Lactobacillus crispatus FY121-Derived Extracellular Vesicles Suppress HPV-Positive Cervical Cancer Cells and Activate RIG-I/MAVS/IRF3 Signaling

High-risk human papillomavirus (HPV) infection is the major cause of cervical cancer (CC), and viral oncogenes E6 and E7 contribute to tumor progression by promoting malignant transformation and impairing innate immune responses. Extracellular vesicles (EVs) derived from beneficial bacteria have emerged as potential regulators of host cellular functions. However, their role in HPV-associated cervical cancer remains unclear. In this study, we isolated and characterized extracellular vesicles derived from Lactobacillus crispatus FY121 (LcEVs) and investigated their effects on HPV-positive cervical cancer cell lines. LcEVs were efficiently internalized by HPV16-positive SiHa and HPV18-positive HeLa cells and significantly suppressed cell proliferation, migration, and invasion. Furthermore, LcEVs reduced HPV E6 and E7 expression at both the transcriptional and protein levels. Mechanistically, LcEV treatment was associated with increased RIG-I/MAVS/IRF3 signaling and enhanced IFN-β transcription and secretion. Transcriptomic analysis further revealed broad alterations in immune-related and cellular functional programs following LcEV treatment. In a co-injection xenograft model, LcEVs reduced tumor establishment and early outgrowth of HPV-positive cervical cancer cells and were accompanied by increased RIG-I/MAVS/IRF3 signaling in tumor tissue. Proteomic profiling identified diverse functional proteins within LcEVs, providing potential molecular candidates underlying their biological effects. Together, these results show that LcEVs suppress the malignant phenotypes of HPV-positive cervical cancer cells and are accompanied by activation of nucleic acid sensing and type I interferon signaling in vitro and in vivo. The identity of the active vesicular cargo and its causal contribution to these effects remain to be established, and bacterial EVs are highlighted as candidate immunomodulatory agents for HPV-related malignancies.