Oncogenic H-Ras Reprograms Madin-Darby Canine Kidney (MDCK) Cell-derived Midbody Remnant Proteins Following Epithelial-Mesenchymal Transition

Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process that allows highly polarized, immotile epithelial cells to transform into motile mesenchymal cells: it is a fundamental cellular process involved in embryonic development, tumour cell metastasis, organ fibrosis and tissue regeneration. To assess the contribution of secreted midbody remnants (MBRs) - a new class of membranous extracellular vesicle (EV) molecularly distinct from exosomes/small EVs - to the EMT process, we conducted a proteomic analysis of MBRs released from Madin-Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). MBRs were harvested from cell culture media in milligram quantities using a continuous culture bioreactor device and purified using sequential centrifugation and buoyant density gradient centrifugation (OptiPrep). Gel-MS/MS protein profiling showed MDCK cell-MBRs reflect their epithelial origin (e.g., enriched CDH1, DSP, THBS1, OLCN, EPCAM proteins) and 21D1 cell-MBRs their mesenchymal phenotype (e.g., HRAS, VIM, MMP14, CDH2, WNT5A and enriched invasive and cell motility proteins). Prominent findings were the unique expression of the immune checkpoint protein NT5E/CD73 (ecto-5′-nucleotidase), and ser/thr kinases LIMK1/K2 in 21D1-MBRs (not present in MDCK cell-MBRs), and enrichment in Wnt signalling network proteins. Collectively, our findings suggest MBRs might play a previously unrecognized role in the EMT process.