Blood- and tissue-derived extracellular vesicle signatures in HPV-positive cervical disease

Background: Extracellular vesicles (EVs) represent promising sources of biomarkers for cervical disease detection. However, optimal EV isolation strategies combining liquid and solid biopsies remain underexplored, particularly in resource-variable clinical settings. Methods: We implemented a dual-source EV isolation approach in a Colombian clinical cohort of HPV-positive women. Plasma samples and solid biopsies were collected from 17 participants (9 squamous intraepithelial lesion [SIL] cases, 8 controls). Plasma-derived EVs were isolated directly, while tissue-derived EVs were obtained from primary fibroblast cultures established through optimized enzymatic and mechanical dissociation protocols. EVs were characterized using nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry immunophenotyping. mRNA and protein cargo were prepared for RNA sequencing and proteomic analysis. Results: The dual-source approach successfully yielded EVs from both plasma and tissue samples with appropriate quality and quantity for downstream analysis. Nanoparticle tracking analysis confirmed expected size distributions, while transmission electron microscopy validated EV morphology from cultured cells. Flow cytometry immunophenotyping identified characteristic EV surface markers. The optimized tissue dissociation workflow generated robust primary cultures capable of producing analyzable EVs. Preliminary mRNA sequencing of plasma-derived EVs revealed candidate molecular signatures that distinguished SIL cases from controls and showed association with lesion grade. Conclusions: This dual-source EV strategy is feasible in Colombian clinical settings and supports the development of liquid biopsy biomarkers for early detection and risk stratification of HPV-related cervical disease. The approach enables comprehensive molecular profiling from complementary biological sources, warranting further validation in larger cohorts.