Phototruncation cell tracking with near-infrared photoimmunotherapy using heptamethine cyanine dye to visualise migratory dynamics of immune cells.

BACKGROUND: Noninvasive in vivo cell tracking is valuable in understanding the mechanisms that enhance anti-cancer immunity. We have recently developed a new method called phototruncation-assisted cell tracking (PACT), that uses photoconvertible cell tracking technology to detect in vivo cell migration. This method has the advantages of not requiring genetic engineering of cells and employing tissue-penetrant near-infrared light. METHODS: We applied PACT to monitor the migration of immune cells between a tumour and its tumour-draining lymph node (TDLN) after near-infrared photoimmunotherapy (NIR-PIT). FINDINGS: PACT showed a significant increase in the migration of dendritic cells (DCs) and macrophages from the tumour to the TDLN immediately after NIR-PIT. This migration by NIR-PIT was abrogated by inhibiting the sphingosine-1-phosphate pathway or Gαi signaling. These results were corroborated by intranodal immune cell profiles at two days post-treatment; NIR-PIT significantly induced DC maturation and increased and activated the CD8+ T cell population in the TDLN. Furthermore, PACT revealed that NIR-PIT significantly enhanced the migration of CD8+ T cells from the TDLN to the tumour four days post-treatment, which was consistent with the immunohistochemical assessment of tumour-infiltrating lymphocytes and tumour regression. INTERPRETATION: Immune cells dramatically migrated between the tumour and TDLN following NIR-PIT, indicating its potential as an immune-stimulating therapy. Also, PACT is potentially applicable to a wide range of immunological research. FUNDING: This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Centre for Cancer Research (grant number: ZIA BC011513 and ZIA BC011506).

Near-infrared photoimmunotherapy targeting Nectin-4 in a preclinical model of bladder cancer.

Enfortumab vedotin (EV), an antibody-drug conjugate (ADC) that targets Nectin-4, has shown promising results in the treatment of bladder cancer. However, multiple resistance mechanisms that are unique to ADCs limit the therapeutic potential of EV in clinical practice. Here, we developed and tested a Nectin-4-targeted near-infrared photoimmunotherapy (NIR-PIT) that utilizes the same target as EV but utilizes a distinct cytotoxic and immunotherapeutic pathway in preclinical models of bladder cancer. NIR-PIT was effective in vitro against luminal subtype human bladder cancer cell lines (RT4, RT112, MGH-U3, SW780, and HT1376-luc), but not against other subtype cell lines (UMUC3 and T24). In vivo, the tumor site was clearly visible by Nectin-4-IR700 fluorescence 24 h after its administration, suggesting the potential as an intraoperative imaging modality. NIR-PIT significantly suppressed tumor growth and prolonged survival in SW780 and RT112 xenograft models. Weekly treatment with NIR-PIT further improved tumor control in RT112 xenograft models. The effectiveness of NIR-PIT was also confirmed in HT1376-luc orthotopic xenograft models. Histological analysis verified that NIR-PIT induced a significant pathologic response. Taken together, Nectin-4-targeted NIR-PIT shows promise as a treatment for luminal subtype bladder cancers.

Intratumoral IL15 Improves Efficacy of Near-Infrared Photoimmunotherapy.

IL15 is a potent inducer of differentiation and proliferation of CD8+ T and natural killer (NK) cells, making it a promising candidate for cancer immunotherapy. However, limited efficacy of systemic monotherapy utilizing intravenous IL15 suggests the needs for alternative routes of administration or combination treatment with other therapies. Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective anticancer treatment that elicits a massive release of tumor antigens and immunogenic signals. Here, we investigated whether intratumoral IL15 can enhance the effectiveness of cancer cell-targeted NIR-PIT using syngeneic murine tumor models. Intratumoral injection of IL15 was more effective than intraperitoneal IL15 in vivo in suppressing tumor growth and inducing intratumoral immune responses. When the efficacy of CD44-targeted NIR-PIT was compared in vivo between IL15-secreting MC38 (hIL15-MC38) and parental MC38 tumors, the hIL15-MC38/NIR-PIT group showed the best tumor growth inhibition and survival. In addition, the hIL15-MC38/NIR-PIT group showed significant dendritic cell maturation and significant increases in the number and Granzyme B expression of tumor-infiltrating CD8+ T, NK, and natural killer T cells compared with the treated parental line. Furthermore, intratumoral IL15 injection combined with CD44-targeted NIR-PIT showed significant tumor control in MC38 and Pan02-luc tumor models. In bilateral tumor models, CD44-targeted NIR-PIT in hIL15-MC38 tumors significantly suppressed the growth of untreated MC38 tumors, suggesting abscopal effects. Mice that achieved complete response after the combination therapy completely rejected later tumor rechallenge. In conclusion, local IL15 administration synergistically improves the efficacy of cancer cell-targeted NIR-PIT probably by inducing stronger anticancer immunity, indicating its potential as an anticancer treatment strategy.

