PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related axis
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Daniela Basso1, Elisa Gnatta1, Andrea Padoan1, Paola Fogar1, Sara Furlanello1, Ada Aita1, Dania Bozzato1, Carlo-Federico Zambon2, Giorgio Arrigoni2,3, Chiara Frasson4, Cinzia Franchin2,3, Stefania Moz1, Thomas Brefort5,6, Thomas Laufer6, Filippo Navaglia1, Sergio Pedrazzoli7, Giuseppe Basso4 and Mario Plebani1
1 Department of Medicine – DIMED, University of Padova, Padova, Italy
2 Department of Biomedical Sciences, University of Padova, Padova, Italy
3 Proteomic Center, University of Padova, Padova, Italy
4 Department of Woman and Child Health, Oncohematology Laboratory, University of Padova, Padova, Italy
5 Eurofins Medigenomix GmbH, Ebersberg, Germany
6 Comprehensive Biomarker Center GmbH (Recently re-named to Hummingbird Diagnostics GmbH), Heidelberg, Germany
7 Association Wirsung Onlus, Padova, Italy
Daniela Basso, email:
Keywords: pancreatic cancer, exosomes, myeloid derived suppressor cells, calcium, SMAD4
Received: March 03, 2017 Accepted: August 04, 2017 Published: September 13, 2017
Tumor genetics and escape from immune surveillance concur in the poor prognosis of PDAC. In this study an experimental model was set up to verify whether SMAD4, deleted in about 55% PDAC and associated with poor prognosis, is involved in determining immunosuppression through Exosomes (Exo). Potential mechanisms and mediators underlying SMAD4-dependent immunosuppression were evaluated by studying intracellular calcium (Fluo-4), Exo-miRNAs (microarray) and Exo-proteins (SILAC). Two PDAC cell lines expressing (BxPC3-SMAD4+) or not-expressing (BxPC3) SMAD4 were used to prepare Exo-enriched conditioned media, employed in experiments with blood donors PBMCs. Exo expanded myeloid derived suppressor cells (gMDSC and mMDSC, flow cytometry) and altered intracellular calcium fluxes in an SMAD4 dependent manner. BxPC3-SMAD4+, but mainly BxPC3 Exo, increased calcium fluxes of PBMCs (p = 0.007) and this increased intracellular calcium trafficking characterized mMDSCs. The analysis of de-regulated Exo-miRNAs and transfection experiments revealed hsa-miR-494-3p and has-miR-1260a as potential mediators of SMAD4-associated de-regulated calcium fluxes. Eleven main biological processes were identified by the analysis of SMAD4-associated de-regulated Exo-proteins, including translation, cell adhesion, cell signaling and glycolysis. A reverse Warburg effect was observed by treating PBMCs with PDAC-derived Exo: BxPC3 Exo induced a higher glucose consumption and lactate production than BxPC3-SMAD4+ Exo. Conclusion: PDAC-derived Exo from cells with, but mainly from those without SMAD4 expression, create an immunosuppressive myeloid cell background by increasing calcium fluxes and glycolysis through the transfer of SMAD4-related differentially expressed miRNAs and proteins.
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