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Published in final edited form as: Cancer Lett. 2024 Jan 23;585:216606. doi: 10.1016/j.canlet.2023.216606

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

Hiroshi Fukushima 1, Seiichiro Takao 1, Aki Furusawa 1, Vladimir Valera Romero 2, Sandeep Gurram 2, Takuya Kato 1, Shuhei Okuyama 1, Makoto Kano 1, Peter L Choyke 1, Hisataka Kobayashi 1
PMCID: PMC10923129  NIHMSID: NIHMS1963620  PMID: 38272345

Abstract

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 hours 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.

Keywords: Bladder cancer, Near-infrared photoimmunotherapy, Nectin-4, Molecular subtype, Preclinical model

1. Introduction

Bladder cancer is the sixth most prevalent cancer in the United States, with an estimated 82,290 new cases and 16,710 deaths in 2023.[1] Bladder cancer is stratified based on the extent of tumor infiltration within the bladder wall: non-muscle invasive bladder cancer (NMIBC) and muscle invasive bladder cancer (MIBC). NMIBC is typically non-fatal and managed through transurethral resection of bladder tumor (TURBT). However, over half of NMIBC patients experience recurrence postoperatively,[2, 3] resulting in the need for repetitive TURBTs and supplementary treatment with Bacille Calmette-Guérin (BCG), which faces serious limited availability worldwide.[4] This requirement for continuous monitoring makes NMIBC one of the most expensive cancers to treat over a patient’s lifetime.[5] Localized MIBC is even more aggressive than NMIBC, and these patients typically have an unfavorable prognosis, with a survival rate of less than 60% at 5 years, even with curative-intent radical cystectomy.[6] The prognosis for patients with metastatic bladder cancer is poor. Immune checkpoint inhibitors have recently been introduced and have shown response rates of approximately 25% in platinum-ineligible patients.[7, 8] Consequently, there is an urgent clinical need to develop alternative therapeutic approaches for treating bladder cancer.

Recently, a paradigm shift in the treatment of metastatic urothelial cancer occurred with the approval of antibody-drug conjugates (ADCs) that selectively deliver cytotoxic payloads to target cells.[9] Enfortumab vedotin (EV), an ADC that targets Nectin-4, has gained significant attention, particularly because Nectin-4 is overexpressed in many bladder cancers[10, 11]. Although EV shows promising results, multiple resistance mechanisms likely underlie EV-resistant tumors that pose a challenge for EV treatment.[12–14]. These mechanisms are unique to ADCs, including payload drug efflux transporters, altered internalization of ADCs, and resistance to cytotoxic payloads.[15, 16] Therefore, the scope of Nectin-4-targeted therapies would be expanded by developing treatments with alternative methods of inducing cancer cell death.

Near-infrared photoimmunotherapy (NIR-PIT) is a cell-selective anti-cancer therapy that destroys cancer cells by NIR-light-induced photochemical reactions within antibody-photoabsorber conjugates (APCs) attached to the cancer cell membrane.[17–20] The monoclonal antibodies that are conjugated to the photoabsorber IRDye700DX (IR700), a silica-phthalocyanine dye, are used to target cancer-specific antigens located on the surface of cancer cells.[21] Upon intravenous infusion the APCs bind to cancer cells within 24 hours. Direct exposure to NIR light causes axial ligand release within the IR700 dye resulting in a transition of IR700 from highly hydrophilic to highly hydrophobic.[22] This photochemical reaction leads to aggregation of APCs, resulting in significant damage to the adjacent cancer cell membrane, with minimal damage to the surrounding normal tissues.[22, 23] Thus, NIR-PIT exerts highly selective cancer cell killing, in contrast to photodynamic therapy, which has not achieved widespread clinical acceptance owing to insufficient specificity of the photosensitizer and the resulting significant local adverse effects.[24] In head and neck cancer, NIR-PIT targeting Epidermal growth factor receptor (EGFR) has been approved for clinical use in Japan and is currently being evaluated in a Phase III clinical trial in the United States. NIR-PIT also holds promise as a clinically feasible modality for treating bladder cancer because the placement of a laser fiber via a cystoscope facilitates targeted NIR light irradiation at any location within the bladder.[19, 25] NIR light directed at non-antigen-bearing normal bladder mucosa should be harmless producing a large therapeutic window. Utilizing a NIR endoscopic camera, IR700 fluorescence enables intraoperative identification of tumor sites, thereby potentially allowing surgeons to optimize tumor detection and meticulously evaluate resection margins, akin to photodynamic diagnosis (PDD), as well as treat residual or recurrent disease.[26, 27] In this study, we aimed to develop Nectin-4-targeted NIR-PIT and assess its efficacy using multiple bladder cancer xenograft models including orthotopic models.

