Insights into extracellular vesicles: From biogenesis and tumor immune modulation to their clinical potential in immunotherapy

Extracellular vesicles (EVs) constitute a key mode of intercellular communication that remodels the complex tumor immune microenvironment by delivering diverse bioactive cargoes between tumor and immune cells. Nucleic acid species, encompassing microRNAs (miRNAs), messenger RNA (mRNAs), and other RNA transcripts, are functionally potent EV cargoes whose selective packaging dictates downstream immune signaling output. This review describes the molecular mechanisms driving selective RNA sorting into EVs, emphasizing sequence-motif recognition and core RNA-binding proteins FMR1 and TRIM25, which govern the enrichment of distinct RNA populations. We illustrate how EV-shuttled RNA molecules elicit context-dependent functional changes across regulatory T cells, myeloid-derived suppressor cells, and macrophages to shape antitumor or pro-tumor immune states. It critically integrates liquid biopsy evidence for EV-borne RNAs and EV-associated programmed death-ligand 1 (PD-L1) as predictive biomarkers of immunotherapy response in melanoma, non-small cell lung cancer, and hepatocellular carcinoma and dissects technical confounders such as non-vesicular RNA contamination and inconsistent isolation protocols. This review further compares nucleic acid loading strategies for native EVs versus cell-membrane-mimetic nanovesicles and explores their applicability for mRNA-based nanovaccines and combinatorial immunotherapy. We highlight underappreciated translational obstacles, noting that harmonized analytical standards, strict encapsulated cargo validation, and independent prospective multicenter trials are essential to translate EV-nucleic acid signatures into personalized cancer immunotherapy.