Background:
Cervical intraepithelial lesions (SIL) are key precancerous findings requiring accurate detection and risk stratification to guide clinical management and prevent progression to cervical cancer. Current screening methods have suboptimal specificity and positive predictive value, leading to overtreatment in some populations and missed lesions in others, a particular challenge in low and middle income countries (LMICs). Extracellular vesicles (EVs), small particles carrying bioactive proteins and nucleic acids, are emerging as promising minimally invasive biomarkers accessible via liquid biopsy. Comprehensive proteomic and transcriptomic characterization of circulating and tissue derived EVs may reveal disease associated signatures capable of discriminating women with cervical lesions from those without.

Objectives:
To identify and characterize differential protein and mRNA expression in blood  and tissue derived extracellular vesicles from women with cervical squamous intraepithelial lesions (cases) versus women without lesions (controls), using mass spectrometry based proteomics and RNA sequencing.

Methods:
This case-control study was conducted as an international collaboration between Instituto Nacional de Cancerología (INC; Bogotá, Colombia), the lead institution providing clinical samples, patient recruitment, and clinical data, and Division of Biomolecular and Cellular Medicine, Karolinska Institutet (Stockholm, Sweden), providing specialized expertise in proteomics and transcriptomics methodology and analysis. We enrolled 25 participants: plasma from 17 individuals (9 with SIL, 8 without SIL) and surgical cervical biopsies from 8 individuals (6 with SIL, 2 without SIL). EVs were isolated from (i) plasma and (ii) primary fibroblast cultures derived from enzymatic dissociation of biopsies. Characterization included nanoparticle tracking analysis (NTA) for size/concentration, immunophenotyping via flow cytometry, and transmission electron microscopy (TEM) for morphology. For transcriptomic analysis, mRNA was extracted from EVs and analyzed by RNA sequencing (HiSeq3000 Illumina); differential mRNA expression was assessed using DESeq2; adjusted p<0.05. For proteomic analysis, proteins extracted from EVs underwent high resolution mass spectrometry using TIMSTOF Bruker, differential protein expression was assessed using limma adjusted p<0.05; fold-change threshold >1.5. 

Results:
EV isolation, immunophenotyping, and morphological characterization confirmed expected size distributions, surface marker expression, and morphology from both plasma and tissue-derived sources; TEM confirmed expected morphology from culture derived EVs, and an optimized dissociation workflow yielded robust primary cultures and consistent EV recovery. Proteomics analysis identified approximately 15 proteins significantly overexpressed in women with SIL compared with controls (fold change range: 1.5–3.0; adjusted p<0.05). At least 3 of these proteins demonstrated consistent differential expression across all sample compartments (plasma derived and tissue-derived cultures), indicating robust, compartment independent biomarker signals. Preliminary RNA sequencing of plasma derived EVs identified potential candidate mRNA signatures associated with SIL; analysis is ongoing to determine the full complement of differentially expressed transcripts, concordance with protein findings, and consistency across both sample types.

?Conclusions/Implications:
Integrated proteomics and transcriptomics successfully identified protein and mRNA signatures associated with SIL. These validated biomarker candidates support development of blood-based multi omics diagnostic assays to improve screening specificity and clinical decision making in cervical cancer prevention programs. This collaborative partnership strengthened advanced biomarker discovery capacity at a Colombian national cancer institute and demonstrated feasibility of integrating proteomics and transcriptomics.