 0.83 compared to the initial value at 0 min. Figure 6(c) displays the diameter change of blood vessels lumen in 60 min with various vessel sizes labeled in Fig. 6(a). Results indicate that all the vessel diameters display shrinking trends despite of the size. The large vessels have decreased in size, while the small vessels or capillaries (< 50 µm in diameter) gradually vanish and cannot be detected at/after 50 min. Figure 6(d) shows the reduction ratio of diameter at various sizes at 60 min or the last time point that it could be detected before vanishing. The reduction in diameter is about 21% for diameter of 30-50 µm, about 20% for diameter of 60-80 µm, about 24% for diameter of 90-110 µm, and about 28% for diameter of 130-150 µm. Results show that the larger vessels filled with blood have more reduction.
Histology images with H&E and CD31 staining are shown in Fig. 7. With H&E staining, the red blood cells do not display a nucleus and can be found in the well-preserved blood vessels as indicated by the red arrows [57]. They can be found in both control and irradiated groups. The blood vessels have well-defined boundary and can be identified easily in both the control and NIR-PIT-treated group indicating the blood vessels are not damaged by NIR-PIT treatment, which is consistent with our in vivo data from conventional structural OCT and SV-OCT. The tumor cells collapsed after NIR light exposure. CD31 is primarily used to demonstrate the presence of endothelial tissue and can also be used to demonstrate hemangiomas. CD31 staining was used to label the endothelial cell of blood vessels in our experimental. Result demonstrates that the blood vessels in the tumors are markedly dilated in the NIR-PIT-treated group compared with the control group (see the black arrow), which is consistent with previously reported results [57].

Discussion
Unlike some fluorescent imaging technology, OCT does not require any contrast agent and has been approved by FDA to image retinal blood vessels in ophthalmic patients in clinic [66,67]. SV-OCT, which relies on calculation of intensity variance over consecutively acquired frames, is a powerful approach for recognizing fluid flow and has advantages for imaging the blood inside the blood vessels [68]. SV-OCT and Doppler-OCT have been used for monitoring a single vessel change during the PIT process in our previous study [57]. Due to the limitation of imaging speed in the previous system, only a small region and a single large peripheral tumor vessel (>150 µm) was observed. Small vessels or capillaries could not be detected. In this study, OCT was applied to monitor the changes of lumen vessels with different sizes during and after NIR-PIT treatment through SV mode and conventional structural OCT mode. The diameters of tumor lumen vessels above 20 µm can be well resolved. Furthermore, we quantitively revealed the change of density of lumen in blood vessels and the alterations of the diameter of lumen in blood vessels during and after NIR-PIT treatment.
The density and diameter of lumen in the blood vessels remained nearly unchanged without LED irradiation as shown in the SV-OCT images from Fig. 3, which indicates that our OCT laser light (with central wavelength at 1310 nm) did not interact with the Pan-mAb and trigger response on the tumor. From Fig. 4 and Fig. 6, the vascular changes were not obvious in the first 10 minutes of LED irradiation, and then the MDLBV of both big vessels and some small vessels/capillaries gradually decreased with time. After LED was turned off, the diameters continued to decrease or vanish, indicating the decrease of blood inside the blood vessels. Results indicate all vessels with different dimensions have changed. The larger blood vessels have shown the greater reduction. Although we only monitored the NIR-PIT process for 60 min, we could distinctly observe the changes of the blood vessels. While on the other hand, histological results (shown in Fig. 7) show the dilation of the blood vessels. Unlike other ablative cancer therapies, both in vivo SV-OCT imaging (Fig. 4) and histology images (Fig. 7) verified that the tumor blood vessels remained intact during and after NIR-PIT treatment. The dilation of the blood vessels may relate to the previously reported super enhanced permeability and retention (SUPR) effect in NIR-PIT treatment [69]. The dilation of the blood vessels and the SUPR effect will increase the permeability of the blood vessels and decrease the intravascular pressure in the blood vessels, and further increase the blood pool volume in the tumor. This process will result in decreased blood flow and blood volume in the tumor blood vessels, which causes the reduction in the signal that we can detect from SV-OCT. If the signal caused by the decreased blood flow and blood volume is below the detection limit of our OCT system, the red blood cells inside the blood vessels cannot be detected anymore, which can explain the vanishing of small blood vessels/capillaries. The vanishing of small blood vessels/capillaries could also be caused by the obstruction of the vessels, since an obstructed vessel could result in the absence of free moving scatters and the vessel would lose its variance contrast relative to static tissue. Indeed, peripheral tumor blood flow after NIR-PIT dramatically slowed down [57]. We realized the limitations of this study. First, the SV-OCT method is sensitive to particles/blood inside the blood vessels, it cannot image the change of blood vessels and blood flow speed directly. On the other hand, the histological results can only indicate the blood vessels size, while it cannot provide change of the same blood vessel before and after NIR-PIT treatment since it is a terminal procedure. The results from SV-OCT and histology cannot verify each other since they are measuring different parameters. To prove our hypothesis above and thoroughly study the change of the blood vessels size, the change of blood flow speed on the same blood vessels before and after NIR-PIT treatment, intravital multiphoton microscope will be used in the next step as we did in the kidney imaging [70,71].
We need to notice some images of the blood vessels in this study are fuzzy, which may mainly come from the motion artifact due to animal breathing and heartbeat although a dorsal window chamber was used to fix the tumor as previously reported [56,62]. Since the tumor cells were inoculated to dorsum of the mouse in this study, the motion artifact from animal breathing and heartbeat is evitable. This motion artifact can also be seen in Fig. 5: some blood vessels were not displayed at 0 min, but were displayed at 30 min and 60 min. In the future, we will try to minimize the imaging artifact by inoculating the tumor to other regions less sensitive to motion artifact such as mouse ear. In terms of OCT system, we will further optimize the B-scan repetition number and adapt new laser with higher imaging speed, which in principle can further reduce the artifacts caused by motion [45,56].

Conclusion
In conclusion, SV-OCT mode and conventional structural OCT mode were applied to monitor the change of tumor vasculatures during and after NIR-PIT treatment. Results show the reduction in both density and diameter of the blood vessels lumen in SV-OCT mode. Dilated vessels have been observed in histological analysis.
Although the study is on a small number of samples, these results indicate that the changes of blood vessels and the alterations of the blood volume inside the blood vessels during the NIR-PIT treatment process. It can aid researchers in deciding the course of therapy, determining the need for treatment.
Funding. National Natural Science Foundation of China (81901787).