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Aim: Evaluation of lymphatic drainage can be challenging to differentiate between separate drainage basins because only one ‘color’ is typically employed in sentinel node studies. This study aimed to test the feasibility of multicolor in vivo lymphangiography using newly developed organic polymer dots. Materials & methods: Biocompatible, purely organic, hydroporphyrin-doped near-infrared-emitting polymer dots were developed and evaluated for in vivo multicolor imaging in mouse lymph nodes. Results & conclusion: The authors demonstrated successful multicolor in vivo fluorescence lymphangiography using polymer dots, each tuned to a different emission spectrum. This allows minimally invasive visualization of at least four separate lymphatic drainage basins using fluorescent nanoparticles, which have the potential for clinical translation.

Background Driver alterations may represent novel candidates for driver gene-guided therapy; however, intrahepatic cholangiocarcinoma (ICC) with multiple genomic aberrations makes them intractable. Therefore, the pathogenesis and metabolic changes of ICC need to be understood to develop new treatment strategies. We aimed to unravel the evolution of ICC and identify ICC-specific metabolic characteristics to investigate the metabolic pathway associated with ICC development using multiregional sampling to encompass the intra- and inter-tumoral heterogeneity. Methods We performed the genomic, transcriptomic, proteomic and metabolomic analysis of 39–77 ICC tumour samples and eleven normal samples. Further, we analysed their cell proliferation and viability. Results We demonstrated that intra-tumoral heterogeneity of ICCs with distinct driver genes per case exhibited neutral evolution, regardless of their tumour stage. Upregulation of BCAT1 and BCAT2 indicated the involvement of ‘Val Leu Ile degradation pathway’. ICCs exhibit the accumulation of ubiquitous metabolites, such as branched-chain amino acids including valine, leucine, and isoleucine, to negatively affect cancer prognosis. We revealed that this metabolic pathway was almost ubiquitously altered in all cases with genomic diversity and might play important roles in tumour progression and overall survival. Conclusions We propose a novel ICC onco-metabolic pathway that could enable the development of new therapeutic interventions.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a mAb conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is a surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, CD25-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Treg), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44- and CD25-targeted NIR-PIT also resulted in some complete remissions. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.

Programmed cell death 1 (PD-1) blockade therapy can result in dramatic responses in some patients with cancer. However, about 15% of patients receiving PD-1 blockade therapy experience rapid tumor progression, a phenomenon termed “hyperprogressive disease” (HPD). The mechanism(s) underlying HPD has been difficult to uncover because HPD is challenging to reproduce in animal models. Near-infrared photoimmunotherapy (NIR-PIT) is a method by which specific cells in the tumor microenvironment (TME) can be selectively depleted without disturbing other cells in the TME. In this study, we partially depleted CD8+ T cells with NIR-PIT by targeting the CD8β antigen thereby temporarily changing the balance of T-cell subsets in two different syngeneic tumor models. PD-1 blockade in these models led to rapid tumor progression compared with controls. CD3ε+CD8α+/CD3ε+CD4+FoxP3+ (Teff/Treg) ratios in the PD-1 and NIR-PIT groups were lower than in controls. Moreover, in a bilateral tumor model, low-dose CD8β-targeted NIR-PIT with anti–PD-1 blockade showed rapid tumor progression only in the tumor exposed to NIR light. In this experiment CD8β-targeted NIR-PIT in the exposed tumor reduced local CD8+ T cells resulting in a regulatory T-cell (Treg)–dominant TME. In conclusion, this reports an animal model to simulate the Treg-dominant TME, and the data generated using the model suggest that HPD after PD-1 blockade therapy can be attributed, at least in part, to imbalances between effector T cells and Tregs in the TME.

Supplementary Figure from Treg-Dominant Tumor Microenvironment Is Responsible for Hyperprogressive Disease after PD-1 Blockade Therapy

Supplementary figures 1 and 2

Supplementary figure S1-S5

Supplementary Fig. S1. Efficacy of PDPN-targeted NIR-PIT for BALB/c 3T3 cells in vitro. Supplementary Fig. S2. Internalization of PDPN-IR700 in MOC1 and BALB/c 3T3 cells. Supplementary Fig. S3. Distribution of PDPN-expressing cells in MOC2 tumors. Supplementary Fig. S4. PDPN expression on hematopoietic cells. Supplementary Fig. S5. In vivo 700 nm fluorescence imaging after the injection of PDPN-IR700. Supplementary Fig. S6. Lymphogram after PDPN-targeted NIR-PIT. Supplementary Fig. S7. The evaluation of lymphatic and blood vessels after PDPN-targeted NIR-PIT in a MOC1 tumor model by multiplex IHC. Supplementary Fig. S8. Efficacy of in vitro PDPN-targeted NIR-PIT against MC38-luc tumors which has no PDPN expression. Supplementary Fig. S9. The difference of MOC2 and MC38-luc tumors in multiplex IHC. Supplementary Fig. S10. The distribution of DIG-labeled anti-PDPN (PDPN-DIG) in MOC1 tumors.

