Abstract 1308: Protein profiling of breast cancer cell-derived extracellular vesicles in breast to bone metastasis

Abstract Breast cancer is the most common cancer among women worldwide, with bone being one of the most common sites for breast cancer metastasis. Despite improvements in systemic therapies for breast cancer, the mechanisms underlying breast cancer metastasis to bone remain incompletely understood thereby limiting the development of effective therapeutic options. Emerging evidence suggests that extracellular vesicles (EVs), nanosized particles secreted by cells including cancer cells, play a pivotal role in organ-specific metastasis. However, relatively little is known regarding the contribution of breast cancer cell-derived EVs to bone metastasis. To begin to investigate the potential role(s) of breast cancer cell-derived EVs in breast to bone metastasis, we first analyzed the protein profiles of EVs derived from parental MDA-MB-231 breast cancer cells (P-EVs) and from bone-seeking MDA-MB-231 breast cancer cells (B-EVs) using iTRAQ (isobaric Tags for Relative and Absolute Quantification) quantitative mass spectrometry. Among a total of 126 identified proteins, 13 were upregulated in B-EVs, 8 were upregulated in P-EVs and 105 were shared by both EVs. Gene Ontology (GO) enrichment analyses and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses identified three key biological processes of interest: osteoclastogenesis, EV-cell attachment and bone extracellular matrix-receptor interactions which may contribute to bone-tropism and pre-metastatic niche preparation. Importantly, confocal microscopy studies demonstrated that B-EVs are preferentially internalized by osteoclast precursor cells. This finding was further confirmed by quantitative flow cytometry which demonstrated significantly higher fluorescent signal intensities for B-EVs compared to P-EVs in these osteoclast precursor cells also suggesting enhanced uptake specificity. Moreover, we found that B-EVs can induce the differentiation of osteoclast precursors into multinucleated osteoclasts in long-term in vitro studies as confirmed by tartrate-resistant acid phosphatase staining, indicating that EVs derived from a bone-seeking subpopulation of breast cancer cells may promote osteoclastogenesis, a process critical for bone resorption. This, in turn, may contribute to the development of a pre-metastatic microenvironment for bone metastasis. Taken together, these studies begin to enhance our understanding of the role of breast cancer cell-derived EVs in breast to bone metastasis and may ultimately offer potential avenues for therapeutic intervention and early detection of bone metastasis. (This work was supported by the Breast Cancer Research Foundation, NIH R21 CA253051-01 and the Nile Albright Research Foundation. The authors acknowledge the gift of the MDA-MB-231 parental cell line and its bone-seeking variant from the Massagué Lab.) Citation Format: Guan Huang, Golnaz Morad, Marsha A. Moses. Protein profiling of breast cancer cell-derived extracellular vesicles in breast to bone metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1308.