Article Figures & Data
Figures
- Figure 1.
Confirmation of CD44 expression as a target for NIR-PIT in MC38-luc cells and evaluation of in vitro NIR-PIT. A, Validation of CD44-IR700 by SDS-PAGE (left: Colloidal Blue staining; right: IR700 fluorescence). Diluted anti-CD44 was used as a control. B, Absorbance curve of CD44–IR700. C, Expression of cell-surface CD44 in MC38-luc cells was examined with flow cytometry. CD44-blocking antibody was added to some wells to validate specific staining. Representative histograms were shown. D, Differential interference contrast (DIC) and fluorescence microscopy images of MC38-luc cells. Change in MC38-luc cellular architecture following 15 minutes of NIR light exposure shown. Scale bars, 20 μm. E, Bioluminescence imaging (BLI) demonstrating luciferase activity in MC38-luc cells following NIR light. F, Quantification of MC38-luc luciferase activity after labeling with CD44-IR700 and treatment with NIR light (n = 5; **, P < 0.01 vs. untreated control, Student t test). G, Membrane permeability of MC38-luc cells, as measured by PI staining, after labeling with CD44–IR700 and treatment with NIR light (n = 5; *, P < 0.05 vs. untreated control; **, P < 0.01 vs. untreated control, Student t test). Each value represents mean ± SEM of five independent experiments.
- Figure 2.
In vivo effect of NIR-PIT and PD-1 mAb in mice bearing unilateral MC38-luc tumors. A, NIR-PIT regimen. Bioluminescence and fluorescence images were obtained at each time point as indicated. B, Light exposure. NIR light was administered to the unilateral right-sided tumor. C, In vivo IR700 fluorescence real-time imaging of tumor-bearing mice in response to NIR-PIT (n ≥ 10). D, In vivo BLI of tumor-bearing mice in response to NIR-PIT. Mice in the PD-1 mAb group also received CD44–IR700 but were not treated with NIR. E, Quantification of luciferase activity in four treatment groups (n ≥ 10; **, P < 0.01 vs. control, Tukey test with ANOVA; #, P < 0.05 vs. PD-1 mAb and NIR-PIT groups, Tukey test with ANOVA). F, Resected tumors (day 10) were stained with H&E and assessed for necrosis and leukocyte infiltration. White scale bars, 100 μm; black scale bars, 20 μm. G, Tumor growth curves (n ≥ 10, **, P < 0.01 vs. control, Tukey test with ANOVA; ##, P < 0.01 vs. PD-1 mAb and NIR-PIT groups, Tukey test with ANOVA). H, Kaplan–Meier survival analysis following NIR-PIT treatment with and without PD-1 mAb (**, P < 0.01 vs. control, Log-rank test; ##, P < 0.01 vs. PD-1 mAb and NIR-PIT groups, log-rank test).
- Figure 3.
Immune correlative and functional effects of NIR-PIT and PD-1 mAb in mice bearing a unilateral MC38-luc tumors. A, MC38-luc tumors (day 10, n = 5/group) treated with NIR-PIT with and without PD-1 mAb and controls were harvested, digested into single-cell suspensions, and analyzed for TIL via flow cytometry. Presented as absolute number of infiltrating cells per 1.5 × 104 live cells analyzed. PD-1 expression shown as inset (MFI). *, P < 0.05; **, P < 0.01; ***, P < 0.001, t test with ANOVA. Representative data from one of two independent experiments shown. B, Multiplex immunofluorescence was used to validate flow-cytometric data. Representative 400 × images shown. Quantification of infiltrating TILs from 5 high power fields (HPF) per tumor, n = 3/group. **, P < 0.01; ***, P < 0.001, t test with ANOVA. C, TILs were isolated from tumors treated as above (n = 5/group) via an IL2 gradient, enriched via negative magnetic selection, and stimulated with irradiated splenocytes pulsed with peptides representing known MHC class I–restricted epitopes from selected TAAs. IFNγ production was determined by ELISA from supernatants collected 24 hours after stimulation. Supernatants from splenocytes (APC) alone, TILs (T) alone, and an MHC class I–restricted epitope from ovalbumin (OVA, SIINFEKL) were used as controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001, t test with ANOVA. D, Flow-cytometric analysis of tumor-infiltrating DCs and macrophages, with quantification of macrophage polarization based on MHC class II expression. **, P < 0.01; ***, P < 0.001, t test with ANOVA. E, Flow-cytometric analysis of tumor-infiltrating neutrophilic-myeloid cells (PMN-myeloid) and regulatory T cells (Tregs). *, P < 0.05; **, P < 0.01, t test with ANOVA. F, Flow-cytometric analysis of PD-L1 expression on CD45.2–CD31–PDGFR– tumor cells and CD45.2+CD31– immune cells. **, P < 0.01 compared with control, t test with ANOVA. N = 5/group.
- Figure 4.
