Research ArticleExperimental Studies
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Near-infrared Photoimmunotherapy Targeting High-risk Human Neuroblastoma Cells Expressing GD2

HIROSHI NOUSO, HIROSHI TAZAWA, TERUTAKA TANIMOTO, MORIMICHI TANI, HINAKO WATANABE, TAKANORI OYAMA, KAZUHIRO NOMA, SHUNSUKE KAGAWA, HISATAKA KOBAYASHI, TAKUO NODA, SHINJI KURODA and TOSHIYOSHI FUJIWARA
Anticancer Research January 2026, 46 (1) 25-38; DOI: https://doi.org/10.21873/anticanres.17921

Abstract

Background/Aim: Neuroblastoma (NB) is a primary malignant tumor of the peripheral sympathetic nervous system in infancy. Despite advances in treatment, the prognosis remains poor for high-risk NB patients. Although immunotherapy using anti-GD2 antibodies is available for high-risk NB, the therapeutic efficacy is insufficient. Near-infrared photoimmunotherapy (NIR-PIT) is an antitumor strategy that induces tumor-specific cytotoxicity by combining an antibody-photoabsorber conjugate (APC) with NIR light irradiation. In this study, we investigated the therapeutic efficacy of GD2-targeted NIR-PIT against human NB cells.

Materials and Methods: GD2 expression was analyzed on the surface of high-risk human NB cells (CHP-134, LA-N-5, IMR-32) and non-high-risk human NB cells (SK-N-SH) by flow cytometry. The APC was synthesized by incubating anti-GD2 antibody and IR700. The cytotoxic effect of GD2-targeted NIR-PIT was evaluated using the XTT assay. The distribution of dead cells within tumor spheres was evaluated using a live/dead assay. The in vivo antitumor effect of GD2-targeted NIR-PIT was assessed using a subcutaneous human NB xenograft tumor model.

Results: GD2 protein was expressed on the surface of CHP-134, LA-N-5, and IMR-32 cells but not SK-N-SH cells. GD2-targeted NIR-PIT significantly suppressed the viability of GD2-positive NB cells but not GD2-negative NB cells, compared to the control and monotherapy groups. GD2-targeted NIR-PIT significantly reduced the volume of GD2-positive CHP-134 tumor spheres by inducing the accumulation of dead cells. Subcutaneous CHP-134 xenograft tumor models demonstrated that GD2-targeted NIR-PIT significantly inhibited tumor growth compared with the control and monotherapy groups.

Conclusion: GD2-targeted NIR-PIT is a promising antitumor strategy for treating high-risk NB tumors expressing GD2.

Keywords:

Introduction

Neuroblastoma (NB) is the most common extracranial solid tumor, accounting for 7% of all pediatric cancers and 15% of all pediatric cancer-related death (1). NB tumors are derived from the sympathetic nervous system in the embryonal period and known to be biologically and clinically heterogeneous (2, 3). Some NB tumors spontaneously regress or mature without treatment, but others behave very aggressively and exhibit resistance to multimodal therapy (3). Many factors, including age at diagnosis, clinical stage of disease, and genetic features (e.g., MYCN status, 11q deletion, DNA ploidy), regulate the prognosis of patients with NB tumors (4). The International Neuroblastoma Risk Group Staging System is used to classify NB tumors into 4 risk groups (very low, low, intermediate, or high) according to specific prognostic factors (4). Patients with low- or intermediate-risk NB typically have excellent outcomes, whereas overall survival of high-risk NB remains <50% despite improvements in therapeutic strategies such as surgery, chemotherapy, radiation, myeloablative therapy, and stem-cell rescue (4). Therefore, novel antitumor modalities for treating high-risk NB tumors are needed to improve currently poor clinical outcomes. An immunotherapy using anti-GD2 antibodies has been developed for treating high-risk NB tumors (5). GD2 is a glycoprotein disialoganglioside uniformly expressed in human NB cells (6, 7). In normal tissues, GD2 expression is weak and limited to neurons, melanocytes, and peripheral pain fibers (6, 7). The primary mechanism of the antitumor effect of anti-GD2 antibodies against NB cells involves complement-dependent and antibody-dependent cellular cytotoxicity (7). As monotherapy with anti-GD2 antibodies has minimal therapeutic effect against NB tumors, a combination therapy involving anti-GD2 antibodies and various cytokines has been developed (7). Recently developed antibody-drug conjugates have shown promise in promoting the antitumor effect of anti-GD2 antibodies (8, 9). Thus, novel antitumor modalities using anti-GD2 antibodies could be a promising approach for treating high-risk NB tumors.

