Introduction
Endometrial carcinomas, the most common tumors of the female genital tract, are usually diagnosed at an early stage with uterine-confined disease and an overall favorable prognosis. However, up to 20% of endometrial carcinomas present as aggressive neoplasms such as high-grade or deeply invasive lesions, at substantial risk of recurrence and death[
1]. Propagation of aggressive tumor cells from the primary lesion is a key event in the process of metastasis and a challenge in oncology. In endometrial cancer, myometrial infiltration, lymph node involvement, and lymphovascular space invasion are current clinical parameters defining the probability of recurrent disease. Nevertheless, early dissemination of tumor cells is usually undetectable in patients by conventional histopathological examination or by standard imaging techniques. Recently, immunocytochemical and molecular assays have been developed for the specific detection of metastatic tumor cells at a cellular level in lymph nodes, peripheral blood or bone marrow, prior to the manifestation of metastasis. Tumor-cell dissemination can proceed at an early stage of tumor development[
2], and detecting circulating tumor cells (CTC) has clinical value in the monitoring and the outcome of metastatic disease. CTC analysis represents an attractive candidate for liquid biopsy in cancer[
3]. Clinically, the presence of CTC above a threshold may have a significant adverse impact on survival. Likewise, changes on CTC quantification during treatment can reflect prognostic significance, the future challenge being whether treatment decision-making should be impacted by CTC levels[
4].
The increasing interest in CTC at the clinical setting is resulting in the development of a number of innovative technologies that include immunoenrichment, microfluidics and filtration devices, combined with semiautomated microscopy or PCR-based detection systems[
5]. We have recently demonstrated that the combination of CTC EpCAM-based immunoisolation, followed by accurate extraction and pre-amplification of RNA from very small number of CTC, provided with a highly sensitive approach to profiling the metastatic tumor cell population in a group of colorectal cancer patients[
6]. In the present study, we adopted a similar approach in high-risk EC patients. CTC immunoisolation plus profiling of a number of genes related to key events in the process of metastasis in EC provided us with an overview of the biology of endometrial CTC. In addition to analyze in immunoisolated CTC the expression of a number of genes involved in signaling pathways related to EC, hormone pathways, stem cell features and epithelial to mesenchymal transition (EMT) markers, we evaluated the efficiency of CTC quantification and its correlation with clinical parameters.
Discussion
We present in this study evidences for the presence of CTC in high-risk EC patients, and further characterized a molecular CTC-phenotype associated with plasticity and stemness features. The major clinical relevance of CTC is that the early detection in patients could be of use for the identification of candidate subjects needing additional systemic therapies after the resection of the primary tumor. Although the aim of these therapies is the prevention of metastasis, the selection of patients is nowadays based on the statistical risk of recurrences, which is not accurate in terms of over-treatment of patients with toxic agents or therapeutic procedures causing serious side effects, in addition to economic costs. An improved ability to target surgical and systemic therapies to well selected high-risk patient populations will increase the likelihood of benefits and decrease the side effects associated with un-necessary treatments[
13]. In addition, the sequential assessment of CTC levels during treatment could provide information at early stages about the therapeutic efficacy of drugs. Likewise, the elimination of these CTC could represent an intermediate endpoint in clinical trials with antitumor drugs. Furthermore, the molecular and functional characterization of CTC will be determinant in the discovery of new molecular tumor markers and in the development of therapies specific for the process of tumor dissemination and metastasis. Overall, CTC represent a potent and promising tool in oncology and, although the clinical role of CTC as a prognosis factor is being recognized, there is still a need for more advanced and precise techniques of detection and robust clinic-pathologic correlations[
14].
