Intratumoral microbiota exhibiting varied responses to neoadjuvant chemotherapy in triple-negative breast cancer revealed by 2bRAD-M

BACKGROUND: Neoadjuvant chemotherapy (NACT) is the conventional therapy for early-stage and locally advanced triple-negative breast cancer (TNBC). Pathological complete response (pCR) serves as an essential indicator for predicting the effectiveness of NACT; however, a limited percentage of patients benefit from NACT. This research examined variations in the tumor microbiota between the pCR and non-pCR groups via 2BRAD sequencing for microbiome (2bRAD-M) technology and assessed the potential of the microbiota as a biomarker for predicting the NACT response. METHODS: A total of 44 TNBC patients were enrolled, of whom 25 achieved pCR and 19 were classified as non-pCR patients. Clinical data were collected, tumor tissue was biopsied as sample, and DNA was extracted. The 2bRAD-M sequencing method was used to analyse the microbial communities and community structures of tumor tissues in both groups, conduct microbial diversity and differential analyses, and perform qualitative and quantitative studies on the microbes inside the tissues. RESULTS: A total of 1,896 microbial species were identified. The microbial diversity in non-pCR tissue exceeded that in pCR patient tissue. The microbial composition of the tumor tissues in both groups was comparable. In the non-pCR group, there were significantly more gram-negative bacteria, such as Klebsiella, Escherichia, Acinetobacter, Vibrio, and Meiothermus, than in the pCR group. Calidithermus chliarophilus, Meiothermus sp.003226535, and Escherichia coli were identified as the three principal species distinguishing the two groups. Seven species were identified as markers to distinguish between non-pCRs and pCRs, with an AUC value of 90.3%. Validation in an independent cohort via qPCR indicated the potential predictive value of this seven-species model, yielding an AUC of 84%. Functional annotation analysis revealed 3,207 differentially expressed COGs and 230 differentially enriched signalling pathways across the non-pCR and pCR microbiomes. In vitro experiments suggested that lipopolysaccharide may contribute to doxorubicin and paclitaxel resistance in MDA-MB-231 cells, a process potentially linked to the reactivation of the PI3K/AKT signaling pathway. CONCLUSION: This exploratory 2bRAD-M microbiome study of pCR and non-pCR tissues from TNBC patients receiving NACT identified significant differences in microbial environments between the two groups. Based on these findings, we developed a predictive model for chemotherapy effectiveness and highlighted the microbiome's potential as a biomarker for the efficacy of NACT in TNBC patients, offering novel insights that may inform future clinical diagnostic strategies.