2. Materials and Methods

Detailed materials and methods are described in the Supplementary File.

3. Results

3.1. Nectin-4 demonstrates high expression in bladder cancer specimens

We evaluated membrane Nectin-4 expression in bladder cancer tissue microarray specimens including 47 NMIBC cases and 100 MIBC cases (Supplementary Table 1) by multiplex immunohistochemistry (IHC) and calculated Nectin-4 H-scores (Fig. 1A). Nectin-4 H-score was significantly higher in NMIBC compared to MIBC (Fig. 1B). When Nectin-4 positivity was determined based on Nectin-4 H-score, membrane Nectin-4 expression was detected in 77 % and 65 % of NMIBC and MIBC patients, respectively (Fig. 1C). Also, Nectin-4 expression was present in 69 % of squamous cell carcinoma variants and 64 % of adenocarcinoma variants (Supplementary Table 2).

Figure 1.

Figure 1.

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Nectin-4 expression in bladder cancer. A–C, Immunohistochemical evaluation of membrane Nectin-4 expression in bladder cancer tissue microarray specimens. Nectin-4 H-score was calculated based on membrane Nectin-4 expression. Nectin-4 staining was classified into one of four groups: negative (H-score 0–14), weak (H-score 15–99), moderate (H-score 100–199), and strong (H-score 200–300). A, Representative images are shown (images; ×200; scale bar, 20 μm). Antibody staining of Nectin-4 is shown in brown. Nuclei are stained with DAPI and shown in blue. B, Comparison of Nectin-4 H score between 47 NMIBC and 100 MIBC cases (unpaired t-test). Median and quartiles are shown as solid and dashed lines, respectively. *, P < 0.05. C, Distribution of Nectin-4 staining category in 47 NMIBC and 100 MIBC cases. D–F, Comparison of Nectin-4 mRNA expression among the consensus molecular subtypes in public clinical cohorts of muscle-invasive bladder cancer [MIBC; TCGA 2017 (D; n = 406) and Sjödahl 2017 (E; n = 243)] and non-muscle-invasive bladder cancer [NMIBC; UROMOL 2020 (F; n = 535)] (one-way ANOVA followed by Tukey’s test). Median and quartiles are shown as solid and dashed lines, respectively. **, P < 0.01 vs. Luminal subtypes; ****, P < 0.0001 vs. Luminal subtypes; Ba/Sq, basal/squamous; LumNS, luminal-nonspecified; LumP, papillary; LumU, luminal-unstable; NE-like, neuroendocrine-like.

Chu et al. reported that luminal subtypes had significantly enriched Nectin-4 expression in MIBC.[11] Therefore, molecular subtyping can be useful in the patient selection for Nectin-4-targeted therapy. Thus, we validated Nectin-4 mRNA expression for consensus molecular subtyping using three publicly available cohorts of MIBC and NMIBC.[28] As is reported in Chu et al.,[11] Nectin-4 mRNA expression was significantly higher in luminal subtypes (luminal papillary, luminal nonspecified, and luminal unstable) in comparison to basal/squamous, NE-like, and stroma-rich subtypes in MIBC (TCGA 2017 and Sjödahl 2017; Fig. 1D and E).[29, 30] In NMIBC (UROMOL 2020), 99 % of the patients were classified into luminal subtypes, and Nectin-4 mRNA expression was significantly higher in luminal subtypes compared to basal/squamous and stroma-rich subtypes (Fig. 1F).[31]

3.2. Cell surface Nectin-4 expression and the specific binding of Nectin-4-IR700 in human bladder cancer cell lines

Conjugated Nectin-4-IR700 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC). In SDS-PAGE, Nectin-4-IR700 had the same approximate molecular weight as unconjugated anti-Nectin-4 antibody but only Nectin-4-IR700 exhibited 700-nm fluorescence (Supplementary Fig. 1A). In SEC analysis, Nectin-4-IR700 showed evidence of absorption at a wavelength of both 280 nm and 689 nm (Supplementary Fig. 1B). These results verified successful conjugation of Nectin-4-IR700.

