Background: Neoadjuvant chemotherapy (NAC) is becoming the standard of care for aggressive breast cancer (BC). In patients with BC, NAC can increase eligibility for breast-conserving surgery, lower the excision volume, and assist in evaluating the disease course. NAC may shrink (residual disease, RD) or even eliminate (pathologic complete response, pCR) the tumor. Patients with RD often have a poor long-term prognosis. In contrast, pCR is associated with better long-term survival outcomes. Approximately 30% of patients with BC show pCR after NAC, while the rest exhibit RD. A critical barrier to improving NAC outcomes in patients with BC is the lack of precise molecular targets and limited understanding of the molecular mechanisms underlying differential treatment outcomes in patients with BC receiving NAC. Thus, there is an urgent clinical need to elucidate the underlying molecular regulation associated with NAC resistance. Studies have shown that cellular stress induces the release of small extracellular vesicles known as exosomes, contributing to drug resistance. In this study, we evaluated the ability of exosomal metabolic profiles to predict NAC response in BC patients. Methods: We collected blood samples of 16 patients with BC who received NAC at Grady Memorial Hospitals, USA. Further, total exosomes were isolated from plasma by an ultracentrifugation method and characterized for their concentration (number/ml), size distribution, and exosomal content. Exosome sizes were validated using nanoparticle tracking analysis. RNA, proteins, and metabolites were isolated from exosomes to perform deep multiplatform analyses (viz. miRNA seq, proteomics, and metabolomics) of exosomal cargo to identify various potential biological determinants of NAC response and evaluate their contributions to the risk of RD in patients with BC after NAC. KEGG and HMDB IDs were used for metabolomics data analyses. Various analyses viz. enriched metabolomic pathway analysis, MS peak to pathway analysis (using GSEA and Mummichog), and network explorer analysis were performed using Metaboanalyst online portal to reveal the metabolic signatures in exosomes of BC patients who exhibited pCR and RD after NAC treatment. Results: Among the 16 patients with BC who received NAC, eight patients attained pCR, and eight had RD. Interestingly, we found that patients with pCR secreted 61% fewer exosomes but had 24% larger-sized exosomes than patients with RD following NAC. Furthermore, significant differences in the loading of exosomal cargo were observed between the two groups. Based on these results, we proposed that the differential RNA, protein, and metabolite profile of exosomes might contribute to the higher drug sensitivity in patients with pCR. Our proteomics and miRNA-seq analyses of exosomes from patients with pCR and RD after NAC did not show any significant differences between the groups. However, metabolite set enrichment analysis using MetaboAnalyst revealed that compared to exosomes from patients with pCR, patients with RD were significantly enriched in various metabolic pathways. These pathways included valine, leucine, and isoleucine biosynthesis; phenylalanine metabolism; one-carbon pool folate; and inositol phosphate metabolism. Additionally, MS peak to pathway analysis revealed differential enrichment of various other metabolic pathways, particularly in patients with RD.Conclusions: These data strongly suggest that exosomal metabolic signatures are associated with differential NAC outcomes in patients with BC. We believe that in-depth studies of these metabolite signatures will give essential clues about differential NAC response in patients with BC.

Citation Format: Shriya Joshi, Chakravarthy Garalapati, Shristi Bhattarai, Darshan Shimoga Chandrashekar, Sooryanarayana Varambally, Gagan Deep, Ritu Aneja. Exosomal metabolic signatures are associated with differential response to neoadjuvant chemotherapy in patients with breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-12-24.