Abstract PR1: Oncogenic driver mutations shape the nature of immune responses in tumors.
Authors: Juric Vladislava , Ruffell Brian , Bishop J. Michael 
Published: August 2014

The composition of tumor-associated leukocytes and their pro- or anti-tumor polarization are heterogeneous among different tumor types. In addition, the abundance of leukocyte subtypes can vary even among tumors of the same type. Understanding what determines this heterogeneity is important since the immune cell contexture of tumors can serve as a strong predictor of clinical outcomes and response to anti-cancer therapy. Given that tumorigenesis is driven by mutations in various types of proto-oncogenes and tumor-suppressor genes, we postulated that the identity of driver mutation could shape the leukocytic composition of tumors. To test this, we used the established mouse models for liver tumorigenesis induced by hydrodynamic transfection of c-MYC (MYC) or myr-AKT1+N-RasV12 (AKT+RAS) oncogenes. The resulting somatic integration and long-term overexpression of MYC or AKT+RAS in the liver leads to development of hepatoblastoma and hepatocellular carcinoma, respectively. We profiled liver-infiltrated CD45+ leukocytes in these two models by staining liver single-cell suspensions with lineage-specific leukocyte markers, followed by polychromatic FACS analysis. A massive increase in the CD11b+/F4/80-/Ly6Ghi/Ly6Cmed fraction of CD45+ leukocytes was detected in the AKT+RAS, but not in the MYC model. Compared to the vehicle-injected controls, AKT+RAS livers exhibited a 3-fold increase in this leukocyte population both in pre-neoplastic stages and in tumors. Overexpression of either oncogenes alone did not trigger such increase, suggesting that CD11b+/F4/80-/Ly6Ghi/Ly6Cmed response resulted from cooperation between AKT and RAS. Consistent with the FACS results, IHC staining of liver tissue sections with the neutrophil marker 7/4 antigen revealed an abundant positive staining both in pre-neoplastic livers and in tumors in the AKT+RAS model, while only a small infiltration of 7/4 antigen-positive cells was observed the MYC model only at tumor stage. To address if CD11b+/F4/80-/Ly6Ghi/Ly6Cmed play a role in tumorigenesis, we treated mice with the rat monoclonal Gr-1 antibody to systemically deplete Ly6G/Ly6C-expressing cells. Treatment with Gr-1 starting on day -1 before oncogene transfection ablated CD11b+/F4/80-/Ly6Ghi/Ly6Cmed cells from the livers and showed a tendency to increase the tumor burden in AKT+RAS mice for nearly 50%, suggesting that these cells act in early stages to suppress AKT+RAS-induced tumorigenesis. In contrast, Gr-1 treatment starting on day 4 after oncogene transfection no longer had an effect on AKT+RAS-induced tumorigenesis. Nevertheless, the same protocol significantly extended survival of mice and reduced tumor burden in the MYC model, suggesting a tumor-promoting role of CD11b+/F4/80-/Ly6Ghi/Ly6Cmed in MYC-induced tumorigenesis. In conclusion, our results demonstrate that the leukocytic composition and the nature of immune responses during tumorigenesis are shaped, at least in part, by tumor-driving mutations. We are currently elucidating the mechanism by which CD11b+/F4/80-/Ly6Ghi/Ly6Cmed cells promote or suppress tumorigenesis driven by MYC and AKT+RAS.

This abstract is also presented as Poster A46.

Citation Format: Vladislava Juric, Brian Ruffell, J. Michael Bishop. Oncogenic driver mutations shape the nature of immune responses in tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR1.