Near-infrared photoimmunotherapy (NIR-PIT) is a novel antitumor therapy that combines an antibody-photoabsorber conjugate (APC) with a water-soluble, silicon-phthalocyanine derivative, IRDye700DX NHS ester (IR700), and a monoclonal antibody targeting tumor-specific activated molecules (10). IR700 is a photoabsorber activated by irradiation with NIR light. IR700 can be covalently conjugated to antibody molecules, which are useful delivery vehicles for PIT due to their high binding specificity and highly accurate target delivery (10). Immediate tumor cell–selective death can be induced by irradiating the APC with NIR light. For instance, an NIR-PIT using a monoclonal antibody targeting epidermal growth factor receptor has been approved in Japan for treating recurrent head and neck cancer. NIR-PIT using an anti-GD2 monoclonal antibody exhibited therapeutic potential against GD2-expressing tumors of neuroectodermal origin (11).

In the present study, we investigated the therapeutic potential of a GD2-targeted NIR-PIT against four human NB cell lines (CHP-134, LA-N-5, IMR-32, and SK-N-SH) (Figure 1). Expression of GD2 protein on the surface of these human NB cells was analyzed by flow cytometry. The in vitro cytotoxic efficacy of the GD2-targeted NIR-PIT approach was assessed by determining the viability of NB cells using the XTT assay. The distribution of dead cells within NB tumor spheres was evaluated using a live/dead cell assay. Moreover, the in vivo antitumor efficacy of the GD2-targeted NIR-PIT approach was assessed using a subcutaneous human NB xenograft tumor model.

Figure 1.
Figure 1.

Schematic illustration of the experimental protocol. Figure was generated using BioRender.

Materials and Methods

Cell lines. Human NB cell lines (CHP-134, LA-N-5, SK-N-SH) were obtained from the RIKEN BioResource Center (Tsukuba, Japan). IMR-32 human NB cells were obtained from the Japanese Cancer Research Resources Bank (Osaka, Japan). Three NB cell lines (CHP-134, IMR-32, LA-N-5) were previously reported as MYCN amplified (12, 13), whereas one NB cell line (SK-N-SH) was reported as non–MYCN amplified (14). CHP-134 and LA-N-5 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). IMR-32 cells were maintained in Eagle’s minimal essential medium with 1% non-essential amino acids and 10% FBS. SK-N-SH cells were maintained in minimal essential medium–alpha supplemented with 10% FBS. All media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were cultured for no longer than 5 months following resuscitation, and all cells were maintained at 37°C in a humidified atmosphere with 5% CO2.

Flow cytometry analysis. To analyze the cell surface expression of GD2 ganglioside, human NB cells were incubated with mouse anti-GD2 monoclonal antibody (mAb) (14G2a; Sigma-Aldrich, St. Louis, MO, USA) or control mouse anti-NK1.1 mAb (PK136; Sigma-Aldrich) for 40 min on ice. The cells were then labeled with fluorescent isothiocyanate–conjugated rabbit anti-mouse IgG secondary antibody (A16161; Invitrogen, Carlsbad, CA, USA) for 30 min and then analyzed using a FACS Array (BD Biosciences, San Jose, CA, USA).

Synthesis of the APC. The procedure for conjugating dyes with mAbs has been reported previously (15). In brief, mouse anti-GD2 mAb (14G2a; Sigma-Aldrich) (1 mg) was incubated with IR700 (LI-COR Biosciences, Lincoln, NE, USA) (66.8 μg) in 100 mM Na2HPO4 (pH 8.5) at 4°C for 2 h. The mixture was then purified on a Sephadex G50 column (PD-10; GE Healthcare, Amersham, UK). The protein concentration was determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA) by measuring the absorbance at 595 nm via spectroscopy. The IR700 concentration was measured by determining the absorbance at 689 nm using a UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The number of fluorophore molecules per mAb was adjusted to approximately 2, and the resulting APC was defined as GD2-IR700.

