Poster Presentation 28th Annual Lorne Proteomics Symposium 2023

Shed microvesicles-derived from MDCK cells following oncogenic H-Ras transformed MDCK cells induce EMT in parental MDCK cells and elicit invasive properties to fibroblast (#176)

Adnan AS Shafiq 1
  1. La Trobe Institute for Molecular Science, Melbourne, VIC, Australia

Introduction
EMT is a process whereby cells transition from epithelial to mesenchymal state. EMT remodels the TME and facilitates invasion and metastasis by reprogramming soluble secretome and exosomes. However, the role of another major class of EVs called the shed microvesicles (sMVs) that arise from plasma membrane remains unknown. Using oncogenic HRAS transformed MDCK (21D1) cells as a model of EMT, we show that sMVs following oncogenic HRAS-mediated EMT are profoundly reprogrammed for their protein profile and function. A salient finding was upregulation of HRAS signal transduction pathway (HRAS, ARAF, phosphorylated MAPK), proteins implicated in invasion and metastasis (ternary complex of FN1, ITGA6/B1, TGM2), and pre-metastatic niche factors (S100A4, S100A9), FN1, TIMP1, CD44), along with EMT hallmark proteins, in sMVs following HRAS-mediated EMT.
Methods
We describe a large-scale production of EVs (sMVs and Exos) (up to 3. 8 mgs) from MDCK and HRAS(G12V) transformed MDCK (21D1 cells) cells using CELLine AD-1000 Bioreactor classic flasks to enable their in-depth protein profiling and functional study. Label-free quantitative mass spectrometry-based protein profiles of MDCK and 21D1-derived sMVs.
Results
Label-free quantitative mass spectrometry-based protein profiles of MDCK and 21D1-derived sMVs revealed that following EMT, cells release sMVs whose protein profile is profoundly reprogrammed. Specifically, we show that reprogrammed sMVs contain mesenchymal proteins implicated in EMT. In addition to that proteomic profiling of 21D1-sMVs and-Exos also show the significant differences in their protein cargo and their uptake conferring fibroblast with invasive capacity.
Conclusion
Our data suggest that sMVs can potentially propagate oncogenic signs within the TME. It is conceivable that sMVs released by cancer cells undergoing EMT potentially activates fibroblast invasion into an extracellular matrix that in turn facilitates their own invasion into surrounding tissues. This study provides molecular insights into sMVs released by cancer cells undergoing HRAS-mediated EMT, a process that is critical to invasion and metastasis.