Oral cavity squamous cell carcinoma (OSCC) is considered one of the most aggressive subtypes of cancer. Anti-CD44 monoclonal antibodies (mAb) are a potential therapy against CD44 expressing OSCC; however, to date the therapeutic effects have been disappointing. Here, a new cancer treatment is described, near-infrared photoimmunotherapy (NIR-PIT), that uses anti-CD44 mAbs conjugated to the photoabsorber IR700DX. This conjugate is injected into mice harboring one of three CD44 expressing syngeneic murine oral cancer cell (MOC) lines, MOC1 (immunogenic), MOC2 mKate2 (moderately immunogenic), and MOC2-luc (poorly immunogenic). Binding of the anti-CD44–IR700 conjugate was shown to be specific and cell-specific cytotoxicity was observed after exposure of the cells to NIR light in vitro. The anti-CD44–IR700 conjugate, when assessed in vivo, demonstrated deposition within the tumor with a high tumor-to-background ratio. Tumor-bearing mice were separated into four cohorts: no treatment; 100 μg of anti-CD44–IR700 i.v. only; NIR light exposure only; and 100 μg of anti-CD44–IR700 i.v. with NIR light exposure. NIR-PIT therapy, compared with the other groups, significantly inhibited tumor growth and prolonged survival in all three cell model systems. In conclusion, these data reveal that anti-CD44 antibodies are suitable as mAb–photoabsorber conjugates for NIR-PIT in MOC cells.

Implications: This study using syngeneic mouse models, which better model the disease in humans than conventional xenografts, suggests that NIR-PIT with anti-CD44–IR700 is a potential candidate for the treatment of OSCC. Mol Cancer Res; 15(12); 1667–77. ©2017 AACR.

Oral cavity squamous cell carcinoma (OSCC) is a common, aggressive, carcinogen-induced malignancy (1). Despite advances in detection and standard therapeutic approaches such as surgery, chemotherapy, and radiation, the prognosis for OSCC has remained poor due to a high rate of locoregional recurrence and development of distant metastasis (1–3). Furthermore, approximately two-thirds of OSCC patients have locoregionally advanced disease at the time of diagnosis, resulting in increased morbidity following surgical ablation and adjuvant therapies (1–3). Thus, there is a need for new therapies in this disease.

Intratumoral heterogeneity has been implicated as a major cause of treatment failure. The presence of cancer stem–like cells in tumors has been proposed as one source of tumor heterogeneity as well as a cause of resistance to chemotherapy and radiation (4–8). CD44 is a well-known marker of cancer stem–like cells and mediates intercellular adhesion, cell orientation, migration, and matrix–cell signaling processes (9–11). CD44 is expressed in various types of cancers, including the majority of head and neck cancers. High expression of CD44 is associated with poor local control rates and resistance to therapy (12–14). Suppression of CD44 can lead to tumor growth delay (10, 12, 15). Thus, CD44 has become an important target for antibody-based therapies. In clinical studies a monoclonal anti-CD44 antibody demonstrated some efficacy in phase 1 studies; however, lethal toxic epidermal necrolysis halted further development (16–18).

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses a targeted monoclonal antibody (mAb)–photoabsorber conjugate (APC) (19). The antibody binds to the appropriate cell surface antigen, and the photoactivatable silica-phthalocyanine dye, IRDye700DX (IR700), induces lethal damage to cell membrane after NIR-light exposure. NIR-PIT has been shown to be effective with a variety of different antibodies (19–25). Moreover, a first-in-human phase I trial of NIR-PIT with an APC targeting epidermal growth factor receptor (EGFR) in patients with inoperable head and neck cancer was approved by the FDA (https://clinicaltrials.gov/ct2/show/NCT02422979).

NIR-PIT induces selective immediate, immunogenic cell death (ICD) unlike conventional cancer therapies that typically induce apoptotic cell death (26). To explore the potential immune activating effects of NIR-PIT, we utilized newly established syngeneic murine oral cancer (MOC) models, consisting of MOC1 (immunogenic), MOC2 mKate2 (moderately immunogenic), and MOC2-luc (poorly immunogenic) cell lines. MOC1 cells display a high somatic gene alteration rate and generate tumors with slow primary tumor growth that do not metastasize, have high MHC class I expression, and show robust effector immune cell infiltration. Conversely, MOC2 cells have fewer genetic alterations, generate aggressive tumors that metastasize early to draining lymph nodes, have very low MHC class I expression, and demonstrate limited effector immune cell infiltration (27–29). Thus, these experiments demonstrate the range of immunologic effects of NIR-PIT in various syngeneic tumor models.

Reagents

Water-soluble, silica-phthalocyanine derivative IRDye 700DX NHS ester was obtained from LI-COR Biosciences. An anti-mouse/human CD44-specific mAb, IM7, was purchased from Bio X Cell. All other chemicals were of reagent grade.

Synthesis of IR700-conjugated anti-CD44 mAb

Anti-CD44 mAb (1.0 mg, 6.7 nmol/L) was incubated with IR700 NHS ester (65.1 μg, 33.3 nmol/L) in 0.1 mol/L Na2HPO4 (pH 8.6) at room temperature for 1 hour. The mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare). The protein concentration was determined with the Coomassie Plus protein assay kit (Thermo Fisher Scientific) by measuring the absorption at 595 nm with UV–Vis (8453 Value System; Agilent Technologies). The concentration of IR700 was measured by absorption at 689 nm to confirm the number of fluorophore molecules per mAb. The synthesis was controlled so that an average of two IR700 molecules was bound to a single antibody. We abbreviate IR700 conjugated to anti-CD44 antibody as anti-CD44–IR700. As a quality control for the conjugate, we performed sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) according to previous reports (19–22). Conjugate was separated by SDS-PAGE with a 4%–20% gradient polyacrylamide gel (Life Technologies). A standard marker (Crystalgen Inc.) was used as a protein molecular weight marker. After electrophoresis at 80 V for 2.5 hours, the gel was imaged with a Pearl Imager (LI-COR Biosciences) using a 700-nm fluorescence channel. We used diluted anti-CD44 antibody as non-conjugated control. The gel was stained with Colloidal Blue staining to determine the molecular weight of conjugate.