Cell viability assay. Cells were seeded in 96-well plates at a density of 1×104 cells/well (CHP-134, LA-N-5, SK-N-SH) or 2×104 cells/well (IMR-32) 48 h before treatment. To optimize the power of NIR light irradiation, the medium was replaced with fresh medium containing GD2-IR700 (5 μg/ml) and incubated for 24 h at 37°C. The cells were then irradiated with NIR light (0-30 J/cm2). By comparison, for optimization of the dose of APC, the medium was replaced with fresh medium containing GD2-IR700 (0-10 μg/ml) and incubated for 24 h. Cells were then irradiated with NIR light (20 J/cm2). Cell viability was evaluated using a Cell Proliferation Kit II (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s protocol.

ATP assay. Cells were seeded in 24-well plates at a density of 1×106 cells/well 48 h before treatment. The medium was replaced with fresh medium containing GD2-IR700 (5 μg/ml) or phosphate-buffered saline (PBS) and incubated for 4 h at 37°C. The cells were then irradiated with NIR light (20 J/cm2), and the supernatant was collected and analyzed using an ENLITEN ATP (adenosine triphosphate) assay (Promega, Madison, WI, USA).

Live/dead cell assay. CHP-134 and LA-N-5 cells were seeded in Corning™ 96-well, round-bottom, ultra-low-attachment microplates at a density of 5×104 cells/well. After 48 h, fresh medium containing GD2-IR700 (final concentration, 5 μg/ml) was added to the wells, and the cells were incubated for an additional 24 h, followed by irradiation with NIR light (30 J/cm2). Live/dead cell assay reagent was added to the culture medium at 1 h after NIR irradiation. Cells that formed spheroids were imaged using a BZ-X700 fluorescence microscope (Keyence, Osaka, Japan). The volume of tumor spheres was calculated at 48, 72, 120, and 192 h after cell seeding.

In vivo subcutaneous NB xenograft tumor model. Animal experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Okayama University School of Medicine (OKU-2018790). CHP-134 cells (1×107 cells per site) were inoculated into the flank of 6-week-old female nude mice (CLEA Japan, Tokyo, Japan). Palpable tumors reaching approximately 50 mm3 in volume were selected for further experiments. Tumor-bearing mice were divided into 4 experimental groups (n=5/group). In the control and NIR groups, mice were injected intraperitoneally with 45 μl of PBS on day 0. In the APC and NIR-PIT groups, mice were injected on day 0 with 45 μl of solution containing 25 μg of GD2-IR700. In the NIR and NIR-PIT groups, the mice were irradiated with NIR light at 50 J/cm2 on day 1 and 100 J/cm2 on day 2 by a 690-nm continuous wave laser (BrixX 690; Omicron, Rodgau-Dudenhofen, Germany) under isoflurane anesthesia. In the NIR-PIT group, 2 of the 5 mice died after general anesthesia on day 2. Tumor size was monitored by measuring tumor length and width using calipers twice each week until 18 days after treatment. Tumor volume was calculated using the following formula: tumor volume (mm3)=L × W2 × 0.5, where L represents tumor length and W represents tumor width.

Statistical analysis. Data are expressed as mean±SD. Student’s t-test was used to compare differences between two groups. One-way analysis of variance followed by Tukey’s multiple-group comparison test was used to compare differences between multiple groups. Statistical significance was defined as a p-value of <0.05.

Results

High-risk human NB cells express GD2 protein on the cell surface. To analyze the therapeutic potential of GD2-targeted NIR-PIT against human NB cells, we evaluated three lines of high-risk human NB cells (CHP-134, LA-N-5, IMR-32) with MYCN amplification (12, 13) and one line of non-high-risk human NB cells (SK-N-SH) without MYCN amplification (14). The expression of GD2 protein on the cell surface was analyzed by flow cytometry. High expression of GD2 protein was observed in all high-risk human NB cells (CHP-134, LA-N-5, IMR-32), whereas the non-high-risk NB cells (SK-N-SH) showed very low expression of GD2 protein (Figure 2A). These results suggest that GD2 protein is a potential therapeutic target in the treatment of high-risk NB.