Near-infrared photoimmunotherapy (NIR-PIT) selectively kills tumor cells to which the photo-absorber dye IR700DX-conjugated antibodies are bound and induces a systemic anti-tumor immune response. NIR-PIT induces immunogenic cell death (ICD), releases damage-associated molecular patterns (DAMPs) molecules from dying tumor cells, and activates dendritic cells (DCs). However, it is unclear whether NIR-PIT affects migration of tumor-infiltrating (Ti)-DCs to draining lymph nodes (dLNs), where a systemic anti-tumor response is induced. Here, we utilized in vivo photolabeling of Ti-DCs in tumors in photoconvertible protein Kikume Green-Red (KikGR) mice to show that NIR-PIT enhanced migration of Ti-DCs including cDC1s, cDC2s, and CD326

V-domain immunoglobulin suppressor of T cell activation (VISTA) is an inhibitory immune checkpoint molecule that is broadly expressed on lymphoid and myeloid cells, including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Near-infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that utilizes an antibody-photoabsorber (IRDye 700DX NHS ester) conjugate to selectively kill target cells after the local application of NIR light. Depletion of VISTA-expressing cells in the tumor microenvironment (TME) using NIR-PIT could enhance anti-tumor immune responses by removing immune suppressive cells. The purpose of this study was to evaluate the anti-tumor efficacy of VISTA-targeted NIR-PIT using two murine tumor models, MC38-luc and LL2-luc. VISTA was expressed on T cells including Tregs and MDSCs in the TME of these tumors. In contrast, CD45 - cells, including cancer cells, did not express VISTA. VISTA-targeted NIR-PIT depleted VISTA-expressing cells ex vivo. In vivo VISTA-targeted NIR-PIT inhibited tumor progression and prolonged survival in both models. After VISTA-targeted NIR-PIT, augmented CD8 + T cell and dendritic cell activation were observed in regional lymph nodes. In conclusion, VISTA-targeted NIR-PIT can effectively treat tumors by decreasing VISTA-expressing immune suppressor cells in the TME. Local depletion of VISTA-expressing cells in the tumor bed using NIR-PIT is a promising new cancer immunotherapy for treating various types of tumors.

Colorectal cancer (CRC) is one of the most common types of cancer and a significant cause of cancer mortality worldwide. Further improvements of CRC therapeutic approaches are needed. BCL2‐associated athanogene 6 (BAG6), a multifunctional scaffold protein, plays an important role in tumor progression. However, regulation of BAG6 in malignancies remains unclear. This study showed that guided entry of tail‐anchored proteins factor 4 (GET4), a component of the BAG6 complex, regulates the intercellular localization of BAG6 in CRC. Furthermore, GET4 was identified as a candidate driver gene on the short arm of chromosome 7, which is often amplified in CRC, by our bioinformatics approach using the CRC dataset from The Cancer Genome Atlas. Clinicopathologic and prognostic analyses using CRC datasets showed that GET4 was overexpressed in tumor cells due to an increased DNA copy number. High GET4 expression was an independent poor prognostic factor in CRC, whereas BAG6 was mainly overexpressed in the cytoplasm of tumor cells without gene alteration. The biological significance of GET4 was examined using GET4 KO CRC cells generated with CRISPR‐Cas9 technology or transfected CRC cells. In vitro and in vivo analyses showed that GET4 promoted tumor growth. It appears to facilitate cell cycle progression by cytoplasmic enrichment of BAG6‐mediated p53 acetylation followed by reduced p21 expression. In conclusion, we showed that GET4 is a novel driver gene and a prognostic biomarker that promotes CRC progression by inducing the cytoplasmic transport of BAG6. GET4 could be a promising therapeutic molecular target in CRC., GET4 stained more intensely in the nuclei and cytoplasm of colorectal cancer tumor cells than those of normal colon.

Near-infrared photoimmunotherapy (NIR-PIT) is a new cancer treatment that utilizes antibody–IRDye700DX (IR700) conjugates. The clinical use of NIR-PIT has recently been approved in Japan for patients with inoperable head and neck cancer targeting human epidermal growth factor receptor (hEGFR). Previously, cytotoxic T-lymphocyte antigen 4 (CTLA4)–targeted NIR-PIT has been shown to strongly inhibit tumor progression and prolonged survival was seen in different tumor models due to enhanced T-cell–mediated antitumor immunity. In this study, combined NIR-PIT targeting CTLA4 expressing cells and cancer cells was investigated in four tumor models including a newly established hEGFR-expressing murine oropharyngeal cancer cell (mEERL-hEGFR). While single molecule–targeted therapy (NIR-PIT targeting hEGFR or CTLA4) did not inhibit tumor progression in poorly immunogenic mEERL-hEGFR tumor, dual (CTLA4/hEGFR)-targeted NIR-PIT significantly suppressed tumor growth and prolonged survival resulting in a 38% complete response rate. After the dual-targeted NIR-PIT, depletion of CTLA4 expressing cells, which were mainly regulatory T cells (Tregs), and an increase in the CD8+/Treg ratio in the tumor bed were observed, suggesting enhanced host antitumor immunity. Furthermore, dual-targeted NIR-PIT showed antitumor immunity in distant untreated tumors of the same type. Thus, simultaneous cancer cell–targeted NIR-PIT and CTLA4-targeted NIR-PIT is a promising new cancer therapy strategy, especially in poorly immunogenic tumors where NIR-PIT monotherapy is suboptimal.