In vivo effect of NIR-PIT and PD-1 mAb in mice bearing bilateral MC38-luc tumors. A, NIR-PIT regimen. Bioluminescence and fluorescence images were obtained at each time point as indicated. B, Light exposure. NIR light was administered to the right-side tumor only in mice bearing bilateral lower flank tumors. The untreated left-side tumor was shielded from NIR light. C, In vivo IR700 fluorescence real-time imaging of tumor-bearing mice in response to NIR-PIT to the right-side tumor only. D, In vivo BLI of tumor-bearing mice in response to combination NIR-PIT and PD-1 mAb. E, Quantification of luciferase activity from each tumor in controls and mice treated with combination NIR-PIT and PD-1 mAb (n = 10, **, P < 0.01, Tukey test with ANOVA). F, Resected tumors (day 10) were stained with H&E and assessed for necrosis and leukocyte infiltration. White scale bars, 100 μm; black scale bars, 20 μm. G, Growth curves of right- and left-side tumors from controls and mice treated with combination NIR-PIT and PD-1 mAb. H, Kaplan–Meier survival analysis from controls and mice treated with combination NIR-PIT and PD-1 mAb (n = 10, **, P < 0.01, Tukey test with ANOVA for growth curves; **, P < 0.01, log-rank test for survival).
- Figure 5.
Immune correlative and functional effects of NIR-PIT and PD-1 mAb in mice bearing bilateral MC38-luc tumors. A, Bilateral MC38-luc tumors (day 10, n = 5/group) treated with PD-1 mAb with or without NIR-PIT and bilateral control tumors were harvested, digested into single-cell suspensions, and analyzed for TIL via flow cytometry. Presented as absolute number of infiltrating cells per 1.5 × 104 live cells analyzed. PD-1 expression shown as inset (MFI). *, P < 0.05; ***, P < 0.001, t test with ANOVA. Representative data from one of two independent experiments shown. B, TILs were extracted from tumors treated as above (n = 5/group) via an IL2 gradient, enriched via negative magnetic selection, and stimulated with irradiated splenocytes pulsed with peptides representing known MHC class I–restricted epitopes from selected TAAs. IFNγ production was determined by ELISA from supernatants collected 24 hours after stimulation. Supernatants from splenocytes (APC) alone, TILs (T) alone, and an MHC class I–restricted epitope from ovalbumin (OVA, SIINFEKL) were used as controls. *, P < 0.05; ***, P < 0.001, t test with ANOVA. C, Flow-cytometric analysis of tumor-infiltrating DCs and macrophages, with quantification of macrophage polarization based on MHC class II expression. **, P < 0.01; ***, P < 0.001, t test with ANOVA. D, Flow-cytometric analysis of tumor-infiltrating PMN-myeloid and Tregs. *, P < 0.05; **, P < 0.01, t test with ANOVA. E, Flow-cytometric analysis of PD-L1 expression on CD45.2–CD31–PDGFR– tumor cells. N = 5/group.
- Figure 6.
In vivo effect of NIR-PIT and PD-1 mAb in mice bearing multiple MC38-luc tumors. A, NIR-PIT regimen. Bioluminescence and fluorescence images were obtained at each time point as indicated. B, Light exposure. NIR light was administered to the caudal right-side tumor only in mice bearing four tumors. All other tumors were shielded from NIR light. C, In vivo IR700 fluorescence real-time imaging of tumor-bearing mice in response to NIR-PIT treatment to the caudal right-side tumor only. D, In vivo BLI of tumor-bearing mice in response to NIR-PIT treatment of the caudal right-side tumor only. E, Quantification of luciferase activity in all tumors from controls and mice treated with combination NIR-PIT and PD-1 mAb. Only the caudal right-side tumor received NIR-PIT treatment (n ≥ 10; **, P < 0.01, Tukey test with ANOVA). F, Resected tumors (day 10) were stained with H&E and assessed for necrosis and leukocyte infiltration. White scale bars, 100 μm; black scale bars, 20 μm. G, Growth curves from controls and treated and untreated tumors from mice receiving combination NIR-PIT and PD-1 mAb. H, Kaplan–Meier survival analysis (n ≥ 10; **, P < 0.01, Tukey test with ANOVA for growth curves; **, P < 0.01, log-rank test for survival).
- Figure 7.
Proposed mechanism of combination treatment with local NIR-PIT and systemic PD-1 mAb. Following NIR-PIT cellular damage, tumor cells undergo ICD with release of innate immune ligands, such as calreticulin (CRT), ATP, and high mobility group box 1 (HMGB1), along with multiple tumor antigens. Following processing and presentation of these antigens by matured APCs such as DCs, a systemic polyclonal T-cell response develops. However, PD-1 expression on activated T cells and PD-L1 expression within the tumor microenvironment leads to adaptive immune resistance and dysfunctional T cells. PD-1 ICB reverses this adaptive immune resistance, resulting in activation of TILs specific for multiple tumor antigens at both primary and distant (abscopal) sites of disease, complete tumor rejection, and immunologic memory.
Additional Files
Supplementary Data
- Video 1 - Supplementary Video 1. NIR-PIT effect for MC38-luc cells. Immediately after exposure to excitation light, cellular swelling, bleb formation, and rupture of vesicles representing necrotic cell death were observed.
- Video 2 - Supplementary Video 2. NIR-PIT effect for LLC cells. Immediately after exposure to excitation light, cellular swelling, bleb formation, and rupture of vesicles representing necrotic cell death were observed.
- Video 3 - Supplementary Video 3. NIR-PIT effect for MOC1 cells. Immediately after exposure to excitation light, cellular swelling, bleb formation, and rupture of vesicles representing necrotic cell death were observed.
- Supplementary Figures S1 - S9 - Supplementary Figures S1 - S9, with legends and supplementary video captions.
- Supplementary Table S1 - qPCR primers.
Volume 7, Issue 3
