ABSTRACT: IFN gamma induced changes in MSC secretome. Secretome was measured by nanoLCMSMS ising a velos pro and the intracellular proteome via a QExactive via TMT labeling of both.
INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606) Rattus (ncbitaxon:10114)
Project description:G-protein coupled receptors (GPCRs) have diverse roles in physiological processes, including immunity. Gs-coupled GPCRs increase while Gi-coupled ones decrease intracellular cAMP. Previous studies suggest that, in epithelial cells, Gs-coupled GPCRs enhance whereas Gi-coupled GPCRs suppress pro-inflammatory immune responses. In order to examine the issue, we chose beta2 adrenergic receptor and GPR40 as representatives of Gs- and Gi- coupled GPCRs, respectively, and examined their effects on TNF-alpha and IFN-gamma-(TNF-alpha + IFN-gamma) induced gene expression by HaCaT. We used microarrays to detail the global changes of gene expression induced by a beta2 adrenergic receptor agonist terbutaline or GPR40 agonist GW9508 pre-treatment in TNF-alpha + IFN-gamma - stimulated HaCaT cells. HaCaT cells were pre-treated with terbutaline or GW9508, TNF-alpha + IFN-gamma were then added, and cultured for another 24 h. Cells were then used for RNA extraction and hybridization on Affymetrix microarrays. We sought to clarify changes in gene expression after 1) TNF-alpha + IFN-gamma, 2) TNF-alpha + IFN-gamma + terbutaline, and 3) TNF-alpha + IFN-gamma + GW9508 treatment. To this end, we set 4 groups of samples; 1) unstimulated group, 2) TNF-alpha + IFN-gamma-stimulated group, 3) TNF-alpha + IFN-gamma + terbutaline-stimulated group, and 4) TNF-alpha + IFN-gamma + GW9508-stimulated group. In each group, HaCaT cells were stimulated in triplicate wells (n=3).
Project description:Background: Glioblastoma (GBM) is an aggressive primary brain tumor with poor prognosis and limited treatments. Interferon-gamma (IFN-γ) plays a complex role in modulating immune and tumor responses, and its long-term impact on GBM remains unclear. Methods: Human GBM cell lines U87 and U251 were subjected to up to 28 days of chronic IFN-γ exposure and assessed for morphology, gene expression, protein dynamics, and cytokine secretion. RNA sequencing, automated Western blotting, and single-cell proteomics were employed to uncover distinct cellular and molecular adaptations to prolonged IFN-γ signaling. Results: Chronic IFN-γ exposure induced significantly divergent phenotypic changes in U87 and U251 cells. U87 cells formed neurosphere-like structures and upregulated STAT1/STAT3 signaling, suggesting an immunogenic response. In contrast, U251 cells displayed elongated morphologies and activated PI3K-Akt and Erk pathways, promoting survival and immune evasion. Differential gene expression analysis highlighted unique temporal profiles, with U251 cells exhibiting higher expression of immune resistance genes such as PD-L1 and VEGF. Secretome profiling further demonstrated contrasting cytokine landscapes: U87 cells secreted pro-inflammatory cytokines, while U251 cells produced immunosuppressive factors. Conclusions: Chronic IFN-γ exposure drives distinct molecular and phenotypic changes in GBM subtypes, reflecting their intrinsic heterogeneity. While IFN-γ enhances immune activation, prolonged exposure may also promote immune escape. These findings emphasize the need for therapeutic strategies combining IFN-γ with immune checkpoint inhibitors and targeted pathway modulation to optimize GBM treatment outcomes.
Project description:Cyclic regeneration of the endometrium, and its repair after parturition or injury, are crucial for successful reproduction. Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSC) facilitate tissue repair via their secretome, which contains growth factors and cytokines that promote wound healing. Despite the implication of MSCs in endometrial regeneration and repair, the mechanisms remain unclear. This study tested the hypothesis that the secretome of MSCs from human BM upregulates human endometrial stromal cell (HESC) proliferation, migration and invasion, and activates pathways to increase HESC motility. MSCs were purchased from ATCC (BM-MSC-1) and cultured from the BM aspirate of a healthy female donor (BM-MSC-2). Indirect co-culture of MSCs and hTERT-immortalized HESCs via a transwell system studied the effect of the BM-MSC secretome on HESC proliferation, migration, and invasion. To study the effect of the MSC secretome on HESC gene expression, HESCs were exposed to the BM-MSC secretome via indirect co-culture for 24 h. Total RNA was extracted from HESCs for RNA sequencing (RNA-Seq). Differentially expressed genes (DEG) and significantly altered pathways were identified. Indirect co-culture of HESCs with BM- MSCs resulted in significant increase in HESC migration and invasion regardless of the source of MSCs. Effects on cellular proliferation varied among the BM-MSC source. Exposure of HESCs to the secretome of BM-MSCs changed the expression of 10,141 genes with FDR < 0.05. There was overlap among 4351 genes between HESCs exposed to BM-MSC-1 and BM-MSC-2, including upregulated expression of cell motility genes common to both BM-MSC exposures. Increased HESC motility by the secretome of BM-MSC appears to be mediated by paracrine and autocrine mechanisms, in part by modifying HESC gene expression. These data support the potential for leveraging the MSC secretome as a novel cell-free therapy in the treatment of disorders of endometrial regeneration.
Project description:The secretome of mesenchymal stromal cells (MSCs) can efficiently stimulate regeneration and therefore is a tempting remedy for “cell-free cellular therapy”. However, the usage of primary MSC cultures as secretome-producers for translation studies has obvious obstacles, including rapid aging of MSC cultures, the need for a large number of verified donors and donor-to-donor variability of secretome content. MSCs immortalization allows to overcome those limitations and to obtain secretome-producing cultures with prolonged lifetime. However, the efficacy and safety of such secretomes are critical issues, which limit their usage as therapeutic agents. In this study we have tested in large detail how the immortalization of MSC cultures affects the content, biological activity and safety of their secretome. MSCs immortalization via overexpression of human TERT gene does not significantly alter the qualitative and quantitative composition of their secretome or its activity according to the results of proteomic analysis, ELISA, qPCR and functional tests in vitro. Moreover, we have demonstrated that the secretome of immortalized MSCs does not contain detectable amounts of telomerase and does not possess any transforming activity. Altogether, our data suggest that immortalized MSC cultures may become a reliable source for obtaining standardized active secretome in large-scale quantities for clinical use.
Project description:Acute myeloid leukemia (AML) cells can shape their niche to their own advantage, perturbing bone marrow stromal and immune landscape. Indeed, AML cells provide the signals, among which inflammatory mediators are crucial, since they are able to subvert mesenchymal stromal cell (MSC) funtions. In particular, IFN-γhigh AML cells hold an inflammatory/immune modulating signature distinct from IFN-γlow cases. We analyzed changes in the gene expression profile of MSCs induced by co-culture with AML cells in vitro. IFN-γhigh but not IFN-γlow AML cells profoundly subverted the MSC transcriptome by inducing immune-modulating pathways, which, intriguingly, included IFN-γ-dependent genes related to regulatory T cell (Treg) differentiation and immune suppression.
Project description:Expression data from HT-29 human colon adenocarcinoma cells treated with IFN-γ for 24 hr Total RNA was isolated from HT-29 cells after 24h stimulation with 200 U ml-1 IFN-γ (Roche). The experiment was done on three biological replicates.
Project description:Transcriptional and lncRNA profiling of human embryonic stem cells derived mesenchymal stem cells within or without IFN-γ treatment