EpCAM-independent enrichment approach for isolation of circulating tumor cells (CTCs) in breast cancer

Background and Objectives: CTCs are essential for establishing metastasis in breast cancer. While many assays currently exist for the enumeration of CTCs, limitations include the reliance on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM) (e.g. CellSearch® system). These approaches may omit CTCs expressing either no or low levels of EpCAM, eventually representing the metastasis-initiating population. In order to successfully capture EpCAM-negative or -low breast cancer cells we have tested different antibodies specific for surface proteins and extracellular matrix (ECM) components for cell lines, spiked samples and blood of metastatic breast cancer patients.

Material and Methods: Surface expression of several markers (e.g. CD49f, Trop2, CK8) was verified on different EpCAM-positive (e.g. MCF-7, SKBR-3, T47D) and -negative (MDA-MB-231) breast cancer cell lines by immunofluorescent staining and flow cytometry. Antibodies and ECM proteins (e.g. hyaluronic acid, collagen type I) were additionally spotted onto glass slides (Schott Nexterion® AL) and binding of tumor cells was investigated via cell adhesion assays (2h, 37 °C, 600rpm). Additionally, captured and EpCAM-depleted fractions from patients' blood could be gathered and analyzed for positive marker expression after capturing with specific antibodies coupled to Dynabeads® and Bio-Adembeads. Captured cells and positive events, respectively, were identified by immunostaining for anti-pan-Cytokeratin ([C11]-FITC), anti-CD45/Alexa Fluor 647 and DAPI, and were visualized/quantified by fluorescent microscopy.

Results: As expected, only marginal binding of EpCAMneg MDA-MB-231 tumor cells to EpCAM-antibodies could be observed. Efficient adhesion/capturing of EpCAMneg/low cells was achieved by hyaluronic acid and immobilized antibodies specific for CD49f and Trop2. Besides that, analysing EpCAM-depleted fractions from 25 metastatic breast cancer patients, we were able to identify additional CKpos/CD45neg events in 69% of the samples [range 1 – 24] applying Trop2, CD49f and/or CK8 magnetic enrichment.

Conclusion: By targeting various cell surface and ECM proteins as capture molecules enrichment of heterogeneous tumor cell populations/EpCAMneg/low expressing CTCs out of patients with metastatic breast cancer can be improved and gathered for further molecular analyses on genomic or protein level.