Abstract PS3-12-12: Association between Intratumoral Microbiota and Immune Microenvironment in Breast Cancer

Abstract Background: Cancer research has revealed intratumoral microbiomes as active modulators of tumor biology, distinct from gut microbiota. In breast cancer—the leading malignancy among Japanese women—these resident bacteria influence immune responses, inflammation, metabolism, and therapy resistance. Bacterial composition varies by tumor subtype and correlates with immunological activity and treatment outcomes. Microbes likely access breast tissue via hematogenous spread or ductal migration, then interact with immune cells, tumor cells, and stroma to shape the tumor microenvironment (TME). However, their clinical significance regarding immune infiltration, gene expression, and interactions with cancer stem cells—which drive tumor initiation, progression, and therapeutic resistance—remains unclear. Our study characterizes breast cancer's intratumoral microbiota and investigates its role in immune landscape formation and transcriptomic profiles. By integrating microbiome and transcriptome analyses from paired tumor, normal, and fecal samples, we aim to elucidate how microbial ecosystems influence breast cancer heterogeneity, cancer stem cell (CSC) biology, and overall tumor behavior. Objective: To investigate how intratumoral microbiota affects the TME and CSC distribution in breast cancer and to examine its relationship with clinical outcomes. Methods: We collected fecal samples, tumor tissues, and adjacent normal tissues from 16 breast cancer patients who underwent surgical resection without neoadjuvant therapy. The median patient age was 65 years (range: 38-91 years). 16S rRNA gene amplicon sequencing was performed for both fecal and tissue samples. RNA sequencing of tumor tissues was conducted to evaluate the expression of cytokines, immune-related genes, and CSC markers. Microbiota profiles were analyzed in conjunction with transcriptomic data to identify potential correlations. Results: Substantial interindividual variation in microbial profiles was observed within tumor tissues. Jaccard similarity analysis of microbial communities between tumor and matched normal tissues demonstrated a moderate positive correlation with intratumoral bacterial load (r = 0.67, p = 0.036). Several bacterial genera showed specific enrichment in tumor tissues compared to patient-matched normal tissues. Comparative analysis of fecal-tumor microbiota revealed shared amplicon sequence variants (ASVs) of 16SrRNA genes in 11 of 16 patients, suggesting multiple potential routes of bacterial translocation, including hematogenous and ductal pathways. Transcriptomic analysis revealed that elevated bacterial load was associated with altered immune signatures, including modifications in regulatory T cell and NK cell activity as assessed by xCell deconvolution analysis. However, CSC-specific signals were limited due to the low fraction of ALDH1-positive cells in our cohort. Gene Set Enrichment Analysis identified differences in hypoxia response, TNF-α signaling, and E2F target pathways that correlated with microbial abundance levels. Conclusion: Our findings suggest that intratumoral microbiota may influence the immune microenvironment and key molecular pathways in breast cancer, potentially affecting disease progression. These results provide foundational evidence for the development of microbiota-targeted therapeutic strategies in breast cancer management. Further studies with larger cohorts and functional validation are warranted to establish causal relationships and clinical implications. Citation Format: A. Yamada, K. Kawashima, M. Sasamoto, H. Fukuoka, M. Oshi, K. Narui, T. M. Dieter, Y. Sekiguchi, T. Ishikawa, I. Endo. Association between Intratumoral Microbiota and Immune Microenvironment in Breast Cancer abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-12-12.