The chemical basis of cytotoxicity of silicon-phthalocyanine-based near infrared photoimmunotherapy (NIR-PIT) and its implications for treatment monitoring.

Near infrared photoimmunotherapy (NIR-PIT) is a new cancer therapy based on the photo-induced ligand release reaction of a silicon-phthalocyanine derivative, IRDye700DX (IR700), that causes rapid cell death. Following exposure to an antibody-IR700-conjugate, cells exposed to NIR light within minutes undergo rapid swelling, blebbing, and finally, bursting. The photo-induced ligand release reaction also induces immediate loss of IR700 fluorescence due to dimerization or aggregation of the antibody-IR700 conjugate allowing for real time monitoring of NIR-PIT therapy.

Near-infrared photoimmunotherapy (NIR-PIT) of bone metastases.

The bones are a common site for metastasis arising from solid tumors such as breast and prostate cancer. Chemotherapy, including immunotherapy, is rarely curative. Radiotherapy with pain palliation can temporize bone metastases but is generally considered a short-term solution and retreatment is difficult. Surgery is often necessary, yet recovery times might exceed life expectancy. Therefore, there is a need to develop new approaches to bone metastases that are effective but minimally invasive. Near-infrared photoimmunotherapy (NIR-PIT) uses antibodies labeled with IRDye700DX (IR700) which is activated by NIR light, resulting in rapid cell membrane damage and immunogenic cell death. NIR-PIT using an anti-epidermal growth factor receptor (EGFR) antibody-IR700 conjugate in patients with recurrent head and neck cancer received qualified approval in Japan in 2020 and is now widely used there. However, no bone metastases have yet been treated. In this study, the efficacy of NIR-PIT for bone metastases was investigated using a bone metastases mouse model successfully established by caudal artery injection of a human triple-negative breast cancer cell line, MDAMB468-GFP/luc. The bone metastatic lesions were treated with NIR-PIT using the anti-EGFR antibody, panitumumab-IR700 conjugate. Bioluminescence imaging and histological evaluation showed that EGFR-targeted NIR-PIT has a therapeutic effect on bone metastatic lesions in mice. In addition, micro-CT showed that repeated NIR-PIT led to repair of metastasis-induced bone destruction and restored bone cortex continuity consistent with healing. These data suggest that NIR-PIT has the potential for clinical application in the treatment of bone metastases.

Evaluating near-infrared photoimmunotherapy for targeting fibroblast activation protein-α expressing cells in vitro and in vivo.

Photoimmunotherapy (PIT), carried out using an Ab conjugated to the near infrared dye IRDye700DX, is achieving significant success in target-specific elimination of cells. Fibroblast activation protein alpha (FAP-α) is an important target in cancer because of its expression by cancer-associated fibroblasts (CAFs) as well as by some cancer cells. Cancer-associated fibroblasts that express FAP-α have protumorigenic and immune suppressive functions. Using immunohistochemistry of human breast cancer tissue microarrays, we identified an increase of FAP-α+  CAFs in invasive breast cancer tissue compared to adjacent normal tissue. We found FAP-α expression increased in fibroblasts cocultured with cancer cells. In proof-of-principle studies, we engineered human FAP-α overexpressing MDA-MB-231 and HT-1080 cancer cells and murine FAP-α overexpressing NIH-3T3 fibroblasts to evaluate several anti-FAP-α Abs and selected AF3715 based on its high binding affinity with both human and mouse FAP-α. After conjugation of AF3715 with the phthalocyanine dye IR700, the resultant Ab conjugate, FAP-α-IR700, was evaluated in cells and tumors for its specificity and effectiveness in eliminating FAP-α expressing cell populations with PIT. Fibroblast activation protein-α-IR700-PIT resulted in effective FAP-α-specific cell killing in the engineered cancer cells and in two patient-derived CAFs in a dose-dependent manner. Following an intravenous injection, FAP-α-IR700 retention was three-fold higher than IgG-IR700 in FAP-α overexpressing tumors, and two-fold higher compared to WT tumors. Fibroblast activation protein-α-IR700-PIT resulted in significant growth inhibition of tumors derived from FAP-α overexpressing human cancer cells. A reduction of endogenous FAP-α+ murine CAFs was identified at 7 days after FAP-α-IR700-PIT. Fibroblast activation protein-α-targeted near infrared PIT presents a promising strategy to eliminate FAP-α+ CAFs.

