A novel method for downstream characterization of breast cancer circulating tumor cells following CellSearch isolation

Abstract

Background: Enumeration of circulating tumor cells (CTCs) obtained from minimally invasive blood samples has been well established as a valuable monitoring tool in metastatic and early breast cancer, as well as in several other cancer types. The gold standard technology for detecting CTCs in blood against a backdrop of millions of leukocytes is the FDA-approved CellSearch system (Janssen Diagnostics), which relies on EpCAM-based immunomagnetic separation. Secondary characterization of these cells could enable treatment selection based on specific targets in these cells, as well as providing a real time window into the metastatic process and offering unique insights into tumor heterogeneity. The objective of this study was to develop a method for downstream characterization of CTCs following isolation with the CellSearch system.

Methods: An in vitro CTC model system focusing on clinically useful treatment predictive biomarkers in breast cancer, specifically the estrogen receptor α (ERα) and the human epidermal growth factor receptor 2 (HER2), was established using healthy donor blood spiked with breast cancer cell lines MCF7 (ERα(+)/HER2(-)) and SKBr3 (ERα(-)/HER2(+)). Following CTC isolation by CellSearch, the captured CTCs were further enriched and fixed on a microscope slide using the in-house-developed CTC-DropMount technique.

Results: The recovery rate of CTCs after CellSearch Profile analysis and CTC-DropMount was 87%. A selective and consistent triple-immunostaining protocol was optimized. Cells positive for DAPI, cytokeratin (CK) 8, 18 and 19, but negative for the leukocyte-specific marker CD45, were classified as CTCs and subsequently analyzed for ERα and HER2 expression. The method was verified in breast cancer patient samples, thus demonstrating its clinical relevance.

Conclusions: Our results show that it is possible to ascertain the status of important predictive biomarkers expressed in breast cancer CTCs using the newly developed CTC-DropMount technique. Downstream characterization of multiple biomarkers using a standard fluorescence microscope demonstrates that important clinical and biological information may be obtained from a single patient blood sample following either CellSearch epithelial or profile analyses.

Trial registration: Clinical Trials NCT01322893.

Figures

Figure 1

Overview of the method. Enriched CTCs were collected after CellSearch analysis using the Profile or Epithelial cell kit. The solution containing CTCs and leukocytes was placed in a magnetic tray. Following incubation, the non-adherent solvent was removed and the ferrofluid-attached cells were re-suspended in a smaller volume of PBS, thus permitting further enrichment. This solution was dropped and fixed on a glass slide before subsequent staining according to protocols 1 or 2. Visualization is possible with a fluorescence or bright-field microscope, depending on the staining method applied. (Drawing of magnetic stand reprinted with permission from IFI CLAIMS Patent Services.)

Figure 2

CD45 staining. Secondary staining of cell line cells spiked into healthy donor blood, from left to right: DAPI counterstain (fluorescent blue), cytokeratins 8, 18, and 19 (CK) stained with Phycoerythrin (red), CD45 stained with AlexaFluor 647 (yellow), and a composite of all channels. The two juxtaposed CTCs (CK-positive) stained negative for CD45, while the leukocytes (white arrows) simultaneously stained positive for CD45 and negative for CK, illustrating methodological selectivity.

Figure 3

ERα staining of MCF7 and SKBr3 cells. Selective ERα staining demonstrated in MCF7 (ERα+) and SKBr3 (ERα−) cells. From left to right: DAPI counterstain (fluorescent blue), cytokeratins 8, 18, and 19 (CK) stained with Phycoerythrin (red), estrogen receptor (ERα) stained with AlexaFluor 488 (green), and a composite of all channels. MCF7 showed positive nuclear staining in AlexaFluor 488 indicating positive ERα expression, while SKBr3 was negative.

Figure 4

HER2-staining of MCF7 and SKBr3. Selective HER2 staining demonstrated in MCF7 (HER2−) and SKBr3 (HER2+) cells. First row: MCF7, from left to right: DAPI counterstain (fluorescent blue), HER2 stained with Liquid Permanent Red (red), and a composite of all channels. Second row: SKBr3, in the corresponding channels. Positive membrane staining was visible in SKBr3 cells only. Additionally, assessment of HER2 staining was also possible using bright-field microscopy, as demonstrated in the lower two rows (third row: MCF7, and fourth row: SKBr3).

Figure 5

Immunostaining of metastatic breast cancer patient blood samples. Representative images of positive ERα and HER2 staining in clinical samples. Row A, from left to right: DAPI counterstain (fluorescent blue), cytokeratins 8, 18, and 19 (CK) stained with Phycoerythrin (red), estrogen receptor (ERα) stained with AlexaFluor 488 (green), and a composite of all channels. This patient sample (no. 4, see Table 3) was collected prior to initiation of therapy, illustrating two clustered ERα+ CTCs, adjacent to a solitary leukocyte located in the lower left corner. This patient was diagnosed with an ERα+ metastasis. Row B, from left to right: DAPI counterstain (fluorescent blue), HER2 stained with Liquid Permanent Red (red). This patient sample (no. 1, see Table 3) was obtained following 6 months of chemotherapy, illustrating HER2+ CTCs identified by combination of fluorescence and bright-field microscopy. This patient was diagnosed with a HER2− primary tumor and HER2− metastasis.