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., We have developed a florescence endoscopy system that can detect the IR700 fluorescence signal and deliver therapeutic NIR light through the endoscope to treat IR700 fluorescent lesions with NIR‐PIT. In this study, we established an oral cancer model using a murine oral squamous cell cancer‐derived MOC2 cell and performed NIR‐PIT using the fluorescence endoscopy system.

Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and conventional chemotherapy and molecular-targeted therapies show limited efficacy. Near-infrared photoimmunotherapy (NIR-PIT) is a new anticancer treatment that selectively damages the cell membrane of cancer cells based on NIR light-induced photochemical reactions of the antibody (Ab)-photoabsorber (IRDye700Dx) conjugate and the cell membrane. TNBC is known to express several adhesion molecules on the cell surface providing a potential new target for therapy. Here, we investigated the therapeutic efficacy of intercellular adhesion molecule-1 (ICAM-1)-targeted NIR-PIT using xenograft mouse models subcutaneously inoculated with two human ICAM-1-expressing TNBC cell lines, MDAMB468-luc and MDAMB231 cells. In vitro ICAM-1-targeted NIR-PIT damaged both cell types in a NIR light dose-dependent manner. In vivo ICAM-1-targeted NIR-PIT in both models showed early histological signs of cancer cell damage, such as cytoplasmic vacuolation. Even among the cancer cells that appeared to be morphologically intact within 2 h post treatment, abnormal distribution of the actin cytoskeleton and a significant decrease in Ki-67 positivity were observed, indicating widespread cellular injury reflected in cytoplasmic degeneration. Such damage to cancer cells by NIR-PIT significantly inhibited subsequent tumor growth and improved survival. This study suggests that ICAM-1-targeted NIR-PIT could have potential clinical application in the treatment of TNBC.

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., A diagnostic imaging method is developed for accurately localizing tumor and IR700 accumulation based on IR700 fluorescence, using weakened therapeutic laser light at a nontherapeutic dose and a commercially available clinical fluorescence camera for indocyanine green (LIGHTVISION).

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.

Near‐infrared photoimmunotherapy (NIR‐PIT) is a novel cancer therapy that employs a combination of infrared light and tumor‐targeted monoclonal antibody‐photoabsorber conjugates to cause both direct tumor necrosis and immunogenic cell death. NIR‐PIT may have potential in the perioperative setting before surgery, and therefore it is important to know the effect of NIR‐PIT on wound healing. Fifty mice were implanted with subcutaneous xenografts of N87 human gastric cancer cells, and tumors were excised after reaching a predetermined size. After excision, 30 mice were split into three groups: Controls, NIR‐PIT 1 day prior to surgery and NIR‐PIT 3 days prior to surgery. The quantity of reactive oxygen species (ROS) in each wound was measured on Postoperative Days 2 and 4, and mice were monitored weekly for 4 weeks for evidence of local tumor recurrence as well as clinical evidence of wound healing complications (eg, dehiscence, infection). The remaining 20 mice (10 controls, 10 treated with NIR‐PIT 1 day prior to surgery) were sacrificed on either Postoperative Day 7 or 14, the skin around wounds were excised, and tensile strength was measured with a digital force gauge. There were no significant differences between treatment and control groups with respect to wound ROS levels, wound tensile strength, local tumor recurrence, or postoperative complication rates (P > .05). In conclusion, neoadjuvant (pre‐operative) NIR‐PIT shows no evidence of adverse wound healing effects, and it is likely a safe adjunctive treatment to surgery. Postoperative use of NIR‐PIT merits investigation., Near‐infrared photoimmunotherapy (NIR‐PIT), which is now under world‐wide phase 3 trial including in Japan, may be useful in the perioperative setting either before or after tumor excision that should be rational because NIR‐PIT could minimize tumor volume and initiate host antitumor immunity for safe and successful outcome. In order to apply this neoadjuvant NIR‐PIT before the surgery, only the concern of surgeons is if wound healing is hampered by preoperative NIR‐PIT. Therefore, in this study, in order to respond high demands from many surgeons, the effect of NIR‐PIT on wound healing was measured using reactive oxygen species monitoring and wound tensile strength measurements and shown to have no difference between treatment and control mice.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700–targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3(+)CD25(+)CD4(+) regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3(+)CD25(+)CD4(+) Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.

