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Context Dependency of Epithelial-to-Mesenchymal Transition for Metastasis

par Revenco, Tatiana
Président du jury Remmelink, Myriam
Promoteur Blanpain, Cédric
Publication Non publié, 2022-03-17

Thèse de doctorat

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Résumé :

We questioned whether EMT is required for metastasis, and we have analyzed metastasis formation in two distinct SCC models, different in terms of SCC ability to undergo EMT. We demonstrated that in spontaneous mouse model in which tumors presented a wide range of EMT (from well differentiated to fully EMT-like) presented high metastatic incidence: 49% (20/70) of mice presented LN metastasis and 28% (34/70) of mice presented lung metastases. Importantly, metastatic incidence was always associated with EMT-like tumors. However, in the second mouse model, that develops mostly well-differentiated tumors, the metastatic incidence was very low: 12% (5/42) of mice developed lung metastases and 10% (4/42) developed lymph node metastases. Importantly, the rare metastases were associated with tumors presenting some degree of EMT, supporting that EMT is needed for metastases arising from skin SCC.To understand whether EMT can occur at the intermediate step between primary tumor and distant metastasis, we analyzed blood from mice based on YFP reporter. We uncovered that most CTCs were EMT-like: 80% of CTCs were EpCAM-negative. This finding suggests that EpCAM marker, a gold standard used to detect CTCs, is not optimal as it doesn’t detect CTCs of EMT phenotype thus underestimating the real number of CTCs. Importantly, we noticed that the presence of CTCs was always associated with lung metastases and indeed can be used to predict prognosis of oncologic disease. Our immunofluorescent analysis of lung and lymph nodes showed that the majority of metastases were of differentiated phenotype. For the lung metastases, 71% (36/51) of them presented epithelial phenotype YFP+/K14+/Vim-, 14% (7/51) presented differentiated epithelial features but did not express K14 or Vimentin, 12% (6/51) presented an EMT hybrid epithelial phenotype YFP+/K14+/Vim+, whereas only 4% (2/51) were purely mesenchymal YFP+/K14-/Vim+. Histological analyses were confirmed by FACS analysis showing that lung metastasis presented lower proportion of YFP+ EpCAM- as compared to the primary tumors and these EpCAM- TCs were mainly composed of early hybrid EMT cells (CD106-/CD51-/CD61-) rather than fully mesenchymal TCs (CD106+/CD51+/CD61+). For LN metastases: 60% (17/28) of metastases were YFP+/K14+/Vim-, 36% (11/28) were composed of hybrid EMT TCs as shown by the YFP+/K14+/Vim+ interspersed between differentiated metastatic cells. FACS analysis of LN metastasis showed lower proportion of YFP+ EpCAM- TCs, with a higher proportion of EpCAM- hybrid EMT TCs (CD106-/CD51-/CD61-). No metastases were composed of mesenchymal cells only. This result supports that MET is important for the metastatic outgrowth in host organs. We also addressed the question of EMT for metastases in transplantation model of skin SCC, where the cancer cells are dissociated from their microenvironment and are subcutaneously transplanted into immunodeficient mice. In sharp contrast to the requirement of EMT in spontaneous model, in our transplantation model we observed that EMT is dispensable for metastatic formation. These findings show that the role of EMT is context-dependent, such as immunity or tumor microenvironment could regulate EMT. Further investigation would be needed to evaluate the mechanisms regulating EMT in the primary tumors.However, as in the spontaneous model, in the transplanted model cancer cells should undergo MET to grow in distant organs. This observation suggests that organ’s microenvironment may play a role in regulating MET for metastatic outgrowth.