Microsatellite instability detection in extracellular vesicles of short-chain fatty acids-treated colorectal cancer cells

Abstract Late-stage diagnosis renders colorectal cancer (CRC) a major health challenge, mostly due to the lack of reliable non-invasive biomarkers. Although microsatellite instability (MSI) is a promising genetic biomarker guiding immunotherapy selection, its detection still relies on invasive sampling. Here, we explored whether short-chain fatty acids (SCFAs), gut microbiota-derived metabolites with established pro-apoptotic and anti-tumorigenic functions, could enhance extracellular vesicle (EV) production and improve the recovery of tumor-derived DNA. EVs represent a promising source of nucleic acids for liquid biopsy applications. Two CRC cell lines with distinct MSI status were exposed to SCFAs, followed by isolation and characterization of small and large EVs. SCFA treatment significantly increased EV production (1.38-fold in RKO and 2.15-fold in SW480), without affecting the expression of known EV markers (i.e., CD9, CD63, Alix). This response was accompanied by caspases detection and a DNA redistribution, with reduced total DNA (cell and cell-free) and increased EV-DNA (17.7-fold in L-EVs and 32.8-fold in s-EVs from SW480 cells). Importantly, we demonstrate, for the first time, the capability to identify MSI-specific alterations in EV-DNA using droplet digital PCR. These findings indicate that SCFA-associated EV release favors the recovery of DNA-rich EVs, highlighting EV-DNA as a promising substrate for non-invasive MSI diagnostics.