Near-Infrared Photoimmunotherapy Targeting Podoplanin-Expressing Cancer Cells and Cancer-Associated Fibroblasts.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that uses an antibody-IRDye700DX (IR700) conjugate that binds to a target followed by the application of NIR light that results in dramatic changes in solubility of the conjugate leading to rapid cell membrane damage and highly immunogenic cell death. NIR-PIT has been used clinically in treating advanced head and neck cancers using an anti-EGFR antibody-IR700 conjugate and has been conditionally approved for clinical use in Japan. NIR-PIT can be employed using a wide range of targeting antibodies. Podoplanin (PDPN), also known as gp38, is a 38 kDa type-1 transmembrane protein associated with lymphatic vessels. In cancer cells and cancer-associated fibroblasts (CAFs), PDPN expression has been widely reported and correlates with poor outcomes in several cancer types. In this study, we evaluated the efficacy of PDPN-targeted NIR-PIT in syngenetic mouse models of cancer. PDPN-targeted NIR-PIT destroyed PDPN-expressing cancer cells and CAFs selectively, suppressing tumor progression and prolonging survival with minimal damage to lymphatic vessels compared with the control group. Interestingly, PDPN-targeted NIR-PIT also exerted a therapeutic effect by targeting CAFs in tumor models which do not express in cancer cells. Furthermore, increased cytotoxic T cells in the tumor bed after PDPN-targeted NIR-PIT were observed, suggesting enhanced host antitumor immunity. Thus, PDPN-targeted NIR-PIT is a promising new cancer therapy strategy for PDPN-expressing cancer cells and CAFs.

[Novel Therapy Targeting the Cancer Microenvironment Using Near-Infrared Photoimmunotherapy Leading to Tumor Immune Activation].

Near-infrared photoimmunotherapy(NIR-PIT)is a novel cancer treatment modality that employs antibody-IRDye700DX (IR700)conjugates. Recently, the clinical application of NIR-PIT has received approval in Japan for patients with inoperable head and neck cancer, specifically targeting the human epidermal growth factor receptor(hEGFR). Furthermore, NIR-PIT extends beyond the scope of tumor antigens and can be employed to eliminate specific host cells that contribute to the creation of immune-permissive environments supporting tumor growth. One of the distinguishing features of NIR-PIT is its ability to selectively eliminate various cell types within the tumor microenvironment(TME)by specifically targeting distinct antigens. By employing podoplanin(PDPN)-targeted NIR-PIT, PDPN-expressing fibroblasts were selectively eradicated, resulting in the suppression of tumor progression and a notable extension of overall survival. Additionally, we investigated the efficacy of depleting myeloid-derived suppressor cells(MDSCs)using NIR-PIT. This approach led to the selective elimination of MDSCs within tumors, and remarkable abscopal effects were observed in bilateral tumor models. Hence, NIR-PIT holds immense promise for the treatment of diverse cancer types by precisely targeting tumor cells, fibroblasts, and immune cells.

Selective depletion of polymorphonuclear myeloid derived suppressor cells in tumor beds with near infrared photoimmunotherapy enhances host immune response.

