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

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with limited therapeutic options and inconsistent response to immune checkpoint inhibitors (ICIs). Emerging evidence indicates that tumor-associated bacteria may shape immune signaling and alter immunotherapy outcomes. Here, we investigated whether Staphylococcus aureus invades TNBC cells, persists intracellularly, and modulates PD-L1 expression. Methods: Using eFluor450-labeled S. aureus for flow cytometry, gentamicin protection assays, CFU quantification, and transmission electron microscopy, we assessed bacterial uptake and persistence in six TNBC cell lines and a non-tumorigenic control. PD-L1, TLR2, and STAT1 activation were evaluated after infection or TLR2 ligand treatment ± IFN-γ. Results: At multiplicity of infection (MOI) of 10, S. aureus internalized into 67% of MDA-MB-468 and 54% of MDA-MB-231, with intermediate uptake in Hs578T (27%) and BT-549 (24%) and only 0.5–9% in low-uptake lines (MDA-MB-453, CAL-51, MCF-12A). High-uptake lines exhibited marked cytotoxicity and reduced proliferation, with MDA-MB-468 showing an 82% drop in viability at 2 h and a 74% decrease after 5 d, whereas low-uptake lines showed minimal impact. Persistence lasted >7 d in MDA-MB-231 but only 3–5 days in others. IFN-γ plus S. aureus significantly amplified PD-L1, with up to a 2.9-fold increase in MDA-MB-468 and 1.5-fold in MDA-MB-231, but no effect in low-uptake lines. TLR2 agonists modestly increased PD-L1 in high-TLR2-expressing lines and synergized with IFN-γ. These effects were accompanied by STAT1 phosphorylation, supporting a TLR2/STAT1 axis linking bacterial sensing to immune checkpoint regulation. Conclusions: Together, these findings identify S. aureus as a modulator of immune signaling in TNBC and highlight the potential for microbial factors to influence ICI responsiveness. Targeting tumor–bacteria interactions may represent a novel strategy to enhance immunotherapy efficacy in breast cancer.