Molecular profiling of circulating tumor cells links plasticity to the metastatic process in endometrial cancer

Abstract

BackgroundAbout 20% of patients diagnosed with endometrial cancer (EC) are considered high-risk with unfavorable prognosis. In the framework of the European Network for Individualized Treatment in EC (ENITEC), we investigated the presence and phenotypic features of Circulating Tumor Cells (CTC) in high-risk EC patients.MethodsCTC isolation was carried out in peripheral blood samples from 34 patients, ranging from Grade 3 Stage IB to Stage IV carcinomas and recurrences, and 27 healthy controls using two methodologies. Samples were subjected to EpCAM-based immunoisolation using the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal) followed by RTqPCR analysis. The phenotypic determinants of endometrial CTC in terms of pathogenesis, hormone receptor pathways, stem cell markers and epithelial to mesenchymal transition (EMT) drivers were asked. Kruskal-Wallis analysis followed by Dunn’s post-test was used for comparisons between groups. Statistical significance was set at p < 0.05.ResultsEpCAM-based immunoisolation positively detected CTC in high-risk endometrial cancer patients. CTC characterization indicated a remarkable plasticity phenotype defined by the expression of the EMT markers ETV5, NOTCH1, SNAI1, TGFB1, ZEB1 and ZEB2. In addition, the expression of ALDH and CD44 pointed to an association with stemness, while the expression of CTNNB1, STS, GDF15, RELA, RUNX1, BRAF and PIK3CA suggested potential therapeutic targets. We further recapitulated the EMT phenotype found in endometrial CTC through the up-regulation of ETV5 in an EC cell line, and validated in an animal model of systemic dissemination the propensity of these CTC in the accomplishment of metastasis.ConclusionsOur results associate the presence of CTC with high-risk EC. Gene-expression profiling characterized a CTC-plasticity phenotype with stemness and EMT features. We finally recapitulated this CTC-phenotype by over-expressing ETV5 in the EC cell line Hec1A and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model of CTC dissemination and homing.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-223) contains supplementary material, which is available to authorized users.

The Landscape of Circulating Tumor Cell Research in the Context of Epithelial-Mesenchymal Transition

“…Importantly, mesenchymal CTCs allowed a more accurate prediction of prognosis than the expression of epithelial markers alone [4] . Clinical significance of mesenchymal CTCs was examined not only in BC, but also in other cancers, including colorectal [43,132] , hepatocellular [133,134] , gastric [135] , prostate [41,136] , bladder [137] , ovarian [138] , endometrial [139] , and non-small cell lung cancer [140] . The majority of data concern the metastatic setting.…”

Liver Metastasis Is Facilitated by the Adherence of Circulating Tumor Cells to Vascular Fibronectin Deposits

“…Orthotopic, intracardiac, and intrasplenic mouse models were generated as described previously (16)(17)(18). Experimental details are included in the Supplementary Methods.…”

Quantitative PCR marker genes for endometrial adenocarcinoma

“…To the best of our knowledge, there are only a few studies describing the presence of CTCs in patients with endometrial adenocarcinoma. In particular, a high-risk group of patients with high-grade endometrial adenocarcinomas (24), demonstrated a correlation of the occurrence of CTCs with stemness was demonstrated in a previous study and CTCs were recognized as possible therapeutic targets (25).…”