Figure 2.
Figure 2.

GD2-targeted NIR-PIT reduces the viability of human NB cells expressing GD2. (A) Expression of GD2 on the surface of high-risk human NB cells (IMR-32, CHP-134, LA-N-5) and non-high-risk human NB cells (SK-N-SH), as assessed by flow cytometry analysis. The MFI was determined by calculating the difference between the MFI of cells incubated with the anti-GD2 antibody versus a control anti-NK1.1 antibody. (B) Human NB cells were preincubated with or without APC (GD2-IR700, 5 μg/ml) for 24 h, and cell viability was quantified using the XTT assay after NIR light irradiation at the indicated doses (0-30 J/cm2). (C) Human NB cells were preincubated with APC (GD2-IR700) at the indicated doses (0-10 μg/ml) for 24 h, and cell viability was quantified using the XTT assay after NIR light irradiation (20 J/cm2). Cell viability was calculated relative to that of the non-treated group, which was set at 1.0. Cell viability data are expressed as the mean±SD (n=6). Statistical significance was determined using Student’s t-test; **p<0.01 (vs. 0). NIR-PIT: Near-infrared photoimmunotherapy; NB: neuroblastoma; MFI: mean fluorescence intensity; APC: antibody-photoabsorber conjugate; IR700: IRDye700DX NHS ester.

Optimization of NIR light irradiation and GD2 Ab-IR700 conditions in treating GD2-positive human NB cells. To determine the optimal power of NIR light against human NB cells, the XTT assay was used to evaluate the viability of GD2-positive human NB cells preincubated with APC (GD2-IR700, 5 μg/ml) for 24 h after NIR light irradiation at different power levels (0-30 J/cm2). NIR light irradiation (>20 J/cm2) significantly suppressed the viability of all human NB cell lines expressing GD2 (Figure 2B). CHP-134 and LA-N-5 cells preincubated with APC were more sensitive to NIR light irradiation (10 J/cm2) than IMR-32 cells (Figure 2B). By contrast, NIR light irradiation (30 J/cm2) alone only slightly decreased the viability of LA-N-5 cells (Figure 2B). These results suggest that 20 J/cm2 is the optimal power of NIR light irradiation for evaluating the effect of GD2-targeted NIR-PIT against GD2-positive human NB cells.

To determine the optimal dose of APC (GD2-IR700) for suppressing human NB cells, the viability of GD2-positive human NB cells preincubated with APC at different doses (0-10 μg/ml) for 24 h after NIR light irradiation (20 J/cm2) was monitored using the XTT assay. Preincubation with APC (>2.5 μg/ml) followed by NIR light irradiation effectively reduced the viability of all GD2-expressing human NB cell lines (Figure 2C). By contrast, treatment with APC (10 μg/ml) suppressed the viability of CHP-134 cells (Figure 2C). These results suggest that preincubation of GD2-positive human NB cells with APC at a dose in the range of 2.5-5 μg/ml is optimal for evaluating the effect of GD2-targeted NIR-PIT.

Cytotoxic effect of GD2-targeted NIR-PIT against GD2-positive and GD2-negative human NB cells. To investigate whether GD2-targeted NIR-PIT affects the viability of GD2-negative human NB cells, the XTT assay was used to monitor the viability of GD2-positive and GD2-negative human NB cells preincubated with APC (GD2-IR700, 5 μg/ml) for 24 h after NIR light irradiation (20 J/cm2). GD2-targeted NIR-PIT significantly reduced the viability of GD2-positive human NB cells compared with the control, APC, and NIR groups, although APC significantly decreased the viability of CHP-134 cells (Figure 3A). By contrast, GD2-negative SK-N-SH cells were resistant to GD2-targeted NIR-PIT (Figure 3B). These results demonstrate the therapeutic potential of GD2-targeted NIR-PIT in treating NB, as evidenced by its cytotoxicity against only GD2-positive human NB cells.

Figure 3.
Figure 3.

