P046
Mycobacteria extracellular vesicles as alternative immunotherapy for Bladder cancer
B Yenew(1,2) R Sorrentino(2) C Venegoni(3) P Miotto(2) C Braccia(4) A Andolfo(4) A Salonia(3) M Alfano(3) D M Cirillo(1,2)
1:Vita-Salute San Raffaele University, Milan, Italy; 2:EBPU, IRCCS San Raffaele Scientific Institute, Milan, Italy; 3:URI, IRCCS San Raffaele Scientific Institute, Milan, Italy; 4:Proteomics and Metabolomics Facility (ProMeFa), IRCCS San Raffaele Scientific Institute, Milan, Italy
Bladder cancer (BC) is the 9th most common cancer worldwide. BCG installation remains the standard treatment for high-grade non-muscle-invasive cases, but its limited efficacy and side effects highlight the need for alternatives. Mycobacterial extracellular vesicles (MEVs) have emerged as promising immunomodulatory agents, providing a unique opportunity for immune-oncology applications. Therefore, we assessed the antitumor capability of different MEVs against BC cell lines (BCaCL). MEVs were isolated from OncoTICE BCG, M. tuberculosis reference strain H37Rv and environmental M. gordonae (MG) by ultracentrifugation; quantified and characterized by Nanosight and BCA assay. Dose-response assays were performed on BCaCLs (T24, 5637) and THP-1-derived macrophages. Cell viability (Calcein-AM) was assessed at 24 and 72 h, and cytokine levels (ELISA) at 24 h post-treatment with MEVs or live BCG. T24 proteins were extracted after 72 h of MEVs treatment (BCG or H37Rv-derived), BCG-infection or untreated cells and analyzed by nLC-MS/MS. MEVs induced dose-dependent cytotoxicity in BCaCLs (12–48% viability reduction) and triggered TNF and IL-12 secretion comparable to BCG infection. IL-6 levels increased by 50–100% relative to BCG-infected cultures. However, MEVs significantly impacted macrophage viability (over 40% reduction). Proteomic analysis identified 228 statistically significant differentially expressed proteins (p< 0.05) across the four groups. MEVs demonstrated cytotoxicity and cytokine induction in BCaCLs, like BCG infection. Their impact on macrophage activation remains under investigation. Proteomic analysis revealed limited differences between MEV-treated and BCG-infected cells in terms of immunological activation, supporting further exploration of MEVs as modulators of immune responses in bladder cancer.