The immune system is recognized as an important factor in regulating the development, progression, and metastasis of cancer. Myeloid-derived suppressor cells (MDSCs) are a major immune-suppressive cell type by interfering with T cell activation, promoting effector T cell apoptosis, and inducing regulatory T cell expansion. Consequently, reducing or eliminating MDSCs has become a goal of some systemic immunotherapies. However, by systemically reducing MDSCs, unwanted side effects can occur. Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed treatment that selectively kills targeted cells without damaging adjacent normal cells. The aim of this study is to evaluate the antitumor efficacy of MDSC-directed NIR-PIT utilizing anti-Ly6G antibodies to specifically destroy polymorphonuclear (PMN)-MDSCs in the tumor microenvironment (TME) in syngeneic mouse models. PMN-MDSCs were selectively eliminated within tumors by Ly6G-targeted NIR-PIT. There was significant tumor growth suppression and prolonged survival in three treated tumor models. In the early phase after NIR-PIT, dendritic cell maturation/activation and CD8+ T cell activation were enhanced in both intratumoral tissues and tumor-draining lymph nodes, and NK cells demonstrated increased expression of cytotoxic molecules. Host immunity remained activated in the TME for at least one week after NIR-PIT. Abscopal effects in bilateral tumor models were observed. Furthermore, the combination of NIR-PIT targeting cancer cells and PMN-MDSCs yielded synergistic effects and demonstrated highly activated host tumor immunity. In conclusion, we demonstrated that selective local PMN-MDSCs depletion by NIR-PIT could be a promising new cancer immunotherapy.

Dual-targeted near-infrared photoimmunotherapy for esophageal cancer and cancer-associated fibroblasts in the tumor microenvironment.

Cancer-associated fibroblasts (CAFs) play a significant role in tumor progression within the tumor microenvironment. Previously, we used near-infrared photoimmunotherapy (NIR-PIT), a next-generation cancer cell-targeted phototherapy, to establish CAF-targeted NIR-PIT. In this study, we investigated whether dual-targeted NIR-PIT, targeting cancer cells and CAFs, could be a therapeutic strategy. A total of 132 cases of esophageal cancer were analyzed for epidermal growth factor receptor (EGFR), human epidermal growth factor 2 (HER2), and fibroblast activation protein (FAP) expression using immunohistochemistry. Human esophageal cancer cells and CAFs were co-cultured and treated with single- or dual-targeted NIR-PIT in vitro. These cells were co-inoculated into BALB/c-nu/nu mice and the tumors were treated with single-targeted NIR-PIT or dual-targeted NIR-PIT in vivo. Survival analysis showed FAP- or EGFR-high patients had worse survival than patients with low expression of FAP or EGFR (log-rank, P < 0.001 and P = 0.074, respectively), while no difference was observed in HER2 status. In vitro, dual (EGFR/FAP)-targeted NIR-PIT induced specific therapeutic effects in cancer cells and CAFs along with suppressing tumor growth in vivo, whereas single-targeted NIR-PIT did not show any significance. Moreover, these experiments demonstrated that dual-targeted NIR-PIT could treat cancer cells and CAFs simultaneously with a single NIR light irradiation. We demonstrated the relationship between EGFR/FAP expression and prognosis of patients with esophageal cancer and the stronger therapeutic effect of dual-targeted NIR-PIT than single-targeted NIR-PIT in experimental models. Thus, dual-targeted NIR-PIT might be a promising therapeutic strategy for cancer treatment.

Quantitative Assessment of the Efficacy of Near-Infrared Photoimmunotherapy with Bioluminescence Imaging.

Near-infrared photoimmunotherapy (NIR-PIT) is a cell-specific cancer therapy in which antibody-photoabsorber conjugates (APCs) are activated by NIR light to induce rapid immunogenic cell death with minimal off-target effects. In preclinical settings, bioluminescence imaging (BLI) is useful to quantitatively assess the efficacy of NIR-PIT for both in vitro and in vivo experiments, especially in the early phase of testing. Here, we describe the detailed methods of the experiments for NIR-PIT and evaluation of its efficacy using BLI.

