Fungal Depletion Bolsters Anti-Tumor Immune Response Elicited by Anti-PD1 Alone and in Combination with Radiation Therapy

Abstract

Pembrolizumab in combination with chemotherapy has become the standard of care treatment for both metastatic and early-stage triple negative breast cancer (TNBC). Clinical trials are currently underway investigating the use of pembrolizumab with radiation in the neoadjuvant setting in early TNBC. Several groups have described a link between the microbiome and the efficacy of chemotherapy and anti-PD1 immune checkpoint inhibitors (ICIs) in preclinical models. Recent work from our lab has shown that targeting commensal fungi in the microbiome enhances the radiation induced antitumor immune response. Therefore, we hypothesized that fungal depletion might positively impact anti-PD1 therapy and combination treatment with anti-PD1 and radiation therapy (RT). This study utilized an orthotopic syngeneic breast tumor model in which the syngeneic cell line E0771 was injected into the mammary fat pad of female C57BL/6 mice. Tumor-bearing mice were then treated with and without the antifungal fluconazole, anti-PD1, and radiation (16 Gy single fraction) using the X-RAD SmART platform with CT guidance. Tumor volumes were compared using 2-way ANOVA and survival curves analyzed using log rank. In a separate set of experiments, tumor-infiltrating immune cells were isolated and analyzed by high-dimensional multiplex flow cytometry. We found that fungal depletion with fluconazole prior to treatment with anti-PD1 reduced the tumor volume and significantly improved survival in comparison to those treated with anti-PD1 alone (P = 0.0016). To identify what changes in the tumor immune microenvironment is driving this increased anti-tumor response, we performed flow cytometry on immune cells isolated from the tumors. We found that the use of fluconazole prior to anti-PD1 treatment reduced the proportion of CD11b+F4/80+ tumor-associated macrophages (TAMs) (P = 0.01) and increased tumor infiltrating cytotoxic T cell population (P = 0.04) when compared with the use of anti-PD1 alone. We also evaluated the effect of fungal depletion on combination therapy with RT and anti-PD1. Strikingly, we found that mice depleted of fungi with fluconazole prior to radiation and anti-PD1 therapy, have decreased tumor burden and significantly increased survival when compared to their fungally-intact counterparts (P = 0.043). Our data indicates that the depletion of the gut fungal populations induces an increased antitumor response following anti-PD1 alone and in combination with radiation. This increased antitumor immune response is associated with an increase in the cytotoxic CD8+ T cell compartment with concomitant decrease in immunosuppressive tumor associated macrophages.