Local Depletion of Immune Checkpoint Ligand CTLA4 Expressing Cells in Tumor Beds Enhances Antitumor Host Immunity

Abstract

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.

Graphical Abstract

CTLA4 is a major immune checkpoint ligand mediating antitumor immune suppression. We describe antitumor therapeutic efficacy after locally depleting intratumoral CTLA4 expressing cells by a specific cell-elimination biotechnology, near-infrared photoimmunotherapy. This treatment dramatically enhances T cell mediated antitumor host immunity in mice that would be easily applied to human patients.

1. Introduction

Immune checkpoint blockade therapies targeting cytotoxic T-lymphocyte antigen 4 (CTLA4) or the programmed death-1 (PD-1) can produce dramatic clinical effects in cancer patients. ^{[1, 2]} The antitumor efficacy of anti-CTLA4 immunotherapy was first reported in 1996, leading to FDA approval of the CTLA4-targeted antibody, Ipilimumab, in 2011.^{[3, 4]} CTLA4 and CD28 share binding the ligands CD80 and CD86. CD28 is constitutively expressed on T cells and interacts with CD80/86 on antigen presenting cells producing co-stimulatory signal, which results in immune activation.^{[5, 6]} On the other hand, CTLA4 is expressed on activated T cells and binds to CD80/86 on antigen presenting cells with higher affinity than CD28 thus which interfering with immune activation during the priming phase of the cancer-immunity cycle.^[7–9] Thus, blockade with an anti-CTLA4 antibody of the binding between CTLA4 and CD80/86 promotes priming in lymph nodes and enhances antitumor immunity.

In addition to anti-CTLA4 immunotherapy, regulatory T cells (Tregs) targeted therapies have also been shown to induce an antitumor effect.^{[10, 11]} There are three key mechanisms by which Tregs normally downregulate antitumor immunity; (i) promoting the CTLA4 axis, (ii) reducing the amount of IL-2 available for effector T cells, and (iii) producing immune-suppressive cytokines, such as IL-10 and TGF-β.^[12] Indeed, anti-CTLA4 immunotherapy is thought to be effective not just because it interferes with the CTLA4 pathway, but also because it leads to depletion of intra-tumoral Tregs.^{[13, 14]} However, systemic cancer immunotherapies, including anti-CTLA4 therapy and Treg depletion, often induce autoimmune adverse events;^{[15, 16]} therefore, more tumor-specific methods would be desirable.

Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer therapy that uses antibody-photoabsorber conjugates (APCs) and NIR light. Approximately 24 hours after intravenous administration, APCs bind to tumor cell surface markers. Once the APCs bind to the cell membrane, exposure to NIR light selectively induces rapid cell-specific, necrotic, and highly immunogenic cell death (ICD).^{[17, 18]} A global phase III clinical trial of NIR-PIT in inoperable head and neck cancers which targets epidermal growth factor receptor (EGFR) is currently underway (https://clinicaltrials.gov/ct2/show/NCT03769506).

In September 2020, the first APC for human use, cetuximab-IR700 (ASP1929) was conditionally approved and registered for clinical use by the Pharmaceuticals and Medical Devices Agency (PMDA) in Japan. NIR-PIT was originally developed to selectively kill cancer cells, but it can be equally applied to other types of cells within the tumor microenvironment. For instance, CD25 is a component of the IL-2 receptor (IL-2R) and is permanently expressed on Tregs but temporally expressed on activated effector cells, such as effector T cells (Teffs) and natural killer (NK) cells.^[19] Since CD25 has been identified on Tregs but not resting effector cells in tumor beds of various tumors,^{[11, 20]} it is a good target for Treg-directed NIR-PIT. CD25-targeted NIR-PIT has been shown to selectively destroy intratumoral Tregs, resulting in antitumor immune activation and tumor growth suppression.^[20]

Likewise, CTLA4-targeted NIR-PIT could selectively deplete such cells in the tumor microenvironment (TME) thus, providing another mechanism to activate anti-cancer immunity. Additionally, CTLA4 is constitutively expressed on immune suppressor cells such as Tregs, therefore, CTLA4-targeted NIR-PIT would deplete immune suppressor cells in the TME thereby activating antitumor immunity. However, using anti-CTLA4 as an agent for NIR-PIT is challenging because most CTLA4 molecules exist intracellularly and not on the cell surface.^[21–23] Here, we assess the efficacy of CTLA4-targeted NIR-PIT in locally depleting CTLA4 expressing cells in the TME and subsequent antitumor effects in murine models.

2. Results

2.1. IR700 was successfully conjugated with anti-CTLA4

To synthesize the APC, IR700 was conjugated to anti-CTLA4 antibody (clone 9D9) and the conjugate (anti-CTLA4-IR700) was analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Anti-CTLA4 and anti-CTLA4-IR700 had approximately the same molecular weight but only anti-CTLA4-IR700 had a fluorescence of 700 nm (Figure 1A). The APC was also evaluated with size-exclusion chromatography (SEC). The majority of the protein, which was detected by its light absorption at 280 nm, also had evidence of light absorption at 689-nm and fluoresed at 700 nm (Figure 1B). These results verified the successful conjugation of the APC.

Figure 1.

Synthesis of anti-CTLA4-IR700 and cell-killing specificity of CTLA4-targeted NIR-PIT. A) Evaluation of anti-CTLA4-IR700 by SDS-PAGE (left: Colloidal Blue staining, right: 700 nm fluorescence). Unconjugated anti-CTLA4 antibody was used as a control. B) Evaluation of anti-CTLA4-IR700 by SEC. The APC demonstrated light absorption at 280 and 689 nm and corresponding fluorescence at 700nm. C) Flow-cytometric analysis of CTLA4 expression on cancer cell lines. Representative histograms are shown. D) PI-stained dead cell % after in vitro NIR-PIT on cancer cell lines (n = 4; unpaired t test; ns, not significant). E) Flow-cytometric analysis of CTLA4 expression of splenocytes from non-tumor bearing mouse. Total (surface and intracellular) CTLA4 was stained. F) Flow-cytometric analysis of CTLA4 expression in spleen T cells. Representative histograms and RFI are shown (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01, ***P < 0.001, ****P < 0.0001). RFI, Relative fluorescence intensity. G) Treg percentages of T cells after ex vivo NIR-PIT for splenocytes analyzed by flow-cytometry (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01, ****P < 0.0001).

