Characterization of the Immune Exhaustion Phenotype in Murine Bladder Cancer Following Radiation

Immunotherapy for bladder cancer has progressed from Bacillus Calmette-Guerin for early stage bladder cancer to checkpoint inhibitors for metastatic disease. While current efforts combine stereotactic ablative radiation (SAR) with immunotherapy to enhance anti-tumor immunity, recent work suggests that SAR treatment of parenchymal tumors increases TIM3+ NK cells in the peripheral blood of patients. To determine if SAR alters TIM-3 in the tumor microenvironment, we developed a mouse model of muscle invasive bladder cancer to characterize the immune exhaustion phenotype after SAR. We hypothesized that ablative doses of radiation would induce the expression of checkpoint inhibitory molecules such as TIM-3 and its ligand Galectin-9 in the tumor microenvironment. Muscle-invasive bladder cancer was induced in Tg(CAG-luc-eGFP) FVB/NJ mice by treatment with N-Butyl-N-(4-hydroxybutyl) nitrosamine (OHBBN) for 14-16 weeks. Primary bladder tumors were surgically resected and subcutaneously transplanted to wildtype FVB/NJ mice to establish mouse derived allografts (MDAs). Harvested MDAs were cultured in vitro for 48 hours and irradiated with 0, 2, 12, or 20 Gy via a small animal radiation research platform (SARRP). Irradiated cells were cultured for 24 or 72 hours and then harvested for RNA extraction and cDNA synthesis. Ifnb1, Cxcl10, CD274, Lgals9, and Havcr2 were analyzed using RT-PCR with Gapdh endogenous control. The relative expression of each gene was calculated using the delta-delta CT method relative to 0 Gy. Cells were stained with 7-AAD, anti-CD45, and anti-TIM-3 fluorophore-conjugated antibodies and analyzed using flow cytometry. Student’s t-test was used to determine statistical significance with p<0.05. Irradiating urothelial cell carcinoma with 12 Gy induces a 4-fold increase in Ifnb1 and a 2-fold increase in Cxcl10 expression at 24 hours. It also induces a 1.5-fold increase in CD274 (PD-L1), and a 2-fold increase in both Havcr2 (TIM-3) and Lgals9 (Galectin 9) expression. TIM-3 protein expression remains elevated at 72 hours as assessed by flow cytometry. None of these changes are seen after 2 Gy or 20 Gy. Ongoing experiments involve irradiation of orthotopic bladder tumors in vivo to study the temporal and spatial aspects of immune exhaustion after SAR. Irradiating murine urothelial cell carcinoma with 12 Gy induces the intratumoral expression of CXCL10, a chemokine known to recruit monocytes, NK cells and T cells. In addition, 12 Gy increases expression of checkpoint inhibitory molecules such as PD-L1, TIM-3, and its ligand Galectin-9. These findings suggest that ablative doses of radiation may modulate the immune exhaustion phenotype of bladder cancer, and highlight the need to investigate options for ablating the Galectin 9-TIM-3 pathway in conjunction with SAR.