Candidate biomarkers of transformed mesenchymal stromal/stem cells by quantitative proteomics and glycoproteomics

Multipotent mesenchymal stromal/stem cells (MSCs) are a key component of the tumour environment and play a role in enabling tumour growth. Genetically transformed human MSCs (hMSCs) may represent the initial cell of sarcoma development. There are currently no reliable methods for discriminating between normal and tumour-promoting hMSCs. Here we aimed at identifying candidate protein biomarkers that are specific to normal and tumourigenic hMSCs and could serve to distinguish both classes. We used transformed hMSC cultures (n=3) and directly compared their membrane protein profile to normal hMSC cultures (n=3) using multiplexing quantitative proteomics (i.e., all proteins) and glycoproteomics (i.e., only glycosylated proteins). Mass spectrometry analysis was conducted to identify and quantify the (glyco)proteins and ingenuity pathway analysis was used to analyse signaling pathways shown to be enriched by altered (glyco)proteins. Quantitative proteomic analysis of membrane proteins identified 2700 proteins and 450 glycoproteins common to all 6 hMSC cultures. Of the 2700 proteins identified, 252 were upregulated in transformed cells and 202 were downregulated. Of the 450 glycoproteins identified, 309 were upregulated in transformed cells and 39 were downregulated, where preliminary examination showed specific glycoprotein glycoform alterations between hMSC classes. Cellular pathway analysis of altered proteins in transformed hMSCs showed activated oncostatin M, NRF2-mediated oxidative stress response and paxilin signaling pathways, as well as repressed integrin, IL-8 and glioma invasiveness signaling pathways. Pathway analysis of altered glycoproteins showed activated Rac signaling and agrin interactions at neuromuscular junction signaling pathways, as well as repressed PTEN and Rho family GTPases signaling pathways. This study yielded potential cell-surface membrane biomarkers that are specific to normal and tumourigenic hMSCs that could serve to distinguish each class, as well as non-nuclear membrane and luminal organelle proteins that may shed further light on the biological mechanisms altered in the transformed hMSCs.