Cell Line-Dependent Internalization, Persistence, and Immunomodulatory Effects of Staphylococcus aureus in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited treatment options. While immune checkpoint inhibitors (ICIs) targeting PD-1/PD-L1 have shown clinical benefit, response rates remain variable, and mechanisms of resistance are not fully understood. Emerging evidence suggests that the tumor microbiome can influence immune responses and therapy outcomes. This study investigates the role of Staphylococcus aureus in modulating PD-L1 expression and immune evasion in TNBC. We examined the internalization, persistence, and functional impact of S. aureus in three TNBC cell lines (MDA-MB-468, MDA-MB-231, MDA-MB-453) and the non-tumorigenic MCF-12A breast epithelial cell line. Bacterial uptake and intracellular survival were assessed using an optimized gentamicin protection assay, colony-forming unit quantification, and transmission electron microscopy for up to seven days. Flow cytometry was used to analyze PD-L1 and TLR2 surface expression, while western blot assessed STAT1 activation. Cells were infected with viable S. aureus or treated with the TLR2 agonists S. aureus lipoteichoic acid and Pam3CSK4, a synthetic triacylated lipopeptide that specifically activates TLR2, alone or in combination with IFN-γ. Our findings demonstrate that S. aureus invades and persists within breast cells in a subtype-dependent manner. Notably, intracellular S. aureus, in the presence of IFN-γ, induces PD-L1 expression, contributing to immune checkpoint regulation. The degree of PD-L1 upregulation varied across TNBC subtypes, with MDA-MB-468 (basal-like) and MDA-MB-231 (mesenchymal stem-like) cells exhibiting the highest response. In MDA-MB-231 cells, which express high levels of TLR2, TLR2 agonists enhanced PD-L1 expression independently of IFN-γ. Across all cell lines, TLR2 agonists in combination with IFN-γ further increased PD-L1 expression, suggesting a synergistic effect between bacterial signaling and inflammatory pathways. These findings provide evidence that S. aureus modulates immune checkpoint pathways in TNBC, potentially contributing to immune evasion and ICI resistance. This highlights the need for further investigation into microbial influences on tumor progression and suggest that targeting bacterial interactions may enhance immunotherapy efficacy in TNBC.