Upregulation of PD-L1 expression in breast cancer cells through the formation of 3D multicellular cancer aggregates under different chemical and mechanical conditions.

Expression of programmed death-ligand 1 (PD-L1) in cancer cells plays an important role in cancer-immune cell interaction. The emerging evidence suggests regulation of PD-L1 expression by several tumor microenvironmental cues. However, the association of PD-L1 expression with chemical and mechanical features of the tumor microenvironment, specifically epidermal growth factor receptor (EGFR) signaling and matrix stiffness, remains elusive. Herein, we determine whether EGFR targeting and substrate stiffness affect the regulation of PD-L1 expression. Breast carcinoma cell lines, MCF7 and MDA-MB-231, were cultured under different conditions targeting EGFR and exposing cells to distinct substrate stiffness to evaluate PD-L1 expression. Furthermore, the ability to form aggregates in short-term culture of breast carcinoma cells and its effect on expression level of PD-L1 was probed. Our results indicated that PD-L1 expression was altered in response to both EGFR inhibition and substrate stiffness. Additionally, a positive association between the formation of multicellular aggregates and PD-L1 expression was observed. MDA-MB-231 cells expressed the highest PD-L1 level on a stiff substrate, while inhibition of EGFR reduced expression of PD-L1. The results suggested that both physical and chemical features of tumor microenvironment regulate PD-L1 expression through alteration of tumor aggregate formation potential. In line with these results, the in-silico study highlighted a positive correlation between PD-L1 expression, EGFR signaling, epithelial to mesenchymal transition related transcription factors (EMT-TFs) and stemness markers in metastatic breast cancer. These findings improve our understanding of regulation of PD-L1 expression by tumor microenvironment leading to evasion of tumor cells from the immune system.