Systemic Antitumor Immunity by PD-1/PD-L1 Inhibition Is Potentiated by Vascular-Targeted Photodynamic Therapy of Primary Tumors

Abstract

Purpose: PD-1/PD-L1 pathway inhibition is effective against advanced renal cell carcinoma, although results are variable and may depend on host factors, including the tumor microenvironment. Vascular-targeted photodynamic (VTP) therapy with the photosensitizer WST11 induces a defined local immune response, and we sought to determine whether this could potentiate the local and systemic antitumor response to PD-1 pathway inhibition.Experimental Design: Using an orthotopic Renca murine model of renal cell carcinoma that develops lung metastases, we treated primary renal tumors with either VTP alone, PD-1/PD-L1 antagonistic antibodies alone, or a combination of VTP and antibodies and then examined treatment responses, including immune infiltration in primary and metastatic sites. Modulation of PD-L1 expression by VTP in human xenograft tumors was also assessed.Results: Treatment of renal tumors with VTP in combination with systemic PD-1/PD-L1 pathway inhibition, but neither treatment alone, resulted in regression of primary tumors, prevented growth of lung metastases, and prolonged survival in a preclinical mouse model. Analysis of tumor-infiltrating lymphocytes revealed that treatment effect was associated with increased CD8⁺:regulatory T cell (Treg) and CD4⁺FoxP3-:Treg ratios in primary renal tumors and increased T-cell infiltration in sites of lung metastasis. Furthermore, PD-L1 expression is induced following VTP treatment of human renal cell carcinoma xenografts.Conclusions: Our results demonstrate a role for local immune modulation with VTP in combination with PD-1/PD-L1 pathway inhibition for generation of potent local and systemic antitumor responses. This combined modality strategy may be an effective therapy in cancers resistant to PD-1/PD-L1 pathway inhibition alone. Clin Cancer Res; 24(3); 592-9. ©2017 AACR.

Figure 1.. Renca cells show increased PD-L1 expression following culture with IFN-γ.

Renca cells upregulate PD-L1 expression after in vitro culture with IFN-γ for 8 hours as assessed by flow cytometry. Unstained= filled, control= solid line, IFN-γ co-cultured=dashed line.

Figure 2.. VTP and PD-1/PD-L1 blockade synergize to reject orthotopic renal tumors and prolong survival.

(a) Treatment scheme. VTP, vascular-targeted photodynamic therapy; IP, intraperitoneal. Renal tumor growth was assessed on day 21 by (b) kidney weight or (c) maximal cross-sectional tumor area; mean ± SEM is shown, data represent results from 2 pooled experiments, n= 15–21 per group, p<0.001 (Kruskal-Wallis). Abs= anti-PD1 + anti-PD-L1 antibodies. (d) Kidneys were harvested on day 21 and stained with H&E and analyzed by light microscopy at 2x and 20x. * represents necrosis and ** represents viable tumor with delineation marked by dotted line. Scale bars = 200 mM. (e) In a separate experiment, mice were treated per the schema in (a) and sacrificed upon development of a large necrotic tumor, loss of ambulation, or labored respiration (n=20 per group), and median survival time post-VTP treatment was compared (Kaplan-Meier).

Figure 3.. Combination therapy with VTP and PD-1/PD-L1 blockade decreases the proportion of regulatory T cells in treated tumors.

Animals were treated with combination VTP and PD-1/PD-L1 blockade per schema in 2a. Kidneys were harvested 1, 3, or 5 days after VTP treatment and analyzed by flow cytometry for infiltrating immune cells. Day 0 indicates untreated Renca tumors. (a) Representative flow cytometry plots of tumor-infiltrating Tconv (CD4+FoxP3−) and CD8+ and Treg (CD4+FoxP3+) cells from a gated CD45+ cell population. (b) Ratios of CD8+:Treg (p=0.15) and (c) Tconv:Treg cells (p=0.07) were calculated. 3 to 5 animals per group were analyzed per timepoint. Mean ± SEM is shown and means were compared using Kruskal-Wallis test.

Figure 4.. VTP of the primary renal tumor plus systemic PD-1/PD-L1 blockade prevents growth of distant lung tumors and increases T cell infiltration in distant tumors.

Animals were treated per schema in Figure 2a. Lungs were harvested on day 21 and analyzed for lung nodules by staining with (a) Fekete’s solution or (b) H&E staining or (c) CD3 immunohistochemistry and analyzed by light microscopy. (d) Absolute number of lung lesions per animal were determined; mean ± SEM is shown. Data represent results from 2 pooled experiments, n= 15–23 per group, **P=0.025. (e) Absolute number of lung lesions from animals not treated with VTP were determined; mean ± SEM is shown, n=9 per group, P=0.8. (f) Lungs were harvested on day 21 and CD3+ cell infiltration was assessed in tumor nodules by immunohistochemistry as in 4c. Percent of tumor-infiltrating CD3+ cells per field (tumor nodules assessed only); mean ± SEM is shown, n=5 per group, p=0.136 (g to i) In a separate group of animals, lungs were harvested 3 days after VTP treatment and analyzed by flow cytometry for infiltrating immune cells (n=5 group). (g) Absolute numbers of CD45+, CD8+, Tconv, and Treg cells per gram of lung. (h) Ratios of CD8+/Treg and Tconv/Treg cells were calculated. (i) Ki-67 expression on tumor-infiltrating CD8+ and Tconv cells was assessed by flow cytometry. Mean ± SEM is shown.

Figure 5.. VTP induces PD-L1 expression in human RCC xenograft tumors.

(a) A-498 and (b) 786-O xenograft flank tumors were harvested prior to VTP (0 hr) or treated with VTP then harvested 5, 24, or 72 hours after treatment and stained with H&E or for PD-L1 by immunohistochemistry. The ratio of PD-L1 positive cells was assessed and expressed as ratio of viable cells per unit area for (c) A-498 tumors and (d) 786-O tumors (n=3–5 per group; p=0.3 and p=0.11, respectively).