Comparison of the Effectiveness of IgG Antibody versus F(ab')2 Antibody Fragment in CTLA4-Targeted Near-Infrared Photoimmunotherapy.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment modality that utilizes antibody-photoabsorber conjugates (APCs) and selectively kills target cells after irradiation with NIR light. Originally, NIR-PIT was targeted against cancer cell surface antigens, but as it became clear that NIR-PIT induced a strong immune response, an effort was made to target selected immune cell populations in the tumor microenvironment to encourage an even stronger immune response. Thus, CD25-targeted NIR-PIT and cytotoxic T-lymphocyte associated protein 4 (CTLA4)-targeted NIR-PIT were developed to kill regulatory T cells (Tregs) in conjunction with cancer-cell-targeted NIR-PIT, in order to amplify the host immune response. It was found that CD25-targeted NIR-PIT, using an antibody with the Fc portion removed, led to better results than the unmodified anti-CD25 antibody-directed NIR-PIT presumably because of a negative effect on activated T cells. The aim of this study was to compare the efficacy of an antibody fragment [anti-CTLA4-F(ab')2] and a whole antibody (anti-CTLA4-IgG) for NIR-PIT. There was no significant difference in NIR-PIT-induced Treg killing between the anti-CTLA4-F(ab')2 and anti-CTLA4-IgG antibodies. Although both the antibody and the antibody fragment resulted in significant tumor growth inhibition, the antibody induced more robust CD8+ T cell activation in ipsilateral lymph nodes and was more effective compared to the antibody fragment. The slower clearance of the anti-CTLA4-IgG APC enhanced antitumor immunity by promoting T cell priming in lymph nodes. In conclusion, unlike the results with CD25 where modified antibodies produced superior results to unmodified antibodies, anti-CTLA4-IgG antibody-based NIR-PIT proved more effective in reducing tumor growth than anti-CTLA4-F(ab')2 antibody-based NIR-PIT.

CD29 targeted near-infrared photoimmunotherapy (NIR-PIT) in the treatment of a pigmented melanoma model.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that utilizes an antibody-photoabsorber-conjugate (AbPC) combined with NIR light. The AbPC is injected and binds to the tumor whereupon NIR light irradiation causes a photochemical reaction that selectively kills cancer cells. NIR-PIT is ideal for surface-located skin cancers such as melanoma. However, there is concern that the pigment in melanoma lesions could interfere with light delivery, rendering treatment ineffective. We investigated the efficacy of CD29- and CD44-targeted NIR-PIT (CD29-PIT and CD44-PIT, respectively) in the B16 melanoma model, which is highly pigmented. While CD29-PIT and CD44-PIT killed B16 cells invitro and invivo, CD29-PIT suppressed tumor growth more efficiently. Ki67 expression showed that cells surviving CD29-PIT were less proliferative, suggesting that CD29-PIT was selective for more proliferative cancer cells. CD29-PIT did not kill immune cells, whereas CD44-PIT killed both T and NK cells and most myeloid cells, including DCs, which could interfere with the immune response to NIR-PIT. The addition of anti-CTLA4 antibody immune checkpoint inhibitor (ICI) to CD29-PIT increased the infiltration of CD8 T cells and enhanced tumor suppression with prolonged survival. Such effects were less prominent when the anti-CTLA4 ICI was combined with CD44-PIT. The preservation of immune cells in the tumor microenvironment (TME) after CD29-PIT likely led to a better response when combined with anti-CTLA4 treatment. We conclude that NIR-PIT can be performed in pigmented melanomas and that CD29 is a promising target for NIR-PIT, which is amenable to combination therapy with other immunotherapies.

Endoscopic near-infrared photoimmunotherapy in an orthotopic head and neck cancer model.

Near-infrared photoimmunotherapy (NIR-PIT) is a cell selective cancer therapy that uses an antibody-photoabsorber (IRDye700DX, IR700) conjugate (APC) and NIR light. NIR-PIT targeting epidermal growth factor receptor (EGFR) in head and neck cancer (HNC) was conditionally approved in Japan in 2020. APC-bound tumors can be detected using endoscopic fluorescence imaging, whereas NIR light can be delivered using endoscopic fiber optics. The aims of this study were: (1) to assess the feasibility of endoscopic NIR-PIT in an orthotopic HNC model using a CD44-expressing MOC2-luc cell line; and (2) to evaluate quantitative fluorescence endoscopic imaging prior to and during NIR-PIT. The results were compared in 3 experimental groups: (1) untreated controls, (2) APC injection without light exposure (APC-IV), and (3) APC injection followed by NIR light exposure (NIR-PIT). APC injected groups showed significantly higher fluorescence signals for IR700 compared with the control group prior to therapeutic NIR light exposure, and the fluorescence signal significantly decreased in the NIR-PIT group after light exposure. After treatment, the NIR-PIT group showed significantly attenuated bioluminescence compared with the control and the APC-IV groups. Histology demonstrated diffuse necrotic death of the cancer cells in the NIR-PIT group alone. In conclusion, endoscopically delivered light combined with quantitative fluorescence imaging can be used to "see and treat" HNC. This method could also be applied to other types of cancer approachable with endoscopy.

