CD82-Mediated Loading of VEGF-A into Extracellular Vesicles Contributes to Ovarian Cancer Evasion of Anti-Angiogenic Therapy
Zhuoye Chen, Conghui Wang, Mingxia Xu, Tingjia Zhu, Yuehan Wang, Xiaodong Cheng, Xinyu WangObjective:
While bevacizumab is a key anti-angiogenic therapy for ovarian cancer, its long-term clinical efficacy is frequently limited by acquired resistance. This study aimed to investigate whether the tetraspanin CD82 mediates the packaging of VEGF-A into extracellular vesicles (EVs) to evade antibody neutralization, and to explore EV inhibition as a potential strategy to overcome this therapeutic resistance.
Methods:
Tumor cells were treated with bevacizumab, and conditioned supernatants were collected. Human umbilical vascular endothelial cells (HUVECs) were treated with the above-mentioned conditioned supernatants for tube formation and migration assays, and angiogenesis was evaluated in vivo using a zebrafish model. Intracellular VEGF-A was detected by WB, while extracellular VEGF-A was analyzed by ELISA and WB. EV secretion was inhibited using GW4869 (a widely established cell-permeable inhibitor of neutral sphingomyelinase that effectively blocks the ceramide-dependent biogenesis and release of EVs), and EV membranes were disrupted by RIPA or Triton X-100. The EVs in the supernatant were extracted with a kit. PCR and WB were used to examine cargo-sorting-related molecules. Protein interactions were analyzed using STRING, and CD82-VEGF-A interactions were validated by immunofluorescence and immunoprecipitation. Patient survival was analyzed using the Kaplan-Meier method.
Results:
Conditioned supernatants from bevacizumab-treated ovarian cancer cells enhanced HUVEC migration, tube formation, and angiogenesis in zebrafish. Bevacizumab increased both intraand extracellular VEGF-A protein levels in ovarian cancer cells, but not in other tumor cell lines. The persistent discrepancy between ELISA-quantified extracellular VEGF-A levels and WB analyses prompted us to hypothesize that a fraction of VEGF-A might be physically shielded within EVs. Inhibition of EV secretion reduced the pro-angiogenic effects of the conditioned supernatant. Disruption of EV membranes increased the detectable levels of VEGF-A. Bevacizumab upregulated CD82, which bound VEGF-A and sequestered it into EVs, enabling escape from bevacizumab neutralization.
Discussion:
The findings provide preliminary evidence that CD82 may facilitate EV-mediated loading of VEGF-A following bevacizumab treatment, potentially contributing to compensatory angiogenic responses. This finding provides a possible explanation for persistent angiogenic signaling despite VEGF blockade and supports further evaluation of the CD82–EV pathway in ovarian cancer.
Conclusion:
This study investigated how CD82 may promote EV-mediated loading of VEGF-A in response to bevacizumab, using in vitro systems and embryonic zebrafish models of ovarian cancer, and provides preliminary insight into compensatory angiogenesis.