Tumors are characterized by intratumoral heterogeneity that makes the development of new treatments difficult. Several studies suggest that exosomes may be an important microenvironmental factor affecting tumor heterogeneity. In this context our goal is to understand if exosomes derived from cell line with highly metastatic potential, may affect the behavior of less aggressive cells and the properties of tumor microenvironment. Our experimental strategy is based on the use of an isogenic model. We used SW480 and SW620 colon carcinoma cell lines derived from, respectively, primary and secondary tumors resected from a single patient, thus showing different metastatic potential. Specifically we evaluated, at morphological and functional level, how exosomes derived from highly metastatic cells influence the biological behavior of both less aggressive and endothelial cells. Finally we performed a proteomic characterization of exosomes by SWATH-MS method. We found that exosomes released by amoeboid SW620 cells are able to induce a mesenchymal-amoeboid transition in SW480 fibroblast-like cells. We observed that after treatment with SW620 exosomes, SW480 cells show non apoptotic membrane blebbing, associated with higher migratory and invasive capabilities. Moreover, the treatment of the HUVECs with SW620 exosomes induce the monolayer permeability, due to destabilization of endothelial junctional systems. Our data indicate that the effects of SW620 exosomes treatment is mediated by RhoA, responsible of cytoskeletal remodeling. Moreover, this data was strongly supported by quantitative proteomic analysis showing an enrichment in SW620 exosomes of RacGap1, activator of RhoA. We demonstrated that, through exosomes, highly metastatic cells spread their malignant behavior to less aggressive cells and affect tumor microenvironment.
I tumori sono caratterizzati da eterogeneità intratumorale e questo rende difficile lo sviluppo di nuovi trattamenti. Diversi studi indicano gli esosomi come importanti componenti che agiscono sulla eterogeneità tumorale. In questo contesto, il nostro scopo è di capire se gli esosomi derivanti da cellule ad alto potenziale metastatico possono influenzare il comportamento delle cellule tumorali meno aggressive e le proprietà dell’endotelio, promuovendo la disseminazione delle cellule tumorali. La nostra strategia sperimentale è basata sull’uso di un modello isogenico. Abbiamo usato cellule di carcinoma del colon, SW620 e SW480, isolate rispettivamente dal tumore primario e dal sito di metastasi dello stesso paziente. In particolare noi abbiamo valutato, a livello morfologico e funzionale, come gli esosomi derivanti dalla linea ad alto potenziale metastatico influenzino il comportamento biologico sia delle cellule meno aggressive che delle cellule endoteliali. Inoltre abbiamo condotto un’analisi proteomica SWATH-MS per caratterizzare gli esosomi . Abbiamo trovato che gli esosomi rilasciati dalle cellule ameboidi, SW620, sono capaci di indurre una transizione nelle SW480 fibroblastoidi, dal fenotipo mesenchimale a quello ameboide. Abbiamo osservato che dopo il trattamento con gli esosomi delle SW620, le cellule SW480 mostrano un blebbing non apoptotico, associato a una maggiore invasione e motilità delle cellule. Inoltre, il trattamento delle HUVECs con gli esosomi delle SW620 induce, rispetto al trattamento con gli esosomi delle SW480, una maggiore permeabilità dell’endotelio, dovuta alla destabilizzazione dei sistemi giunzionali delle cellule endoteliali. I nostri dati indicano che gli effetti del trattamento con gli esosomi derivanti dalla linea metastatica , sono mediati da RhoA, responsabile del rimodelllamento del citoscheletro. Inoltre, questo dato è stato fortemente supportato dai risultati di proteomica quantitativa che indicano un arricchimento di RacGap1, attivatore di RhoA, negli esosomi delle SW620. In conclusione, questo lavoro di tesi dimostra come, attraverso gli esosomi , le cellule ad alto potenziale metastatico possano influenzare il fenotipo delle cellule meno aggressive e del microambiente tumorale.
Schillaci, O.Metastatic colon cancer exosomes spread malignant properties in tumor microenvironment affecting the behavior of both tumor and endothelial cells.
Metastatic colon cancer exosomes spread malignant properties in tumor microenvironment affecting the behavior of both tumor and endothelial cells
SCHILLACI, Odessa
Abstract
Tumors are characterized by intratumoral heterogeneity that makes the development of new treatments difficult. Several studies suggest that exosomes may be an important microenvironmental factor affecting tumor heterogeneity. In this context our goal is to understand if exosomes derived from cell line with highly metastatic potential, may affect the behavior of less aggressive cells and the properties of tumor microenvironment. Our experimental strategy is based on the use of an isogenic model. We used SW480 and SW620 colon carcinoma cell lines derived from, respectively, primary and secondary tumors resected from a single patient, thus showing different metastatic potential. Specifically we evaluated, at morphological and functional level, how exosomes derived from highly metastatic cells influence the biological behavior of both less aggressive and endothelial cells. Finally we performed a proteomic characterization of exosomes by SWATH-MS method. We found that exosomes released by amoeboid SW620 cells are able to induce a mesenchymal-amoeboid transition in SW480 fibroblast-like cells. We observed that after treatment with SW620 exosomes, SW480 cells show non apoptotic membrane blebbing, associated with higher migratory and invasive capabilities. Moreover, the treatment of the HUVECs with SW620 exosomes induce the monolayer permeability, due to destabilization of endothelial junctional systems. Our data indicate that the effects of SW620 exosomes treatment is mediated by RhoA, responsible of cytoskeletal remodeling. Moreover, this data was strongly supported by quantitative proteomic analysis showing an enrichment in SW620 exosomes of RacGap1, activator of RhoA. We demonstrated that, through exosomes, highly metastatic cells spread their malignant behavior to less aggressive cells and affect tumor microenvironment.File | Dimensione | Formato | |
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