Introduction: Cigarette smoke (CS) is the most well-established risk factor for bladder cancer. Although some evidence indicates that E-cigarettes (E-cig) may be a safer alternative to traditional CS, their full safety profile remains largely unproven. Extracellular vesicles (EVs) are recognized as intriguing entities in cancer pathogenesis. We reported that EVs derived from bladder cancer cells drive the malignant transformation of normal urothelial recipient cells. This model allows us to determine the impact of various genotoxic stresses present in the bladder environment including CS, E-cigs, and cancer EVs that drive the malignant transformation of normal urothelial cells and to study the underlying oncogenesis mechanisms.
Methods: E-Cigs, including unflavored and Icy menthol (12mg nicotine/mL, VistaVapors, Inc.) and research cigarettes (3R4F, University of Kentucky) were used. Cigarette smoke extract (CSE) was prepared by flowing CS though phenol red-free DMEM medium. Cell lines, including TCCSUP bladder cancer cells, and non-malignant urothelial SV-HUC cells were used. EVs were purified through serial centrifugation, and the released EV number was determined by nanoparticle tracking analysis. The carcinogenicity of interplays among CSE, E-cigs, and EVs was determined for DNA damage and colony formation assay. To reflect this scenario in a clinical setting, patient-derived bladder organoids were applied and tumorigenicity was further confirmed by a nude mouse xenograft model.
Results: We demonstrated that chronic exposure to naïve TCCSUP EVs induced SV-HUC cells’ malignant transformation as shown by increased colony formation and tumor growth in nude mice. CSE and mentholated E-Cigs, but not unflavored E-Cigs, induced double-strand DNA (dsDNA) breaks in normal SV-HUC cells, suggesting menthol is the major insult from E-Cig treatment. More critically, CSE and mentholated E-Cigs induce TCCSUP cells releasing EVs, and those EVs significantly cause more dsDNA breaks than naïve EVs. Thus, the interplays among cancer EVs, CSE and E-cigs form a positive feedback oncogenesis loop.
Conclusions: Investigation of tumorigenesis regulated by cancer EVs in response to CS and E-cigs provides a novel view of field cancerization present in post-TURBT smokers and establishes CS vs. E-cigs’ carcinogenicity in the urinary bladder. The results may lead to new therapeutic strategies and dispel the unsubstantiated claims that E-cigs are safe. Source of