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 naïve CD8+ T cells in abscopal tumors compared to the other groups.Conclusions: Cryoablation in combination with anti-CTLA4 increased T cell activation in abscopal tumors and blood after treatment, while the combination with anti-PD-L1 increased naïve CD8+ T cells. The observed differences are in accordance with the distinct mechanism of action for each drug. Further studies will investigate each strategy in long-term survival experiments. The goal is to identify and develop predictive biomarkers for efficacy of cryoablation in combination with ICIs that can be translated to the clinic. Citation Format: Flavia Sardela de Miranda, Rachel Babcock, Maribel Castro, Sonia Y Khan, Carsen Roach, Thomas Hintelmann, Kathryn Furr, Chang H Lee, Geetha P Boligala, Fahmida Rasha, Luis Brandi, Harvinder S Gill, Kevin Pruitt, Rakhshanda L Rahman. Breast cancer cryoablation in combination with anti-CTLA-4 increases T cell activation in a murine tumor model [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B18.

Introduction: Cryoablation is a non-surgical approach that uses rapid freeze-thaw cycles to kill cancer cells while preserving tumor-derived antigens. Our recent preclinical study in mice bearing bilateral high-risk metastatic breast tumors revealed cryoablation of the primary tumor enriched tumor-infiltrating lymphocytes in the non-treated abscopal tumor compared to resection, and prolonged overall mouse survival. Thus, it is clinically relevant to define the specific mechanisms by which cryoablation promotes antitumor immunity in abscopal tumors compared to resection. Here, we used RNA-sequencing (RNA-Seq) to identify immune gene signatures associated with enhanced abscopal effect following cryoablation versus resection in a murine model of metastatic breast cancer. Methods: BALB/c mice were implanted orthotopically and bilaterally with 4T1-12B (luciferase-expressing) cells. Two weeks after, left tumors were resected or cryoablated (tumor remained in mouse); right tumors were removed one week posttreatment to assess the abscopal immune response by RNA-Seq. Left tumors resected at two weeks were used as baseline controls. FASTQ files were trimmed and aligned to GRCm39; read counts were analyzed with edgeR and resulting differentially expressed genes with Ingenuity Pathway Analysis. Results: Cryoablation and resection uniquely affected the overall gene signatures and global activation of distinct signaling pathways in abscopal tumors compared to baseline and to each other. Since we observed differences in immune-related pathways, we further examined changes in the tumor immune landscape. Genes of various T cell populations and B cells were more upregulated in abscopal tumors following cryoablation compared to resection, suggesting cryoablation uniquely affects lymphocyte status. Within immune pathways in abscopal tumors following primary tumor cytoablation compared to resection, we found similar activation of Th1 signaling and greater activation of nitric oxide and reactive oxygen species production in macrophages, as well as Th2, NK cell, and IL-7 signaling. By contrast, crosstalk between dendritic cells and NK cells, PD-1/PD-L1 signaling, phagosome formation, and TREM1 signaling were less activated. Within these pathways, genes that promote angiogenesis and immunosuppression (Cxcl2) or metastasis (Cx3cr1) were downregulated in abscopal tumors following cryoablation compared to resection, whereas genes involved in T and NK cell-mediated cytolysis (perforin 1, Prf1), and tumor suppression (Stat6 and Nlrp12), were upregulated. Summary: We identified important pathways and genes involved in tumorigenesis and immune function, which may serve as biomarkers of the abscopal effect induced by cryoablation. Our findings suggest cryoablation reduces immunosuppression while enhancing T cell infiltration and cytotoxicity within abscopal tumors compared to resection. Future in silico and in vivo approaches will further define mechanisms by which cryoablation shapes the abscopal tumor immune landscape. Citation Format: Rachel L. Babcock, Dalia Martinez-Marin, Flavia Sardela de Miranda, Sonia Y. Khan, Maribel Castro, Isabel Castro-Piedras, Kevin Pruitt, Michael W. Melkus, Rakhshanda Layeequr Rahman. Identification of immune gene signatures as potential biomarkers of abscopal effect post-cryoablation of breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B09.