2.2. CTLA4-targeted NIR-PIT selectively destroys CTLA4 expressing Treg cells among - splenocytes but not cultured cancer cell lines

To evaluate the expression of CTLA4 in cultured cancer cell lines, MC38-luc, LL/2-luc, and MOC2-luc cell lines were incubated with Alexa-647-labeled anti-CTLA4 (clone 9D9) or isotype control (anti-CTLA4-Alexa647 or control-Alexa647) and analyzed with flow cytometry. Although CTLA4 was slightly expressed as an intracellular molecules, it was not expressed on the surface of these cancer cell lines (Figure 1C; Figure S1, Supporting information). The cytotoxicity of CTLA4-targeted NIR-PIT against the cancer cell lines was evaluated with flow cytometry. In vitro CTLA4-targeted NIR-PIT did not increase the percentage of propidium iodide (PI)-stained (dead) cells, verifying that surface CTLA4 expression is necessary for the cell-killing with NIR-PIT (Figure1D).

Next, we assessed CTLA4 expression in splenocytes, which revealed > 90% of CTLA4^hi cells were CD45⁺CD3⁺ T cells (Figure 1E; Figure S2, Supporting information). We further investigated T cell-specific CTLA4 expression in splenic tissue. Surface and total (i.e., both surface and intracellular) CTLA4 expression was assessed with flow cytometry. Although the fluorescent shift was small, CD4⁺Foxp3⁺ Tregs showed higher relative fluorescence intensity (RFI) of surface CTLA4 compared with CD8⁺ T cells or CD4⁺Foxp3⁻ T cells (Figure 1F). CD4⁺Foxp3⁺ Tregs also showed higher total expression of CTLA4 than CD8⁺ T cells or CD4⁺Foxp3⁻ T cells. The average RFI surface/total ratio of Tregs was 7.45% suggesting that CTLA4 was predominantly internalized in splenic Tregs. In order to test the specificity of CTLA4-targeted NIR-PIT, we performed ex vivo CTLA4-targeted NIR-PIT against splenocytes. In spite of the low surface CTLA4 expression, ex vivo CTLA4-targeted NIR-PIT selectively destroyed CD4⁺Foxp3⁺ Tregs, sparing other CD3⁺ T cells, in a light dose-dependent manner, while administration of APC or NIR light alone were statistically identical to the untreated control (Figure 1G).

2.3. In vivo fluorescence imaging after the injection of anti-CTLA4-IR700

The biodistribution of intravenously injected APC was assessed with quantitative fluorescence. Anti-CTLA4-IR700 fluorescence in the TME was clearly detected as early as 1 hour after intravenous injection (Figure S3, Supporting information). The average fluorescence intensity peaked within 24 hours of injection, then gradually decreased over the following days for all three tumor types. The target-to-background ratio (TBR) also peaked within 24 hours and did not decrease for several days. These results suggest that the optimal time for NIR light exposure was approximately one day post-injection of APC.

2.4. Intra-tumoral CTLA4 expressing cells were mainly T cells

CTLA4 expression of intra-tumoral cells was investigated with flow cytometry. Single cell suspensions of tumor tissues were made and stained with the anti-CTLA4-Alexa647. Among CTLA4^hi cells, the majority were CD45⁺CD3⁺ T cells with smaller populations of CD45⁺CD3⁻ non-T hematopoietic cells (Figure S4, Supporting information). Among CD3⁺ T cells, CD4⁺Foxp3⁺ Treg cells in all 3 tumor lines showed significantly higher RFI than other T cell types (Figure 2A). Intra-tumoral Tregs showed higher surface expression of CTLA4 than splenic Tregs. The total CTLA4 expression (Figure 2A) and CTLA4^hi percentage (Figure 2B) were also higher in intra-tumoral Tregs than other types of T cells. The average RFI surface/total ratio of Tregs was 5-15%. Surface CTLA4 expression on tumor cells, endothelial cells, B cells, NK cells, myeloid cells, macrophages, or dendritic cells was undetectable (Figure S5, Supporting information).

Figure 2.

CTLA4 was expressed by Treg cells more than other T cells in tumor tissue. A) CTLA4 expression in T lymphocyte populations in MC38-luc, LL/2-luc, and MOC2-luc tumors were analyzed by flow cytometry. Representative histograms for surface or total (surface + intracellular) CTLA4 expression in CD8⁺ T cells (CD3⁺CD8⁺), CD4⁺Fopx3⁻T cells (CD3⁺CD4⁺Foxp3⁻), and CD4⁺Foxp3⁺ Tregs (CD3⁺CD4⁺Foxp3⁺) and relative fluorescence intensity (RFI) of CTLA4 are shown (n = 4; one-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ****P < 0.0001). B) CTLA4^hi percentage was calculated based on the total expression. The CTLA4^hi gates are shown in histograms in A (n = 4; one-way ANOVA followed by Tukey’s test; ****P < 0.0001).

2.5. CTLA4-targeted NIR-PIT selectively depletes CTLA4 expressing cells

Three hours following CTLA4-targeted NIR-PIT, selective depletion of CTLA4 expressing cells was assessed in tumors, tumor draining lymph nodes, and spleens (Figure 3A). CTLA4^hi cells were selectively depleted from the TME but not regional lymph nodes or the spleen, confirming that the effect of NIR-PIT is localized to the site of treatment (Figure 3B). Some CD45⁺CD3⁻ non-T hematopoietic CTLA4^hi cells were also depleted although the effect was not statistically significant. This is likely because the CTLA4^hi population constituted less than 1% of the CD45⁺CD3⁻ non-T hematopoietic cells. To assess if CTLA4-targeted NIR-PIT directly destroys tumor cells, MC38-luc tumors were harvested after treatment (Figure S6, Supporting information). Since MC38-luc expresses CD44 on cell surface, CD44-targeted NIR-PIT-treated tumors were also examined for comparison. CD44-targeted NIR-PIT induced cellular swelling and vacuolation of the tumor cells, while no obvious morphological changes were seen in CTLA4-targeted NIR-PIT-treated tumor cells, confirming that CTLA4-targeted NIR-PIT was selective for surface CTLA4 expressing TME cells and not tumors.

