Research Projects

Intercellular communication via transfer of biological information through extracellular vesicles (EVs) plays crucial role in various physiological and pathological processes including cancer metastasis by modulating the tumor microenvironment. Exosomes are nanosized small extracellular vesicles (sEVs) of endocytic origin, the biogenesis of which involves formation of intraluminal vesicles (ILVs) during endosome maturation into multivesicular bodies (MVBs). MVBs eventually fuse with the plasma membrane, exposing the ILVs into extracellular space as exosomes. The process of cargo sorting during ILV biogenesis involves various pathways, of which the ceramide-dependent or neutral sphingomyelinase 2 (nSMase2)-mediated path remained poorly explored. nSMase2 hydrolyses sphingomyelin into ceramide which helps in ILV formation. A recent study reported that nSMase2 regulates exosome secretion by counteracting V-ATPase-mediated endosomal acidification and lysosomal degradation of MVB. However, metastatic cargo proteins that are preferentially secreted through this route is not explored particularly via any high-throughput investigation. Using iTRAQ-based proteomics (Ghosh et. al., 2024 The FEBS Jrnl., revision submitted), we attempted to explore the metastatic cargoes for their selective preferences towards ceramide-dependent exosomal route (sEV-cer) in the context of variable CRC stages. Primary (SW480) and metastatic (SW620) CRC cells were treated with specific nSMase2 blocker GW4869 and sEV proteins were subjected to quantitative proteomic profiling. 1781 sEV proteins were identified, of which 22.8% (406) and 17.01% (303) were found to be depleted in GW4869-treated sEVs from SW480 and SW620 cells respectively. These depleted proteins represent sEV-cer cargoes for respective cell types (Cargo-Cer-SW480 and Cargo-Cer-SW620). Cargo-Cer-SW480 overrepresented integrin signaling and Cargo-Cer-SW620 overrepresented integrin and PDGF signaling pathways. Interestingly the uniquely overrepresented Cargo-Cer-SW480 and Cargo-Cer-SW620 were biologically connected within integrin signaling pathway which is implicated in formation and sustenance of secondary metastatic niche. Proposed study will assess the protein level of these cargo-Cer in respective sEVs, followed by investigating their downstream signaling effect in cargo-Cer-recipient cells by in vitro co-culture and sEV treatment experiments. This will strengthen the hypothesis whether these cargo-Cer help to establish a secondary metastatic niche, via transfer of biological cues over variable metastatic stages in CRC. We further propose to explore a clinical correlation between the levels of these cargo-Cer and the blood cholesterol profiles of CRC patients, since higher blood (secreted) cholesterol is known to be associated with sEV-cer. Proven the hypothesis, proposed study will potentially contribute towards development of therapeutic strategies and prognostic test(s) for restraining CRC metastasis.