Background/aim Kinesin family member 15 (KIF15) participates in the transport of macromolecules in essential cellular processes. In this study we evaluated the clinical relevance of KIF15 expression in hepatocellular carcinoma (HCC). Materials and methods Association between KIF15 expression and clinical outcomes in HCC patients was analyzed using three independent cohorts. Localization of KIF15 expression was assessed by immunohistochemical analysis. Co-culture experiments were performed using healthy donor peripheral blood mononuclear cells (PBMC) and HCC cell lines. Results Immunohistochemical analysis showed that KIF15 was mainly expressed in inflammatory monocytes around cancer cells. Multivariate analysis indicated high KIF15 expression was an independent poor prognostic factor for survival. HCC cells with high expression of minichromosome maintenance protein 2 (MCM2) were located close to KIF15-expressing inflammatory monocytes. The proliferation ability of HCC cells was increased by co-culture with PBMC. Conclusion High KIF15 expression in inflammatory monocytes in tumor tissues may serve as a prognostic marker for poor outcome in HCC.

Programmed cell death 1 (PD-1) blockade therapy can result in dramatic responses in some patients with cancer. However, about 15% of patients receiving PD-1 blockade therapy experience rapid tumor progression, a phenomenon termed “hyperprogressive disease” (HPD). The mechanism(s) underlying HPD has been difficult to uncover because HPD is challenging to reproduce in animal models. Near-infrared photoimmunotherapy (NIR-PIT) is a method by which specific cells in the tumor microenvironment (TME) can be selectively depleted without disturbing other cells in the TME. In this study, we partially depleted CD8+ T cells with NIR-PIT by targeting the CD8β antigen thereby temporarily changing the balance of T-cell subsets in two different syngeneic tumor models. PD-1 blockade in these models led to rapid tumor progression compared with controls. CD3ε+CD8α+/CD3ε+CD4+FoxP3+ (Teff/Treg) ratios in the PD-1 and NIR-PIT groups were lower than in controls. Moreover, in a bilateral tumor model, low-dose CD8β-targeted NIR-PIT with anti–PD-1 blockade showed rapid tumor progression only in the tumor exposed to NIR light. In this experiment CD8β-targeted NIR-PIT in the exposed tumor reduced local CD8+ T cells resulting in a regulatory T-cell (Treg)–dominant TME. In conclusion, this reports an animal model to simulate the Treg-dominant TME, and the data generated using the model suggest that HPD after PD-1 blockade therapy can be attributed, at least in part, to imbalances between effector T cells and Tregs in the TME.

[Image: see text] Near-infrared photoimmunotherapy (NIR-PIT) employs molecularly targeted antibodies conjugated with a photoabsorbing silicon-phthalocyanine dye derivative which binds to cancer cells. Application of NIR light following binding of the antibody–photoabsorber conjugates (APCs) results in ligand release on the dye, dramatic changes in solubility of the APC–antigen complex, and rapid, irreversible cell membrane damage of cancer cells in a highly selective manner, resulting in a highly immunogenic cell death. Clinically, this process results in edema after treatment mediated by reactive oxygen species (ROS). Based on the chemical and biological mechanism of NIR-PIT cytotoxicity and edema formation, in order to minimize acute inflammatory edema without compromising therapeutic effects, l-sodium ascorbate (l-NaAA) was administered to quench harmful ROS and accelerate the ligand release reaction. l-NaAA suppressed acute edema by reducing ROS after NIR-PIT yet did not alter the therapeutic effects. NIR-PIT could be performed safely under existence of l-NaAA without side effects caused by unnecessary ROS production.

Near-infrared photoimmunotherapy (NIR-PIT) is a cell-specific cancer therapy that uses an antibody-photoabsorber (IRDye700DX, IR700) conjugate (APC) and NIR light. Intravenously injected APC binds the target cells, and subsequent NIR light exposure induces immunogenic cell death only in targeted cells. Panitumumab and cetuximab are antibodies that target human epidermal growth factor receptor (hEGFR) and are suitable for NIR-PIT. In athymic nude mouse models, panitumumab-based NIR-PIT showed superior therapeutic efficacy compared to cetuximab-based NIR-PIT because of the longer half-life of panitumumab-IR700 (pan-IR700) compared with cetuximab-IR700 (cet-IR700). Two light exposures on two consecutive days have also been shown to induce superior effects compared to a single light exposure in the athymic nude mouse model. However, the optimal regimen has not been assessed in immunocompetent mice. In this study, we compared panitumumab and cetuximab in APCs for NIR-PIT, and single and double light exposures using a newly established hEGFR-expressing cancer cell line derived from immunocompetent C57BL/6 mice (mEERL-hEGFR cell line). Fluorescence imaging showed that the decline of pan-IR700 was slower than cet-IR700 confirming a longer clearance time. Among all the combinations tested, mice receiving pan-IR700 and double light exposure showed the greatest tumor growth inhibition. This group was also shown to activate CD8