Cell culture

MOC1 cells stably expressing CD44 antigen, MOC2 cells expressing CD44 and luciferase or CD44 and mKate2 (far-red fluorescent protein TagFP635) were used in this study. We abbreviate one cell line as MOC2-luc and the other MOC2-mKate. Possessing fluorescence with excitation/emission maxima at 588 and 633 nm, mKate2 is almost 3-fold brighter than TagFP635 at physiological pH 7.5. The mKate2 protein is characterized by complete and fast chromophore maturation at 37°C with maturation half-time < 20 minutes. It is also more photostable under both wide-field and confocal illumination than other monomeric far-red proteins (30, 31). High mKate2 expression was confirmed with 10 passages. All MOC cells were cultured in HyClone Iscove's Modified Dulbecco's Medium (IMDM; GE Healthcare Life Sciences)/HyClone Ham's Nutrient Mixture F12 (GE Healthcare Life sciences) at a 2:1 mixture with 5% fetal bovine serum (Life Technologies), 1% penicillin/streptomycin (Life Technologies), 5 ng/mL epidermal growth factor (EGF; EMD Millipore Corporation), 400 ng/mL hydrocortisone (Sigma-Aldrich), and 5 mg/mL insulin (Sigma-Aldrich) in tissue culture flasks in a humidified incubator at 37°C in an atmosphere of 95% air and 5% carbon dioxide (27, 32). Cells were maintained in culture for no more than 30 passages, authenticated by exome sequencing by others (33), and tested for mycoplasma.

Flow cytometry

To verify in vitro anti-CD44–IR700 binding, fluorescence from cells after incubation with APC was measured using a flow cytometer (FACS Calibur, BD Biosciences) and CellQuest software (BD Biosciences). MOC1 and MOC2-luc cells (2 × 105) were seeded into 12-well plates and incubated for 24 hours. Medium was replaced with fresh culture medium containing 10 μg/mL of anti-CD44–IR700 and incubated for 6 hours at 37°C. To validate the specific binding of the conjugated antibody, excess antibody (100 μg) was used to block 10 μg of APCs.

Fluorescence microscopy

To detect the antigen specific localization and effect of NIR-PIT, fluorescence microscopy was performed (BX61; Olympus America, Inc.). Ten thousand MOC1 and MOC2-luc cells were seeded on cover-glass-bottomed dishes and incubated for 24 hours. Anti-CD44–IR700 was then added to the culture medium at 10 μg/mL and incubated for 6 hours at 37°C. After incubation, the cells were washed with phosphate buffered saline (PBS). The filter set to detect IR700 consisted of a 590 to 650 nm excitation filter, a 665 to 740 nm band pass emission filter. Transmitted light differential interference contrast (DIC) images were also acquired.

In vitro NIR-PIT

The cytotoxic effects of NIR-PIT with anti-CD44–IR700 were determined by flow cytometric propidium iodide (PI; Life Technologies) staining, which can detect compromised cell membranes. Two hundred thousand MOC1 and MOC2-luc cells were seeded into 12-well plates and incubated for 24 hours. Medium was replaced with fresh culture medium containing 10 μg/mL of anti-CD44–IR700 and incubated for 6 h at 37°C. After washing with PBS, PBS was added, and cells were irradiated with a red light-emitting diode (LED), which emits light at 670 to 710 nm wavelength (L690-66-60; Marubeni America Co.) at a power density of 50 mW/cm2 as measured with an optical power meter (PM 100, Thorlabs). Cells were scratched 1 hour after treatment. PI was then added in the cell suspension (final 2 μg/mL) and incubated at room temperature for 30 minutes, followed by flow cytometry. Each value represents mean ± SEM of five experiments.

For bioluminescence imaging (BLI), either 200,000 MOC2-luc cells were seeded into 12-well plates or 20 million cells were seeded onto a 10-cm dish; both were preincubated for 24 hours. After replacing the medium with fresh culture medium containing 10 μg/mL of anti-CD44–IR700, the cells were incubated for 6 hours at 37°C in a humidified incubator. After washing with PBS, phenol-red-free culture medium was added. Then, cells were exposed with a LED or a NIR laser which emits light at a 685 to 695 nm wavelength (BWF5-690-8-600-0.37; B&W TEK INC.). The output power density in mW/cm2 was measured with an optical power meter (PM 100, Thorlabs). For luciferase activity, 150 μg/mL D-luciferin-containing media (Gold Biotechnology) was administered to PBS-washed cells 1 hour after NIR-PIT and images were obtained on a BLI system (Photon Imager; Biospace Lab). Regions of interest (ROI) were placed on each entire well, and the luciferase activity (photons/min) was then calculated using M3 Vision Software (Biospace Lab).

Animal and tumor models

All in vivo procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the local Animal Care and Use Committee. Six- to eight-week-old female C57BL/6 mice (strain #000664) were purchased from The Jackson Laboratory. The lower part of the body of the mice was shaved for irradiation and image analysis. During the procedure, mice were anesthetized with inhaled 3%–5% isoflurane and/or via intraperitoneal injection of 1 mg of sodium pentobarbital (Nembutal Sodium Solution, Ovation Pharmaceuticals Inc.). In order to determine tumor volume, the greatest longitudinal diameter (length) and the greatest transverse diameter (width) were measured with an external caliper. Tumor volumes were based on caliper measurements and were calculated using the following formula; tumor volume = length × width2 × 0.5. Body weight was also measured. Mice were monitored daily for general health. Skin color was observed for signs of technology and general health indices, including weight loss and appetite loss, were assessed. Tumor volumes were measured three times a week until the tumor volume reached 2,000 mm3, whereupon the mice were euthanized with inhalation of carbon dioxide gas.

