The Tumor Microenvironment In Cholangiocarcinoma Is Dominated By An Immunosuppressive Infiltrate

Abstract Purpose: To characterize the dynamics of the immune response to cholangiocarcinoma (CCA) in a genetically engineered mouse model of CCA in order to identify immune pathways susceptible to targeted immunotherapies. Experimental Design: Genetically modified mice with targeted Kras activation and loss of p53 (Alb-Cre/LSL-KRASG12D /p53Lox/+) in the liver spontaneously develop CCA recapitulating the histopathologic features of human disease. To evaluate the in vivo immune response to CCA, tumors from Kras-p53 mice and normal livers were excised and processed for histologic examination, flow cytometry, and gene expression analysis. Immunohistochemistry (IHC) and flow cytometry were performed on myeloid and T cell markers to distinguish immune cell subsets. Cell lines established from isolated CCA tumors were used to assess the functional impact between the immune system and tumor cells in co-culture in-vitro studies. Results: Kras-p53 murine hepatic tumors were highly desmoplastic with a heavy fibrotic stromal compartment. IHC analysis demonstrated a prominent inflammatory leukocyte infiltrate compared to normal liver. Flow cytometry of single cell tumor suspensions showed the immune reaction was dominated by CD11b+ monocytic (Ly6C+) and granulocytic (Ly6G+) myeloid cells, and Foxp3+ regulatory T cells (T reg). In vitro studies revealed tumor educated myeloid cells expressed higher levels of genes associated with an immunosuppressive phenotype, including Arginase 1 (p=<0.007). Invasion assays analyzing the migration potential of CCA cell lines co-cultured with CD11b+ cells showed increased invasive properties in these tumor cells when compared to naive controls (p=<0.0001). Additionally, increased tumor-initiating properties were seen by qRT-PCR in tumor cell lines having undergone similar co-culture experiments. These data suggest the immune response to CCA is predominantly immunosuppressive and tumor supportive. Conclusion: CCA tumors from Alb-Cre/LSL-KRASG12D /p53Lox/+ mice have a prominent immunosuppressive infiltrate recapitulating features of the immune reaction in human disease. Thus, Kras-p53 mice provide an ideal model to test targeted immunotherapy for the treatment of CCA. Citation Format: Nathania M. Figueroa, Brian Belt, Ankit Patel, Booyeon Han, Margaret Hill, William Alexander, Michael O'Dell, Aditi Murthy, Kelli Connolly, Scott A. Gerber, Aram Hezel, David Linehan. The tumor microenvironment in cholangiocarcinoma is dominated by an immunosuppressive infiltrate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2951. doi:10.1158/1538-7445.AM2017-2951