Abstract 4032: Modulation of tumor PD-L1 expression by epithelial-mesenchymal phenotypic plasticity

Epithelial-mesenchymal transition (EMT) is a molecular and cellular program in which epithelial cells lose their well-differentiated phenotype and adopt mesenchymal traits. This process occurs during the progression of cancer to metastatic disease; tumor cells undergoing EMT acquire invasive characteristics, and mesenchymal features can be highly enriched among circulating tumor cells. EMT has also been associated with the resistance to multiple therapeutic agents, and may ultimately lead to relapse. Emerging research indicates that tumor cells undergoing EMT may also exhibit resistance to killing by innate natural killer (NK) cells and antigen-specific cytotoxic T lymphocytes (CTLs). Furthermore, tumor cells can evade immune destruction by upregulating the checkpoint molecule PD-L1. Given the contribution of tumor cell phenotypic plasticity to the susceptibility of tumor cells to immune attack, we have evaluated the role of EMT in the expression of PD-L1. Using several different cell line model systems, we devised an mRNA-based EMT scoring method to rank tumor cells according to their phenotypic characteristics. Our model systems included groups of isogenic cell lines in which phenotype modulation was achieved via 1) TGF-β1 treatment; 2) manipulation of EMT genes by transfection; 3) isolation of single cell clones; and 4) selection with chemotherapy. Scoring of the cell lines revealed the existence of distinct epithelial and mesenchymal phenotypes, as well as an intermediate (also termed ‘hybrid’ or ‘metastable’) phenotype in which markers of both differentiation states are co-expressed within the same cell line. Analysis of PD-L1 at the mRNA and protein level (antibody clone MIH1) demonstrated that significant changes in PD-L1 expression take place in response to tumor phenotypic modulation. Using multiple systems, it was observed that acquisition of a distinct mesenchymal phenotype associated with increased levels of PD-L1 expression, compared to isogenic tumor cells exhibiting an epithelial phenotype (for example, with tumor cells induced into the mesenchymal phenotype via TGF-β1 treatment or E-cadherin knockdown). In contrast, acquisition of an intermediate tumor phenotype characterized by co-expression of epithelial and mesenchymal features resulted in significant loss of PD-L1 expression. This phenomenon was observed in tumor cells exposed to chemotherapy in which acquisition of mesenchymal markers took place in the presence of sustained levels of epithelial markers (including E-cadherin). Altogether, our results highlight the importance of tumor phenotypic plasticity on PD-L1 expression. We plan to utilize these various models to understand the mechanism(s) of PD-L1 regulation by phenotype modulation and also to evaluate the potential predictive value of tumor cell phenotype, in conjunction with PD-L1 levels, on the susceptibility of tumor cells to killing by immune cells. Citation Format: Justin M. David, Charli L. Dominguez, Jeffrey Schlom, Claudia Palena. Modulation of tumor PD-L1 expression by epithelial-mesenchymal phenotypic plasticity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4032.