Figure 3.

Selective CTLA4^hi cell depletion by CTLA4-targeted NIR-PIT. CTLA4^hi cells within MC38-luc tumor, tumor draining lymph node, and spleen were assessed with flow cytometry 3 hours after the light exposure. CTLA4 was stained with anti-CTLA4 (clone UC10-4B9) after fixation and permeabilization. A) Representative dot plots to show CTLA4 expression in live cells, and CTLA4 and CD3 expressions among CD45⁺ live cells. B) The percentages of CTLA4^hi cells among total live cells, CD45⁺CD3⁺ T cells and CD45⁺CD3⁻ non-T hematopoietic cells (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ***P < 0.001, **** P < 0.0001; ns, not significant).

2.6. CTLA4-targeted NIR-PIT inhibits the tumor growth

In vivo therapeutic efficacy of CTLA4-targeted NIR-PIT was evaluated in MC38-luc, LL/2-luc and MOC2-luc tumor-bearing mice. Three groups were compared as follows: no treatment (control), intravenous (IV) APC injection without NIR light exposure (APC-IV), and APC injection followed by NIR light exposure (NIR-PIT). The treatment regimen and the imaging schedule are shown in Figure 4A. Tumors were established on the right flank and exposed to NIR light (Figure 4B). All APC-injected mice showed clear 700-nm fluorescent signal at the tumor site one day post-injection prior to therapeutic NIR light exposure (Figure 4C). This signal immediately decreased after therapeutic NIR light exposure, indicating photobleaching of the conjugated IR700 which correlates with treatment efficacy. The early phase of therapeutic efficacy was evaluated with bioluminescence imaging (BLI) (Figure 4D). In all three tumor types, the NIR-PIT treatment group showed significantly lower intensity on BLI compared with the control group (Figure 4E). The intensity of the BLI signal decreased slowly in the APC-IV group but became statistically significant 4-5 days after injection, at which time no significant difference between the APC-IV group and the NIR-PIT group was discernable in any of the tumor models. However, the NIR-PIT treatment group suppressed tumor growth significantly more than the APC-IV group, which also suppressed tumor growth compared with the control group in all the cancer models (Figure 4F). CTLA4-targeted NIR-PIT eradicated 80%, 70%, and 40% of established MC38-luc, LL/2-luc, and MOC2-luc tumors respectively, which led to significantly prolonged survival compared to the control group (Figure 4G). The NIR-PIT treatment groups also achieved significantly prolonged survival compared with the APC-IV groups in MC38-luc and MOC2-luc but not in LL/2-luc, likely because administration of the APC alone cured 3 of 10 LL/2-luc tumors.

Figure 4.

Efficacy of in vivo CTLA4-targeted NIR-PIT. CTLA4-targeted NIR-PIT was performed on MC38-luc, LL/2-luc and MOC2-luc mouse tumor models. A) Treatment schedule. B) Diagram of NIR-light exposure. The red circle indicates where NIR light was irradiated i.e., NIR-light was exposed only to the tumor. C) Fluorescent imaging before and after NIR-PIT in MC38-luc tumor bearing mouse. D) Bioluminescence imaging (BLI) before (day 6) and after (day 8-11) NIR-PIT in MC38-luc tumor bearing mice. E) Luciferase activity calculated from BLI (n = 10; repeated measures two-way ANOVA followed by Tukey’s test; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; vs. control group). F) Tumor volume curves (n = 10; repeated measures two-way ANOVA followed by Tukey’s test; *P < 0.05, ****P < 0.0001). G) Survival curves (n = 10; log-rank test with Bonferroni correction; **P < 0.01, ***P < 0.001; ns, not significant).

To assess treatment efficacy against poorly immunogenic tumors, CTLA4-targted NIR-PIT was performed on MOC2 tumors (Figure S7, Supporting information). Although the NIR-PIT treatment group experienced significantly suppressed tumor growth and prolonged survival, no mice were cured.

2.7. CTLA4-targeted NIR-PIT selectively depletes Tregs which activates antitumor host immunity

CD4⁺Foxp3⁺ Tregs were significantly reduced after CTLA4-targeted NIR-PIT resulting in an increase in the ratio of CD4⁺Foxp3⁻ :CD4⁺Foxp3⁺ (CD4⁺ non-Treg/Treg ratio) (Figure 5A). In contrast, no obvious difference was seen among splenic T cell populations in the three experimental groups, suggesting that depletion of Treg cells was limited to the treatment site.

Figure 5.

Selective cell depletion immediately after the CTLA4-targeted NIR-PIT against MC38-luc tumors. T cell populations after the therapy were analyzed by flow cytometry. A) Treg population in tumors and spleens 3 hours after light exposure. The dot plots show the representative examples of CD4 and Foxp3 expressions in the CD3⁺ T cells. Scatter plots show the percentage of Tregs in CD3⁺ cells and ratios of non-regulatory/regulatory CD4⁺ T cells (CD4⁺Foxp3⁻/CD4⁺Foxp3⁺) (n = 4; one-way ANOVA followed by Tukey’s test; **P < 0.01; ***, P < 0.001; ns, not significant). B) CD8⁺ T cell populations in the tumors 3 hours after light exposure. The dot plots show the representative examples of CD8 and IFN-γ expressions in the CD3⁺ T cells. Scatter plots show the percentage of intra-tumoral CD8⁺ T cells among total cells and IFN-γ⁺ cells among CD8⁺ T cells (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05; ns, not significant).

