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Combined use of EpCAM and FR? enables the high-efficiency capture of circulating tumor cells in non-small cell lung cancer.

ABSTRACT: Circulating tumor cells (CTCs) provide a new approach for auxiliary diagnosis, therapeutic effect evaluation, and prognosis prediction for cancer patients. The epithelial cell adhesion molecule (EpCAM)-based separation method (CellSearch) showed good clinical use in multiple types of cancer. Nevertheless, some non-small cell lung cancer (NSCLC) tumor cells have a lower expression of EpCAM and are less frequently detected by CellSearch. Here, we present a highly sensitive immunomagnetic separation method to capture CTCs based on two cell surface markers for NSCLC, EpCAM and Folate receptor alpha (FR?). Our method has been demonstrated to be more efficient in capturing NSCLC cells (P?

SUBMITTER: Chen L

PROVIDER: S-EPMC5775318 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Combined use of EpCAM and FRα enables the high-efficiency capture of circulating tumor cells in non-small cell lung cancer.

Chen Luojun L Peng Min M Li Na N Song Qibin Q Yao Yi Y Xu Bin B Liu Huali H Ruan Peng P

Scientific reports 20180119 1

Circulating tumor cells (CTCs) provide a new approach for auxiliary diagnosis, therapeutic effect evaluation, and prognosis prediction for cancer patients. The epithelial cell adhesion molecule (EpCAM)-based separation method (CellSearch) showed good clinical use in multiple types of cancer. Nevertheless, some non-small cell lung cancer (NSCLC) tumor cells have a lower expression of EpCAM and are less frequently detected by CellSearch. Here, we present a highly sensitive immunomagnetic separatio ...[more]

PMID: 29352248

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Project description:IntroductionThe exclusion of circulating tumor cells (CTCs) that have lost epithelial antigens during the epithelial-to-mesenchymal transition (EMT) process by using Epithelial Cell Adhesion Molecule (EpCAM) based capture methods is still a matter of debate. In this study, cells obtained after depletion procedure from blood samples of squamous cell lung cancer (SQCLC) patients were identified based on morphology and characterized with the combination of FISH assessment and immunophenotypic profile.Materials and methodsFive mL blood samples, collected from 55 advanced SQCLC patients, were analyzed by a non-EpCAM-based capture method. After depletion of leukocytes and erythroid cells, the negative fraction was characterized by both FISH using a fibroblast growth factor receptor 1 (FGFR1) probe and by immunocytochemistry. Thirty healthy donors were also tested.ResultsBased on morphology (nuclear dimension ≥10 μm, shape and hypercromatic aspect) suspicious circulating cells clearly distinguishable from contaminant leukocytes were observed in 49/55 (89%) SQCLC patients. Thirty-four of the 44 (77%) samples evaluable for FGFR1 FISH showed ≥ 6 FGFR1 gene copy number on average per cell. Vimentin expression involved 43% (18/42) of pooled circulating SQCLC cells, whereas only 29% (14/48) were EpCAM positive. Confocal microscopy confirmed the localization of FGFR1 probe in suspicious circulating cells. Suspicious circulating elements were also observed in healthy donors and did not show any epithelial associated antigens. A significantly lower number of suspicious circulating cells in healthy donors compared to SQCLC patients was found.ConclusionsAmong the heterogeneous cell population isolated by depletion procedure, the coexistence of cells with epithelial and/or mesenchymal phenotype suggests that EMT may participate to transendothelial invasion and migration of tumor cells in advanced SQCLC. The finding of cells with neither EpCAM or EMT phenotype, retrieved after non-EpCAM-based systems, underlines the presence of suspicious elements in the blood of both SQCLC patients and healthy donors. Further phenotyping and molecular analyses are necessary to fully characterize these circulating elements.

Project description:Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components.

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Combined use of EpCAM and FR? enables the high-efficiency capture of circulating tumor cells in non-small cell lung cancer.

Publications

Combined use of EpCAM and FRα enables the high-efficiency capture of circulating tumor cells in non-small cell lung cancer.

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