In vivo fluorescence imaging

In vivo IR700 fluorescence images were obtained with a Pearl Imager (LI-COR Biosciences) with a 700-nm fluorescence channel. A ROI was placed on the tumor and the average fluorescence intensity of IR700 signal was calculated for each ROI using a Pearl Cam Software (LI-COR Biosciences). For mKate2 fluorescence imaging, a Maestro Imager (CRi) with a band-pass excitation filter from 575 to 605 nm and a long-pass emission blue filter over 645 nm was used. The tunable emission filter was automatically increased in 10-nm increments from 630 to 850 nm at a constant exposure time (500 ms). The spectral fluorescence images consisted of autofluorescence spectra and spectra from mKate2, which were then unmixed from each other based on the characteristic spectral pattern of mKate2 using a Maestro software (CRi).

In vivo bioluminescence imaging

For in vivo BLI, D-luciferin (15 mg/mL, 200 μL) was injected intraperitoneally, and the mice were analyzed on a BLI system (Photon Imager) for luciferase activity. ROIs were set to include the entire tumor in order to quantify BLI. ROIs were also placed in the adjacent non-tumor region as background. Average luciferase activity of each ROI was calculated.

In vivo fluorescence imaging studies

MOC1 cells (2.0 × 106) and MOC2-luc cells (1.5 × 105) were injected subcutaneously in the right dorsum of the mice. Tumors were studied after they reached volumes of approximately 50 mm3. Serial dorsal fluorescence images of IR700 were obtained with a Pearl Imager using a 700-nm fluorescence channel before and 0, ½, 1, 2, 3, 4, 5, 6, 9, 12, 24, 48, 72, 96, 120, 144, and 168 hours after i.v. injection of 100 μg of anti-CD44–IR700 via the tail vein. Pearl Cam Software (LI-COR Biosciences) was used for analyzing IR700 fluorescence intensities. ROIs were placed on the tumor and the adjacent non-tumor region as background (left dorsum). Average fluorescence intensity of each ROI was calculated. Target-to-background ratios (TBR; fluorescence intensities of target/fluorescence intensities of background) were also calculated (n = 10).

In vivo NIR-PIT in a unilateral tumor model

MOC1 cells (2.0 × 106) and MOC2-luc cells (1.5 × 105) were injected subcutaneously in the right dorsum of the mice. Tumors were studied after they reached volumes of approximately 50 mm3. To examine the therapeutic effect of in vivo NIR-PIT on both cells, tumor-bearing mice were randomized into 4 groups of at least 8 animals per group for the following treatments: (i) no treatment (control); (ii) 100 μg of anti-CD44–IR700 i.v., no NIR light exposure (APC i.v. only); (iii) NIR light exposure only, NIR light was administered at 50 J/cm2 on day 0 and 100 J/cm2 on day 1 (NIR light only); (iv) 100 μg of anti-CD44–IR700 i.v., NIR light was administered at 50 J/cm2 on day 0 and 100 J/cm2 on day 1 (NIR-PIT). Serial IR700 fluorescence images, as well as white light images, were obtained before and after each NIR light exposure (day 0 and day 1), and day 2 using a Pearl Imager with a 700-nm fluorescence channel. BLI also obtained before NIR light exposure (day 0), day 1, and day 2 using Photon Imager.

MOC2 mKate2 cells (1.5 × 105) were injected subcutaneously in the right dorsum of the mice. Tumors were studied after they reached volumes of approximately 50 mm3. To examine the therapeutic effect of in vivo NIR-PIT on MOC2 mKate2 cells, tumor-bearing mice were randomized into 2 groups of at least 10 animals per group for the following treatments: (i) no treatment (control); (ii) 100 μg of anti-CD44–IR700 i.v., NIR light was administered at 50 J/cm2 on day 0 and 100 J/cm2 on day 1 (NIR-PIT). Serial IR700 fluorescence images, as well as white light images, were obtained before and after each NIR light exposure (day 0 and day 1), day 2, and day 3 using a Pearl Imager with a 700-nm fluorescence channel. The mKate2 fluorescence images also obtained before NIR light exposure (day 0), day 1, day 2, and day 3 using a Maestro Imager.

In vivo NIR-PIT in bilateral tumors model

MOC1 cells (2.0 × 106) and MOC2-luc cells (1.5 × 105) were injected subcutaneously in both dorsa of the mice. Tumors were studied after they reached volumes of approximately 50 mm3. Tumor-bearing mice were randomized into 2 groups of at least 8 animals per group for the following treatments: (i) no APC, NIR light was administered at 100 J/cm2 only to the right-sided tumor on day 0; (ii) 100 μg of anti-CD44–IR700 i.v., NIR light was administered at 100 J/cm2 only to the right-sided tumor on 1 day after injection (day 0). The untreated left-sided tumor was shielded from light exposure by covering it with aluminum foil to prevent direct NIR light exposure. Serial IR700 fluorescence images, as well as white light images, were obtained before and after NIR light exposure (day 0), day 1, and day 2 using a Pearl Imager with a 700-nm fluorescence channel. BLI also obtained before NIR light exposure (day 0), day 1, and day 2 using the Photon Imager.

MOC2 mKate2 cells (1.5 × 105) were injected subcutaneously in both dorsa of the mice. Tumors were studied after they reached volumes of approximately 50 mm3. Tumor-bearing mice were randomized into 2 groups of at least 8 animals per group for the following treatments: (i) no APC, NIR light was administered at 100 J/cm2 only to the right-sided tumor on day 0; (ii) 100 μg of anti-CD44–IR700 i.v., NIR light was administered at 100 J/cm2 only to the right-sided tumor one day after injection (day 0). The untreated left-sided tumor was shielded from light exposure by covering it with aluminum foil to prevent direct NIR light exposure. Serial IR700 fluorescence images, as well as white light images, were obtained before and after each NIR light exposure (day 0 and day 1), day 2, and day 3 using a Pearl Imager with a 700-nm fluorescence channel. The mKate2 fluorescence images were also obtained before NIR light exposure (day 0), day 1, day 2, and day 3 using a Maestro Imager.