We next analyzed CD8⁺ T cells. Although the percentage of total CD8⁺ T cells compared to intratumoral total cells was not significantly different among the three experimental groups at 3 hours after light exposure, IFN-γ⁺ activated CD8⁺ T cells were significantly fewer in number in the NIR-PIT group compared with the other groups (Figure 5B), suggesting that CTLA4 expressing activated CD8⁺ T cells were also depleted by CTLA4-targeted NIR-PIT. Nevertheless, 1 day after the therapy, the percentage of CD69 positive intratumoral CD8⁺ T cells and NK cells were significantly higher in the NIR-PIT group compared with the control group, suggesting CTLA4-targeted NIR-PIT resulted in the activation of CD8⁺ T cells and NK cells (Figure 6A). Moreover, 3 days after the therapy, the percentage of CD69⁺ or CD25⁺ cells among CD8⁺ T cells within tumor draining lymph nodes was significantly higher in the NIR-PIT group compared with the other two groups (Figure 6B), suggesting that newly activated CD8⁺ T cells were expanding after NIR-PIT. To analyze the distribution of T cells in the TME after NIR-PIT, tumors were harvested 5 days after treatment and analyzed with multiplex immunohistochemical staining (Figure 6C). The densities of CD8⁺ cells, CD4⁺Foxp3⁻ cells, and CD4⁺Foxp3⁺ cells were quantified both in tumor and stroma. Both stromal and intratumoral CD8⁺ cell density was significantly higher in the NIR-PIT group compared with the other two groups. Granzyme B was expressed in the intratumoral CD8⁺ T cells after CTLA4-targeted NIR-PIT verifying that these cells were differenticiated into cytotoxic effector cells (Figure S8, Supporting information). A higher CD8⁺/CD4⁺Foxp3⁺ ratio is known to be an indicator of a robust antitumor immune response;^[24] the intra-tumoral CD8⁺/CD4⁺Foxp3⁺ ratio was significantly higher in the NIR-PIT group than in the other two groups. These results suggest CTLA4-targeted NIR-PIT induced a strong T cell-mediated antitumor immune reaction.

Figure 6.

Immune cell response after the CTLA4-targeted NIR-PIT. A) Early phase activation shows increased percentages of CD69 positive intratumoral CD8⁺ T cells and NK cells by flow cytometry 1 day after therapy (n = 5; one-way ANOVA followed by Tukey’s test; *P < 0.05; ns, not significant). B) The expression of activation markers was analyzed in the regional lymph nodes 3 days after therapy. CD69⁺ and CD25⁺ percentages among CD8⁺ T cells were calculated (n = 5-6; one-way ANOVA followed by Tukey’s test; **P < 0.01; ***P < 0.001; ns, not significant). C) Multiplex immunohistochemical staining of tumors 5 days after the therapy. Representative pictures are shown. Scatter plots show the CD8⁺ cell density within stroma or tumor and the CD8⁺/CD4⁺Foxp3⁺ cell ratio within tumor (n = 3; one-way ANOVA followed by Tukey’s test; *, P < 0.05; **, P < 0.01, ***, P < 0.001; ns, not significant). White dashed line represents tumor border; Scale bar = 100 μm; pCK, pan cytokeratin.

2.8. CTLA4-targeted NIR-PIT impairs intra-tumoral blood perfusion

Based on BLI results, CTLA4-targeted NIR-PIT resulted in killing of tumor cells within one day of treatment. We hypothesized that intra-tumoral blood flow might be reduced which would explain this rapid response. To compare the post-treatment blood perfusion, albumin-IR800 was intravenously injected 24 hours after light exposure. The fluorescent signal at the tumor was clearly detected as early as 5 minutes after the injection in the control group and APC-IV group but not in the NIR-PIT group (Figure S9, Supporting information). The target-to-background fluorescence ratio in the NIR-PIT treated group was significantly lower at all the measured timepoints compared with the control group. These results suggested CTLA4-targeted NIR-PIT acutely impairs intra-tumoral blood perfusion.

2.9. CTLA4-targeted NIR-PIT shows an abscopal effect

To assess potential abscopal effects of CTLA4-targeted NIR-PIT, a bilateral MC38-luc tumor model was employed with unilateral treatment. The treatment regimen and imaging schedule are shown in Figure 7A. MC38-luc cancer cells were inoculated into both dorsi of each mouse but only one side was exposed to NIR light (Figure 7B). The left-sided tumor was completely covered with aluminum foil during light exposure to block any light absorption. After light exposure, the 700-nm fluorescence of the treated tumor immediately decreased, while the fluorescence of the contralateral untreated tumor was unchanged, which verified the absence of light exposure to the left-sided tumor (Figure 7C). Luciferase activity in both tumors was compared using BLI in the right and left sided tumors (Figure 7D). Luciferase activity was significantly decreased not only in the treated tumor but also in the untreated tumor after unilateral NIR-PIT treatment compared with the APC-IV group (Figure 7E). Also, tumor growth rates were significantly decreased bilaterally in the NIR-PIT treatment group compared with APC-IV group (Figure 7F). In the NIR-PIT treatment group 40% of mice demonstrated bilateral complete response (CR) and achieved significantly prolonged survival compared with the APC-IV group (Figure 7G).

Figure 7.

In vivo effect of NIR-PIT in a bilateral MC38-luc tumor model. A) Treatment regimen. B) Diagram of NIR-light exposure. NIR-light was exposed only to right-sided tumors; the contralateral tumor was completely covered with aluminum foil during the light exposure. C) Fluorescent imaging shows 700-nm fluorescence before and after NIR-PIT in MC38-luc tumor bearing mouse. D) Bioluminescence imaging (BLI) before (day 6) and after (day 8-11) NIR-PIT. E) Luciferase activity calculated from BLI (n = 10; repeated measures two-way ANOVA followed by Tukey’s test; **P < 0.01, ****P < 0.0001; vs. the same sided tumor of APC-IV group). F) Tumor volume curves (n = 10; repeated measures two-way ANOVA followed by Tukey’s test; ****P < 0.0001; ns, not significant). G) Survival curves (n = 10; log-rank test; ***P < 0.001).

2.10. CTLA4-targeted NIR-PIT results in immunologic memory

To assess the presence of immunologic memory, mice with complete responses of their MC38-luc tumor after CTLA4-targeted NIR-PIT were re-inoculated with the MC38-luc cells six weeks after the initial NIR-PIT on the contralateral dorsum (Figure S10A,B, Supporting information). The mice that previously had complete responses to the initial treatment also resisted re-engraftment, whereas control mice were readily engrafted (Figure S10C,D, Supporting information). These results suggest the development of immunologic memory after CTLA4-targeted NIR-PIT.

