A comparative study of inhibitors targeting extracellular vesicle biogenesis
Background
Tumor cells release high numbers of extracellular vesicles (EVs) which play a critical role in tumor progression. Using ultracentrifugation, three different EV populations can be purified that differ in size and cargo: large oncosomes (LOs), large EVs (lEVs) and small EVs (sEVs). However, markers for the different populations, especially lEVs and LOs, are scarce and their biogenesis mechanisms remain largely unexplored.
Methods
EVs from MCF-7 (breast cancer), HT-29 (colorectal cancer) and H1975 (lung cancer) cells were isolated by differential ultracentrifugation and characterized by electron microscopy, NTA and mass spectrometry. Identified markers were validated by immunoblotting and ten inhibitors selected that potentially target these factors. Their maximum tolerated doses were determined in the three cancer cell lines using MTT- and AnnexinV/7-AAD-based viability assays. Changes in EV secretion and cargo upon inhibitor treatment were analyzed by NTA, Lowry assay and immunoblotting.
Results
Proteomics revealed distinct signatures for sEVs and lEVs, with lEVs showing an enrichment of cytoskeleton-associated proteins. Consequently, ten chemical inhibitors mainly affecting this compartment were selected and tested for their effect on the secretion of the distinct EV subpopulations. None of the inhibitors showed major effects on EV size, but changes were observed particularly in LO and lEV numbers. In terms of EV cargo, not only the expression of certain EV marker proteins was altered, but the total protein amount loaded onto LOs and lEVs was either diminished, or increased. Overall, inhibitors targeting Ras/Raf/ERK signalling, HDAC6 or ARF6 provoked the most significant differences in the secretion of lEVs and LOs, suggesting their involvement in the biogenesis of these EV subpopulations.
Conclusion
This study identified fundamental and cell-line specific differences in the biogenesis of all three investigated EV subpopulations. Although some inhibitors distinctly altered the number and/or cargo of the EVs, identifying general inhibitors of EV secretion remains a challenge.
Keywords
inhibitors, large extracellular vesicles, EV biogenesis, proteomics
Funding/Acknowledgments
This work was funded by the Wilhelm-Sander Foundation (project 2022.139.1). We would like to thank Annette Westermann and Buket Celik for their excellent technical support.
Authors
Allegra Angenendt1,2 (Corresponding Author: Allegra.Angenendt[at]ukmuenster[dot]de), Janes Efing1,2, Uwe Hansen3, Jürgen Eirich4, Iris Finkemeier4, Annalen Bleckmann1,2, Kerstin Menck1,2