A Novel Localization in Human Large Extracellular Vesicles for the EGF-CFC Founder Member CRIPTO and Its Biological and Therapeutic Implications

Abstract

Tumor growth and metastasis strongly rely on cell–cell communication. One of the mechanisms by which tumor cells communicate involves the release and uptake of lipid membrane encapsulated particles full of bioactive molecules, called extracellular vesicles (EVs). EV exchange between cancer cells may induce phenotype changes in the recipient cells. Our work investigated the effect of EVs released by teratocarcinoma cells on glioblastoma (GBM) cells. EVs were isolated by differential centrifugation and analyzed through Western blot, nanoparticle tracking analysis, and electron microscopy. The effect of large EVs on GBM cells was tested through cell migration, proliferation, and drug-sensitivity assays, and resulted in a specific impairment in cell migration with no effects on proliferation and drug-sensitivity. Noticeably, we found the presence of the EGF-CFC founder member CRIPTO on both small and large EVs, in the latter case implicated in the EV-mediated negative regulation of GBM cell migration. Our data let us propose a novel route and function for CRIPTO during tumorigenesis, highlighting a complex scenario regulating its effect, and paving the way to novel strategies to control cell migration, to ultimately improve the prognosis and quality of life of GBM patients.

CRIPTO promotes extracellular vesicle uptake and activation of cancer associated fibroblasts

“…Recent studies have identified CRIPTO in tumor-derived EVs (TEVs) in several cancer types 20, 24 . To determine whether CRIPTO was likely to be present in EVs within the TME of breast cancers in our system, we reanalyzed MDA-MB-468 xenograft tumors for expression and localization of CRIPTO and the canonical EV marker CD81 25 .…”

“…Recent studies have identified CRIPTO in TEVs in several cancer types 18,22 . To determine whether CRIPTO was likely to be present in EVs within the TME of breast cancers in our system, we reanalyzed MDA-MB-468 xenograft tumors for expression and localization of CRIPTO and the canonical EV marker CD81 23 Next we asked whether CRIPTO overexpression could be leveraged to promote delivery of selected payloads to recipient cells.…”

Inflammatory, Oxidative Stress and Small Cellular Particle Response in HUVEC Induced by Debris from Endoprosthesis Processing

“…We have performed the isolation of SCPs from the media in which the cells had grown by differential ultracentrifugation. We have used a protocol that is common for harvesting small extracellular vesicles [58]. We have assessed samples by interference light microscopy and visualized them by cryogenic electron microscopy [59].…”

Assessment of Small Cellular Particles from Four Different Natural Sources and Liposomes by Interferometric Light Microscopy

“…SCPs were isolated by differential centrifugation, using a protocol widely used for the isolation of small extracellular vesicles [ 63 ]. Briefly, the cells were removed by low-speed centrifugation (300× g , 10 min, 4 °C, centrifuge Centric 260R with rotor RA 6/50 (Domel, Železniki, Slovenia)), using 50 mL conical centrifuge tubes (ref.…”