Cell surface Nectin-4 expression was analyzed in vitro in human bladder cancer cell lines using flow cytometry. As shown in Fig. 2A, Nectin-4 expression was detected in all luminal subtype cell lines (MGH-U3, RT112, RT4, SW780, HT1376-luc), but not in basal (UMUC3) and mixed (T24) subtype cell lines.[11, 32] Nectin-4 expression was especially high in SW780, a luminal subtype NMIBC-derived cell line, and HT1376-luc, a luminal subtype MIBC-derived cell line (Fig. 2B). Similarly, cell surface Nectin-4 expression was also detected in vivo in tumors established from the five luminal subtype cell lines (Supplementary Fig. 2). Furthermore, in vitro binding of Nectin-4-IR700 to HT1376-luc or SW780 cells was evaluated (Supplementary Fig. 3). Both cells exhibited fluorescence indicating binding of Nectin-4-IR700, which was totally blocked by the addition of an excess of unconjugated anti-Nectin-4 antibody. This indicates the binding of Nectin-4-IR700 to Nectin-4-expressing cancer cells was specific.

Figure 2.

Figure 2.

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In vitro Nectin-4 expression and efficacy of Nectin-4-targeted NIR-PIT in human bladder cancer cell lines. A, Flow-cytometric analysis of in vitro Nectin-4 expression on the cell surface of human bladder cancer cell lines. B, The relative fluorescence intensity (RFI) of Nectin-4 for each cell line (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test). RFI was calculated as the ratio of the median fluorescence intensity of anti-Nectin-4 antibody to that of the isotype control. **, P < 0.01; ****, P < 0.0001 vs. UMUC3. C, Cell viability of each cell line after in vitro Nectin-4-targeted NIR-PIT measured by MTT assay (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test). The value of absorbance in the treated group was normalized to the untreated control. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 vs. UMUC3. D, Microscopic observation of HT1376-luc cells before and after in vitro Nectin-4-targeted NIR-PIT (images, ×100; scale bar, 20 μm). The insets (a–d) displayed on the right side show representative images of bleb formation (white-filled arrowhead) and propidium iodide (PI)-stained cells (white hollow arrowhead). DIC, differential interference contrast. E, Cell membrane damage of HT1376-luc cells induced by in vitro Nectin-4-targeted NIR-PIT was assessed with the dead cell count using PI staining (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test). ****, P < 0.0001 vs. untreated control. F and G, Cell surface expression of calreticulin (F) and HSP70 (G) in HT1376-luc cells after in vitro Nectin-4-targeted NIR-PIT was measured by flow cytometry (n = 4; mean ± SEM; one-way ANOVA followed by Tukey’s test. ****, P < 0.0001 vs. untreated control.

