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

Abstract

Background: About 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.

Methods: CTC 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.

Results: EpCAM-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.

Conclusions: Our 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.

Figure 1

Immunoisolation of CTC in high-risk EC patients. (A) Representative primary carcinoma of a patient included in the study, demonstrating positivity for membrane EpCAM staining in epithelial tumor cells. (B) GAPDH expression levels normalized to CD45 in CTC isolated from the group of controls (white box, n = 27), as background of unspecific immunoisolation, and from the group of high-risk EC patients (grey box, n = 34) (Mann–Whitney test, ***p < 0.001). (C) CD45 expression levels in CTC isolated from controls and patients; similar expression denoted equivalent degree of unspecific immunoisolation.

Figure 2

Gene expression profiling in endometrial CTC. (A) Significant expression levels of genes involved in signaling pathways reported altered in EC, (B) hormone pathways and (C) stem cell features, in CTC from high-risk EC patients compared to the background of unspecific immunoisolation. White boxes represent the gene expression levels in the group of healthy controls, light grey boxes those corresponding to FIGO Stages I and II EC patients while dark grey boxes those corresponding to FIGO Stages III-IV EC patients and recurrences. (Kruskal-Wallis test, *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3

Plasticity phenotype characterizes CTC in EC. Almost all genes related to EMT assessed in CTC from high-risk EC patients presented significant expression compared to the background of unspecific immunoisolation. White boxes represent the gene expression levels in the group of healthy controls, light grey boxes those corresponding to FIGO Stages I and II EC patients while dark grey boxes those corresponding to FIGO Stages III-IV EC patients and recurrences. (Kruskal-Wallis test, *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4

ETV5 recapitulates the EMT phenotype found in CTC and the metastasis potential in an EC mouse model. (A) CTC-gene expression profiling in Hec1A and Hec1A-ETV5 cell lines by RT-qPCR. The results were represented as the fold change in gene expression relative to GAPDH gene expression (2^-ΔΔCt). (B) Representative luminiscence examples of athymic nude mice inoculated with Hec1A (upper panels) or Hec1A-ETV5 (lower panels) cells by intracardiac injection. Luminescence images were acquired for 1 min at ventral (left image) and dorsal (right image) positions. (C) Extent of dissemination evaluated as number of metastasis and (D) as luminescence quantification of metastasis.