The breast tissue microbiome has been increasingly recognized as a potential contributor to breast cancer development and progression. However, inconsistencies in microbial composition across studies have hindered the identification of definitive microbial signatures. We conducted a systematic review and meta-analysis of 11 studies using 16S rRNA sequencing to characterize the bacterial microbiome in 1260 fresh breast tissue samples, including normal, mastitis-affected, benign, cancer-adjacent, and cancerous tissues. Studies published until 31 December 2023 were included if they analyzed human breast tissue using Illumina short-read 16S rRNA sequencing with sufficient metadata, while non-human samples, non-breast tissues, non-English articles, and those lacking metadata or using alternative sequencing methods were excluded. We also incorporated microbiome data from The Cancer Genome Atlas breast cancer (TCGA-BRCA) cohort to enhance our analyses. Our meta-analysis identified Proteobacteria, Firmicutes, Actinobacteriota, and Bacteroidota as the dominant phyla in breast tissue, with Staphylococcus and Corynebacterium frequently detected across studies. While microbial diversity was similar between cancer and cancer-adjacent tissues, they both exhibited a lower diversity compared to normal and mastitis-affected tissues. Variability in bacterial genera was observed across primer sets and studies, emphasizing the need for standardized methodologies in microbiome research. An analysis of TCGA-BRCA data confirmed the dominance of Staphylococcus and Corynebacterium, which was associated with breast cancer proliferation-related gene expression programs. Notably, high Staphylococcus abundance was associated with a 4.1-fold increased mortality risk. These findings underscore the potential clinical relevance of the breast microbiome in tumor progression and emphasize the importance of methodological consistency. Future studies to establish causal relationships, elucidate underlying mechanisms, and assess microbiome-targeted interventions are warranted.

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.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options and high resistance to chemotherapy. Doxorubicin is commonly used, but its efficacy is limited by variable sensitivity and resistance. Bacopaside II, a saponin compound, has shown anti-cancer potential. This study evaluates the effects of doxorubicin and bacopaside II, both individually and in combination, across TNBC subtypes to explore mechanisms of resistance and enhanced drug efficacy. Methods: The growth-inhibitory effects of doxorubicin and bacopaside II were assessed in four TNBC cell lines. IC50 values were determined using dose–response assays, and doxorubicin accumulation was measured via spectral flow cytometry. ATP-binding cassette (ABC) transporter expression (ABCB1, ABCC1, ABCC3, and ABCG2) was analyzed for correlations with drug sensitivity. In silico docking assessed the binding affinity of bacopaside II to ABC transporters. A 3D culture model simulated drug-resistant TNBC, and combination effects were evaluated with live-cell imaging. Results: Doxorubicin sensitivity varied across TNBC molecular subtypes, correlating to intracellular accumulation. Bacopaside II inhibited growth across subtypes, inducing apoptosis in sensitive cells and necrosis in resistant cells. Bacopaside II increased doxorubicin accumulation, independent of P-glycoprotein (ABCB1), possibly through interactions with other ABC transporters. In drug-resistant 3D cultures, bacopaside II maintained efficacy and enhanced doxorubicin accumulation, counteracting ABC transporter-mediated resistance. The doxorubicin and bacopaside II combination showed synergistic growth inhibition. Conclusions: Bacopaside II enhances doxorubicin efficacy in TNBC by increasing drug accumulation and overcoming ABC transporter-mediated resistance, suggesting its potential as an adjuvant in TNBC treatment. These findings support further investigation of bacopaside II, particularly for resistant TNBC subtypes.

The breast tissue microbiome has been increasingly recognized as a potential contributor to breast cancer development and progression. However, inconsistencies in microbial composition across studies have hindered the identification of definitive microbial signatures. To address these gaps, we conducted a systematic review and meta-analysis of 11 studies employing 16S rRNA sequencing to characterize the bacterial microbiome across 1,260 fresh breast tissue samples, including normal, mastitis-affected, benign, cancer-adjacent, and cancerous tissues. We also incorporated microbiome data from The Cancer Genome Atlas breast cancer (TCGA-BRCA) cohort to enhance our analyses. Our meta-analysis identified Proteobacteria, Firmicutes, Actinobacteriota, and Bacteroidota as the dominant phyla in breast tissue, with Staphylococcus and Corynebacterium frequently detected across studies. While microbial diversity was similar between cancer and cancer-adjacent tissues, both exhibited lower diversity compared to normal and mastitis-affected tissues. Variability in bacterial genera was observed across primer sets and studies, emphasizing the need for standardized methodologies in microbiome research. Analysis of TCGA-BRCA data confirmed the dominance of Staphylococcus and Corynebacterium, which was associated with breast cancer proliferation-related gene expression programs. Notably, high Staphylococcus abundance was associated with a 4.1-fold increased mortality risk. These findings underscore the potential clinical relevance of the breast microbiome in tumor progression and emphasize the importance of methodological consistency. Future studies to establish causal relationships, elucidate underlying mechanisms, and assess microbiome-targeted interventions are warranted.

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.

Background: Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options and poor clinical outcomes. Emerging evidence suggests that the tumor-associated microbiome may influence disease progression and therapy response. Methods: We investigated how the Gram-negative bacterium Pseudomonas aeruginosa and Gram-positive bacterium Staphylococcus aureus, together with their cell wall components lipopolysaccharide (LPS) and lipoteichoic acid (LTA), modulate doxorubicin (DOX) efficacy in TNBC cells. Using gentamicin protection combined with flow cytometry of eFluor 450-labeled bacteria and CFU quantification, we assessed bacterial uptake, persistence, and effects on drug response in MDA-MB-468, MDA-MB-231, and MDA-MB-453 cells. Results: Both bacteria entered TNBC cells and survived for several days in a cell line-dependent manner. Notably, bacterial infection and purified cell wall ligands (LPS and LTA) significantly increased DOX accumulation and enhanced cytotoxicity in MDA-MB-468 and MDA-MB-231, but not in MDA-MB-453. The similar effects of LPS and LTA implicate Toll-like receptor signaling (TLR2 and TLR4) in modulating drug uptake. Conclusions: These findings demonstrate that bacterial infection and associated ligands can enhance doxorubicin uptake and cytotoxicity in TNBC cells, implicating TLR signaling as a potential contributor. Our results highlight the importance of host–microbe interactions in shaping chemotherapy response and warrant further investigation into their therapeutic relevance.