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Proteomic Analysis of Exosomes Secreted from Human Alpha-1 Antitrypsin Overexpressing Mesenchymal Stromal Cells.

ABSTRACT: Extracellular vesicles (EVs) mediate many therapeutic effects of stem cells during cellular therapies. Bone marrow-derived mesenchymal stromal cells (BM-MSCs) were manufactured to overexpress the human antiprotease alpha-1 antitrypsin (hAAT) and studied to compare the EV production compared to lentivirus treated control MSCs. The goal of this study was to compare protein profiles in the EVs/exosomes of control and hAAT-MSCs using unbiased, high resolution liquid chromatography and mass spectrometry to explore differences. Nanoparticle tracking analysis (NTA) showed that the particle size of the EVs from control MSCs or hAAT-MSCs ranged from 30 to 200 nm. Both MSCs and hAAT-MSCs expressed exosome-associated proteins, including CD63, CD81, and CD9. hAAT-MSCs also expressed high levels of hAAT. We next performed proteomic analysis of EVs from three healthy donor cell lines. Exosomes collected from cell supernatant were classified by GO analysis which showed proteins important to cell adhesion and extracellular matrix organization. However, there were differences between exosomes from control MSCs and hAAT-MSCs in cytokine signaling of the immune system, stem cell differentiation, and carbohydrate metabolism (p < 0.05). These results show that hAAT-MSC exosomes contain a different profile of paracrine effectors with altered immune function, impacts on MSC stemness, differentiation, and prevention of cell apoptosis and survival that could contribute to improved therapeutic functions.

SUBMITTER: Wei H

PROVIDER: S-EPMC8773149 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:INTRODUCTION: Studying cancer tumors' microenvironment may reveal a novel role in driving cancer progression and metastasis. The biological interaction between stromal (mesenchymal) stem cells (MSCs) and cancer cells remains incompletely understood. Herein, we investigated the effects of tumor cells' secreted factors as represented by a panel of human cancer cell lines (breast (MCF7 and MDA-MB-231); prostate (PC-3); lung (NCI-H522); colon (HT-29) and head & neck (FaDu)) on the biological characteristics of MSCs. METHODS: Morphological changes were assessed using fluorescence microscopy. Changes in gene expression were assessed using Agilent microarray and qRT-PCR. GeneSpring 12.1 and DAVID tools were used for bioinformatic and signaling pathway analyses. Cell migration was assessed using a transwell migration system. SB-431542, PF-573228 and PD98059 were used to inhibit transforming growth factor ? (TGF?), focal adhesion kinase (FAK), and mitogen activated protein kinase kinase (MAPKK) pathways, respectively. Interleukin-1? (IL1?) was measured using ELISA. RESULTS: MSCs exposed to secreted factors present in conditioned media (CM) from FaDu, MDA-MB-231, PC-3 and NCI-H522, but not from MCF7 and HT-29, developed an elongated, spindle-shaped morphology with bipolar processes. In association with phenotypic changes, genome-wide gene expression and bioinformatics analysis revealed an enhanced pro-inflammatory response of those MSCs. Pharmacological inhibitions of FAK and MAPKK severely impaired the pro-inflammatory response of MSCs to tumor CM (approximately 80% to 99%, and 55% to 88% inhibition, respectively), while inhibition of the TGF? pathway was found to promote the pro-inflammatory response (approximately 3-fold increase). In addition, bioinformatics and pathway analysis of gene expression data from tumor cell lines combined with experimental validation revealed tumor-derived IL1? as one mediator of the pro-inflammatory phenotype observed in MSCs exposed to tumor CM. CONCLUSIONS: Our data revealed tumor-derived IL1? as one mediator of the pro-inflammatory response in MSCs exposed to tumor CM, which was found to be positively regulated by FAK and MAPK signaling and negatively regulated by TGF? signaling. Thus, our data support a model where MSCs could promote cancer progression through becoming pro-inflammatory cells within the cancer stroma.

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Proteomic Analysis of Exosomes Secreted from Human Alpha-1 Antitrypsin Overexpressing Mesenchymal Stromal Cells.

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