3.3. Efficacy of in vitro Nectin-4-targeted NIR-PIT across molecular subtyping

We examined the cytotoxic efficacy of in vitro Nectin-4-targeted NIR-PIT across molecular subtyping. In the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay, cell viability was significantly decreased after Nectin-4-targeted NIR-PIT in all luminal subtype cell lines compared to UMUC3 (Fig. 2C). Cytotoxic efficacy of NIR-PIT was the highest in HT1376-luc cells, which had the highest Nectin-4 expression (Fig. 2C). Additionally, cytotoxic efficacy of NIR-PIT was augmented in a light dose-dependent manner (Supplementary Fig. 4A). Microscopically, HT1376-luc cells showed cell morphologic changes such as cellular swelling and bleb formation within 30 minutes following in vitro Nectin-4-targeted NIR-PIT (Fig. 2D). Propidium iodide (PI) staining gradually increased in the nucleus of HT1376-luc cells, corresponding with cell morphologic changes (Fig. 2D). This represents that the cytotoxic mechanism of Nectin-4-targeted NIR-PIT is based on cell membrane damage. Quantitative measurement of cell membrane damage was performed in vitro via PI flow cytometry after Nectin-4-targeted NIR-PIT in HT1376-luc cells. The percentage of PI-positive cells significantly increased after NIR-PIT compared to the control (Fig. 2E). Nevertheless, Nectin-4-targeted NIR-PIT did not increase Annexin V-positive/PI-negative apoptosis cells (Supplementary Fig. 4B), suggesting that Nectin-4-targeted NIR-PIT kills cancer cells by causing necrotic cell membrane damage, but not by activating the apoptotic pathway. Moreover, expression of damage-associated molecular patterns (DAMPs), which indicate immunogenic cell death, were examined in vitro immediately after Nectin-4-targeted NIR-PIT against HT1376-luc cells. Cell surface Calreticulin and Heat shock protein 70 (HSP70) expression were significantly increased after NIR-PIT (Fig. 2F and G).

3.4. Delivery of anti-Nectin-4 antibody to the tumor and biodistribution of Nectin-4-IR700 in vivo

To evaluate the delivery of anti-Nectin-4 antibody to cancer cells in vivo, either Nectin-4-digoxigenin (DIG) or isotype-DIG was infused into SW780 and HT1376-luc tumor-bearing mice, then intratumoral DIG distribution was analyzed by multiplex IHC (Supplementary Fig. 5). In both tumors, most pan-cytokeratin (pCK)-positive cancer cells showed positivity for Nectin-4 and, therefore, Nectin-4-DIG was detected on the cell surface of pCK positive cancer cells. Some occasional isotype-DIG-positive cells were detected in the tumor tissue, and this phenomenon might be ascribed to non-specific Fc receptor binding. These results indicated that there was specific delivery and binding of anti-Nectin-4 antibody to the surface of cancer cells in vivo. Next, serial in vivo fluorescent imaging of Nectin-4-IR700 was performed after injection in SW780 and HT1376-luc tumor-bearing mice (Supplementary Fig. 6). The intra-tumor fluorescence intensity of Nectin-4-IR700 peaked 24 hours after injection, followed by a gradual decline over time in both models. TBR of Nectin-4-IR700 increased up to 24 hours post injection and remained stable thereafter in both models during the observation period. Fluorescence intensity and TBR of Nectin-4-IR700 tended to be higher in SW780 tumors compared to HT1376-luc tumors, which was consistent with in vivo Nectin-4 expression levels (Supplementary Fig. 2). To achieve higher fluorescence intensity at the tumor site and the highest TBR, NIR light irradiation was administered 24 hours after the injection of Nectin-4-IR700 in in vivo studies.