Histological analysis

To detect the antigen-specific microdistribution in the tumor, fluorescence microscopy was performed. Tumor xenografts were excised from the right flank xenograft without treatment, 24 hours after injection of anti-CD44–IR700 (APC i.v. only) and 24 hours after NIR-PIT. Extracted tumors were frozen with optimal cutting temperature (OCT) compound (SAKURA Finetek Japan Co.) and frozen sections (10 μm thick) were prepared. Fluorescence microscopy was performed using the BX61 microscope with the following filters: excitation wavelength 590 to 650 nm, emission wavelength 665 to 740 nm long pass for IR700 fluorescence. DIC images were also acquired. To evaluate histological changes, light microscopy study was also performed using Olympus BX61. Tumor xenografts were excised from mice without treatment, 24 hours after injection of anti-CD44–IR700 (APC i.v. only) and 24 hours after NIR-PIT. Tumors were also excised from mice with bilateral flank tumors (both treated right-sided tumors and untreated left-sided tumors) 24 hours after NIR-PIT of the right tumor. Extracted tumors were also placed in 10% formalin, and serial 10-μm slice sections were fixed on a glass slide with H&E staining.

Statistical analysis

Data are expressed as means ± SEM from a minimum of five experiments, unless otherwise indicated. Statistical analyses were carried out using GraphPad Prism version 7 (GraphPad Software). The Student t test was used to compare the treatment effects with that of control in vitro. To compare intensity and TBR between MOC1 and MOC2-luc tumors at all time points and to compare tumor growth in a unilateral tumor model of MOC2 mKate2, the Mann–Whitney test was used. For multiple comparisons, a one-way analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons was used. The cumulative probability of survival based on volume (2,000 mm3) were estimated in each group with a Kaplan–Meier survival curve analysis, and the results were compared with use of the log-rank test. A P value of < 0.05 was considered statistically significant.

In vitro characterization of MOC cell lines

As defined by SDS-PAGE, the band of anti-CD44–IR700 was almost the same molecular weight as the non-conjugated control mAb and fluorescence intensity was identical with the band of naked anti-CD44 antibody (Fig. 1A). After a 6 hours of incubation with anti-CD44–IR700, MOC1 and MOC2-luc cells showed high fluorescence signal, which was confirmed with flow cytometry (Fig. 1B and C) and fluorescence microscopy (Fig. 1D). These signals were completely blocked by adding excess anti-CD44 mAb (Fig. 1B and C), indicating that anti-CD44–IR700 binds specifically. The IR700 fluorescence signals of MOC2-luc cells were higher than MOC1 cells (Fig. 1B–D).

In vitro NIR-PIT

Immediately after exposure, NIR light induced cellular swelling, bleb formation, and rupture of vesicles representing necrotic cell death (Supplementary Videos S1 and S2). Most of these morphologic changes were observed within 15 minutes of light exposure (Fig. 1D), indicating rapid induction of necrotic cell death. Based on incorporation of PI, percentage of cell death increased in a light dose dependent manner (Fig. 1E and F). There was no significant cytotoxicity associated with NIR light alone in the absence of APC and with APC alone without NIR light. Percentage cell death in MOC2-luc cells was higher than in MOC1 cells at the same light doses. For instance, when exposed to 4J of NIR light the percentage of cell death in MOC2-luc cells was over 80% (Fig. 1F), while it was less than 60% in MOC1 cells (Fig. 1E). Bioluminescence showed a decrease of luciferase activity in a light dose-dependent manner (Fig. 1G) and in relative luciferase activity in MOC2-luc cells (Fig. 1H).

In vivo fluorescence imaging studies

Moderate fluorescence intensity from anti-CD44–IR700 was observed in MOC1 with high fluorescence intensity seen in MOC2 tumors 1 day after APC injection, after which the fluorescence in both cell types gradually decreased (Fig. 2A and B). Similarly, the TBR of anti-CD44–IR700 in both tumors was high 24 hours after APC injection, following which the TBR decreased over the following days (Fig. 2C). The highest TBR was observed 1 day after APC injection.

In vivo NIR-PIT for MOC1 tumor in a unilateral tumor model

The NIR-PIT regimen and imaging protocol are depicted in Fig. 3A. One day after injection of anti-CD44–IR700, the tumors were exposed to 50 J/cm2 of NIR light. IR700 tumor fluorescence signal decreased due to dispersion of fluorophore from dying cells and partial photo-bleaching, while in the absence of NIR light, the IR700 fluorescence gradually decreased over the following days (Fig. 3B). Tumor growth was significantly inhibited in the NIR-PIT treatment group compared with the other groups (P < 0.001) (Fig. 3C), and significantly prolonged survival was achieved in the NIR-PIT group (P < 0.001 vs. other groups; Fig. 3D). No significant therapeutic effect was observed in the other groups. There was no skin necrosis or toxicity attributable to the APC in any group.

Almost all fluorescence disappeared 24 hours after 100 J/cm2 of NIR-PIT in the histological fluorescence image (Fig. 4B). Hematoxylin and eosin (H&E) staining of NIR-PIT–treated MOC1 tumors revealed diffuse necrosis and microhemorrhage, with scattered clusters of live but damaged tumor cells, while no obvious damage was observed in the tumor receiving only anti-CD44–IR700 but no light (Fig. 4C).

