Supplementary Material for: TLR9 Downregulation in Breast Cancer: Its Role in Tumor Immunity, Inflammatory Response, and Cellular Senescence

Toll-like receptor 9 (TLR9) is primarily expressed in human dendritic and B cells and recognizes double-stranded DNA motifs from pathogens to initiate an inflammatory response. Recent studies have revealed TLR9's involvement beyond its conventional role in the immune response, notably during the tumorigenesis of various cancers such as head and neck, cervical, and ovarian cancers. In this study, immunohistochemistry (IHC) analysis demonstrated significantly lower TLR9 levels in breast cancer tumors compared to normal breast tissue epithelium. This downregulation was also observed in several transformed breast cancer cell lines compared to untransformed breast epithelial cell lines. Furthermore, MDA-MB-361 breast cancer cells expressing exogenous TLR9 exhibited reduced colony growth and an increase in senescence marker IL-6, pro- inflammatory cytokine CCL2, CXCL1 chemokine; and growth factor GM-CSF. These findings support TLR9's regulatory role in mitigating breast cancer and highlight its critical connection between the innate immunity and tumor cell growth.