2.11. CTLA4-targeted NIR-PIT has no impact on established tumors in athymic nude mice

To assess if the therapeutic effect of CTLA4-targeted NIR-PIT was T cell dependent, efficacy was tested in athymic nude mice. Treatment and imaging regimens are shown in Figure 8A. Right-sided tumors were established and exposed to NIR-light (Figure 8B) with expected loss of fluorescence due to quenching confirming therapeutic light exposure (Figure 8C). No significant difference in BLI intensity was shown between any two groups at the any measured timepoints (Figure 8D,E). The NIR-PIT treatment group in athymic nude mice did not suppress tumor growth (Figure 8F) or prolong survival (Figure 8G). These results confirm that a robust T cell repertoire is necessary for successful CTLA4-targeted NIR-PIT.

Figure 8.

In vivo efficacy of CTLA4-targeted NIR-PIT against MC38-luc tumor in athymic nude mice. A) Treatment regimen. B) Diagram of NIR-light exposure. The red circle indicates where NIR light was irradiated i.e. only to the tumor. C) Fluorescent imaging demonstrates 700-nm fluorescence before and after NIR-PIT in MC38-luc tumor bearing mouse. D) Bioluminescence imaging (BLI) before (day 5) and after (day 7-10) NIR-PIT. E) Luciferase activity calculated from BLI (n = 13; repeated measures two-way ANOVA; ns, not significant). F) Tumor volume curves (n = 13; repeated measures two-way ANOVA; ns, not significant). G) Survival curves (n = 13; log-rank test with Bonferroni correction; ns, not significant).

3. Discussion

We have demonstrated that CTLA4-targeted NIR-PIT causes a T cell mediated antitumor effect in four syngeneic tumor models of cancer. This effect depends on two cooperating mechanisms. First, CTLA4-targeted NIR-PIT selectively eliminates all CTLA4^hi cells within the treated TME, yet spares neighboring lymph nodes, achieving a complete local blockade of the CTLA4 axis. The role of intratumoral CTLA4 in this process is still unclear. Reportedly, about 90% of CTLA4 molecules exist intracellularly because of constitutive endocytosis, which is rapid and the majority of surface CTLA4 molecules are internalized within 5 minutes.^[23] Despite the short exposure window, CTLA4-targeted NIR-PIT killed CTLA4 expressing cells even though their surface expression was low and completely eliminated the CTLA4 axis within minutes of light exposure. Second, although CTLA4-targeted NIR-PIT selectively depleted CTLA4^hi cells among Tregs, CD8⁺ T cells and CD4⁺Foxp3⁻ T cells, the dominant effect was in Tregs due to their higher expression of CTLA4.^{[13, 25]} We observed an increase in the ratio of Foxp3⁻/Foxp3⁺ CD4⁺ T cells in the TME after CTLA4-targeted NIR-PIT. This shift in balance of effector T cells to Tregs favors antitumor activity and correlates with better outcomes after immunotherapy.^[26] As a result of abrogating these two immunosuppressive pathways, CTLA4-targeted NIR-PIT induced T cell activation in regional lymph nodes and stimulated infiltration of intra-tumoral CD8⁺ T cells overcoming the initial depletion of CTLA4^hi populations of intra-tumoral CD8⁺ T cells. Once a tumor was eradicated with CTLA4-targeted NIR-PIT, the same cancer cells were rejected upon re-inoculation, suggesting the development of immunologic memory. In contrast, CTLA4-targeted NIR-PIT showed no therapeutic effect in athymic nude mice, suggesting that the antitumor effect is T cell dependent. No histological changes were detected in tumor cells one hour after therapy, suggesting no direct cytotoxic antitumor effect, further supporting a T cell mediated immune response as the mechanism of action. The acute reduction of intra-tumoral perfusion after CTLA4-targeted NIR-PIT is an interesting aspect of the treatment. It has been reported that rapid depletion of intra-tumoral Tregs by CD25-targeted NIR-PIT activates CD8⁺ T and NK cells producing IFN-γ which in turn results in tumor vessel regression.^[27] Theoretically, a similar regression could occur with CTLA4-targeted NIR-PIT via IFN-γ production. An alternative explanation for the reduction in blood perfusion during CTLA4-targeted NIR-PIT is that unbound APC within the light exposure area could produce reactive oxygen species (ROS) causing damage to blood vessels.^[28] In this study, luciferase activity as measured by BLI decreased as early as one day after light exposure for all three tumors. This early effect might be due to the rapid decrease in intra-tumoral vessel blood flow which precedes the immune response.

CTLA4-targeted NIR-PIT induced an abscopal effect in over half the animals. In bilateral tumor models, when one tumor was treated, the untreated tumors also shrank, an effect also seen in CD25-targeted NIR-PIT.^{[20, 29]} This effect is likely mediated by activated CD8⁺ T cells migrating into untreated tumors, a concept supported by multiplex immunohistochemistry performed in this study showing that CTLA4-targeted NIR-PIT recruited a large number of CD8⁺ T cells. Thus, this therapy holds promise as a potential cancer vaccine therapy.

Administration of anti-CTLA4-IR700 without NIR light exposure significantly suppressed tumor growth yet did not significantly prolong survival compared to the control group for all tumor models. The anti-CTLA4-IR700 injected as APC alone may act as immune checkpoint inhibitor promoting T cell priming, however, only one dose of 50 μg was administered, which is lower than the dose used in previous reports that showed efficacy in mouse models.^{[4, 25]} Furthermore, IR700 conjugation to the antibody shortened the in vivo half-life of the APC compared with the unconjugated antibody. Therefore, the blockade of CTLA4 pathway with this dose of APC is not likely to have major effect. Additionally, anti-CTLA4 used in this study was the clone 9D9, which is a murine IgG2b, which induces minimal antibody-dependent cellular cytotoxicity (ADCC) whereas the same clone with IgG2a Fc portion depletes intra-tumoral Treg cells and eradicates the majority of established MC38 and CT26 colon cancers.^[25] Thus, although a small effect was seen with the low dose APC alone, the same dose combined with NIR light resulted in profound depletion of CTLA4^hi cells. The ability to use a low dose of the antibody lowers the possibility of systemic adverse events.