In vivo NIR-PIT for MOC1 tumor in bilateral tumors model

Local CD44-targeted NIR-PIT was performed only on right-sided tumors in mice bearing bilateral tumors. Like the unilateral model, after exposure of the right sided tumor to 100 J/cm2 of NIR light, IR700 tumor fluorescence signal decreased due to dying cells and partial photo-bleaching, while the IR700 fluorescence of the untreated left-sided tumors did not change (Supplementary Fig. S1B). Tumor growth was significantly inhibited in the treated tumors compared with the other tumors (P < 0.001 vs. control tumors) (Supplementary Fig. S1C). There were no significant differences between the two groups in survival owing to the continued growth of left sided tumors which determined survival (Supplementary Fig. S1D). H&E staining of NIR-PIT–treated tumors revealed diffuse necrosis and microhemorrhage, with scattered clusters of live but damaged tumor cells (Supplementary Fig. S1E). On the other hand, no obvious damage was observed in control tumors including contralateral untreated left-sided tumors (Supplementary Fig. S1E).

In vivo NIR-PIT for MOC2-luc tumor in a unilateral tumor model

The NIR-PIT regimen and imaging protocol are depicted in Fig. 5A. NIR-PIT resulted in decreases in bioluminescence in the treated tumor (Fig. 5B) but increases in bioluminescence in other tumor groups due to rapid tumor growth. In contrast, luciferase activity decreased 1 and 2 days after NIR-PIT (P < 0.05 vs. other groups; Fig. 5C). After exposure to 50 J/cm2 of NIR light, IR700 tumor fluorescence signal markedly decreased compared to tumors receiving anti-CD44–IR700 but no NIR light (Fig. 5D). Tumor growth was significantly inhibited in the NIR-PIT treatment group (P < 0.001; Fig. 5E), and significantly prolonged survival was achieved in the NIR-PIT group (P < 0.001 vs. other groups; Fig. 5F). No significant therapeutic effect was observed in the control groups.

In the histological fluorescence image, high fluorescence intensity was shown in MOC2-luc tumors 24 hours after anti-CD44–IR700 injection. The IR700 fluorescence signals of MOC2-luc tumor were higher compared with the signals of MOC1 tumor (Fig. 6B; MOC2-luc tumor vs. Fig. 4B; MOC1 tumor). Fluorescence almost completely disappeared 24 hours after 100 J/cm2 of NIR-PIT (Fig. 6B). H&E staining of NIR-PIT–treated MOC2-luc tumors revealed diffuse necrosis and microhemorrhage, with scattered clusters of live but damaged tumor cells, whereas no obvious damage was observed in the tumor receiving only anti-CD44–IR700 but no light (Fig. 6C).

In vivo NIR-PIT for MOC2-luc tumor in bilateral tumors model

The treatment and imaging regimen is shown in Supplementary Fig. S2A. Local CD44-targeted NIR-PIT was performed only on the right-sided tumors in mice bearing bilateral tumors. NIR-PIT resulted in decreases in bioluminescence of right-sided tumor (Supplementary Fig. S2B) while luciferase activity of both tumors in the control group and untreated left-sided tumors of the NIR-PIT group increased due to rapid tumor growth. In contrast, luciferase activity of treated right-sided tumors decreased 1 and 2 days after NIR-PIT (P < 0.001 vs. other tumors for 1 day, P < 0.01 vs. other tumors for 2 days, respectively) (Supplementary Fig. S2C). After exposure to 100 J/cm2 of NIR light, IR700 tumor fluorescence signal of treated right-sided tumors decreased, while the IR700 fluorescence of untreated left-sided tumors slightly decreased after NIR-PIT (Supplementary Fig. S2D). Tumor growth was significantly inhibited in the treated right-sided tumors compared with the other tumors (P < 0.01 vs. untreated left-sided tumors, P < 0.001 vs. control tumors, respectively; Supplementary Fig. S2E). Moreover, tumor growth was also significantly inhibited in the untreated left-sided tumors compared with the tumors in the control group (P < 0.05 vs. both tumors of the control group; Supplementary Fig. S2E). Significantly prolonged survival was achieved in the NIR-PIT group (P < 0.01 vs. control group; Supplementary Fig. S2F). There was no toxicity attributable to the APC in the NIR-PIT group. H&E staining of NIR-PIT–treated MOC2-luc tumors revealed diffuse necrosis and microhemorrhage, with scattered clusters of live but damaged tumor cells (Supplementary Fig. S2G). On the other hand, only the tumor surface area was damaged in untreated left-sided tumors (Supplementary Fig. S2H).

In vivo NIR-PIT for MOC2 mKate2 tumor in a unilateral tumor model

The NIR-PIT regimen and imaging protocol are depicted in Supplementary Fig. S3A. In IR700 fluorescence imaging, 1 day after injection of anti-CD44–IR700, the tumors showed higher fluorescence intensity (Supplementary Fig. S3B). In mKate2 fluorescence real-time imaging, the tumor treated by NIR-PIT showed decreasing mKate2 fluorescence signal (Supplementary Fig. S3C). Tumor growth was significantly inhibited in the NIR-PIT treatment group compared with the control group (P < 0.001) (Supplementary Fig. S3D), and significantly prolonged survival was achieved in the NIR-PIT group (P < 0.001 vs. control group; Supplementary Fig. S3E).

In vivo NIR-PIT for MOC2 mKate2 tumor in the bilateral tumor model

The treatment and imaging regimen is shown in Supplementary Fig. S4A. Local CD44-targeted NIR-PIT was performed only on right-sided tumors in mice bearing bilateral tumors (Supplementary Fig. S4B). In mKate2 fluorescence real-time imaging, the tumor treated by NIR-PIT showed decreasing mKate2 fluorescence signal, while the mKate2 fluorescence of control tumors including untreated left-sided tumors did not change (Supplementary Fig. S4C). Tumor growth was significantly inhibited in the treated right-sided tumors (P < 0.001 vs. other control tumors; Supplementary Fig. S4D). Moreover, tumor growth was also significantly inhibited in the untreated left-sided tumors compared with the tumors in the control group (P < 0.05 vs. both tumors of control group; Supplementary Fig. S4D). Significantly prolonged survival was achieved in the NIR-PIT group (P < 0.01 vs. control group; Supplementary Fig. S4E).