From our results, we can conclude that EpCAM-based immune-enrichment followed by RT-qPCR analysis is a reliable method to effectively isolate CTC from high-risk EC patients and to potentially distinguish high-risk from low-risk patients. Previous efforts to analyze and evaluate CTC in EC included the assessment of a six gene panel in blood samples[
15]. Although this methodology represents a semi-quantitative evaluation of CTC, it demonstrated an added value compared to the gold standard CellSearch technology for the positive detection of CTC (see Additional file
1). In addition to the clinical utility of CTC as a surrogate marker in the management of high-risk and metastatic cancer patients, the challenge stands on the possibility of a therapeutic approach targeting these metastatic CTC with the aim of controlling and/or eradicating the source of recurrences. The advantage of combining CTC immunoisolation and RT-qPCR analysis consists in the possibility to identify and characterize biomarkers specific of this subpopulation of metastatic cells. This approach has the limitation to determine whether the increased levels in biomarker expression upon CTC immunoisolation is related to an enhanced expression or to an augmentation in the number of CTC. Nevertheless, in addition to their potential in terms of diagnosis/prognosis and follow-up of patients, these biomarkers provide with phenotypic clues on the biology of CTC that may be determinant in the identification of new therapeutic strategies aiming to specifically control and/or eradicate the metastatic dissemination in EC[
16]. From a clinical perspective, the management of metastatic EC has been recently considered as a poly-chemotherapy adjuvant regimen for patients with high risk of relapse, and as a palliative regimen for patients with disseminated disease or with extrapelvic recurrence not responding to hormone treatment. In addition, endometrial carcinomas are considered as chemoresistant and the most active drugs (platinum salts, doxorubicin, anthracyclines and paclitaxel) present relative rate responses ranging from 25% in monotherapy to 57% in poly-chemotherapy, with median survival of 12–15 months. In this scenario, chemotherapy has shown limited utility and there is a clear need for the development of new rationale therapies focused on metastasis[
17]. To this regard, our profiling of endometrial CTC has pointed out at a number of pathways relevant to the metastatic process and that could be targeted. For instance, the expression of
STS, which is associated with higher availability of estrogens in tumor cells[
18], can provide a growth advantage to CTC and its inhibition can be used to block such event and decrease the risk of recurrences. A second important marker highly expressed in CTC was
PIK3CA, which confirms the role of this kinase pathway as a potential target in high-risk and metastatic disease[
19‐
21].
A main feature observed in the molecular profiling of CTC in EC corresponded to the EMT phenotype, with almost all analyzed genes related to plasticity being significantly expressed in CTC (
ETV5, NOTCH1, SNAI1, TGFB1, ZEB1, ZEB2). EMT is a dynamic process whereby epithelial cells lose polarity and cell-cell contacts, undergo dramatic remodeling of the cytoskeleton, acquire a migratory phenotype and a mesenchymal-like gene expression program. Both invasion and metastasis may be critically dependent on the acquisition by the incipient cancer cell of EMT features[
22,
23]. Interestingly, both EMT and the PIK3CA pathway have been closely linked in the promotion of metastasis, particularly in EC[
24,
25]. Our results also reinforce a role for ETV5 in the process of EMT and EC dissemination[
11]. ETV5 up-regulation in Hec1A cells recapitulated in vitro the plasticity phenotype found in high-risk patients, and demonstrated an advantage in the promotion of metastasis in an in vivo mouse model that mimic CTC dissemination and homing.
Likewise, and concerning
ALDH and
CD44 as stem-cell genes identified in this profiling, we observed a concordance between the presence of endometrial CTC and recurrent disease. Whether these CTC include a subpopulation of tumor cells with a stem cell-like phenotype (Cancer Initiating Cells[
26]) or whole CTC population must be considered responsible for recurrences with more or less efficiency, has yet to be addressed. In addition to its plasticity phenotype, CTC triggering micrometastasis in the target organs must own the capacity to survive in the blood flow, to home and to regenerate a tumor mass with similar characteristics as the primary lesion in the tissue recipient of metastasis. Recent studies on CTC in breast cancer have demonstrated that a subset of isolated CTC express stem cell markers such as those analysed in our study[
27,
28]. The evidence that tumor dissemination to the blood circulation is an early event and that the process of metastasis is an ineffective process with only a small number of CTC ending up in micrometastasis, support the hypothesis of a CTC phenotype with plasticity and stemness features with the capacity to develop metastasis[
29]. This concept linking EMT and stem cell features in the process of tumor dissemination has also been addressed in EC[
30], in association with a micro-RNA signature of EMT in endometrial carcinosarcoma mainly represented by the down-regulation of members of the miR-200 family[
31]. Remarkably, the balanced expression of ZEB factors and miR-200 is considered as a molecular motor of cellular plasticity, in particular is a driving force for cancer progression towards metastasis by controlling the state of cancer stem cells[
32,
33]. The results we obtained both in CTC and in paired carcinoma and lymph-node tissue samples demonstrating the potency of
ZEB2 within the CTC-phenotype reinforce the need of future investigations examining stem-like features in CTC to obtain relevant information about this cancer subpopulation responsible of metastases.
