Mice with targeted KrasG12D and loss of p53 in the liver: A robust model for studying immunologic interventions for treating cholangiocarcinoma

Abstract

281 Background: Inflammation in the tumor microenvironment—chronically elevated cytokines, chemokines, and inflammatory cells—promotes tumorigenesis. Cholangiocarcinoma (CC) is the most common liver malignancy within the biliary tree with few treatment options and a poor 5-year survival. CC tumors are characterized with a dense tumor stroma and abundant inflammatory immune cell infiltrate, yet little is known about the immune dynamics underlying the disease. Here, we characterize immune signaling pathways in human CC and a spontaneous mouse model of the disease to identify potential targets susceptible to immune based therapies. Methods: Histology and immunohistochemistry (IHC) were performed on archived human specimens and tissue microarrays of 52 CC specimens constructed under an IRB approved protocol to identify the prognostic significance of cytokine and immune markers. Peripheral blood, bone marrow, and tissue from mice with targeted activation of KrasG12D and loss of p53 (Kras-p53-/-) in the liver that spontaneously developed CC tumors versus normal controls were collected and analyzed by histology, IHC, quantitative gene expression, and flow cytometry based studies. Results: High levels of inflammatory leukocytes in human CC were predominantly of monocytic and granulocytic origin including macrophages (TAM) and granulocytic-myeloid derived suppressor cells (G-MDSC) respectively. Malignant cells expressed high levels of CCL2 and TAM stained positive for its cognate receptor CCR2. In addition, CD8+ and CD4+ T cells expressing PD-1 were associated with PD-L1+ tumor cells. Kras-p53-/- mice developed CC tumors histologically similar to human disease: tumors had high levels of cytokines and induced myelopoiesis leading to significantly more monocytes and granulocytes in the bone marrow, blood, and spleen compared to normal liver controls. Tumors also expressed higher levels of CCL2 and PD-L1 with more CCR2+ TAMs and PD-1+ T cells compared to controls. Conclusions: Human CC utilizes immune signaling pathways and Kras-p53-/- mice recapitulate the immune dynamics of the disease representing a model to study immune based therapies for treating CC.