NIR-PIT using anti-CD44–IR700 conjugate demonstrates efficacy in treating CD44-expressing syngeneic mouse models of OSCC (Figs. 3–6 and Supplementary Figs. S1–S4). NIR-PIT with anti-CD44–IR700 led to rapid cell death in vitro (Fig. 1; Supplementary Videos S1 and S2) and tumor growth reduction with survival improvements (Figs. 3, 5 and Supplementary Fig. S3). Importantly, in immunologic tumors such as MOC2 tumors, treatment effects could be measured in the non-irradiated tumors (Supplementary Figs. S2 and S4).

There are several preconditions for successful NIR-PIT. First, there must be adequate uptake and retention of the APC in the target tumor. In vivo fluorescence imaging clearly showed the cell lines taking up the APC (Fig. 2). Thus, the conjugation of IR700 to the antibody minimally alters the pharmacokinetics of antibody.

Another prerequisite for successful NIR-PIT is that the treatment is tumor specific to avoid side effects. The anti-CD44 antibody has demonstrated clinical activity as monotherapy; however, it is associated with significant skin toxicity, including a case of lethal epidermal necrolysis (16–18). Recently, it was reported that a first-in-human phase I clinical trial with a new mAb targeting the constant region of CD44 showed an acceptable safety profile in patients with advanced, CD44-expressing solid tumors, however, this study was terminated early due to modest clinical efficacy (best response was stable disease) and lack of a clinical and/or pharmacodynamics dose–response relationship (34). NIR-PIT has several advantages because it is minimally invasive, has few side effects, and can be used repeatedly (35). Moreover, the dose of the APC needed for NIR-PIT is much lower than naked antibody monotherapy doses, those posing lower risks of systemic side effects.

The models used in this study are syngeneic and therefore cast light on the immunologic consequences of NIR-PIT. MOC models are carcinogen-induced, fully syngeneic on a C57BL/6 genetic background, and consist of cell lines with genetic alterations that mirror human OSCC (28, 32, 33, 36). Cells treated with NIR-PIT undergo rapid volume expansion, leading to rupture of the cell membrane and extrusion of cell contents into the extracellular space (Fig. 1; Supplementary Videos S1 and S2), also known as an immunogenic cell death (ICD), in contrast to most other treatments that result in apoptosis (26, 37–39). After NIR-PIT signaling, moieties including calreticulin, ATP, and HMGB1 effectively promote maturation of immature dendritic cells (26) which are capable of digesting the cancer cell debris. These effects were in evidence in this study as non-treated tumors showed responses and this resulted in overall prolonged survival. Looking toward the future, NIR-PIT, using antibodies targeting immune-suppressor cells could result in rapid activation of cytotoxic CD8+T- and NK cells both locally and at other untreated sites in the body (23).

It is noteworthy that no contralateral therapeutic effect was observed in bilateral tumors models of MOC1 (Supplementary Fig. S1). On the other hand, MOC2-luc and MOC2 mKate2 showed a response to treatment in contralateral tumors (Supplementary Figs. S2 and S4). However, IR700 fluorescence signals in untreated left-sided tumors were also slightly decreased after contralateral NIR-PIT despite shielding the left-sided tumors from light exposure by covering them with aluminum foil (Supplementary Figs. S2D and S4B). Moreover, pathological analysis of untreated left-sided tumors of the MOC2-luc revealed that cellular necrosis and microhemorrhage, identical in quality to the NIR-PIT treated side, were observed albeit limited to the tumor surface (Supplementary Fig. S2H). These results suggest the possibility that despite shielding, there was internal scattering of light within the mouse body, resulting in a mild NIR-PIT effect.

NIR-PIT shows highly target-specific cytotoxicity, and NIR light can be easily delivered to lesions in the mouth, such as CD44 expressing cancers of the pharynx or larynx. On the other hand, an obvious limitation of NIR-PIT is the inability to deliver NIR light to tumors located deep to the surface. NIR light can penetrate several centimeters into tissue (40), but eventually skin, fat and other organs will absorb NIR light before it reaches deeper tumor tissue. There are several potential solutions to this problem. For instance, NIR light could be delivered to a tumor while it is exposed as, for instance, during surgery. Alternatively, light could be administered endoscopically (e.g., bronchus, bowel, ureter, etc.) or via needle trocars in organs. Another caveat of this study is that we performed one injection of the APC with one or two exposures of NIR light because C57BL/6 mice started to have skin pigmentation within 7 days after shaving and therefore were not suitable for the experiments as the pigmentation could result in excessive absorption of light. This objection can be overcome by fractionating the dose of APC followed by repeated light exposures (41, 42). It would be desirable to extend these studies to multiple doses of the APC and light.

NIR-PIT using anti-CD44–IR700 can induce significant therapeutic responses after only a single injection of the conjugate and NIR light exposure in three CD44 expressing syngeneic mouse models of OSCC. Thus, NIR-PIT utilizing CD44 as the targeting antigen for the APC might be new treatment modality for OSCC, especially after initial diagnosis or after locoregional recurrence.

No potential conflicts of interest were disclosed.