3.5. In vivo efficacy of Nectin-4-targeted NIR-PIT in subcutaneously inoculated bladder cancer xenograft models

To evaluate the therapeutic efficacy of in vivo Nectin-4-targeted NIR-PIT in NMIBC, we subcutaneously inoculated SW780 cells (luminal subtype NMIBC-derived cell line) into mice and treated tumors by NIR-PIT (Fig. 3A and 3B).[32] A 700-nm fluorescent signal was clearly detected at the tumor site prior to NIR light exposure. This signal began to decay immediately after NIR light irradiation, suggesting photobleaching of IR700 (Fig. 3C and 3D). In the NIR-PIT group, tumor growth was significantly inhibited compared to the Control and APC-IV groups (Fig. 3E). The NIR-PIT group showed significantly longer survival compared to the Control and APC-IV groups (Fig. 3F). Tumor growth and survival were not significantly different between the Control and APC-IV groups. Additionally, we analyzed the therapeutic efficacy of in vivo Nectin-4-targeted NIR-PIT in subcutaneously inoculated HT1376-luc (luminal subtype MIBC-derived cell line) tumors (Supplementary Fig. 7).[11] NIR-PIT significantly suppressed tumor growth and improved survival, suggesting it had a therapeutic effect in luminal subtype MIBC. The histology of SW780 tumors 24 hours after NIR-PIT demonstrated cytoplasmic vacuolation on hematoxylin and eosin (HE) stains after NIR-PIT (Fig. 3G). Furthermore, multiplex IHC demonstrated that whereas lactate dehydrogenase A (LDHA) was localized in the cytoplasm of cancer cells in the Control group, release of LDHA into the extracellular matrix was observed after NIR-PIT (Fig. 3H), suggesting necrotic cell death.[33] Ki-67 positive cancer cells were significantly decreased after NIR-PIT compared to the Control and APC-IV groups, representing decrease in proliferation (Fig. 3I and Supplementary Fig. 8).[34] In addition to a reduction in Ki-67 positivity, NIR-PIT elicited significant changes in the actin cytoskeleton of cancer cells; actin filaments exhibited aggregated patterns near the cell membrane in most cancer cells in the NIR-PIT group, in contrast to their uniform distribution across the entire cytoplasm of cancer cells in the Control group (Fig. 3J), suggesting actin cytoskeleton disruption after NIR-PIT.

Figure 3.

Figure 3.

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Efficacy of in vivo Nectin-4-targeted NIR-PIT in the SW780 subcutaneous xenograft model. A, Treatment schedule. B, Diagram of NIR light irradiation. Subcutaneous SW780 tumors were located at the right dorsum. The red circle represents where NIR light was irradiated. C, Representative 700-nm fluorescence images before and after NIR-PIT (A.U., arbitrary units). D, Changes in 700-nm fluorescence intensity at the tumor site before and after NIR-PIT (n = 11; mean ± SEM; repeated measures two-way ANOVA followed by Sidak’s test); ****, P < 0.0001. E, Tumor volume curves (n = 11; mean ± SEM; repeated measures two-way repeated measures ANOVA followed by Tukey’s test); ****, P < 0.0001 vs. the Control group. F, Survival curves (n = 11, log-rank test with Bonferroni correction); ****, P < 0.0001; ns, not significant; CR, complete response. G, HE staining of tumor sections 24 hours after NIR-PIT (images; ×400; scale bar, 20 μm). H, Immunohistochemical evaluation of lactate dehydrogenase-A (LDHA) expression in SW780 tumors 24 hours after NIR-PIT. Representative pictures of LDHA expression (images; ×400; scale bar, 20 μm). The inset shows examples of LDHA leakage into the extracellular space, which suggests necrotic cell death (white-filled arrowhead). Antibody staining of LDHA and pan-cytokeratin (pCK) is shown in orange and cyan, respectively. Nucleus is stained with DAPI and shown in white. I, Immunohistochemical evaluation of Ki-67 expression in SW780 tumors 24 hours after NIR-PIT. The percentage of Ki-67 positive cancer cells was compared among the three groups (n = 3; one-way ANOVA followed by Tukey’s test); **, P < 0.01; ns, not significant. J, Immunohistochemical evaluation of actin cytoskeleton in SW780 tumors 24 hours after NIR-PIT. Representative pictures of β-actin expression (images; ×400; scale bar, 20 μm). Antibody staining of β-actin is shown in magenta. Nucleus is stained with DAPI and shown in white.

3.6. Weekly administration of Nectin-4-targeted NIR-PIT improved in vivo tumor control in a NMIBC xenograft model with low Nectin-4 expression

Given the standard therapeutic course of NMIBC, one potential clinical application of Nectin-4-targeted NIR-PIT would involve the utilization of intraoperative NIR-PIT immediately after TURBT, followed by subsequent weekly NIR-PIT in the adjuvant setting. Thus, we examined whether weekly administration of Nectin-4-targeted NIR-PIT can result in more sustained tumor control in a NMIBC xenografts model (Fig. 4A and 4B). In this experiment, we used RT112 xenografts that had relatively low Nectin-4 expression in vitro and in vivo (Fig. 2B and Supplementary Fig. 2). A cohort of mice underwent NIR-PIT only once (One-time NIR-PIT), whereas, in another cohort, mice were treated once-per-week for three weeks with NIR-PIT (Weekly NIR-PIT). Although tumor growth was significantly inhibited in the group treated only once compared to the Control group (Fig. 4C), tumor growth was even more effectively controlled in the group undergoing three treatments (Fig. 4C). Furthermore, weekly NIR-PIT provided significantly longer survival compared to the control and single treatment groups (Fig. 4D).

