Abstract B18: Breast cancer cryoablation in combination with anti-CTLA-4 increases T cell activation in a murine tumor model

Introduction: Triple-negative and HER2+ breast cancers (BCs) are high-risk subtypes that spread easily and are hard to treat. Thus, newer therapeutic approaches that can prevent local recurrence and metastatic spread of disease are required. Cryoablation, a technique that kills tumor cells through rapid freeze/thaw cycles, preserving tumor-associated antigens, is approved to treat small low-risk breast tumors but has not been as successful for high-risk BCs. A promising area of BC cryoablation research is its combinational use with immune checkpoint inhibitors (ICIs) to enhance the anti-tumor immune response and to generate distant tumor cell targeting – the abscopal effect. Using a murine model of high-risk metastatic BC, we investigated cryoablation in conjunction with anti-CTLA4 and anti-PD-L1.Methods: BALB/c mice were bilaterally transplanted in the mammary fat pad with 4T1-12b-luciferase-expressing metastatic BC cells. Two weeks after transplant, all mice had their left tumors cryoablated; 24 hours pre- and post-cryoablation, mice received an intraperitoneal injection containing PBS (control, n=5) or 100 µg of either anti-CTLA4 (n=5) or anti-PD-L1 (n=5). Right tumors were not manipulated and represented distant metastatic tumors to examine the immune abscopal effect. Mice were sacrificed one-week post-cryoablation; cryoablated (left) and abscopal (right) tumors, peripheral blood and spleen were collected and processed to obtain isolated cells for flow cytometry analysis of immune cell populations.Results: In vivo fluorescence imaging and mouse necropsies revealed cryoablated tumors undergoing necrosis. A trend of reduced tumor weights was observed in abscopal tumors of ICI-treated groups. Flow cytometry analysis showed that the frequency of total T cells was similar across all groups in both the cryoablated and abscopal tumors, spleens, and blood. However, when examining different activation states of T cell subsets, we found anti-CTLA4 treated mice had increased T cell activity with higher percent of effector and effector memory CD4+ T cells in the abscopal tumors compared to the non-treated group, where similar trends were observed for the CD8+ T cells. Additionally, a higher percent of activated, effector, and effector memory CD4+ and CD8+ T cells were found in the blood of mice treated with anti-CTLA4, compared to the non-treated mice. Interestingly, we did not observe broad T cell activation with anti-PD-L1 treatment but found increased levels of