Conception and design: T. Nagaya, P.L. Choyke, C. Allen, H. Kobayashi

Development of methodology: T. Nagaya, P.L. Choyke, H. Kobayashi

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T. Nagaya, Y. Nakamura, S. Okuyama, H. Kobayashi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T. Nagaya, Y. Maruoka, P.L. Choyke, H. Kobayashi

Writing, review, and/or revision of the manuscript: T. Nagaya, F. Ogata, P.L. Choyke, C. Allen, H. Kobayashi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Nagaya, F. Ogata, P.L. Choyke, C. Allen, H. Kobayashi

Study supervision: P.L. Choyke, H. Kobayashi

This research was supported by the Intramural Research Program of the NIH, NCI, Center for Cancer Research (ZIA BC011513).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Kademani
D
. 
Oral cancer
.
Mayo Clin Proc
2007
;
82
:
878
87
.
2.
Haddad
RI
,
Shin
DM
. 
Recent advances in head and neck cancer
.
N Engl J Med
2008
;
359
:
1143
54
.
3.
Rogers
SN
,
Brown
JS
,
Woolgar
JA
,
Lowe
D
,
Magennis
P
,
Shaw
RJ
, et al
Survival following primary surgery for oral cancer
.
Oral Oncol
2009
;
45
:
201
11
.
4.
Jamieson
CH
,
Ailles
LE
,
Dylla
SJ
,
Muijtjens
M
,
Jones
C
,
Zehnder
JL
, et al
Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML
.
N Engl J Med
2004
;
351
:
657
67
.
5.
Lee
IC
,
Chuang
CC
,
Wu
YC
. 
Niche mimicking for selection and enrichment of liver cancer stem cells by hyaluronic acid-based multilayer films
.
ACS Appl Mater Interfaces
2015
;
7
:
22188
95
.
6.
Ma
Y
,
Liang
D
,
Liu
J
,
Axcrona
K
,
Kvalheim
G
,
Stokke
T
, et al
Prostate cancer cell lines under hypoxia exhibit greater stem-like properties
.
PLoS One
2011
;
6
:
e29170
.
7.
Prince
ME
,
Ailles
LE
. 
Cancer stem cells in head and neck squamous cell cancer
.
J Clin Oncol
2008
;
26
:
2871
5
.
8.
Singh
SK
,
Clarke
ID
,
Terasaki
M
,
Bonn
VE
,
Hawkins
C
,
Squire
J
, et al
Identification of a cancer stem cell in human brain tumors
.
Cancer Res
2003
;
63
:
5821
8
.
9.
Muntimadugu
E
,
Kumar
R
,
Saladi
S
,
Rafeeqi
TA
,
Khan
W
. 
CD44 targeted chemotherapy for co-eradication of breast cancer stem cells and cancer cells using polymeric nanoparticles of salinomycin and paclitaxel
.
Colloids Surf B Biointerfaces
2016
;
143
:
532
46
.
10.
Ponta
H
,
Sherman
L
,
Herrlich
PA
. 
CD44: from adhesion molecules to signalling regulators
.
Nat Rev Mol Cell Biol
2003
;
4
:
33
45
.
11.
Tovuu
LO
,
Imura
S
,
Utsunomiya
T
,
Morine
Y
,
Ikemoto
T
,
Arakawa
Y
, et al
Role of CD44 expression in non-tumor tissue on intrahepatic recurrence of hepatocellular carcinoma
.
Int J Clin Oncol
2013
;
18
:
651
6
.
12.
Chen
J
,
Zhou
J
,
Lu
J
,
Xiong
H
,
Shi
X
,
Gong
L
. 
Significance of CD44 expression in head and neck cancer: a systemic review and meta-analysis
.
BMC Cancer
2014
;
14
:
15
.
13.
de Jong
MC
,
Pramana
J
,
van der Wal
JE
,
Lacko
M
,
Peutz-Kootstra
CJ
,
de Jong
JM
, et al
CD44 expression predicts local recurrence after radiotherapy in larynx cancer
.
Clin Cancer Res
2010
;
16
:
5329
38
.
14.
Motegi
A
,
Fujii
S
,
Zenda
S
,
Arahira
S
,
Tahara
M
,
Hayashi
R
, et al
Impact of expression of CD44, a cancer stem cell marker, on the treatment outcomes of intensity modulated radiation therapy in patients with oropharyngeal squamous cell carcinoma
.
Int J Radiat Oncol Biol Phys
2016
;
94
:
461
8
.
15.
Okada
H
,
Yoshida
J
,
Sokabe
M
,
Wakabayashi
T
,
Hagiwara
M
. 
Suppression of CD44 expression decreases migration and invasion of human glioma cells
.
Int J Cancer
1996
;
66
:
255
60
.
16.
Rupp
U
,
Schoendorf-Holland
E
,
Eichbaum
M
,
Schuetz
F
,
Lauschner
I
,
Schmidt
P
, et al
Safety and pharmacokinetics of bivatuzumab mertansine in patients with CD44v6-positive metastatic breast cancer: final results of a phase I study
.
Anticancer Drugs
2007
;
18
:
477
85
.
17.
Sauter
A
,
Kloft
C
,
Gronau
S
,
Bogeschdorfer
F
,
Erhardt
T
,
Golze
W
, et al
Pharmacokinetics, immunogenicity and safety of bivatuzumab mertansine, a novel CD44v6-targeting immunoconjugate, in patients with squamous cell carcinoma of the head and neck
.
Int J Oncol
2007
;
30
:
927
35
.
18.
Tijink
BM
,
Buter
J
,
de Bree
R
,
Giaccone
G
,
Lang
MS
,
Staab
A
, et al
A phase I dose escalation study with anti-CD44v6 bivatuzumab mertansine in patients with incurable squamous cell carcinoma of the head and neck or esophagus
.
Clin Cancer Res
2006
;
12
:
6064
72
.
19.
Mitsunaga
M
,
Ogawa
M
,
Kosaka
N
,
Rosenblum
LT
,
Choyke
PL
,
Kobayashi
H
. 
Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules
.
Nat Med
2011
;
17
:
1685
91
.
20.
Nagaya
T
,
Nakamura
Y
,
Sato
K
,
