The effect of jetPEI loaded siRNA molecules against PD-L1 on inhibition of stemness and activated T-lymphocytes differentiation to induced regulatory T-lymphocytes

Abstract

Triple Negative Breast Cancer Cell (TNBC) is known as invasive tumor with high incidence of distant metastasis and poorer prognosis than other subtypes. High PD-L1 expression by TNBC cells have immunosuppressive effect on tumor infiltrating T-Lymphocytes (TILs). Although several FDA-approved anti-PD-1/PD-L1 axis are suggested as the therapeutic methods, immune-related adverse events (irAEs) induced by these therapies in many cases it has been become a critical problem. Furthermore, siRNAs as regulator of gene expression was used to directly target PD-L1 on breast cancer cells. Materials and Methods: We initiate this study by bioinformatic analysis of TCGA and CCLE databases and after determining the appropriate cell line, to evaluate the effect of PD-L1 specific siRNA on survival and proliferation of MDA-MB-231 cell line. The cytotoxic effects of PD-L1-siRNA on MDA-MB-231 cells were determined using MTT assay. In the following, qRT-PCR and western blotting were used to determine the effect of PD-L1-siRNA on PD-L1 mRNA and protein expression, respectively. Wound healing assay, colony formation were used to evaluate the migration rate and self-renewal ability after transfection with PD-L1-siRNA. Flow cytometric studies were also used to determine the rate of induction of apoptosis using Annexin V-FITC/PI and DAPI staining. Moreover, cell cycle arrest and surface expression of PD-L1 was determined by flowcytometry. Finally, qRT-PCR was used to study the effect of PD-L1 silencing on the expression levels of metastasis, stemness and apoptosis related genes. In the second phase of this study, co-culture system was designed in order to investigate the effect of PD-L1 suppression in breast cancer cells on cytokine genes profile in T lymphocytes. Additionally, flowcytometery analysis was also performed to investigate their differentiation into regulatory lymphocytes. Results: The results of bioinformatic analysis showed that the expression of PD-L1 in TNBC patients was higher than other breast cancer subtypes. The results also showed that among several breast cancer cell lines, MDA-MB-231 cells line have the highest PD-L1 expression among the other TNBC or non-TNBC cell lines. The expression of PD-L1 mRNA and protein reduced after silencing PD-L1 in MDA-MB-231 cancer cells. PD-L1-siRNA reduced the migration and self-renewal capacity of transfected cells. Silencing of PD-L1 leads to cell cycle arrest in sub G1 and G1. Furthermore, the use of PD-L1-siRNA reduced the expression of c-Myc, MMP-9, CD44 gene expression wich is related to proliferation, invasion and metastasis of cancer cells.