Alternative splicing, regulated by DEAD‐Box Helicase (DDX) families, plays an important role in cancer. However, the relationship between the DDX family and cancer has not been fully elucidated. In the present study, we identified a candidate oncogene DDX56 on Ch.7p by a bioinformatics approach using The Cancer Genome Atlas (TCGA) dataset of colorectal cancer (CRC). DDX56 expression was measured by RT‐qPCR and immunochemical staining in 108 CRC patients. Clinicopathological and survival analyses were carried out using three CRC datasets. Biological roles of DDX56 were explored by gene set enrichment analysis (GSEA), and cell proliferation in vitro and in vivo, cell cycle assays, and using DDX56‐knockdown or overexpressed CRC cells. RNA sequencing was carried out to elucidate the effect of DDX56 on mRNA splicing. We found that DDX56 expression was positively correlated with the amplification of DDX56 and was upregulated in CRC cells. High DDX56 expression was associated with lymphatic invasion and distant metastasis and was an independent poor prognostic factor. In vitro analysis, in vivo analysis and GSEA showed that DDX56 promoted proliferation ability through regulating the cell cycle. DDX56 knockdown reduced intron retention and tumor suppressor WEE1 expression, which functions as a G2‐M DNA damage checkpoint. We have identified DDX56 as a novel oncogene and prognostic biomarker of CRC that promotes alternative splicing of WEE1.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that targets cancer cells using a monoclonal antibody-photon absorber conjugate (APC) that is bound to the target cell surface. Subsequent application of low levels of NIR light results in immediate cancer cell death. The anti-tumor effect of NIR-PIT in immunocompromised mice depends on immediate cancer cell death; therefore, the efficacy increases in a light-dose-dependent manner. However, NIR-PIT also induces a strong anti-tumor immune activation in immunocompetent mice that begins soon after therapy. Thus, it may be possible to reduce the light dose, which might otherwise cause local edema while maintaining therapeutic efficacy. In this study, we determined the optimal dose of NIR light in NIR-PIT based on a comparison of the therapeutic and adverse effects. Either one of two monoclonal antibodies (mAbs) against human epidermal growth factor receptor (hEGFR), Cetuximab or Panitumumab, were conjugated with a photo-absorbing chemical, IRDye700DX (IR700), and then injected in hEGFR-expressing mEERL (mEERL-hEGFR) tumor-bearing C57BL/6 immunocompetent mice or A431-GFP-luc tumor-bearing athymic immunocompromised mice. NIR light was varied between 0 to 100 J/cm2 one day after administration of APC. In an immunocompromised mouse model, tumor growth was inhibited in a light-dose-dependent manner, yet extensive local edema and weight loss were observed at 100 J/cm2. On the other hand, in an immunocompetent mouse model using the mEERL-hEGFR cell line, maximal tumor response was achieved at 50 J/cm2, with a commensurate decrease in local edema. In this study, we show that a relatively low dose of NIR light is sufficient in an immunocompetent mouse model and avoids side effects seen with higher light doses required in immunocompetent mice. Thus, light dosing can be optimized in NIR-PIT based on the expected immune response.

Background near-infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that uses antibody-photoabsorber (IRDye700DX, IR700) conjugates (APCs) which bind to target cells and are photoactivated by NIR light inducing rapid necrotic cell death. NIR-PIT targeting human epidermal growth factor receptor (hEGFR) has been shown to destroy hEGFR expressing human tumor cells and to be effective in immunodeficient mouse models. NIR-PIT can also be targeted to cells in the tumor microenvironment, for instance, CD25-targeted NIR-PIT can be used to selectively deplete regulatory T cells (Tregs) within a tumor. The aim of this study was to evaluate the combined therapeutic efficacy of hEGFR and CD25-targeted NIR-PIT in a newly established hEGFR expressing murine oropharyngeal cell line (mEERL-hEGFR). Methods panitumumab conjugated with IR700 (pan-IR700) was used as the cancer cell-directed component of NIR-PIT and anti-CD25-F(ab′)2-IR700 was used as the tumor microenvironment-directed component of NIR-PIT. Efficacy was evaluated using tumor-bearing mice in four groups: (1) non-treatment group (control), (2) pan-IR700 based NIR-PIT (pan-PIT), (3) anti-CD25-F(ab′)2-IR700 based NIR-PIT (CD25-PIT), (4) combined NIR-PIT with pan-IR700 and anti-CD25- F(ab′)2-IR700 (combined PIT). Findings the combined PIT group showed the greatest inhibition of tumor growth. Destruction of cancer cells likely leads to an immune response which is amplified by the loss of Tregs in the tumor microenvironment. Interpretation combined hEGFR and CD25-targeted NIR-PIT is a promising treatment for hEGFR expressing cancers in which Treg cells play an immunosuppressive role.

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/cm2. 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.

