Abstract 2005: ROR1-specific extracellular vesicle tetraspanin-8 levels predict distant metastasis in triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC), comprising 15-20% of all breast cancer cases, is the most aggressive subtype, with limited efficacy from targeted therapies and immunotherapies. Metastasis drives TNBC-related mortality, emphasizing the urgent need for predictive methods to identify high-risk cases early, guide clinical management, and facilitate timely interventions. Extracellular vesicles (EVs) play a pivotal role in cancer progression and metastasis, offering a promising avenue for metastasis research. However, isolating tumor-derived EVs from the total EV population remains a significant clinical challenge, underscoring the necessity for precise detection strategies. Methods: EVs were isolated from MCF-10A cells, MCF-7 cells, and MDA-MB-231 cells using ultracentrifugation, followed by morphological characterization and surface marker protein validation. Mass spectrometry and UniProt gene ontology analyses were conducted to identify TNBC-specific EV proteins, classified by molecular functions or biological processes. TSPAN8 expression in breast cancer cells was analyzed using the CCLE and TCGA databases. Cholesterol-PEG-Biotin and Streptavidin-APC (Cholesterol-PEG-APC) were employed to anchor EV membranes, enabling concurrent detection of quantum dot-labeled EV proteins and membrane signals. Results: ROR1, a transmembrane protein expressed in TNBC cells and EVs, was identified as a specific capture protein for TNBC-derived EVs. Database analysis revealed elevated TSPAN8 expression in highly metastatic TNBC MDA-MB-231 cells and in the tissue of TNBC-LAR subtype, where TSPAN8 plays a decisive role in metastasis. Using ROR1 to capture MDA-MB-231 EVs demonstrated that the fluorescence intensity from quantum dot-labeled QD605 anti-TSPAN8 and APC increased with EV concentration. Furthermore, higher average EV-TSPAN8/APC levels were associated with greater metastatic risk in patients. Correlation analysis using the TCGA database showed a positive relationship between ROR1 and TSPAN8 expression. VCAM1 was identified within the interaction network of ROR1 and TSPAN8, with positive correlations to both, providing a theoretical basis for leveraging ROR1-specific EV-TSPAN8 detection in metastasis research. Conclusions: ROR1 effectively captures tumor-derived EVs and holds significant potential for enhancing EV-TSPAN8 detection, providing a promising strategy for predicting metastasis in TNBC. Evaluating ROR1-specific EV-TSPAN8 levels in TNBC blood samples and its link to disease progression warrants further investigation. Additionally, exploring its clinical utility across diverse TNBC subtypes and treatment regimens could refine its application, ultimately improving the precision of early metastasis prediction in TNBC. Research Sponsor: National Natural Science Foundation of China (No. 82202603). Citation Format: Yinchao Zhang, Linze Xu, Jin Zhang, Dandan Wu, Yang Liu. ROR1-specific extracellular vesicle tetraspanin-8 levels predict distant metastasis in triple-negative breast cancer [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 2005.