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PDAC-derived exosomes enrich the microenvironment in MDSCs in a SMAD4-dependent manner through a new calcium related
Daniela Basso1, Elisa Gnatta1, Andrea Padoan1
1Department of Medicine - DIMED, University of Padova, Padova, Italy.
Abstract:
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.
Insights
Pancreatic ductal adenocarcinoma (PDAC) exosomes, particularly those lacking SMAD4, promote immune suppression. These exosomes increase myeloid-derived suppressor cells and alter calcium signaling and glycolysis via miRNA and protein transfer.
Area of Science:
- Cancer Biology
- Immunology
- Molecular Biology
Background:
- Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor prognosis due to tumor genetics and immune evasion.
- SMAD4 gene deletion, observed in ~55% of PDAC cases, correlates with poor prognosis.
- Exosomes (Exo) are implicated in intercellular communication and immune modulation in cancer.
Purpose of the Study:
- To investigate the role of SMAD4 in PDAC-mediated immunosuppression via exosomes.
- To identify mechanisms and mediators of SMAD4-dependent immunosuppression.
Main Methods:
- Utilized two PDAC cell lines (BxPC3 expressing SMAD4 and BxPC3 lacking SMAD4) to generate exosome-enriched conditioned media.
- Assessed exosome effects on peripheral blood mononuclear cells (PBMCs), including myeloid-derived suppressor cell expansion (flow cytometry) and intracellular calcium flux (Fluo-4).
- Analyzed exosomal microRNAs (miRNAs) via microarray and exosomal proteins via SILAC (Stable Isotope Labeling by Amino acids in Cell culture).
Main Results:
- PDAC-derived exosomes expanded myeloid-derived suppressor cells and altered intracellular calcium fluxes in a SMAD4-dependent manner.
- Exosomes from SMAD4-deficient cells (BxPC3) significantly increased PBMC calcium fluxes, a characteristic enhanced in monocytic MDSCs (mMDSCs).
- Identified hsa-miR-494-3p and hsa-miR-1260a as potential mediators of SMAD4-associated calcium flux dysregulation.
- Analysis of SMAD4-associated exosomal proteins revealed involvement in translation, cell adhesion, signaling, and glycolysis.
- PDAC exosomes induced a reverse Warburg effect in PBMCs, with SMAD4-deficient exosomes showing higher glucose consumption and lactate production.
Conclusions:
- PDAC-derived exosomes, especially those lacking SMAD4, induce an immunosuppressive myeloid cell environment.
- This immunosuppression is mediated by increased calcium fluxes and glycolysis, driven by the transfer of SMAD4-related differentially expressed miRNAs and proteins.
- SMAD4 status influences exosome cargo and function, impacting immune cell behavior and potentially contributing to PDAC's poor prognosis.
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