CD25-targeted NIR-PIT also has been shown to be an effective tumor treatment, however, the presence of residual APC after the light exposure might block IL-2/IL-2R binding on activated effector cells resulting in their inhibition.^[29] In this regard, CTLA4-targeted NIR-PIT would potentially be a superior strategy, as residual anti-CTLA4-IR700 might enhance antitumor immunity by promoting the T cell priming.

Several previous reports emphasize that CTLA4 is also expressed on other types of the cells besides T cells, such as some human cancer cells or myeloid-derived suppressor cells (MDSCs).^{[30, 31]} If correct, CTLA4-targeted NIR-PIT could destroy these cells within tumors and enhance the host immune response. MDSCs are another type of immunosuppressive cell population.^[32–34] In mice, MDSCs are thought to have the surface phenotype of CD11b⁺Gr1⁺; yet there is no definitive surface marker to distinguish them from myeloid cells.^{[34, 35]} In this study, a small population of CTLA4^hiCD45⁺CD3⁻ non-T hematopoietic cells were identified which might include MDSCs, and this population of cells was depleted with CTLA4-targeted NIR-PIT. Thus, the antitumor efficacy of CTLA4-targeted NIR-PIT could partially be mediated via elimination of MDSCs from TME that could be potentially more important in human cancer patients than in mouse models.

Tumor-targeted NIR-PIT has been successful with a variety of antibodies including epithelial growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), mesothelin, programmed death-ligand 1 (PD-L1), and prostate-specific membrane antigen (PSMA).^[36] Tumor-targeted NIR-PIT has been shown to enhance TIL response by inducing immunogenic cell death on target cancer cells followed by the release of multiple tumor associated antigens.^[37] Therefore, the efficacy of tumor-targeted NIR-PIT is further enhanced when concurrently combined with immune regulatory NIR-PIT such as CD25-targeted NIR-PIT.^[38] Similarly, CTLA4-targeted NIR-PIT could also induce additive effects when it is combined with tumor-targeted NIR-PIT especially for poorly immunogenic tumors. Although CTLA4-targeted NIR-PIT eradicated MC38-luc, LL/2-luc, and MOC2-luc tumors at a frequency as 80%, 70%, and 40%, respectively, it failed in MOC2 tumors. This might be because of poor immunogenicity of MOC2 tumor.^[39] MOC2-luc cells were thought to have increased immunogenicity compared with MOC2 cells because of luciferase expression.^[40] The difference of therapeutic efficacy between the MOC2 and the MOC2-luc tumors provided further evidence that the efficacy of CTLA4-targeted NIR-PIT is immune system dependent. Combined NIR-PIT targeting both tumor cells and CTLA4 expressing cells might show superior effect to either one alone.

There are several limitations to this study. First, we used only one clone of anti-mouse-CTLA4. The results may differ if other clones of anti-CTLA4 are used. Second, we used subcutaneous syngeneic tumor modes and not orthotopic models that are generally considered to be more realistic at evaluating in vivo efficacy of therapy. Also, in subcutaneous tumor models, NIR light is easily accessible, this treatment may not be feasible when the tumor is located deep inside the body where NIR light could not be delivered with a frontal diffuser. In such cases, NIR light could be delivered with a fiber optic diffusers inserted directly or via endoscope.^[41] Nonetheless, this is the first study to demonstrate the efficacy of CTLA4-targeted NIR-PIT to selectively deplete intra-tumoral CTLA4^hi cells resulting in activation of host immunity.

4. Conclusion

CTLA4-targeted NIR-PIT was highly effective in eradicating murine cancers in three unique models. The mechanism of action involves activation of effector T cells and is not observed in athymic nude mice, lacking a T cell repertoire. The T cell activation produced both abscopal effects and long-term immunologic memory which prevented re-inoculation of the tumor. When combined with tumor-targeted NIR-PIT, CTLA4-targeted NIR-PIT which could be performed at the same time and is anticipated to have at least additive anti-tumor effects. CTLA4-targeted NIR-PIT is therefore, a promising antitumor therapy.

5. Experimental Section

Reagents

A water soluble, silica-phthalocyanine derivative IRDye 700DX NHS ester was obtained from LI-COR Biosciences (Lincoln, NE, USA). Anti-mouse CTLA4 antibody (9D9), and its corresponding mouse IgG2b isotype control (MPC-11), were purchased from Bio X Cell (West Lebanon, NH, USA). All other chemicals were of reagent grade.

Synthesis of IR700-conjugated anti-CTLA4

Anti-CTLA4 (1 mg, 6.8 nmol) was incubated with 5-fold molar excess of IR700 NHS ester in phosphate buffer (pH 8.5) at room temperature for 1 hour. The mixture was purified with a Sephadex G25 column (PD-10; GE Healthcare, Piscataway, NJ, USA). The concentration of IR700 was determined with absorption at 689 nm using UV-Vis (8453 Value System; Agilent Technologies, Santa Clara, CA, USA). The protein concentration was confirmed with Coomassie Plus protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA) by measuring the absorption at 595 nm. The number of IR700 per antibody was calculated to be four. The quality of APC was evaluated with SDS-PAGE with a 4-20% gradient polyacrylamide gel (Life Technologies, Gaithersburg, MD, USA). Non-conjugated antibody and ultrapure water were used for controls. After electrophoresis at 80 V for 2.5 h, the gel was observed with a Pearl Imager (LI-COR Biosciences) using the 700-nm fluorescence channel. The gel was then stained with Colloidal Blue to compare the molecular weight of the conjugate to that of non-conjugated antibody. The APC was also evaluated with SEC. SEC analysis was performed on a Nexera XR UHPLC system (Shimadzu Co., Kyoto, Japan). Approximately 10 μg of protein was loaded onto a TSKgel SuperSW 3000 (4.6 mm × 30 cm, 5 μm) column (Tosoh Bioscience, Inc., South San Francisco, CA, USA) and eluted using an isocratic flow (37 min, 0.25 mL min⁻¹) of 200 mM sodium phosphate with 10% of acetonitrile at pH 6.8. The absorption of elute was monitored at a wavelength of 280 and 689 nm. The fluorescence of elute was also monitored at an excitation wavelength of 689 nm and the emission wavelength of 700 nm.