Figure 4.

Figure 4.

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Efficacy of in vivo weekly Nectin-4-targeted NIR-PIT in the RT112 subcutaneous xenograft model. A, Treatment schedule. In the Weekly NIR-PIT group, once-weekly NIR-PIT was performed three times in total. B, Diagram of NIR light irradiation. Subcutaneous RT112 tumors were located at the right dorsum. The red circle represents where NIR light was irradiated. C, Tumor volume curves (n = 7; mean ± SEM; repeated measures two-way repeated measures ANOVA followed by Tukey’s test); *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the Control group; †, P < 0.05 vs. the One-time NIR-PIT group. D, Survival curves (n = 7, log-rank test with Bonferroni correction); **, P < 0.01; ***, P < 0.001; CR, complete response.

3.7. In vivo Nectin-4-targeted NIR-PIT showed significant tumor control in a MIBC orthotopic xenograft model

We developed a HT1376-luc orthotopic xenograft model to further evaluate therapeutic efficacy of in vivo Nectin-4-targeted NIR-PIT against luminal subtype MIBC. We used multiple imaging studies to characterize this model (Fig. 5A). The bladder showed 700-nm fluorescence, which was colocalized with bioluminescence. IR700 fluorescence was also evaluated by LIGHTVISION, a commercially available camera for indocyanine green (ICG) capable of also detecting IR700 fluorescence.[27] LIGHTVISION detected fluorescence in the bladder similar to the Pearl Imager. Furthermore, we observed the inner surface of the bladder by fluorescence endoscopy (Fig. 5B). Endoscopic study showed a high fluorescence lesion in the bladder, which suggests the presence of Nectin-4-positive bladder tumor.

Figure 5.

Figure 5.

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Characterization of the HT1376-luc orthotopic xenograft model. A, In vivo fluorescence and bioluminescence imaging of HT1376-luc orthotopic bladder tumor model showed colocalization of fluorescence and bioluminescence. 700-nm fluorescence images were obtained by Pearl Imager and LIGHTVISION. B, The representative white light and 700-nm fluorescence images of the bladder mucosa by cystoscopy. White dotted line represents the outline of the high fluorescence lesion that suggests the presence of a tumor.

Next, we treated HT1376-luc orthotopic xenografts by Nectin-4-targeted NIR-PIT (Fig. 6A and 6B). NIR light irradiation induced photobleaching of the 700-nm fluorescence signal at the treatment site (Fig. 6C). Bioluminescence imaging was utilized to assess the tumor growth rate after NIR-PIT (Fig. 6D). The luciferase activity in the NIR-PIT group initially decreased after NIR-PIT, however, signal gradually recovered but remained significantly lower than the Control group (Fig. 6E). The bladder was harvested and its weight was measured two weeks after NIR-PIT (Fig. 6F and 6G). The weight of the bladder was significantly lower in the NIR-PIT group compared to the Control group (Fig. 6F). Also, we analyzed the histology of HT1376-luc orthotopic tumors 24 hours after NIR-PIT. HE staining showed cytoplasmic vacuolation in the majority of cancer cells after NIR-PIT (Fig. 6H). Moreover, multiplex IHC revealed extracellular leakage of LDHA (Fig. 6I), a significant decrease in Ki-67 positivity among cancer cells (Fig. 6J and Supplementary Fig. 9A), and aberrant distribution of actin cytoskeleton (Supplementary Fig. 9B) in tumors treated with NIR-PIT.

Figure 6.

Figure 6.