Finally, it should be note that although EpCAM expression was found consistent in primary endometrial carcinomas, its proposed modulation and eventual loss during EMT adds controversy to the efficiency of enrichment of CTC owning a plasticity phenotype[
34]. To this regard, we analyzed the expression of EpCAM both in the epithelial endometrial cancer cell line HEC1A and its mesenchymal counterpart Hec1A-ETV5, and found similar levels of EpCAM expression irrespective of their EMT phenotype (Additional file
5). Moreover, EpCAM-based immunoisolation of these HEC1A and HEC1A-ETV5 cells lines rendered similar efficiencies (75% versus 64%, respectively). From these results, it seems reasonable to speculate that cells detaching from the primary lesion and incorporating into the blood stream recapitulate a metastable epithelial–mesenchymal phenotype that may be maintained during their way to those distant sites where this CTC will home and end up in the generation of micrometastasis. The dissociation of tumor cells from the epithelial layer and the penetration through the basement membrane into the adjacent connective tissue, are the initial events in the multistep process that characterizes metastasis[
35]. We are additionally conducting further studies with other immunoisolating antigens.
Methods
Patient samples
Peripheral blood samples collected just before initiation of treatment from 34 EC patients and 27 controls were processed for CTC immunoisolation and accurate RNA extraction as described[
7]. Patients participating in the study were surgically staged according to FIGO and recruited between March 2012 - October 2013 in Vall d’Hebron University Hospital (Barcelona, Spain), University Hospital of Santiago de Compostela (Santiago de Compostela, Spain), Arnau de Vilanova Hospital (Lleida, Spain), MD-Anderson Cancer Center Madrid (Madrid, Spain), Fundacion Dexeus (Barcelona, Spain) and Haukeland University Hospital (Bergen, Norway), and included high-risk endometrial carcinomas ranging from Grade 3 Stage IB carcinomas to metastatic Stage IV carcinomas and recurrences (Table
1). Control group included a set of 27 healthy women with absence of a previous cancer episode and with an age range similar to patients. Informed consent approved by the relevant ethical committee was signed by all patients. In addition, fresh-frozen tissue from primary tumor and paired affected lymphatic nodes from 6 EC patients were provided by Tumor Bank of the Vall d’Hebron University Hospital Biobank (Barcelona, Spain) with appropriate ethics approval.
EpCAM immunohistochemistry
EpCAM expression was checked in whole paraffin-embedded sections of primary endometrial carcinomas from patients prospectively subjected to evaluation of CTC. Sections were dried for 1 h at 65°C before pre-treatment procedure of deparaffinization, rehydration and epitope retrieval in the Pre-Treatment Module, PT-LINK (DAKO) at 95°C for 20 min in Citrate buffer (10 mM), Low pH, endogenous peroxidase was blocked before staining with antibodies against Epithelial Related Antigen (clone MOC-31, dil. 1:50; DAKO, Denmark). After incubation, the reaction was visualized with the EnVision FLEX Detection Kit (DAKO) using diaminobenzidine chromogen as a substrate.
Positivity ranged from 75% to 100% with a mean of 93%.
CTC immunoisolation and quantitative real-time polymerase chain reaction (RT-qPCR)
CTC immunoisolation with the CELLection™ Epithelial Enrich kit (Invitrogen, Dynal, Oslo, Norway), RNA extraction and RT-qPCR were carried out as previously described[
7]. After EpCAM-based immune-enrichment of CTC following manufacturer’s protocol, RNA purification was performed with QiampViral kit (Qiagen, Valencia, CA, USA), optimized for very low cellularity samples. cDNA was synthesized using SuperScriptIII chemistry (Invitrogen, Carlsbad, CA, USA) according to the user’s guide and subjected to pre-amplification with TaqMan®PreAmp Master Mix kit (Applied Biosystems, Foster City, CA, USA) for 14 reaction cycles before proceeding to RTqPCR, to provide with optimal detection rates. TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA, USA) for 35 selected genes (Additional file
2; plus
GAPDH as housekeeping gene and
CD45 as a marker of non-specific isolation) were used to measure the gene expression levels in CTC isolated from patients in comparison to the background of hematogenous cells unspecifically immunoisolated from the group of healthy controls. Values were analyzed using StepOne Software v.2.1 (Applied Biosystems, Foster City, CA, USA), normalized to
CD45 and represented as (40–ΔCt), whereby ΔCt = duplicate mean (CtTARGET – Ct
CD45).
Cell lines and cell culture
The human endometrial carcinoma cell lines Hec1A and Hec1A stably expressing the ETV5 transcription factor (Hec1A-ETV5) were maintained in McCoy’s 5A Medium (Gibco, Grand Island, NY, USA) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in 5% CO
2, Hec1A-ETV5 cells further selected with Geneticin (500 μg/ml; Gibco, Grand Island, NY, USA). These cells were previously generated and thoroughly characterized[
11,
12].