Diagnostic imaging in near-infrared photoimmunotherapy using a commercially available camera for indocyanine green.

Near-infrared photoimmunotherapy (NIR-PIT) is a new type of cancer treatment, which was recently approved in Japan for patients with inoperable head and neck cancer. NIR-PIT utilizes antibody-IRDye700DX (IR700) conjugates and NIR light at a wavelength of 690 nm. NIR light exposure leads to physicochemical changes in the antibody-IR700 conjugate cell receptor complex, inducing rapid necrotic cell death. Just as fluorescence guided surgery is useful for surgeons to resect tumors completely, real-time information of tumor locations would help clinicians irradiate NIR light more precisely. IR700 is a fluorescence dye that emits at 702 nm; however, there is no clinically available device optimized for detecting this fluorescence. On the other hand, many indocyanine green (ICG) fluorescence imaging devices have been approved for clinical use. Therefore, we investigated whether LIGHTVISION, one of the clinically available ICG cameras, could be employed for tumor detection. We hypothesized that irradiation with even low-power 690-nm laser light, attenuated by 99% with a neutral-density filter, could be detected with LIGHTVISION without fluorescence decay or therapeutic effect because of the long emission tail of IR700 beyond 800 nm (within the detection range of LIGHTVISION). We demonstrated that the LIGHTVISION camera, originally designed for ICG detection, can detect the tail of IR700 fluorescence in real time, thus enabling the visualization of target tumors.

Fluorescence Imaging of Tumor-Accumulating Antibody-IR700 Conjugates Prior to Near-Infrared Photoimmunotherapy (NIR-PIT) Using a Commercially Available Camera Designed for Indocyanine Green.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses antibody-IRDye700DX (IR700) conjugates and was recently approved in Japan for patients with inoperable head and neck cancer. Exposure of the tumor with NIR light at a wavelength of 690 nm leads to physicochemical changes in the antibody-IR700 conjugate-cell receptor complex, resulting in increased hydrophobicity and damage to the integrity of the cell membrane. However, it is important that the tumor be completely exposed to light during NIR-PIT, and thus, a method to provide real-time information on tumor location would help clinicians direct light more accurately. IR700 is a fluorophore that emits at 702 nm; however, there is no clinically available device optimized for detecting this fluorescence. On the other hand, many indocyanine green (ICG) fluorescence imaging devices have been approved for clinical use in operating rooms. Therefore, we investigated whether LIGHTVISION, one of the clinically available ICG cameras, could be employed for NIR-PIT target tumor detection. Due to the limited benefits of adding IR700 molecules, the additional conjugation of IRDye800CW (IR800) or ICG-EG4-Sulfo-OSu (ICG-EG4), which has an overlapping spectrum with ICG, to trastuzumab-IR700 conjugates was performed. Conjugation of second NIR dyes did not interfere the efficacy of NIR-PIT. The dual conjugation of IR800 and IR700 to trastuzumab clearly visualized target tumors with LIGHTVISION by detecting emission light of IR800. We demonstrated that the conjugation of second NIR dyes enables us to provide a real-time feedback of tumor locations prior to NIR-PIT.

Fibroblast activation protein targeted near infrared photoimmunotherapy (NIR PIT) overcomes therapeutic resistance in human esophageal cancer.