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Efficacy of in vivo Nectin-4-targeted NIR-PIT in the HT1376-luc orthotopic xenograft model. A, Treatment schedule. B, Diagram of NIR light irradiation. The red circle represents where NIR light was irradiated. C, Representative 700-nm fluorescence images before and after NIR-PIT (A.U., arbitrary units). D, Representative bioluminescence images before and after NIR-PIT. E, Luciferase activity after NIR-PIT (n = 11–12; mean ± SEM; repeated measures two-way ANOVA followed by Sidak’s test); *, P < 0.05. F, Comparison of bladder weight between the Control and NIR-PIT groups (n = 11–12; mean ± SEM; unpaired t-test); ***, P < 0.001. G, Pictures of resected bladder (scale bar, 10 mm). H, HE staining of tumor sections 24 hours after NIR-PIT (M, Muscle; L, Lumen; scale bar, 20 μm). I, Immunohistochemical evaluation of lactate dehydrogenase-A (LDHA) expression in HT1376-luc orthotopic tumors 24 hours after NIR-PIT. Representative pictures of LDHA expression (images; ×400; scale bar, 20 μm). Antibody staining of LDHA and pan-cytokeratin (pCK) is shown in orange and cyan, respectively. Nuclei are stained with DAPI and shown in white. J, Immunohistochemical evaluation of Ki-67 expression in HT1376-luc orthotopic tumors 24 hours after NIR-PIT. The percentage of Ki-67 positive cancer cells was compared between the Control and NIR-PIT groups (n = 3; unpaired t-test); **, P < 0.01.

4. Discussion

In this study, we demonstrate the ability of Nectin-4-targeted NIR-PIT to kill bladder cancer cells both in vitro and in vivo. In vitro, Nectin-4-targeted NIR-PIT demonstrated more cytotoxic effects in luminal subtype cell lines compared to other subtypes. This finding is supported by the higher Nectin-4 expression in luminal subtypes among the consensus molecular subtypes in transcriptome analyses. Nectin-4-targeted NIR-PIT significantly suppressed tumor growth and improved survival in in vivo murine xenograft models of luminal subtype NMIBC and MIBC. Weekly administration of NIR-PIT further improved in vivo tumor control in NMIBC xenograft models with low Nectin-4 expression. Moreover, Nectin-4-targeted NIR-PIT was also effective in MIBC orthotopic xenografts. Histological evaluation including multiplex IHC confirmed that NIR-PIT caused extensive histological damage to cancer cells. Taken together, our results provide a preclinical rationale for treating luminal subtype bladder cancer patients with Nectin-4-targeted NIR-PIT in a clinical setting.

Our results also suggest that Nectin-4-IR700 may assist in detecting bladder tumors during TURBT. In this study, orthotopic bladder tumors clearly showed IR700 fluorescence. Of note, IR700 fluorescence was successfully detected by LIGHTVISION, a commercially available camera for ICG. The extended emission tail of IR700 exceeding 800 nm falls within the detectable range of LIGHTVISION.[27] Using LIGHTVISION, tumors can be visualized through the excitation by low-output-power NIR light irradiation at a wavelength of around 690 nm, devoid of any fluorescence degradation.[27] Thus, ICG fluorescent endoscope systems could be clinically applicable in the intraoperative detection of bladder tumors. Fluorescence imaging of Nectin-4-IR700 may contribute to more accurate detection of positive surgical margins during TURBT. Recently, PDD has been used to aid the detection of bladder tumors during TURBT.[35] A photosensitizer such as 5-aminolevulinic acid and hexaminolevulinate is administered, and bladder tumors can be detected after excitation by blue light. However, blue light has low tissue penetration, limiting its utility to submucosal tumor cells. In contrast, IR700 is excited with NIR light, which can penetrate up to 1–2 cm below the tissue surface.[36] Therefore, Nectin-4-IR700 may overcome a limitation of PDD and might be applied as a diagnostic tool for bladder cancer.

