Abstract B09: Identification of immune gene signatures as potential biomarkers of abscopal effect post-cryoablation of breast cancer
Authors: Babcock Rachel L. , Martinez-Marin Dalia , de Miranda Flavia Sardela , Khan Sonia Y. , Castro Maribel , Castro-Piedras Isabel , Pruitt Kevin , Melkus Michael W. , Rahman Rakhshanda Layeequr 
Published: December 2022

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.