Smad-4 related and unrelated soluble factors and exosomes cooperate in determining PDAC immunoevasion

Genetic aberrations, including Smad4 inactivation, and tumor-piloted immunevasion, characterized by MDSCs expansion and DCs inhibition, concur in worsening PDAC prognosis. Our aims were to verify whether PDAC affects immune cells in a Smad4-dependent manner and whether tumor-derived exosomes (Ex) play a part. 1%FCS-Conditioned media (CM) from BxPC3 (Smad4 HD) and Smad4-transfected BxPC3 (BxPC3-Smad4+) were used. PBMC from 22 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 (100000 g). 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). 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). DCs were reduced by BxPC3-Smad4+/CM (Median:4;IQR:2-7) and BxPC3/CM (M:3;IQR:2-8) with respect to control (M:19;IQR8-32)(p<0.01). This inhibitory effect was replicated by Ex-free fraction (p<0.05). With respect to control (M:10;IQR:6-18 and M:14;IQR:9-25), BxPC3/CM (M:26;IQR:20-34 and M:7;IQR:4-12) and BxPC3-Smad4+/CM (M:23;IQR:18-36 and M:8;IQR:6-12) expanded mMDSCs, but reduced gMDSCs (p<0.005 and p<0.05 respectively). All effects on MDSCs were not reproduced by ex-free or ex-enriched media. mMDSCs/gMDSCs ratio was higher in BxPC3/CM (M:5.3;IQR:1.8-6.2), than in control (M:0.51;IQR:0.23-1.3)(p<0.005) or BxPC3-Smad4+/CM(M:3;IQR:1.8-4)(p<0.05). Conclusions: Smad4 modulate PDAC effects on immune cells. PDAC inhibition of CD4+ T-cells is Smad4-dependent and Ex-mediated. PDAC cells inhibition of DCs is Smad4-independent and soluble factors-mediated. MDSCs expansion and imbalance is in part Smad4-dependent and requires the co-presence of exosomes and soluble factors.