Improved ovarian cancer EMT-CTC isolation by immunomagnetic targeting of epithelial EpCAM and mesenchymal N-cadherin

Abstract

Epithelial cell adhesion molecule (EpCAM)-targeted capture remains the most common isolation strategy for circulating tumor cells (CTCs). However, epithelial-to-mesenchymal transition (EMT) leads to decreased epithelial EpCAM expression affecting the optimal CTC capture. In this study, we tested a cohort of ovarian cancer cell lines using flow cytometry to identify N-cadherin as the additional immunomagnetic cell surface target for ovarian cancer cell isolation. Combined immunomagnetic targeting of mesenchymal N-cadherin and epithelial EpCAM enriched CTCs from advanced ovarian cancer patient blood approximately three times more efficiently than targeting of EpCAM alone. We also show that more EMT-phenotype CTCs are captured by including N-cadherin targeting into CTC isolation protocols. However, after N-cadherin-based CTC isolation, in some blood samples of healthy individuals, we also observed the presence of cells expressing markers common to CTCs. Our data show that these “false positives” can be largely distinguished from CTCs as circulating endothelial cells (CECs) by vascular endothelial–cadherin co-staining. CEC counts are highly variable in patients and healthy controls. Our data demonstrate that a combination of EpCAM with N-cadherin-targeted isolation can improve CTC detection and widen the EMT-phenotype spectrum of captured CTCs.

Establishment of novel long-term cultures from EpCAM positive and negative circulating tumour cells from patients with metastatic gastroesophageal cancer

“…The negative selection used for CTC culture (CD45 depletion), rather than the positive selection used for CTC enumeration (EpCAM capture) was critical to detect these cells. The optimal isolation method of CTCs continues to evolve in the face of these issues, and while more emphasis has been given to epithelial to mesenchymal transition (EMT) phenotype CTCs in recent years 41 , the data presented here highlight the persistent challenges identifying the optimal markers to isolate CTCs.…”

Preclinical and Clinical Evaluation of Magnetic-Activated Cell Separation Technology for CTC Isolation in Breast Cancer

“…High levels of phenotypic heterogeneity among CTCs has been reported (57,67), and while most current CTC detection methods only target EpCAM and/or CK to enrich epithelial CTCs, they may fail to recognize other CTC phenotypes that lack expression of these markers. Since the present study was conducted, increased isolation yields have been shown using an antibody against the mesenchymal marker N-cadherin (68), and using an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (69). Unfortunately, no commercially available isolation method combining epithelial and mesenchymal markers was available at the time of the present study.…”

The Prospect of Identifying Resistance Mechanisms for Castrate-Resistant Prostate Cancer Using Circulating Tumor Cells: Is Epithelial-to-Mesenchymal Transition a Key Player?

“…For PCa, increased CTC counts are associated with earlier disease progression and shorter OS, with enumeration of PCa CTCs using the CellSearch CTC platform gaining FDA approval as a prognostic indicator [175]. While common CTC isolation and analysis techniques favour epithelial CTCs, there have been numerous advances in improving capture, detection, and analysis of EMT-CTCs by screening for epithelial and mesenchymal marker expression [176][177][178][179][180][181]. Equally, as Table 1 shows, several major signaling pathways implicated in EMT have, to some extent, been analysed in CTC samples.…”

“…Protein detection is usually limited to immunocytostaining which relies on antibody-based detection and the number of microscope channels available with 3 usually dedicated to detection of a CTC marker (often cytokeratin), a nuclear marker such as DAPI, and exclusion of a blood cell marker usually CD45. Nevertheless, some studies have detected additional proteins such as EMT markers [21,22,176] or posttranslational modifications such as phosphorylation of pFAK, pPI3K, pSRC, pEGFR, and pAkt [53,[201][202][203][204]].…”