Antibiotic driven microbiota disruption impairs HER2 targeted therapy efficacy

Up to 60% of cancer patients require antibiotics during therapy, yet retrospective studies indicate that antibiotic exposure can worsen outcomes across cancers. In breast cancer (BC), HER2+ patients show notably reduced survival after antibiotic use, an effect attributed to microbiota disruption, though mechanisms remain unclear. Our previous work demonstrated the role of microbiota signaling in chemo and immunotherapy efficacy. But HER2+ BC relies on targeted therapies, thus the impact of antibiotics on these agents requires further investigation. To address this, we used 16S rRNA sequencing of cecal content from mice bearing HER2+ TUBO tumors to assess whether an anti-HER2 antibody or neratinib, a HER2 tyrosine kinase inhibitor, each induces distinct microbiota shifts. Anti-HER2 increased Oscillospiraceae , Rikenellaceae , Ruminococcaceae , and Lachnospiraceae , whereas neratinib increased only the latter two Families. However, alpha diversity metrics remained unchanged. To study how these microbes contribute to treatment, we first depleted the gut microbiota using a broad-spectrum antibiotic cocktail (ABX). We observed the partial and complete loss of anti-HER2 and neratinib efficacy, respectively, on TUBO tumors. A clinically relevant antibiotic, such as ciprofloxacin (cipro), also fully halted neratinib response, whereas amoxicillin/clavulanate (am/clav) caused partial impairment. Because both antibiotics and neratinib were administered orally, we quantified plasma neratinib by LC-MRM mass spectrometry and ruled out antibiotic-induced blockade of neratinib absorption. Cipro and am/clav significantly reduced alpha diversity to a lesser extent than ABX, with cipro showing the most dramatic effect. Families enriched by HER2 therapies, including Lachnospiraceae and Ruminococcaceae, were targeted by cipro and am/clav. Notably, both antibiotics increased Akkermansia , a Family previously linked to favorable immunotherapy outcomes, suggesting that HER2-targeted therapies may rely on microbiota features, like Lachnospiraceae or Ruminococcaceae , distinct from those supporting checkpoint blockade. Spectral flow cytometry analysis of the tumor immune infiltration revealed that HER2 therapies triggered NK, CD8+, and CD4+ T cell infiltration. Antibiotic-treated tumors showed enrichment of pro-tumor macrophages and neutrophils with no significant NK cell recruitment. Microbiota alpha diversity was positively correlated with tumor NK and CD8+ T cell content, which, in turn, were associated with treatment outcomes. Overall, our findings demonstrate that antibiotic-driven disruption of gut microbiota composition and diversity impairs immune mechanisms linked to the efficacy of HER2+ BC therapy. These results highlight the potential of microbiota-based strategies to mitigate antibiotic-associated therapeutic resistance in HER2+ BC.