Abstract 6750: Highly multiplexed immunoassays enable discovery of multimarker EV surface signatures as novel biomarkers

Abstract Extracellular vesicles (EVs) are lipid bilayer particles that mediate cell communication by carrying molecules that reflect their cell of origin. Their enrichment in disease states and accessibility via minimally invasive sampling make them promising biomarkers. EV heterogeneity remains a challenge as single surface markers are often insufficient to define disease-related EV subpopulations, underscoring the need for technologies enabling single-vesicle resolution and multiplexed detection of composite marker signatures. We developed a highly multiplexed immunoassay technique to count combinations of three surface markers on individual, intact EVs. EVs are captured on magnetic beads using antibodies against tetraspanin proteins broadly expressed on EV surfaces, enabling selective EV enrichment and automation. Plasma is pre-processed using a mixed-mode resin that removes 99.5% of proteins while recovering 60% of EVs, followed by immune-depletion of platelet-derived EVs to improve sensitivity for non-platelet EVs. A panel of barcoded antibodies for different cell-surface proteins is used to bind EV surface antigens. When three antibodies co-localize on an EV, barcodes associated with the antibodies are linked to form a DNA product encoding the three target antigens. A library of probes targeting 63 EV surface markers, selected to distinguish circulating EVs from major cell lineages, enables broad phenotyping. DNA products are PCR-amplified and sequenced, quantifying thousands of 3-marker combinations on individual EVs. We applied our technique to 366 plasma samples provided by collaborators from those with colorectal (CRC), breast, and gastric cancer, alongside 47 healthy controls and cell-line-derived EV samples. Technical precision assessed using replicate controls yielded average inter-plate and intra-plate CVs of 19% and 16%, respectively. Spiking defined EV populations into plasma established a detection limit of 1 part in 107. Across all cancer types, differential expression analysis identified elevated 3-marker combinations involving cancer-associated markers (e.g., CEA, CD10, CD13 and N-cadherin) relative to controls. Selected EV populations were validated using ultrasensitive electrochemiluminescence (ECL) assays, showing strong correlation with sequencing results (Pearson’s r 0.8). In two longitudinal CRC groups, elevated levels of several 3-marker EV signatures were significantly associated with shorter overall and progression-free survival. This multiplexed single-EV assay offers a powerful, sensitive platform for high-throughput profiling of disease-associated EV populations. Its modular design enables flexible selection of marker panels tailored to specific biological questions. Additionally, it can be integrated with ultrasensitive ECL immunoassays for cost-effective, large-scale validation of selected EV signatures. Citation Format: Lucie Hebert, Misk Al-Ameen, Evan A. Gizzie, Collin Nelson, James H. Butler, J. Chris Brady, Jeffrey L. Franklin, Richard J. Cote, Robert J. Coffey, Andrew B. Nixon, David Aaron Routenberg. Highly multiplexed immunoassays enable discovery of multimarker EV surface signatures as novel biomarkers abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 6750.