GD2-targeted NIR-PIT reduces the viability of GD2-expressing human NB cells. (A, B) GD2-positive human NB cells (CHP-134, LA-N-5, IMR-32) (A) and GD2-negative human NB cells (SK-N-SH) (B) were preincubated with fresh medium containing APC (GD2-IR700, 5 μg/ml) or PBS for 24 h, and cell viability was quantified using the XTT assay after NIR light irradiation (20 J/cm2). Cell viability was calculated relative to that of the non-treated group, which was set at 1.0. Cell viability data are expressed as the mean±SD (n=6). Statistical significance was determined using one-way analysis of variance followed by Tukey’s multiple-group comparison test; **p<0.01. NIR-PIT: Near-infrared photoimmunotherapy; APC: antibody-photoabsorber conjugate; IR700: IRDye700DX NHS ester.

GD2-targeted NIR-PIT induces immunogenic cell death (ICD) of GD2-positive human NB cells. ICD is associated with the release of damage-associated molecular patterns and ATP from dying cells (16). NIR-PIT has been shown to induce ICD (17). To investigate the therapeutic potential of GD2-positive NIR-PIT to induce ICD of NB cells, ATP release was monitored in GD2-positive and GD2-negative human NB cells preincubated with APC (GD2-IR700, 5 μg/ml) for 24 h and then irradiated with NIR light (20 J/cm2). GD2-targeted NIR-PIT significantly increased the level of extracellular ATP in cultures of GD2-positive human NB cells (Figure 4A). However, neither APC nor NIR alone affected the level of extracellular ATP in cultures of GD2-positive human NB cells (Figure 4A). By contrast, GD2-negative human NB cells were less sensitive to GD2-targeted NIR-PIT (Figure 4B). These results suggest that GD2-targeted NIR-PIT has therapeutic potential to induce ICD in GD2-positive human NB cells.

Figure 4.
Figure 4.

GD2-targeted NIR-PIT induces immunogenic cell death in human NB cells expressing GD2. (A, B) GD2-positive human NB cells (CHP-134, LA-N-5, IMR-32) (A) and GD2-negative human NB cells (SK-N-SH) (B) were preincubated with fresh medium containing APC (GD2-IR700, 5 μg/ml) or PBS for 4 h and irradiated with NIR light (20 J/cm2). The supernatants were then collected, and the level of extracellular ATP was determined using an ENLITEN ATP assay. Data are expressed as the mean±SD (n=3). Statistical significance was determined using one-way analysis of variance followed by Tukey’s multiple-group comparison test; **p<0.01. NIR-PIT: Near-infrared photoimmunotherapy; NB: neuroblastoma; APC: antibody-photoabsorber conjugate: IR700: IRDye700DX NHS ester; PBS: phosphate-buffered saline; ATP: adenosine triphosphate.

Inhibitory effect of GD2-targeted NIR-PIT in the formation of tumor spheres in GD2-positive human NB. A three-dimensional (3D) spheroid model was used to evaluate the therapeutic potential of combination therapy involving chemotherapy and anti-GD2 antibody for treating high-risk NB (18). To evaluate the therapeutic potential of GD2-targeted NIR-PIT against tumor sphere–forming, GD2-expressing human NB cells, GD2-positive CHP-134 cells were seeded in low-attachment 96-well plates to form tumor spheres and then treated with APC (GD2-IR700, 5 μg/ml) for 24 h with NIR light irradiation (30 J/cm2). The distribution of dead cells in tumor spheres composed of CHP-134 and LA-N-5 cells was analyzed using live/dead cell assays. APC treatment resulted in the appearance of red-colored dead cells, primarily in the internal areas of tumor spheres (Figure 5A and B). NIR light irradiation also resulted in the appearance of red-colored dead cells, primarily on the surface of tumor spheres (Figure 5A and B). By comparison, the number of red-colored dead cells on the surface of tumor spheres was greater following GD2-targeted NIR-PIT (Figure 5A and B). Analysis of the volume of tumor spheres on days 0, 1, 3, and 6 after APC treatment and NIR light irradiation revealed that GD2-targeted NIR-PIT significantly reduced the volume of CHP-134 tumor spheres compared with the control, APC, and NIR groups (Figure 6). These results suggest that GD2-targeted NIR-PIT has therapeutic potential to suppress the formation of tumor spheres by GD2-positive human NB cells.