Background A simple measure of immune cytolytic activity (CYT) base on mRNA expression levels of two genes, GZMA and PRF1, was recently reported. Here, we aimed to evaluate the CYT score's potential as a measure of antitumor immunity and predictor of clinical outcome in gastric cancer (GC) patients. Materials and Methods We evaluated the correlations between tumor‐infiltrating immune cells and the CYT score in 238 GC samples from The Cancer Genome Atlas (TCGA). Next, we investigated CYT score associations with molecular subtypes, somatic mutation load, and immune checkpoint molecules in GC samples from TCGA and Asian Cancer Research Group (ACRG). Moreover, we evaluated the clinical significance of the CYT score calculated by reverse transcription (RT)‐quantitative PCR (qPCR) data in 123 GC samples and the association of the CYT score with the response to anti‐PD‐1 therapy in 7 GC samples from Kyushu University Hospital. Results The CYT score positively correlated with the proportions of tumor‐infiltrating CD8+ T cells and macrophages and negatively correlated with the proportion of regulatory T cells in GC tissues. A high CYT score was associated with common immune checkpoint molecules, a high mutation, the Epstein–Barr virus subtype, and the microsatellite instability subtype in GC. Moreover, a low CYT score was a poor prognosis factor in patients with GC. Finally, the CYT score was higher in a responder to anti‐PD‐1 therapy compared to nonresponders. Conclusion The CYT score reflects antitumor immunity and predicts clinical outcome in GC patients., High CYT score was associated with tumor‐infiltrating CD8+ T cells, EBV, MSI‐H, high tumor mutation load, and upregulated immune checkpoint molecules in gastric cancer. Moreover, the CYT score was associated with prognosis and response to the anti‐PD‐1 antibody in gastric cancer patients.

Targeting mutated oncogenes is an effective approach for treating cancer. The 4 main driver genes of pancreatic ductal adenocarcinoma (PDAC) are KRAS, TP53, CDKN2A, and SMAD4, collectively called the “big 4” of PDAC, however they remain challenging therapeutic targets. In this study, ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 (ASAP2), one of the ArfGAP family, was identified as a novel driver gene in PDAC. Clinical analysis with PDAC datasets showed that ASAP2 was overexpressed in PDAC cells based on increased DNA copy numbers, and high ASAP2 expression contributed to a poor prognosis in PDAC. The biological roles of ASAP2 were investigated using ASAP2‐knockout PDAC cells generated with CRISPR‐Cas9 technology or transfected PDAC cells. In vitro and in vivo analyses showed that ASAP2 promoted tumor growth by facilitating cell cycle progression through phosphorylation of epidermal growth factor receptor (EGFR). A repositioned drug targeting the ASAP2 pathway was identified using a bioinformatics approach. The gene perturbation correlation method showed that niclosamide, an antiparasitic drug, suppressed PDAC growth by inhibition of ASAP2 expression. These data show that ASAP2 is a novel druggable driver gene that activates the EGFR signaling pathway. Furthermore, niclosamide was identified as a repositioned therapeutic agent for PDAC possibly targeting ASAP2., In this study, we identified ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 (ASAP2) as a potentially druggable driver gene using bioinformatics analysis in pancreatic ductal adenocarcinoma (PDAC), which is one of the most lethal cancers worldwide. Then, we showed that ASAP2 promoted cell migration and proliferation by facilitating cell cycle progression through phosphorylation of EGFR. Finally, we identified niclosamide, an antiparasitic drug, as a repositioned therapeutic agent for PDAC, possibly targeting ASAP2.

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.

Simple Summary Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective cancer treatment that employs an antibody photoabsorber conjugate (APC) composed of a targeting monoclonal antibody (mAb) conjugated with a photoactivatable phthalocyanine-derivative dye. APC bound cancer cells are selectively destroyed upon NIR light exposure. Since NIR-PIT can induce selective immunogenic cell death, even in poorly immunogenic tumors, NIR-PIT has the potential to improve immunogenicity of tumor cells that could further enhance anti-tumor host immunity when combined with immune checkpoint inhibition. In this study, we showed that cancer-targeting NIR-PIT converted a minimally immunogenic tumor (MOC2-luc) into a highly immunogenic tumor by increasing the number and density of activated CD8+ T cells infiltrating into the tumor, leading to improved efficacy of the anti-PD-1 immune-checkpoint inhibitor. Abstract Near-infrared photoimmunotherapy (NIR-PIT) is a highly selective cancer treatment that employs an antibody photoabsorber conjugate (APC) composed of a targeting monoclonal antibody (mAb) conjugated with a photoactivatable phthalocyanine-derivative dye. Once injected and allowed to bind to a tumor, the APC is activated by local near-infrared light which kills cancer cells and induces a strong immune response in the tumor microenvironment by unmasking of new tumor antigens emerging from damaged tumor cells. Due to its ability to incite an immune reaction, even in poorly immunogenic tumors, NIR-PIT has the potential to enhance immunogenicity in tumors especially after immune checkpoint inhibition. In this study, we employ a poorly immunogenic MOC2-luc syngeneic tumor model and evaluate the efficacy of cancer-targeting CD44-targeted NIR-PIT. Increased infiltration of CD8+ T cells observed after NIR-PIT suggested an enhanced immune environment. Next, we evaluated tumor progression and survival after the combination of CD44-targeted NIR-PIT and short-term administration of an anti-PD1 immune checkpoint inhibitor (ICI) to further activate CD8+ T cells. Additionally, in mice in which the tumors were eradicated by this combination therapy, a re-challenge with fresh MOC2-luc cells demonstrated failure of tumor implantation implying acquired long-term immunity against the cancer cells. Combination therapy decreased tumor progression and prolonged survival significantly. Therefore, we concluded that NIR-PIT was able to convert a minimally immunogenic tumor unresponsive to anti-PD-1 ICI into a highly immunogenic tumor responsive to anti-PD-1 ICI, and this therapy was capable of inducing long-term immunity against the treated cancer.