Cancer-associated fibroblasts (CAFs) have an important role in the tumor microenvironment. CAFs have the multifunctionality which strongly support cancer progression and the acquisition of therapeutic resistance by cancer cells. Near-infrared photoimmunotherapy (NIR-PIT) is a novel cancer treatment that uses a highly selective monoclonal antibody (mAb)-photosensitizer conjugate. We developed fibroblast activation protein (FAP)-targeted NIR-PIT, in which IR700 was conjugated to a FAP-specific antibody to target CAFs (CAFs-targeted NIR-PIT: CAFs-PIT). Thus, we hypothesized that the control of CAFs could overcome the resistance to conventional chemotherapy in esophageal cancer (EC). In this study, we evaluated whether EC cell acquisition of stronger malignant characteristics and refractoriness to chemoradiotherapy are mediated by CAFs. Next, we assessed whether the resistance could be rescued by eliminating CAF stimulation by CAFs-PIT in vitro and in vivo. Cancer cells acquired chemoradiotherapy resistance via CAF stimulation in vitro and 5-fluorouracil (FU) resistance in CAF-coinoculated tumor models in vivo. CAF stimulation promoted the migration/invasion of cancer cells and a stem-like phenotype in vitro, which were rescued by elimination of CAF stimulation. CAFs-PIT had a highly selective effect on CAFs in vitro. Finally, CAF elimination by CAFs-PIT in vivo demonstrated that the combination of 5-FU and NIR-PIT succeeded in producing 70.9% tumor reduction, while 5-FU alone achieved only 13.3% reduction, suggesting the recovery of 5-FU sensitivity in CAF-rich tumors. In conclusion, CAFs-PIT could overcome therapeutic resistance via CAF elimination. The combined use of novel targeted CAFs-PIT with conventional anticancer treatments can be expected to provide a more effective and sensible treatment strategy.

Real-time IR700 Fluorescence Imaging During Near-infrared Photoimmunotherapy Using a Clinically-approved Camera for Indocyanine Green.

Background/Aim: Near-infrared photoimmuno-therapy (NIR-PIT) is a newly approved cancer phototherapy. We aim to confirm whether a clinically approved camera for indocyanine green (ICG) could monitor IR700 fluorescence in real time during exposure to therapeutic NIR light. Materials and Methods: An NIR camera, LIGHTVISION, designed to image ICG fluorescence, was used. A431-GFP/luc tumor-bearing mice were exposed to therapeutic NIR light and real-time fluorescence imaging (RT-FI) was obtained and measured with LIGHTVISION. Bioluminescence imaging (BLI) was performed to confirm cell death. Results: RT-FI during NIR-PIT revealed an initial rapid loss of fluorescence, followed by a plateau which occurred at a light dose of approximately 30 J/cm 2 . Correlation between BLI and IR700 fluorescence loss showed that loss of fluorescence was associated with increased cell death. Conclusion: The efficacy of NIR-PIT could be monitored non-invasively and in real-time using weak fluorescence at wavelengths much longer than the peak fluorescence of IR700. This technique can achieve precise light dosimetry that allows us to decide on the optimal exposure.

Real-Time Fluorescence Imaging Using Indocyanine Green to Assess Therapeutic Effects of Near-Infrared Photoimmunotherapy in Tumor Model Mice.

BACKGROUND: Near-infrared photoimmunotherapy (NIR-PIT) is a cancer therapy that causes an increase in tumor perfusion, a phenomenon termed the super-enhanced permeability and retention effect. Currently, in vivo treatment efficacy of NIR-PIT is observable days after treatment, but monitoring would be improved by more acute detection of intratumor change. Fluorescence imaging may detect increased tumor perfusion immediately after treatment. METHODS: In the first experiment, athymic nude mouse models bearing unilateral subcutaneous flank tumors were treated with either NIR-PIT or laser therapy only. In the second experiment, mice bearing bilateral flank tumors were treated with NIR-PIT only on the left-sided tumor. In both groups, immediately after treatment, indocyanine green was injected at different doses intravenously, and mice were monitored with the Shimadzu LIGHTVISION fluorescence imaging system for 1 hour. RESULTS: Tumor-to-background ratio of fluorescence intensity increased over the 60 minutes of monitoring in treated mice but did not vary significantly in control mice. Tumor-to-background ratio was highest in the 1 mg kg-1 and 0.3 mg kg-1 doses. In mice with bilateral tumors, tumor-to-untreated tumor ratio increased similarly. CONCLUSIONS: Acute changes in tumor perfusion after NIR-PIT can be detected by real-time fluorescence imaging.