Figure 5.
Figure 5.

GD2-targeted NIR-PIT induces the death of sphere-forming GD2-positive human NB cells. (A, B) CHP-134 (A) and LA-N-5 (B) cells were seeded in ultra-low attachment, round-bottom 96-well plates at a density of 5×104 cells/well. After 48 h, fresh medium containing GD2-IR700 (final concentration, 5 μg/ml) was added, and the cells were incubated for an additional 24 h and then irradiated with NIR light (30 J/cm2). The distributions of green-colored live and red-colored dead cells within tumor spheres composed of CHP-134 (A) or LA-N-5 (B) cells were determined using the live/dead cell assay after preincubation with APC and NIR light irradiation. Scale bars: 1.0 mm. NIR-PIT: Near-infrared photoimmunotherapy; NB: neuroblastoma; IR700: IRDye700DX NHS ester; APC: antibody-photoabsorber conjugate.

Figure 6.
Figure 6.

GD2-targeted NIR-PIT reduces the formation of tumor spheres by GD2-positive human NB cells. CHP-134 cells were seeded in ultra-low attachment, round-bottom 96-well plates at a density of 5×104 cells/well. After 48 h, fresh medium containing GD2-IR700 (final concentration, 5 μg/ml) was added, and the cells were incubated for an additional 24 h and then irradiated with NIR light (30 J/cm2). The volume of tumor spheres in each group was determined on days 0, 1, 3, and 6 after treatment. Data are expressed as the mean±SD (n=3). Statistical significance was determined using one-way analysis of variance followed by Tukey’s multiple-group comparison test; **p<0.01. NIR-PIT: Near infrared photoimmunotherapy; NB: neuroblastoma; IR700: IRDye700DX NHS ester.

GD2-targeted NIR-PIT suppresses the growth of tumors composed of GD2-expressing human NB cells. To assess the in vivo antitumor effect of GD2-targeted NIR-PIT against human NB tumors expressing GD2, we used a xenograft tumor model of GD2-positive CHP-134 cells. Tumor-bearing mice were injected intraperitoneally with APC (GD2-IR700, 25 μg/mouse) and then irradiated at 50 and 100 J/cm2 twice for 2 days. APC treatment significantly suppressed the growth of CHP-134 tumors compared to the control group, whereas NIR light irradiation alone did not affect tumor growth (Figure 7). By contrast, GD2-targeted NIR-PIT significantly suppressed the growth of CHP-134 tumors compared with the control, APC, and NIR groups (Figure 7). These results suggest that GD2-targeted NIR-PIT has therapeutic potential to suppress the growth of GD2-positive human NB tumors.

Figure 7.
Figure 7.

In vivo antitumor effect of GD2-targeted NIR-PIT in a subcutaneous CHP-134 tumor model. CHP-134 cells (1×107 cells/site) were inoculated into the flanks of 6-week-old female nude mice. One day before NIR light irradiation, the mice were injected intraperitoneally with 45 μl of solution containing either APC (GD2-IR700, 25 μg) or PBS. The mice were irradiated with NIR light at 50 J/cm2 on day 1 and 100 J/cm2 on day 2. Tumor size was monitored by measuring tumor length and width using calipers twice each week until 18 days after treatment. Data are expressed as the mean±SD (n=3 or n=5). Statistical significance was determined using one-way analysis of variance followed by Tukey’s multiple-group comparison test; **p<0.01. NIR-PIT: Near-infrared photoimmunotherapy; APC: antibody-photoabsorber conjugate; IR700: IRDye700DX NHS ester; PBS: phosphate-buffered saline.

