Costa, C.; Gray, S.; Pinton, G.; Moro, L.; Del Grosso, E.; Bellan, C.; Addi, L.; Lombardi, R.; Bruzzese, f.; De Biase, D.; Pucci, B.; Di Gennaro, E.; Ascierto, P. A.; Gravina, G. L.; Mutti, L.

Background: Mesothelioma (Me) is an aggressive cancer with limited response to conventional therapies. The tumor harsh microenvironment contributes to immune escape and therapy resistance and the effects of ICIs on Me are still unclear. Adenosine, an immunosuppressive molecule produced from AMP by the enzyme CD73, accumulates in hypoxic tumor areas. Elevated CD73 and adenosine receptor A2B (A2Br) levels on Me cells are linked to worse patient outcomes, indicating their important role in disease progression and potential as targets for treatment. Aim: This study characterizes the Me-ME (micro environment) and evaluates the efficacy of TT-4 (A2B inhibitor) and AB680 (CD73 inhibitor), alone or with aPD-1, using 3D models in vitro and in vivo. Methods: CD73 and A2B receptor levels were quantified in tumor and normal samples using qRT-PCR and IHC. Cells lines were treated with CoCl2 to mimic hypoxia, then CD73, A2Br and related markers were analyzed. MSTO-211H and REN cells were silenced for CD73, grown as spheroids and adenosine release was measured. Co-culture spheroids of MSTO-211H and Jurkat cells were treated with AMP and CD73 inhibitor, then analyzed for viability and immune markers. An orthotopic Me model was established by injecting AB1-B/c-LUC cells and monitored by in vivo imaging. Proteomic analysis of spheroids was conducted to identify proteins and pathways involved. Results: Hypoxia boosts CD73 and A2Br expression in Me cells, leading to adenosine production via CD73. In 3D co-cultures, AB680 lowered Me cell viability and enhanced activation of Jurkat T cells. In mice, combining aPD-1 therapy treatment with A2B or CD73 inhibitors was more effective than treatment with aPD-1 therapy. strongly The combination of A2B or CD73 with aPD-1 inhibitors caused a further reduction in tumor growth. Proteomics identified 93 proteins influenced by adenosine signaling through A2B. Conclusion: Targeting the adenosine pathway alongside PD-1 blockade offers a promising new immunotherapy strategy for Me.