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 whetherStaphylococcus aureusinvades TNBC cells, persists intracellularly, and modulates PD-L1 expression.Methods:Using eFluor450-labeledS. aureusfor 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. aureusinternalized 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 plusS. aureussignificantly 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 identifyS. aureusas 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.
背景：三阴性乳腺癌（TNBC）是一种侵袭性亚型，治疗选择有限且对免疫检查点抑制剂（ICIs）的反应不一致。新出现的证据表明，肿瘤相关细菌可能影响免疫信号传导并改变免疫治疗效果。本研究旨在探究金黄色葡萄球菌是否能够侵入TNBC细胞、在细胞内持续存在并调节PD-L1表达。 方法：通过使用eFluor450标记的金黄色葡萄球菌进行流式细胞术、庆大霉素保护实验、菌落形成单位定量及透射电子显微镜观察，评估了六种TNBC细胞系及一种非致瘤性对照细胞系对细菌的摄取和持续存在情况。在感染或TLR2配体处理（联合或不联合IFN-γ）后，评估了PD-L1、TLR2及STAT1的活化状态。 结果：在感染复数（MOI）为10时，金黄色葡萄球菌被内化至67%的MDA-MB-468细胞和54%的MDA-MB-231细胞中；Hs578T（27%）和BT-549（24%）细胞摄取水平中等，而低摄取细胞系（MDA-MB-453、CAL-51、MCF-12A）仅摄取0.5–9%。高摄取细胞系表现出显著的细胞毒性和增殖减缓，其中MDA-MB-468细胞在2小时内存活率下降82%，5天后下降74%，而低摄取细胞系影响甚微。金黄色葡萄球菌在MDA-MB-231细胞中持续存在超过7天，在其他细胞系中仅持续3–5天。IFN-γ联合金黄色葡萄球菌显著增强PD-L1表达，在MDA-MB-468细胞中最高增加2.9倍，在MDA-MB-231细胞中增加1.5倍，但在低摄取细胞系中无此效应。TLR2激动剂在TLR2高表达细胞系中适度增加PD-L1，并与IFN-γ产生协同作用。这些效应伴随STAT1磷酸化，支持了连接细菌感知与免疫检查点调控的TLR2/STAT1轴。 结论：综上，本研究证实金黄色葡萄球菌是TNBC中免疫信号的调节因子，并凸显了微生物因素影响ICI反应性的潜力。靶向肿瘤-细菌相互作用可能成为增强乳腺癌免疫治疗效果的新策略。
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