Discussion

The prognosis of patients with high-risk NB remains poor, even though several antitumor modalities specific for high-risk NB are available, including chemotherapy and immunotherapy (19). In this study, we demonstrated that a new approach, GD2-targeted NIR-PIT, exhibits therapeutic antitumor effects against GD2-positive human NB cells. Several lines of high-risk NB cells (CHP-134, LA-N-5, IMR-32) expressed detectable levels of GD2 protein on the cell surface, whereas non-high-risk NB cells (SK-N-SH) showed very low GD2 expression. GD2-targeted NIR-PIT significantly reduced the viability of GD2-positive NB cells but had no effect on GD2-negative NB cells. This effect was mediated by the induction of ICD. GD2-targeted NIR-PIT decreased the volume of GD2-positive NB tumor spheres by inducing the death of tumor cells. In GD2-positive, CHP-134 xenograft tumor model mice, GD2-targeted NIR-PIT significantly suppressed tumor growth compared with either APC or NIR light irradiation monotherapy. Taken together, these results suggest that GD2-targeted NIR-PIT is a promising antitumor strategy that exhibits a profound antitumor effect against GD2-positive high-risk NB.

High-risk NB cells exhibited high expression of GD2 protein on the cell surface (Figure 2), suggesting GD2 as a potential target in the treatment of high-risk NB. With regard to the relationship between GD2 expression and NB progression, Valentino et al. showed that high levels of circulating GD2 at diagnosis are associated with rapid NB progression and poor prognosis (20). McNerney et al. demonstrated that TH-MYCN transgenic mice develop GD2-expressing NB tumors that are highly comparable to human NB tumors due to enforced MYCN expression (21). TH-MYCN transgenic mice are responsive to anti-GD2 antibody therapy (21), suggesting a possible relationship between GD2 expression and progression of high-risk NB with MYCN overexpression. Although the mechanism underlying GD2 activation in high-risk NB cells remains unclear, GD2 protein appears to be an attractive therapeutic target in the treatment of high-risk NB using NIR-PIT.

In our study, GD2-targeted NIR-PIT reduced the viability of GD2-expressing high-risk NB cells, whereas GD2-negative NB cells were less sensitive to GD2-targeted NIR-PIT (Figure 3). These findings suggest that GD2-targeted NIR-PIT is a promising antitumor strategy for treating GD2-positive high-risk NB. Consistent with our findings, Inagaki et al. reported that tumor cells of neuroectodermal origin, including NB cells, exhibit high expression of GD2 protein and are thus sensitive to GD2-targeted NIR-PIT (11). GD2 protein is expressed by a variety of solid tumors, including small-cell lung cancer, melanoma, Ewing sarcoma, osteosarcoma, glioma, breast cancer, and bladder cancer (6). Moreover, prostate cancer cells also exhibit the expression of GD2 protein on the cell surface (22). Although NIR-PIT targeting the prostate specific membrane antigen (PSMA) has been shown to be effective against human prostate cancer cells (23), GD2-targeted NIR-PIT would be effective for treating GD2-positive prostate cancer cells. As NIR light can penetrate tissue to a depth of approximately 2 cm from the surface (24), GD2-targeted NIR-PIT may be a promising antitumor strategy for targeting various types of superficial solid tumors, including high-risk NB.

In the present study, GD2-targeted NIR-PIT induced the secretion of ATP from GD2-positive high-risk NB cells but not GD2-negative human NB cells (Figure 4). These findings suggest that the antitumor effect of GD2-targeted NIR-PIT involves the induction of ICD. This is consistent with another study that demonstrated NIR-PIT promotes antitumor immunity via the induction of ICD (17). Hirakawa et al. demonstrated the feasibility of combining NIR-PIT with immune checkpoint inhibitors (ICIs) in patients with advanced head and neck cancer (25). Therefore, combination therapy with GD2-targeted NIR-PIT and ICIs may be a promising antitumor strategy for treating patients with high-risk NB.

NIR-PIT promotes the migration of immune cells within tumors and tumor-draining lymph nodes and expands multiclonal, tumor-infiltrating, cytotoxic T lymphocytes (26). As we used immunodeficient mice to obtain GD2-positive human NB tumors in the present study (Figure 5), it will be necessary to use immunocompetent mice bearing GD2-positive NB tumors in future studies to evaluate the antitumor immune response in GD2-targeted NIR-PIT. TH-MYCN transgenic mice have been shown to develop GD2-expressing NB tumors (21), so these mice may be a more suitable model for evaluating the therapeutic efficacy of GD2-targeted NIR-PIT for inducing antitumor immune responses against GD2-expressing high-risk NB cells.

