The proteo-transcriptome of extracellular vesicles and particles is largely preserved after cryopreservation

Extracellular vesicles and particles (EVPs) are small lipid-bilayer membrane structures containing proteins, lipids, and nucleic acids that play crucial roles in tumorigenesis, metastasis, and immunomodulation. The extent to which frozen tissues faithfully reflect the biology of fresh tissues remains unclear. To address this issue in the context of EVPs, we analyzed proteo-transcriptomic differences in EVP cargo between fresh and frozen human tissues. First, we extracted EVPs from tumor and adjacent normal tissues from cancer patients with a combination of ultrafiltration and size-exclusion chromatography. Next, we profiled the protein and RNA contents using spectroscopy methods. Further, we used Olink and mixed-effect models to investigate the differences between fresh and frozen protein contents. Our results show that cryopreservation has little effect on protein concentration, particle size, and proteo-transcriptomics of EVPs. These findings support the feasibility of using frozen specimens for EVPs research, potentially expanding access to large biobanks that house frozen specimens while mitigating reliance on fresh tissue samples, and in doing so, overcoming a major obstacle in human research.