Abstract A011: Identifying surface biomarkers from colorectal cancer-derived extracellular vesicles using a bead-based approach
Authors: Debattista Jessica , Grech Laura , Scerri Christian , Buhagiar Malcolm , Grech Godfrey 
Published: November 2024

Colorectal cancer (CRC) accounts for the second most common cause of cancer-related mortality globally. The search for prognostic biomarkers subserving to detected cancer at an early stage, to improve tumor classification and to stratify therapeutic patient management groups is actively ongoing. Surgical resections and chemotherapeutics remain a mainstay in the cure and control of solid tumors. Despite their usefulness in the clinical scenario, shortcomings are well recognized, leading researchers to investigate for a circulating source of tumor-derived materials in biofluids, including mostly circulating tumor DNA (ctDNA), circulating tumor cells (CTCs) and extracellular vesicles (EVs). Characterization of EVs surface markers and assessment of their molecular cargo, can lead to the identification of early metastatic colorectal cancer signatures. Cell surface markers of three CRC cell lines (HCT116, DLD1 and LOVO) were characterized using flow cytometry. EVs were isolated from 48-hour serum-deprived cell lines using the polymer pull down method (20% Polyethelene glycol (PEG)). The concentration and particle size distribution of isolated EVs was checked by the NanoSight NS300. Avidin-coated Polystyrene beads were conjugated with CD9 biotin antibodies; and were used to immunoprecipitated (IP) CD9+ EVs. Following a 2-hour incubation and a series of washing steps, the PE-labelled antibodies of interest (CD9, CD63, EpCAM, Vim, CD44, CD133 and CD29) were added and read using xMAP technology. To increase the number of surface markers that can be read in tandem, IP was multiplexed using EpCAM, CD9 and CD63. This was followed by the isolation of EVs from plasma of metastatic CRC patients (N=3) using size exclusion chromatography (Izon qEV1 columns-35nm in conjunction with the automatic fraction collector) and polymer pull down. The concentration and size were assessed by Nanosight NS300 and surface markers of tumor-derived EVs were assessed using the immunoprecipitation technique on the Luminex system. Comparison of signals between EVs isolated from cell lines and matched originator cells showed that cell surface signatures in EVs match closely with their originator cells. The mean fluorescence intensities quantified cell surface marker expression in the different exosome-bead complexes. Interestingly, one of the metastatic cancer patients with invasive colorectal adenocarcinoma and lung metastasis showed EpCAM and Vimentin positivity suggesting epithelial-mesenchymal plasticity. These results show the importance of multiplexing both at the level of immunoprecipitation and at end-measurement, given the dynamics of cell surface marker changes during disease progression. The use of the mesenchymal marker; Vimentin in a panel for cell surface markers stems from studies on biotinylated cell surface markers on various cancer cell lines and identification of Vimentin in such models. Interestingly we find Vimentin on EVs surface in this study.

Citation Format: Jessica Debattista, Laura Grech, Christian Scerri, Malcolm Buhagiar, Godfrey Grech. Identifying surface biomarkers from colorectal cancer-derived extracellular vesicles using a bead-based approach [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A011.