Immunoblotting Analysis of Intracellular PD-L1 Levels in Interferon-γ-Treated Ovarian Cancer Cells Stably Transfected with Bcl3 shRNA.

Expression of the immune checkpoint programmed death ligand 1 (PD-L1, CD274) is increased in many types of cancer, including ovarian cancer (OC), but the mechanisms that regulate the PD-L1 expression are not fully understood. In addition to binding to PD-1 on T cells, thus inhibiting T cell-mediated antitumor responses, PD-L1 has also tumor-intrinsic effects that include increased cancer cell survival and proliferation, and that might be in part mediated by the intracellular PD-L1. In this chapter, we describe a protocol for the analysis of the intracellular PD-L1 protein levels in OC cells by immunoblotting. Our results show that interferon-γ (IFNγ) induces the intracellular levels of PD-L1 and the proto-oncogene Bcl3 in OC cells. However, the PD-L1 expression is significantly decreased in OC cells stably transfected with Bcl3 shRNA, demonstrating that the IFNγ-induced PD-L1 expression in OC cells is mediated by Bcl3. These data identify the IFNγ-Bcl3-PD-L1 axis as a novel therapeutic target in OC, and suggest that targeting Bcl3 may provide a novel strategy to regulate the PD-L1 expression, and especially the tumor-intrinsic PD-L1 effects mediated by the intracellular PD-L1 in OC cells.