Abstract

Tetraspanins are highly conserved transmembrane proteins that interact with various partner molecules to form tetraspanin-enriched microdomains. These domains influence extracellular vesicle (EV) biogenesis, cargo sorting, and cellular uptake. Although CD9, CD63, and CD81 are commonly known as their markers, it remains unclear whether they perform similar functions or if each uniquely influences EVs proteome and lipidome differently. This study utilizes proteomic and lipidomic profiling of CD9, CD63, and CD81-deficient DU145 prostate cancer cells. Our results demonstrate that the absence of these tetraspanins leads to specific molecular alterations in cells and as previously observed in extracellular vesicles. Comparative proteomics identify CD81 as a primary regulator of protein selection during EV biogenesis. Across all knockout models, the proteome shifted toward an invasive phenotype, noted by the upregulation of Epithelial-to-Mesenchymal related proteins and the downregulation of adhesion molecules. Additionally, lipidomic analysis via HPLC-MS/MS revealed significant alterations, primarily within the glycosphingolipid class. This analysis provides new insights into how individual tetraspanins influence cargo selection and their composition in this cellular model. …víceméně