High-risk human NB cell lines exhibiting MYCN amplification, including CHP-134 and LA-N-5, have been shown to form tumor spheres containing a large proportion of CD133-positive cancer stem-like cells and chemoresistant tumor cells (27). In our study, dead cells appeared following GD2-targeted NIR-PIT, and the volume of tumor spheres composed of GD2-positive NB cells decreased (Figure 5 and Figure 6). These findings suggest that GD2-targeted NIR-PIT is effective against chemoresistant tumor cells. Takahashi et al. demonstrated that NIR-PIT targeting human epidermal growth factor receptor 2 (HER2) with a clinically used anti-HER2 antibody is effective against cisplatin-resistant small-cell lung cancer (28). Moreover, NIR-PIT has been shown to induce super-enhanced permeability and retention effects, which improves the delivery of chemotherapeutic drugs into tumor tissues (29). Thus, combination therapy involving GD2-targeted NIR-PIT and chemotherapy may be a promising antitumor strategy against NB tumors.

Treatment with both APC and GD2-targeted NIR-PIT significantly suppressed the growth of GD2-positive CHP-134 cells (Figure 7). Moreover, the antitumor effect of GD2-targeted NIR-PIT was superior to that of APC treatment alone (Figure 7). These findings suggest that GD2-positive CHP-134 tumors are sensitive to anti-GD2 antibodies and GD2-targeted NIR-PIT. With regard to the therapeutic role of anti-GD2 antibodies against GD2-positive CHP-134 cells, Durbas et al. showed that treatment with an anti-GD2 antibody downregulates the PI3K/Akt/mTOR signaling pathway in CHP-134 cells (30). By contrast, in a study examining the therapeutic effect of a photoabsorber-conjugated anti-GD2 antibody against GD2-positive human tumors, Wellens et al. found that an anti-GD2 antibody conjugated with the NIR fluorescent dye IRDye800CW bound to GD2-expressing human NB cells. The dye-conjugated antibody functioned as a novel imaging system in a xenograft tumor model of patient-derived NB organoids (31), suggesting a potential role for GD2-IR800 in fluorescence-guided surgery (FGS). Therefore, anti-GD2 antibodies conjugated with IR700 could become novel platforms for the treatment of high-risk NB tumors by combining NIR-PIT and FGS.

In conclusion, we demonstrated that GD2-targeted NIR-PIT using an IR700-conjugated anti-GD2 antibody in conjunction with NIR light irradiation reduces the viability, sphere-forming capacity, and growth of human NB tumor cells expressing GD2. Taken together, our results suggest that GD2-targeted NIR-PIT represents a potentially useful novel therapeutic approach for the treatment of GD2-expressing high-risk NB tumors.

Acknowledgements

We thank Tomoko Sueishi, Yuko Hoshijima, and Tae Yamanishi for their excellent technical support.

Footnotes

  • Authors’ Contributions

    Conception and design: H.T., S.K., T.F.; Development of methodology: H.N., T.T., M.T.; Acquisition of data: H.N., T.T., M.T.; Analysis and interpretation of data: H.N., T.T., M.T., H.W.: Writing, review, and/or revision of the manuscript: H.N., H.T., T.F.; Study supervision: H.T., T.O., K.N., S.K., H.K., T.N., K.S., T.F.

  • Conflicts of Interest

    The Authors disclosed no potential conflicts of interest.

  • Funding

    This study was supported, in part, by the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant JP19K18054 to T.T.).

  • Artificial Intelligence (AI) Disclosure

    No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.

  • Received September 2, 2025.
  • Revision received October 23, 2025.
  • Accepted October 31, 2025.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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Near-infrared Photoimmunotherapy Targeting High-risk Human Neuroblastoma Cells Expressing GD2
HIROSHI NOUSO, HIROSHI TAZAWA, TERUTAKA TANIMOTO, MORIMICHI TANI, HINAKO WATANABE, TAKANORI OYAMA, KAZUHIRO NOMA, SHUNSUKE KAGAWA, HISATAKA KOBAYASHI, TAKUO NODA, SHINJI KURODA, TOSHIYOSHI FUJIWARA
Anticancer Research Jan 2026, 46 (1) 25-38; DOI: 10.21873/anticanres.17921
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