Near infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that induces necrotic/immunogenic cell death. It employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by NIR light. Tumor-targeting NIR-PIT is also at least partly mediated by a profound immune response against the tumor. Cytotoxic T-lymphocyte antigen-4 (CTLA4) is widely recognized as a major immune checkpoint protein, which inhibits the immune response against tumors and is therefore, a target for systemic blockade. We investigated the effect of combining tumor-targeted NIR-PIT against the cell-surface antigen, CD44, which is known as a cancer stem cell marker, with a systemic CTLA4 immune checkpoint inhibitor in three syngeneic tumor models (MC38-luc, LL/2, and MOC1). CD44-targeted NIR-PIT combined with CTLA4 blockade showed greater tumor growth inhibition with longer survival compared with CTLA4 blockade alone in all tumor models. NIR-PIT and CTLA4 blockade produced more complete remission in MOC1 tumors (44%) than NIR-PIT and programmed cell death protein 1 (PD-1) blockade (8%), which was reported in our previous paper. However, the combination of NIR-PIT and CTLA4 blockade was less effective in MC38-luc tumors (11%) than the combination of NIR-PIT and PD-1 blockade (70%). Nonetheless, in many cases ineffective results with NIR-PIT and PD-1 blockade were reversed with NIR-PIT and CTLA4 blockade.

Simple Summary Near infrared photoimmunotherapy is a newly developed and highly selective cancer treatment that employs a monoclonal antibody conjugated to a photo-absorber dye, IRDye700DX, which is activated by 690 nm light. Cancer cell-targeted near infrared photoimmunotherapy selectively induces rapid necrotic/immunogenic cell death only on target cancer cells and this induces antitumor host immunity including re-priming and proliferation of multi-chronal T-cells that can react with cancer-specific antigens. Interleukin-15 is a type-I cytokine that activates natural killer-, B- and T-cells while having minimal effect on regulatory T-cells that lack the interleukin-15 receptor. Therefore, interleukin-15 administration combined with cancer cell-targeted near infrared photoimmunotherapy could further inhibit tumor growth by increasing antitumor host immunity. In tumor-bearing immunocompetent mice receiving this combination therapy, significant tumor growth inhibition and prolonged survival was demonstrated compared with either single therapy alone, and tumor infiltrating CD8+ T-cells increased in number in combination-treated mice. Interleukin-15 enhances therapeutic effects of cancer-targeted near infrared photoimmunotherapy. Abstract Near infrared photoimmunotherapy (NIR-PIT) is a newly developed and highly selective cancer treatment that employs a monoclonal antibody (mAb) conjugated to a photo-absorber dye, IRDye700DX, which is activated by 690 nm light. Cancer cell-targeted NIR-PIT induces rapid necrotic/immunogenic cell death (ICD) that induces antitumor host immunity including re-priming and proliferation of T cells. Interleukin-15 (IL-15) is a cytokine that activates natural killer (NK)-, B- and T-cells while having minimal effect on regulatory T cells (Tregs) that lack the IL-15 receptor. Here, we hypothesized that IL-15 administration with cancer cell-targeted NIR-PIT could further inhibit tumor growth by increasing antitumor host immunity. Three syngeneic mouse tumor models, MC38-luc, LL/2, and MOC1, underwent combined CD44-targeted NIR-PIT and short-term IL-15 administration with appropriate controls. Comparing with the single-agent therapy, the combination therapy of IL-15 after NIR-PIT inhibited tumor growth, prolonged survival, and increased tumor infiltrating CD8+ T cells more efficiently in tumor-bearing mice. IL-15 appears to enhance the therapeutic effect of cancer-targeted NIR-PIT.

