SMAD-4 related and unrelated soluble factors and exosomes co-operate in determining pancreatic adenocarcinoma (PDAC) immunoevasion

Genetic aberrations, including Smad4 inactivation, and tumor-piloted immunoevasion, characterized by myeloid derived suppressive cells (MDSCs) expansion and dendritic cells (DCs) inhibition, concur in worsening PDAC prognosis. Objective To verify whether PDAC affects immune cells in a Smad4-dependent manner and whether tumor-derived exosomes (Ex) play a part. Methods 1%FCS-Conditioned media (CM) from BxPC3 (Smad4 HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+) were used. Peripheral blood mononuclear cells from 10 blood donors were cultured (4 days) in unfractioned control medium, BxPC3/CM and BxPC3-Smad4+/CM, and in Ex-enriched (pellet), or Ex-free (supernatant) media (ultracentrifugation at 100000 g for 1h). CD4+,CD8+,CD4+CD25+ T-cells, DCs (CD11b+CD14+DR-) and two MDSCs subsetes (CD11b+CD14-DR+=mMDSCs; CD11b+CD14-DR-=gMDSCs) were FACS analysed. Exosomes enrichement and deprivation were confirmed by western blot (Alix and tsg 101). Results BxPC3-Smad4+/CM decreased CD4+ and CD8+ with respect to control (p<0.05) and BxPC3/CM (p<0.05). Ex-enriched BxPC3-Smad4+/CM replicated the inhibitory effects on CD4+ cells (p<0.05). With respect to control, BxPC3-Smad4+/CM reduced mMDSCs (p<0.05) and expanded gMDSCs (p<0.05). The effect on mMDSCs was not reproduced by Ex-free or Ex-enriched media. By contrast, the whole media stimulatory effect on gMDSCs was reproduced by Ex-enriched (p<0.05), not by Ex-free media (p:ns). Ex-enriched BxPC3/CM also expanded gMDSC (p<0.05), while Ex-free BxPC3/CM reduced the same cellular population (p<0.05). Whole BxPC3/CM was ineffective on gMDSC (p:ns). DCs were unaffected by both BxPC3 and BxPC3-Smad4+ entire and fractionated CM. Conclusions Smad4-associated exosomes modulate PDAC effects on immune cells. PDAC inhibition of CD4+ T-cells is Smad4-dependent and Ex-mediated. Exosomes transfer also PDAC-associated signals leading to gMDSC expansion, which is counteracted by soluble mediators released by cells with Smad4 HD.