The Molecular Determinants of Tumor Cell Modulation of Immune Selection

Abstract

Over the past century, the identification of molecules that tumor cells utilize to manipulate the activation or survival of immune cells has proven difficult. Expression analyses have helped to discover the downregulation of PD-L1, TGF- β, and FasL in the tumor microenvironment. Mutation analyses have helped identify novel tumor-specific or –associated antigens presented to the cell surface through major histocompatibility (MHC) complexes. Unfortunately, these techniques often fail to recognize the complex cross-talk occurring between cells in the tumor microenvironment to influence the anti-cancer immune response. By employing an RNAi screen in vivo, we have parsed how gene knockdown in tumor cells affects the ability of the immune system to identify and eliminate malignant cells. To identify tumor cell derived immune modulators, the EO771 breast adenocarcinoma cell line derived from a C57Bl/6 mice was transduced via lentiviral vectors with a barcoded genome-wide murine shRNA library and engrafted in immune-competent and immune-deficient mice. By analyzing the relative shRNA representation in tumors grown in the presence of a functional adaptive immune system compared to those grown in immune-deficient mice, we discovered a subset of tumor-based genes whose knock-down affects immune recognition. Pathway analysis identified enrichment of shRNAs targeting previously identified immune regulators, including the TGF-βR pathway and MHC class I antigen processing. By engrafting shRNA tumor cell lines targeting gene candidates in immune-competent and –deficient mice, we identified that CD47, Tex9, Pex14, and Sgpl1 play putative roles in T cell-dependent recognition and elimination of EO771 tumors. CD47, a known inhibitor of target cell phagocytosis by macrophages, also regulated the adaptive immune response. Two previously understudied molecules, Pex14 and Sgpl1, appear to have immune inhibitory functions, as their respective knockdown suppressed EO771 tumor growth and increased survival of immunocompetent mice. Tex9 serves as a potential tumor antigen or immune stimulant, as reduction of expression enhances tumor growth when EO771 tumors are grown in immunocompetent mice. Therefore, this functional in vivo screening approach enabled the discovery of CD47, Pex14, Sgpl1, and Tex9 as novel tumor-based modulators of anti-tumor adaptive immunity. To the best of our knowledge, we are the first to report the successful utilization of an in vivo functional genomics approach to identify novel tumor-based mediators of immune regulation.