Cell culture

Luciferase expressing murine cancer cell lines, MC38-luc (colon cancer), LL/2-luc (lung cancer), MOC2-luc (oral cancer) were used for this study. Parental MOC2 cells were also used. MC38 was a kind gift from Dr. Claudia Palena, NCI, and luciferase was transduced with RediFect Red-Fluc lentivirus (PerkinElmer, Waltham, MA, USA). LL/2-luc was purchased from Imanis Life Sciences (Rochester, MN, USA). MOC2 was purchased from Kerafast (Boston, MA, USA) and luciferase was transduced as same as MC38-luc. MC38-luc and LL/2-luc cells were cultured in RPMI1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin (Thermo Fisher Scientific). MOC2 and MOC2-luc cells were cultured in the mixture of IMDM medium and Ham’s Nutrient Mixture F12 Media (at a ratio of 2:1, GE Health Healthcare Life Sciences) supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, 5 ng mL⁻¹ insulin (MilliporeSigma, Burlington, MA, USA), 40 ng mL⁻¹ hydrocortisone (MilliporeSigma), and 3.5 ng mL⁻¹ human recombinant EGF (MilliporeSigma). All cells were cultured in a humidified incubator at 37°C in an atmosphere of 95% air and 5% CO₂.

CTLA4 expression on cancer cell lines in vitro

Anti-CTLA4 (9D9) or an isotype control (murine IgG2b) was conjugated with Alexa Fluor 647 NHS Ester (Thermo Fisher Scientific). The conjugation was performed with the same method as that used in IR700 synthesis. We abbreviate the Alexa647-conjugated anti-CTLA4 and Alexa647-conjugated isotype control as anti-CTLA4-Alexa647 and control-Alexa647, respectively. One million cells of each cancer cell line were incubated with the anti-CTLA4-Alexa647 or control-Alexa647 and LIVE/DEAD Fixable Dead Cell Stain (Thermo Fisher Scientific) for 1 hour at 4°C. The fluorescence of the cells was then analyzed with a flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA, USA) and FlowJo software (FlowJo LLC, Ashland, OR, USA).

In vitro NIR-PIT against cancer cell lines

Two hundred thousand cells of each cancer cells were seeded into each corner well of 12-well plates. After one day, the cells were incubated with 10 μg mL^-1 of anti-CTLA4-IR700 for 1 hour at 37°C. After washing with PBS, phenol-red-free medium was added. The cells were then exposed to NIR light (690 nm, 150 mW cm⁻², 50 J cm⁻²) with an ML7710 laser system (Modulight, Tampere, Finland). One hour after the light exposure, the cells were collected with 0.05% trypsin-added PBS and stained with 1 μg mL⁻¹ PI. The percentage of PI-stained cells was analyzed with flow cytometry.

Animals and tumor models

All in vivo procedures were conducted in compliance with the Guide for the Care and Use of Laboratory Animal Resources (1996), US National Research Council, and approved by the local Animal Care and Use Committee. Six- to eight-week-old female C57BL/6 mice and homozygote athymic nude mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and Charles River Laboratories (Wilmington, MA, USA), respectively. MC38-luc (1 × 10⁶), LL/2-luc (5 × 10⁵), MOC2-luc (1 × 10⁶), or MOC2 (5 × 10⁵) cells were inoculated into the right or both side of the dorsum. The hair overlying the tumor site was removed before NIR-PIT and imaging studies for C57BL/6 mice. Tumor volume was calculated from the greatest longitudinal diameter (length) and the greatest transverse diameter (width) as length × width² × 0.5. Tumor volumes were measured three times a week until the volume reached 2000 mm³, whereupon the mice were euthanized with CO₂.

In vivo Fluorescence Imaging

Anti-CTLA4-IR700 (50 μg) was injected via lateral tail vein 6 days after inoculation of the tumor. Serial dorsal fluorescence images were taken with the 700 nm fluorescence channel of a Pearl Imager (LI-COR Bioscience). The images were obtained before and 1, 3, 6, 9, 12, 24, 48, 72, and 96 hours after the APC administration. The images were analyzed with Pearl Cam Software (LI-COR Bioscience). Regions of interest (ROIs) were drawn on the tumor and the non-tumoral region of the contralateral side. TBR was calculated as (Mean fluorescence intensity of the tumor)/(Mean fluorescence intensity of the non-tumoral region of the contralateral side).

Ex vivo NIR-PIT

Spleens were extracted from non-tumor bearing mice and single cell suspensions were prepared by gently squeezing the cells out by pushing the spleen using the back of the plunger of a syringe. Red blood cells (RBCs) were removed by incubating with RBC lysis buffer (BioLegend, San Diego, CA, USA). Ten million splenocytes were incubated with 10 μg mL⁻¹ of anti-CTLA4-IR700 for 2 hours at 37°C. After washing the cells with PBS, NIR light (690 nm, 150 mW cm⁻²) was applied at 0, 25, or 50 J cm⁻². After 3 hours, the percentage of each live T cell population was analyzed with a flow cytometer after staining with LIVE/DEAD Fixable Dead Cell Stain (Thermo Fisher Scientific).

In vivo NIR-PIT

Tumor-bearing mice were randomized into 3 groups as follows: (i) no treatment (control), (ii) intravenous administration of anti-CTLA4-IR700 (50 μg) without NIR light exposure (APC-IV), and (iii) intravenous administration of anti-CTLA4-IR700 (50 μg) followed by NIR light exposure (NIR-PIT). The APC was injected 6 and 5 days after inoculation of cancer cells into C57BL/6 and athymic nude mice, respectively. NIR light (690 nm, 150 mW cm⁻², 50 J cm⁻²) was exposed on the next day. Upon NIR light exposure, a piece of aluminum foil with a hole of approximately half inch diameter was placed over the mouse and NIR light was irradiated onto the tumor through the hole to ensure that the NIR light exposure is limited in tumor site. Acute treatment efficacy was evaluated with BLI analysis, in which D-luciferin (15 mg mL⁻¹ for MC38-luc and MOC2-luc, 3 mg mL⁻¹ for LL/2-luc, 200 μL; Gold Biotechnology, St. Louis, MO, USA) was injected intraperitoneally and luciferase activity was analyzed with a Photon Imager and M3 Vision Software (Biospace Lab, Paris, France). ROIs were drawn to include the entire tumor and the counts per minute of relative light units were calculated. BLI images were continually recorded until the onset of depilation-induced skin pigmentation precluded accurate measurement.