Nectin-4 is an excellent target for molecular photoimmunotherapy [37]. Nectin-4 was detected in approximately 80–90% of NMIBC surgical specimens. Moreover, almost all NMIBC tumors were categorized as luminal subtypes which constitute the majority cell type. These results suggest that the majority of NMIBC patients are good candidates for Nectin-4-targeted NIR-PIT. After TURBT, NIR laser fiber can be placed via a cystoscope, followed by NIR light irradiation to the bladder. NIR-PIT can be utilized as an adjuvant therapy to eradicate residual cancer cells. Given the scarcity of BCG worldwide,[4] NIR-PIT may have a role as an alternative to BCG therapy, especially in high-risk NMIBC and carcinoma in situ. Because of the simplicity and minimally invasive nature of NIR-PIT, administering weekly NIR-PIT treatments by transurethral NIR irradiation in an outpatient clinic setting may be a viable and practical approach after TURBT. Our results confirmed the effectiveness of weekly administration of NIR-PIT in NMIBC preclinical models.

Furthermore, Nectin-4-targeted NIR-PIT is also promising as a treatment for MIBC, especially for the luminal subtype. Recently, bladder-preservation therapy (BPT) that includes maximal TURBT and chemoradiotherapy has emerged as an alternative therapy for localized MIBC,[38] but further refinement of BPT is mandatory to improve its outcomes. As shown in this study, NIR-PIT triggered substantial release of DAMPs including calreticulin and HSP70 indicating that stimulates a local immune response. NIR-PIT combined with immune checkpoint inhibitors resulted in 50–70% of complete response in multiple syngeneic mouse cancer models. Therefore, the combination of Nectin-4-targeted NIR-PIT and immune checkpoint inhibitors might be a more potent BPT strategy against localized MIBC.

This study has several limitations. First, we used human cell line-derived xenografts in immunodeficient mice. Therefore, the effect of Nectin-4-targeted NIR-PIT on host immunity was not evaluable in this study. Our previous studies showed that anti-cancer immune responses were highly induced by NIR-PIT targeting EGFR,[39] CD29,[40] and CD44.[41] Thus, Nectin-4-targeted NIR-PIT is likely to have similar immune effects. Second, we did not evaluate percutaneous toxicities as reported with EV. EV sometimes induces severe cutaneous toxicities because of high Nectin-4 expression and release of the drug payload in keratinocytes. Given that cytotoxic effects of NIR-PIT are limited to the irradiated site, such adverse events are less likely to occur. This point should be evaluated in human clinical trials. Third, we used a frontal light diffuser to irradiate NIR light in this study because cylindrical light diffuser fibers are too large to insert through the murine urethra. In humans, cylindrical light diffuser fibers are easily inserted into the bladder. Finally, further refinement of NIR-PIT is necessary to improve its therapeutic efficacy, especially in molecular subtypes other than luminal subtypes and metastatic MIBC. Because bladder cancer cells highly express EGFR, Human epidermal growth factor receptor 2 (HER2), and Trophoblast cell surface antigen 2 (TROP2) as well as Nectin-4,[13, 37, 42] the utilization of custom-made cocktails of APCs targeting these cancer-specific antigens might be a good approach to treat all subtypes. Additionally, NIR-PIT targeting cancer-associated fibroblasts might be an effective treatment in the stromal-rich subtype.[43]

In conclusion, Nectin-4-targeted NIR-PIT showed significant efficacy in vitro, using human luminal subtype bladder cancer cell lines. Moreover, Nectin-4-targeted NIR-PIT has demonstrated significant in vivo tumor control in murine xenograft models of luminal subtype bladder cancer, including orthotopic models. Therefore, Nectin-4-targeted NIR-PIT is a promising therapy for luminal subtype bladder cancer and is a good candidate for clinical translation.

Supplementary Material

1

Highlights.

  • Nectin-4 is highly expressed in bladder cancer, especially in luminal subtypes.

  • We developed Nectin-4-targeted NIR-PIT as a theranostic modality in bladder cancer.

  • NIR-PIT was effective in vitro in luminal subtype bladder cancer cell lines.

  • NIR-PIT was effective in bladder cancer xenografts including orthotopic models.

  • Nectin-4-targeted NIR-PIT is a promising therapy of luminal subtype bladder cancer.

Funding:

This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (award recipient: Hisataka Kobayashi, grant number: ZIA BC 011513).

Footnotes

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Declaration of competing interest: The authors declare no potential conflicts of interest.

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