Senescence‐Associated Exosome Exchange Activates Myofibroblast Phenotype in Mesenchymal Stem Cells

Abstract

Introduction Bone marrow derived mesenchymal stem cells (MSCs) are recruited and accumulate in primary tumors due to their natural tropism for inflammatory tissues. MSCs can undergo senescence as a result of ionizing radiation therapy. Senescent cells are often resistant to apoptosis and rapidly develop a senescence-associated secretory phenotype (SASP), which is linked to increased secretion of pro-inflammatory cytokines and matrix degradation enzymes. The potency of inflammatory SASP factors is widely accepted in many tumor models. Senescence also affects the number and content of secreted exosomes. Exosomes are nanovesicles that can transfer important protein, RNA, and miRNA during cell-cell communication. Previous studies have also shown that senescence-associated exosomes (SA-EXOs) can serve as key mediators of disease progression. The objective of this study is to understand how radiation-induced senescent cells and SA-EXOs induce myofibroblast phenotypes in neighboring stromal cells. We hypothesize that SA-EXOs play critical roles in cell-cell communication to promote persistent fibroblast activation that drives local matrix remodeling for subsequent tumor metastasis. Methods MSCs from female donors were treated with gamma-irradiation of 15Gy and senescence was confirmed 10 days later based on positive staining for β-Galactosidase and increased gene expression of senescent markers (Fig 1a-b). All exosome populations were harvested using standard ultracentrifugation methods. miRNA microarrays were carried out according to the manufacturer's published protocol. Results Our proteomics data showed that proteins crucial in exosome secretion and biogenesis pathways were elevated in senescent MSCs compared to normal MSCs. This was further validated using a gene expression panel that probed for markers of the same pathways (Fig 2a-b). Dynamic Light Scattering techniques revealed that senescent MSCs secreted 2-fold more exosomes than normal MSCs (Fig 3). Exosome miRNA microarrays showed 27 differentially expressed miRNAs (non-SA-EXOs vs. SA-EXOs)—22 upregulated and 5 downregulated. Many of the miRNAs were involved in mechanosensitive pathways. Finally, we treated normal MSCs with SA-EXOs and stained for alpha smooth muscle actin (α-SMA) and ki-67. α-SMA is a protein that is elevated in senescence and myofibroblasts; ki-67 is a proliferative marker. SA-EXOs increased α-SMA and ki-67 expression in normal MSCs (Fig 4) compared to control groups. Discussion/Conclusions We showed that radiation-induced senescent MSCs secreted elevated amounts of SA-EXOs that possess unique miRNA content and that SA-EXO exchange with neighboring MSCs increased α-SMA. This highlights that these exosomes are important SASP factors that can induce myofibroblast phenotypes, which can play a large role in promoting tumor progression.