Evaluation of The Effects of siRNA-Mediated Silencing of CTLA-4 on the Activation of Breast Cancer Cell Lysate-Pulsed Dendritic Cells

Abstract

Breast cancer is the most common cancer in women and the second leading cause of their death. Today, the use of immunotherapies in the treatment of advanced cancers with poor response to conventional treatments such as chemotherapy has shown remarkable progress in clinical trials. Since immunotherapy using dendritic cells (DCs) in cancer treatment is very important and can activate immune system responses against tumor roots, investigating ways to improve DC efficacy can be effective in immunotherapies. One of the methods of immunotherapy is the use of dendritic cell (DC) vaccines, which have shown promising results in activating specific anti-tumor immune responses in cancer treatment through the activation of immune system responses. However, the expression of some immune checkpoint molecules such as CTLA-4 on DCs reduces their efficacy. Therefore, in this study, the effects of silencing or inhibition of CTLA-4 gene expression, which reduces the performance of DCs in the tumor microenvironment, were investigated on the expression of genes related to their function in human monocyte-derived DCs. Materials and Methods: Firstly, peripheral blood mononuclear cells (PBMCs) were obtained using the Ficoll separation method. Then, monocytes were separated from PBMCs by their adhesive property to plastic and differentiated into DCs in the presence of GM-CSF and IL-4 cytokines. The resulting DCs were then loaded with breast tumor lysates and the expression of CTLA-4 gene was silenced using siRNA. Finally, flow cytometry was used to evaluate the differentiation and maturation of DCs, and real-time PCR was used to investigate the expression of genes involved in the pathways of DC activation or inhibition. Results: The results showed that inhibition of the CTLA-4 gene in breast cancer-loaded mDCs significantly enhances the expression of HLA-DR, CD40, and CD86. Moreover, CTLA-4 inhibition in mDCs led to an increase in the production of inflammatory cytokines IL-12 and IL-10 (not significantly) and a decrease in TGF-β, IDO-1, STAT3 production.