Radiation and Cancer Biology

SS 43 - Biology 8 - Radiation and the Immune Response Session II

318 - Characterization of the Immune Exhaustion Phenotype In Murine Bladder Cancer Following Radiation

Wednesday, October 24
3:55 PM - 4:05 PM
Location: Room 004

Characterization of the Immune Exhaustion Phenotype In Murine Bladder Cancer Following Radiation
H. M. McGee1,2, H. Anastos2,3, M. Galsky2, J. Sfakianos3, M. E. Daly4, A. M. Monjazeb4, R. G. Stock1, M. Buckstein1, and D. Mulholland2; 1Department of Radiation Oncology, Icahn School of Medicine at Mount Sinai, New York, NY, 2Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 3Department of Urology, Icahn School of Medicine at Mount Sinai, New York, NY, 4Department of Radiation Oncology, University of California Davis Comprehensive Cancer Center, Sacramento, CA

Purpose/Objective(s): 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.

Materials/Methods: 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.

Results: 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.

Conclusion: 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.

Author Disclosure: H.M. McGee: Honoraria; Society for Immunotherapy of Cancer Workshop. Advisory Board; AstraZeneca Immuno-Oncology Advistory Board. Travel Expenses; Society for Immunotherapy of Cancer "Sparkathon". Committee Member; ASTRO Science Council. H. Anastos: None. M. Galsky: Research Grant; Prostate Cancer Foundation. J. Sfakianos: None. M.E. Daly: Research Grant; NIH, Department of Defense, American Cancer Society. Associate Editor; IJROBP, Practical Radiation Oncology. A.M. Monjazeb: Research Grant; Genentech, Transgene, Incyte, Merck. R.G. Stock: Honoraria; BARD. D. Mulholland: None.

Heather McGee, MD, PhD

Mount Sinai Dept of Radiation Oncology

Disclosure:
Employment
MOUNT SINAI RADIATION ONCOLOGY: RESIDENT: Employee

Compensation
AstraZeneca Immuno-Oncology Advistory Board: Advisory Board; Society for Immunotherapy of Cancer "Sparkathon": Travel Expenses; Society for Immunotherapy of Cancer Workshop: Honoraria

Leadership
ASTRO Science Council: Committee Member

Biography:
I am currently one of the chief residents in Radiation Oncology at Icahn School of Medicine at Mount Sinai. My research is focused on the immune response to radiation, and how it differs based on the site irradiated. My clinical interests are in thoracic malignancies, gastrointestinal malignancies, bladder cancer and sarcoma.

I was raised in Davis, California and attended the University of California, Berkeley, where I studied Biochemistry and Molecular Biology. After graduation, I moved to England and received a M. Phil in Immunology at the University of Cambridge. I then completed my MD/PhD in Immunobiology at Yale University, where I worked with Dr. Richard Flavell to study the role of the cytokine Interleukin-22 in wound healing and tissue repair. I was an intern in general surgery at University of California, San Francisco. During radiation oncology residency, I served on the planning committee for the ASTRO/SITC/NCI Science Workshop on “Incorporating Radiation Oncology into Immunotherapy.” Additionally, I was a member of the first annual SITC “Sparkathon” for Young Investigators in Immunotherapy. I am currently performing basic science research on how radiation to different organs leads to unique changes in the tumor immune microenvironment in a murine model of urothelial cell carcinoma, and studying changes in the TCR repertoire and immune exhaustion phenotype after SBRT for HCC in a translational study. I am looking forward to pursuing a career as a physician-scientist. In my free time, I enjoy spending time with my fiancé, doing yoga and running.

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