Context: Genetic aberrations, inactivation, and tumor-piloted 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: Two pancreatic cancer cell lines were used: BxPC3 (Smad4 Homozygous Deletion, HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+). 1%FCS- Conditioned media (CM) were obtained from these cell lines. 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/CM and BxPC3-Smad4+/CM decreased 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.
SMAD-4 RELATED AND UNRELATED SOLUBLE FACTORS AND EXOSOMES CO-OPERATE IN DETERMINING PDAC IMMUNOEVASION
GNATTA, ELISA;AITA, ADA;FOGAR, PAOLA;PADOAN, ANDREA;BOZZATO, DANIA;MOZ, STEFANIA;GRECO, ELIANA;ZAMBON, CARLO-FEDERICO;PLEBANI, MARIO;BASSO, DANIELA
2014
Abstract
Context: Genetic aberrations, inactivation, and tumor-piloted 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: Two pancreatic cancer cell lines were used: BxPC3 (Smad4 Homozygous Deletion, HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+). 1%FCS- Conditioned media (CM) were obtained from these cell lines. 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/CM and BxPC3-Smad4+/CM decreased 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.
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