Quantitative proteomics identifies possible flow of metastatic cues between progressive stages of colorectal cancer via transfer of ceramide‐dependent exosomal cargoes

Cancer metastasis is largely influenced by cell-cell communication, to which exosomes play a vital role. Exosomes are small extracellular vesicles (sEVs) that originate as intraluminal vesicles (ILVs) within multivesicular bodies (MVBs) during endosome maturation. ILV formation depends on several pathways, including that of ceramide synthesis by neutral sphingomyelinase 2 [nSMase2]. Colorectal cancer (CRC)-derived sEVs are reported to carry a diverse range of metastatic cargo proteins; however, segregation of them in the ceramide-dependent sEV pool (sEV^Cer) remains unexplored. The current study aimed to identify the metastatic proteins that are secreted through sEV^Cer, from CRC cells of variable metastatic potentials. Primary (SW480) and metastatic (SW620) CRC cells were treated with nSMase2 blocker and sEVs were isolated, followed by extraction of the sEV proteins for a quantitative proteomic profiling using isobaric tags for relative and absolute quantitation (iTRAQ). In total, 1781 proteins were identified with unused protein score > 1.3. Of these identified proteins, 22.8% and 17.01% were found to be depleted within sEVs of the treated SW480 and SW620 cells, respectively. These depleted protein pools represented the cargo that are preferentially secreted through sEV^Cer in respective cell types (Cargo^Cer-SW480 and Cargo^Cer-SW620). Cargo^Cer-SW480 overrepresented integrin signaling pathway members and Cargo^Cer-SW620 overrepresented integrin as well as platelet-derived growth factor (PDGF) signaling pathway members. Interestingly, the uniquely overrepresented Cargo^Cer-SW480 and Cargo^Cer-SW620 were biologically connected, rendering possible transfer of metastatic cues via sEV^Cer. Overall, this study identified Cargo^Cer and their dynamics over progressive CRC stages, and thereby opens up a new research direction for exploring the flow of metastatic cues through uptake and release of sEV^Cer.