Flow-cytometric analysis of intra-tumoral cells, lymph node cells, and splenocytes

To evaluate the CTLA4 expression of intra-tumoral cells, established untreated tumors were harvested at an approximate volume of 200 mm³. To assess the depletion of the targeted cells after NIR-PIT, tumors were harvested 3 hours after NIR light exposure. Tumor draining lymph nodes and spleens were also analyzed to evaluate for systemic effects. To assess the immune reaction in the regional lymph nodes, an ipsilateral inguinal lymph node was harvested 3 days after NIR-PIT. The tumors were digested with collagenase type IV (1 mg mL⁻¹, Thermo Fisher Scientific) and DNase I (20 μg mL⁻¹, MilliporeSigma), then dissociated and filtered with 70 μm cell strainer (Corning, Corning, NY, USA). The cells were stained with antibodies, purchased from either BioLegend [anti-CD3e (145-2C11), anti-CD8α (53-6.7), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD25 (PC61.5), anti-F4/80 (BM8), anti-CD31 (390), anti-CD45 (30-F11), and anti-I-A/I-E (M5/114.15.2)] or from Thermo Fisher Scientific [anti-CD4 (RM4-5), anti-CD19 (1D3), anti-CD69 (H1.2F3), anti- Gr1 (RB6-8C5), anti-NK1.1 (136), and anti-CTLA4 (UC10-4B9)]. To distinguish live from dead cells, cells were also stained with LIVE/DEAD Fixable Dead Cell Stain (Thermo Fisher Scientific). For the staining of Foxp3, the cells were fixed and permeabilized with Foxp3 Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) followed by the incubation with anti-Foxp3 (FJK-16s; Thermo Fisher Scientific). To assess surface and total CTLA4 expression, the cells were stained with anti-CTLA4-Alexa647/control-Alexa647 before and after the permeabilization, respectively. For comparison, relative fluorescence intensity (RFI) was calculated from mean fluorescence intensity (MFI) as (MFI of anti-CTLA4-Alexa647)/(MFI of control-Alexa647). To evaluate IFN-γ expression, tumors were put in Brefeldin A (BioLegend) added medium immediately after the harvest, cut in about 1 mm³ pieces, and incubated for 4 hours at 37°C. Then, single cell suspension was made and the cells were stained with surface markers, fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher Scientific), and stained with anti-IFN-γ (XMG1.2; Thermo Fisher Scientific).

Histological analysis

Tumors were harvested 1 hour after NIR light exposure. Extracted tumors were fixed with 10% formalin, embedded in paraffin, thinly sliced and stained with hematoxylin and eosin (H-E) staining.

Multicolor immunofluorescence staining

Multicolor immunofluorescence staining was performed to analyze the tumor-infiltrating lymphocytes (TILs) using Opal 7-color Automation IHC Kit (Akoya Bioscience, Menlo Park, CA, USA) and Bond RXm auto stainer (Leica Biosystems, Wetzlar, Germany). The staining was performed according to the Opal 7 color protocol of the manufacturer with the following modification: (i) antigen retrieval was performed with BOND ER2 solution (Leica Biosystems) for 20 minutes and (ii) the ImmPRESS HRP anti-Rabbit (Peroxidase) Polymer Detection Kit (Vector Laboratories, Burlingame, CA, USA) was used instead of anti-mouse/human secondary antibody. The sections were stained with DAPI and the following antibodies: anti-CD8 (EPR20305; Abcam, Cambridge, UK; 1:500 dilution), anti-CD4 (EPR 19514; Abcam; 1:1,000 dilution), anti-Foxp3 (1054C; Novus Biologicals, Centennial, CO, USA; 1:1,000 dilution), anti-pan cytokeratin (rabbit poly; Bioss Antibodies, Woburn, MA, USA; 1:250 dilution), and anti-Granzyme B (rabbit poly; Abcam; 1:1,000 dilution). Stained slides were mounted with VECTASHIELD Hardest Antifade Mounting Medium (Vector Laboratories) and imaged with Mantra Quantitative Pathology Workstation (Akoya Biosystems). The obtained images were analyzed with inForm Tissue Finder software (Akoya Biosystems). Tissue area was divided into “Stroma” and “Tumor” based on the expression of pan cytokeratin. Cells were classified into CD8⁺ cells, CD4⁺Foxp3⁻ cells, CD4⁺Foxp3⁺ cells, and other cells. Five pictures were obtained for each specimen, and tissue area and cell count were summed for each tissue category. Cell density was calculated as cell counts per square millimeter.

In vivo blood perfusion analysis

Bovine albumin (Thermo Fisher) was conjugated with IRDye 800CW NHS ester (IR800; LI-COR Biosciences) using the same methods used for IR700. We abbreviate the conjugate as albumin-IR800. One day after light exposure, 50 μg of albumin-IR800 in 200 μL of PBS was intravenously injected. Serial dorsal 800-nm fluorescence images were obtained with the 800-nm channel of a Pearl Imager. The images were taken before and 5, 10, 15 minutes after the injection. TBR was calculated with the same way of in vivo fluorescence study for IR700.

Statistical analysis

Data are expressed as means ± SEM. Statistical analysis was performed with GraphPad Prism (GraphPad Software, La Jolla, CA, USA). Samples size (n) for each expriment is discribed in each figure legend. For one-time measurement, a two-tailed unpaired t test (two groups) or a one-way analysis of variance (ANOVA) followed by Tukey’s test (three or more groups) was used. For comparison of luciferase activity and tumor volumes, a repeated measures two-way ANOVA followed by Tukey’s test was used. The cumulative probability of survival based on tumor volume (2000 mm³) was estimated with the Kaplan-Meier survival curve analysis, and the results were compared with log-rank test with Bonferroni correction. p-value of less than 0.05 was considered significant.