Background/aim The cytolytic activity (CYT) score is a new index of cancer immunity calculated from the mRNA expression levels of GZMA and PRF1. We assessed the clinical significance of the CYT score in HCC. Materials and methods The calculated CYT scores of peripheral blood cells (GSE24759), cell lines (CCLE) and HCC tissues (TCGA, GSE14520 and Kyushu cohorts) were assessed. Then, immunohistochemical analysis (IHC) of GZMA and PRF1 was performed. Results The CYT scores of HCC tissues were lower than those of non-cancerous tissues. The 5-year recurrence-free survival of patients with low CYT scores was significantly shorter than that of patients with high CYT scores. Multivariate analysis indicated that the CYT score was an independent prognostic factor for RFS in TCGA and GSE14520 cohorts. Conclusion CYT score could be a useful prognostic biomarker in HCC, possibly through reflecting the host immune status.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly-developed, highly-selective cancer treatment, which utilizes a monoclonal antibody conjugated to a photoabsorbing dye, IRDye700DX (IR700). The antibody conjugate is injected into the patient and accumulates in the tumour. Within 24 h of injection the tumour is exposed to NIR light which activates the conjugate and causes rapid, selective cancer cell death. A global phase III clinical trial of NIR-PIT in recurrent head and neck squamous cell cancer (HNSCC) patients is currently underway. Conditional clinical approval for NIR-PIT in recurrent HNSCC has been granted in Japan as of September 2020. Not only does NIR-PIT induce highly selective and immediate cancer cell killing, but it also stimulates highly active anti-tumour immunity. While monotherapy with NIR-PIT has proven effective it is likely that combinations with immune-checkpoint inhibitors or additional NIR-PIT targeting immune suppressive cells in the tumour microenvironment will further improve results. In this review, we discuss the translational aspects of NIR-PIT especially in HNSCC, and potential future applications.

Simple Summary Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses an antibody-photoabsorber (IRDye700DX) conjugate (APC) that is activated by NIR light irradiation. A major benefit of NIR-PIT is that only APC-bound cancer cells that are exposed to NIR light are killed by NIR-PIT; thus, minimal damage occurs in adjacent normal cells. NIR-PIT has now been applied to many cancers expressing various cell-surface target proteins using monoclonal antibodies designed to bind to them. Moreover, NIR-PIT is not limited to tumor antigens but can also be used to kill specific host cells that create immune-permissive environments in which tumors grow. Moreover, multiple targets can be treated simultaneously with NIR-PIT using a cocktail of APCs. NIR-PIT has great potential to treat a wide variety of cancers by targeting appropriate tumor cells, immune cells, or both, and can be augmented by other immunotherapies. Abstract Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that uses an antibody-photoabsorber (IRDye700DX) conjugate (APC) that is activated by NIR light irradiation. In September 2020, the first APC and laser system were conditionally approved for clinical use in Japan. A major benefit of NIR-PIT is that only APC-bound cancer cells that are exposed to NIR light are killed by NIR-PIT; thus, minimal damage occurs in adjacent normal cells. These early trials have demonstrated that in addition to direct cell killing, there is a significant therapeutic host immune response that greatly contributes to the success of the therapy. Although the first clinical use of NIR-PIT targeted epidermal growth factor receptor (EGFR), many other targets are suitable for NIR-PIT. NIR-PIT has now been applied to many cancers expressing various cell-surface target proteins using monoclonal antibodies designed to bind to them. Moreover, NIR-PIT is not limited to tumor antigens but can also be used to kill specific host cells that create immune-permissive environments in which tumors grow. Moreover, multiple targets can be treated simultaneously with NIR-PIT using a cocktail of APCs. NIR-PIT can be used in combination with other therapies, such as immune checkpoint inhibitors, to enhance the therapeutic effect. Thus, NIR-PIT has great potential to treat a wide variety of cancers by targeting appropriate tumor cells, immune cells, or both, and can be augmented by other immunotherapies.

Near-infrared photoimmunotherapy (NIR-PIT) is a cancer treatment that utilizes antibody-photoabsorber (IR700) conjugates to selectively kill target cells by exposing them to NIR light. Cytotoxic T-lymphocyte antigen 4 (CTLA4) is a major immune checkpoint ligand mediating antitumor immune suppression. Local depletion of CTLA4 expressing cells in the tumor bed with NIR-PIT could enhance antitumor immune responses by both blocking the CTLA4-axis and depleting immune suppressive cells. The aim of this study is to evaluate the antitumor efficacy of CTLA4-targeted NIR-PIT using four murine tumor models, MC38-luc, LL/2-luc, MOC2-luc, and MOC2. The CTLA4-targeted NIR-PIT depletes intratumoral CTLA4 expressing cells which are mostly regulatory T cells. In vivo CTLA4-targeted NIR-PIT yields complete responses in 80% of MC38-luc, 70% of LL/2-luc and 40% of MOC2-luc tumors prolonging survival in all cases. After CTLA4-targeted NIR-PIT, activation and infiltration of CD8(+) T cells within the tumor microenvironment is observed. In conclusion, CTLA4-targeted NIR-PIT can effectively treat tumors by blocking the CTLA4-axis as well as by eliminating CTLA4-expressing immune suppressor cells, resulting in T cell mediated antitumor immunity. Local CTLA4-expressing cell depletion in tumor beds using NIR-PIT could be a promising new cancer immunotherapy for safely treating a variety of tumor types.

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.