To monitor non-invasively tumor grafts of Hec1A and Hec1A-ETV5 cells, these cells were infected with lentiviruses bearing pLenti CMV V5-LUC Blast (w567-1) (Addgene, Cambridge, MA, USA) to constitutively express the luciferase reporter gene, as previously described[
36]. Stable infected cells expressing luciferase were selected with Blasticidine S HCl (3 μg/ml; Invitrogen, CA, USA).
RNA extraction and real-time PCR
Paired frozen tumors and metastatic lymph node samples were disrupted and homogenized using a TissueLyser II (Qiagen, Valencia, CA, USA) and total RNA was extracted using the AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. Alternatively, total RNA was isolated from Hec1A and Hec1A-ETV5 cells using High Pure RNA Isolation Kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer.
cDNA synthesis was carried out using MuLV reverse transcriptase Kit (Applied Biosystems, Foster City, CA, USA) following the instructions provided by the manufacturer. Real-time PCR was performed using Applied Biosystem 7500 Real-Time PCR Machine and data were analyzed with StepOne Software v.2.1 (Applied Biosystems, Foster City, CA, USA). GAPDH was used as an internal normalization control. The results were represented as fold change in gene expression relative to GAPDH gene expression (2-ΔΔCt).
In vivo assay and bioluminescent imaging
Six Five-week-old female athymic Nude-Foxn1nu mice were purchased from Harlan Laboratories (Indianapolis, IN). Mice were divided in 2 groups and either Hec1A or Hec1A-ETV5 stably expressing luciferase cells (5×105 cells in 100 μl of sterile PBS) were inoculated into animals by intracardiac injection under 2% isoflurane/air anesthesia. A successful intracardiac injection was indicated on day 0 by images showing systemic bioluminescence distributed throughout the animal. Only mice with evidence of a satisfactory injection continued in the experiment. Three weeks after cells injection and before sacrifice, IVIS system (Xenogen Corporation) coupled to Living Imaging software 4.2 (Xenogen Corporation) were used to detect tumor metastases by bioluminescent imaging. For non-invasive bioluminescence tumour imaging, luciferin (Firefly Luciferin, Caliper Lifescience Corp, Hopkinton, MA, USA) was used as the substrate for the luciferase expressing tumor cells and injected intraperitoneally at a concentration of 150 mg/kg in PBS. Mice were housed and maintained under specific pathogen-free conditions and used in accordance with institutional guidelines approved by the Use Committee for Animal Care.
Statistical analysis
Statistical analyses were conducted using SPSS (Chicago, version 15.00 for Windows) and GraphPad Prism 4.00 software (GraphPad Softwares Inc, San Diego, CA, USA). Mann–Whitney and Kruskal-Wallis non-parametric tests were used to determine the differences between conditions. For Kruskal-Wallis analysis we used Dunn’s post-test. Alternatively, Wilcoxon signed test was used to determine the differences in relative gene expression between paired frozen tumors and metastatic lymph node samples. Statistical significance was set at p < 0.05.
Acknowledgements
We thank the patients for their willingness to participate in the study. Antonio Diaz-Lopez is a Sara Borrell Fellowship; Laura Muinelo-Romay is supported by ISCIII as Responsible of the Liquid Biopsy Analysis Unit (IDIS); Javier Mariscal is a fellowship from Escola de Doutoramento Internacional Campus Vida. IISC RETIC- RD12/0036/0007 and RD12/0036/0035.
Financial support
ISCIII PI11/00873; Fundación Asociación Española Contra el Cancer (AECC), Grupos Estables 2011; InveNNta (Innovation in Nanomedicine), co-financed by the European Union (EU) through the Operational Programme for Cross-border Cooperation, Spain-Portugal (POCTEP 2007-2013), European Regional Development Fund (ERDF); Helse Vest, Research Council of Norway, Norwegian Cancer Society and Harald Andersens legat (H.B.S.); L. Alonso-Alconada is recipient of fellowship from the Basque Government (Spain).
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Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
LAA, LMR, KM, ADL, CK, JM, EC, JC and XD carried out the immunoisolation, processing and analysis of samples; EW, DH, LC, AC, participated in the design of the study and critical revision of the manuscript; JT, AGM, LC, JC, MV, EO and MC contributed to the design and organization of human sample collection; HWN, TB, JSG, AR, JR, RLL, HBS, FA, XMG, GMB and MA made substantial contributions to conception and design of the study, interpretation of data and drafting and critical revision of the manuscript. All authors read and approved the final manuscript.