Harada
T
,
Choyke
PL
,
Hodge
JW
, et al
Near infrared photoimmunotherapy with avelumab, an anti-programmed death-ligand 1 (PD-L1) antibody
.
Oncotarget
2017
;
8
:
8807
17
.
21.
Nagaya
T
,
Nakamura
Y
,
Sato
K
,
Harada
T
,
Choyke
PL
,
Kobayashi
H
. 
Near infrared photoimmunotherapy of B-cell lymphoma
.
Mol Oncol
2016
;
10
:
1404
14
.
22.
Nagaya
T
,
Nakamura
Y
,
Sato
K
,
Zhang
YF
,
Ni
M
,
Choyke
PL
, et al
Near infrared photoimmunotherapy with an anti-mesothelin antibody
.
Oncotarget
2016
;
7
:
23361
9
.
23.
Sato
K
,
Sato
N
,
Xu
B
,
Nakamura
Y
,
Nagaya
T
,
Choyke
PL
, et al
Spatially selective depletion of tumor-associated regulatory T cells with near-infrared photoimmunotherapy
.
Sci Transl Med
2016
;
8
:
352ra110
.
24.
Sato
K
,
Watanabe
R
,
Hanaoka
H
,
Harada
T
,
Nakajima
T
,
Kim
I
, et al
Photoimmunotherapy: comparative effectiveness of two monoclonal antibodies targeting the epidermal growth factor receptor
.
Mol Oncol
2014
;
8
:
620
32
.
25.
Watanabe
R
,
Hanaoka
H
,
Sato
K
,
Nagaya
T
,
Harada
T
,
Mitsunaga
M
, et al
Photoimmunotherapy targeting prostate-specific membrane antigen: are antibody fragments as effective as antibodies?
J Nucl Med
2015
;
56
:
140
4
.
26.
Ogawa
M
,
Tomita
Y
,
Nakamura
Y
,
Lee
MJ
,
Lee
S
,
Tomita
S
, et al
Immunogenic cancer cell death selectively induced by near infrared photoimmunotherapy initiates host tumor immunity
.
Oncotarget
2017
;
8
:
10425
36
.
27.
Judd
NP
,
Allen
CT
,
Winkler
AE
,
Uppaluri
R
. 
Comparative analysis of tumor-infiltrating lymphocytes in a syngeneic mouse model of oral cancer
.
Otolaryngol Head Neck Surg
2012
;
147
:
493
500
.
28.
Moore
EC
,
Cash
HA
,
Caruso
AM
,
Uppaluri
R
,
Hodge
JW
,
Van Waes
C
, et al
Enhanced tumor control with combination mTOR and PD-L1 inhibition in syngeneic oral cavity cancers
.
Cancer Immunol Res
2016
;
4
:
611
20
.
29.
Shah
S
,
Caruso
A
,
Cash
H
,
Waes
CV
,
Allen
CT
. 
Pools of programmed death-ligand within the oral cavity tumor microenvironment: variable alteration by targeted therapies
.
Head Neck
2016
;
38
:
1176
86
.
30.
Shcherbo
D
,
Merzlyak
EM
,
Chepurnykh
TV
,
Fradkov
AF
,
Ermakova
GV
,
Solovieva
EA
, et al
Bright far-red fluorescent protein for whole-body imaging
.
Nat Methods
2007
;
4
:
741
6
.
31.
Shcherbo
D
,
Murphy
CS
,
Ermakova
GV
,
Solovieva
EA
,
Chepurnykh
TV
,
Shcheglov
AS
, et al
Far-red fluorescent tags for protein imaging in living tissues
.
Biochem J
2009
;
418
:
567
74
.
32.
Judd
NP
,
Winkler
AE
,
Murillo-Sauca
O
,
Brotman
JJ
,
Law
JH
,
Lewis
JS
 Jr
, et al
ERK1/2 regulation of CD44 modulates oral cancer aggressiveness
.
Cancer Res
2012
;
72
:
365
74
.
33.
Onken
MD
,
Winkler
AE
,
Kanchi
KL
,
Chalivendra
V
,
Law
JH
,
Rickert
CG
, et al
A surprising cross-species conservation in the genomic landscape of mouse and human oral cancer identifies a transcriptional signature predicting metastatic disease
.
Clin Cancer Res
2014
;
20
:
2873
84
.
34.
der Houven van Oordt
CW
,
Gomez-Roca
C
,
Herpen
CV
,
Coveler
AL
,
Mahalingam
D
,
Verheul
HM
, et al
First-in-human phase I clinical trial of RG7356, an anti-CD44 humanized antibody, in patients with advanced, CD44-expressing solid tumors
.
Oncotarget
2016
;
7
:
80046
58
.
35.
Wilson
BC
,
Patterson
MS
. 
The physics, biophysics and technology of photodynamic therapy
.
Phys Med Biol
2008
;
53
:
R61
109
.
36.
Moore
E
,
Clavijo
PE
,
Davis
R
,
Cash
H
,
Van Waes
C
,
Kim
Y
, et al
Established T cell-inflamed tumors rejected after adaptive resistance was reversed by combination STING activation and PD-1 pathway blockade
.
Cancer Immunol Res
2016
;
4
:
1061
71
.
37.
Kerr
JF
. 
Shrinkage necrosis: a distinct mode of cellular death
.
J Pathol
1971
;
105
:
13
20
.
38.
Willingham
MC
. 
Cytochemical methods for the detection of apoptosis
.
J Histochem Cytochem
1999
;
47
:
1101
10
.
39.
Ziegler
U
,
Groscurth
P
. 
Morphological features of cell death
.
News Physiol Sci
2004
;
19
:
124
8
.
40.
Henderson
TA
,
Morries
LD
. 
Near-infrared photonic energy penetration: can infrared phototherapy effectively reach the human brain?
Neuropsychiatr Dis Treat
2015
;
11
:
2191
208
.
41.
Mitsunaga
M
,
Nakajima
T
,
Sano
K
,
Choyke
PL
,
Kobayashi
H
. 
Near-infrared theranostic photoimmunotherapy (PIT): repeated exposure of light enhances the effect of immunoconjugate
.
Bioconjug Chem
2012
;
23
:
604
9
.
42.
Nagaya
T
,
Sato
K
,
Harada
T
,
Nakamura
Y
,
Choyke
PL
,
Kobayashi
H
. 
Near infrared photoimmunotherapy targeting EGFR positive triple negative breast cancer: optimizing the conjugate-light regimen
.
PLoS One
2015
;
10
:
e0136829
.