Detection of EpCAM-Negative and Cytokeratin-Negative Circulating Tumor Cells in Peripheral Blood

Abstract

Enrichment of rare circulating tumor cells (CTCs) in blood is typically achieved using antibodies to epithelial cell adhesion molecule (EpCAM), with detection using cytokeratin (CK) antibodies. However, EpCAM and CK are not expressed in some tumors and can be downregulated during epithelial-to-mesenchymal transition. A micro-fluidic system, not limited to EpCAM or CK, was developed to use multiple antibodies for capture followed by detection using CEE-Enhanced (CE), a novel in situ staining method that fluorescently labels the capture antibodies bound to CTCs. Higher recovery of CTCs was demonstrated using antibody mixtures compared to anti-EpCAM. In addition, CK-positive breast cancer cells were found in 15 of 24 samples (63%; range 1-60 CTCs), while all samples contained additional CE-positive cells (range 1-41; median = 11; P = .02). Thus, antibody mixtures against a range of cell surface antigens enables capture of more CTCs than anti-EpCAM alone and CE staining enables the detection of CK-negative CTCs.

Figure 1

Diagram of the CEE microchannel. (a) Top view of the channel showing the inlet where sample is loaded and the outlet that is attached to a syringe pump to draw sample through the channel. (b) Bottom view shows the area where 9,000 posts are located in the silicone block and the channel sealed with the bottom cover slip. The total volume of the microchannel is 24 μL. A standard microscope slide is added for stability during handling but is removed to visualize cells. The microchannel is inverted on a microscope and the captured cells viewed through the coverslip.

Figure 2

Capture of T24 and SKOV cells spiked into blood. A: the percentage capture of T24 cells using anti-EpCAM antibody. B: T24 capture % using anti-EpCAM and TROP-2 antibodies. C: SKOV capture % using anti-EpCAM antibody; D: SKOV capture % using anti-EpCAM and TROP-2 antibodies. By FACS T24 cells were shown to contain 4,000 and 60,000 EpCAM and TROP-2 antigens, respectively; SKOV cells were shown to contain 66.000 and 12,000 EpCAM and TROP-2 antigens, respectively. Antibody capture is less efficient with low-antigen expression on the cells, but increases in an additive manner when antibodies are used in combination.

Figure 3

Capture and stain of SKOV cells with EpCAM and with an antibody mixture. (a) The relative stain intensity of these cells on a microscope slide using anti-mouse-AlexaFluor488 when the cells were preincubated with anti-EpCAM antibody only, or with an antibody mixture of anti-HER2/neu, anti-CD44 and anti-CD28. (b) The FACS profile of the respective antigens of EpCAM or of the 3-antibody mixture present on each cell. (c) The percentage capture of SKOV cells when preincubated with anti-EpCAM or the antibody mixture. This shows that much lower antigen levels are necessary for good cell capture than for good staining intensity. Antibody mixtures improve CE staining efficiency.

Figure 4

Immunofluorescent staining. (a) A LnCAP cell spiked into blood and captured on the microchannel, stained for CK (green) and also nuclear stained with DAPI (blue). A small WBC is seen with only the nucleus stained blue. (b) A cluster of CTCs from a clinical lung cancer sample captured on the microchannel that are stained for CK. These cells were CD45-negative and DAPI-positive (not shown). (c) A cluster of cells from lung cancer showing triple staining with CK (green), CD45 (red), and DAPI (blue). Three CK-positive CTCs are shown with 2 smaller WBCs stained positive for CD45.

Figure 5

The use of CEE-Enhanced to improve detection of cells on the microchannel. (a) A clinical breast cancer CTC stained for CK and nuclear-stained with DAPI. This cell is weakly CK positive. (b) The same cell after subsequent stain with CE labeled with the same AlexaFLuor-488 fluorophore in order to enhance the stain intensity. (c) SKOV cell spiked into blood and recovered on the microchannel using an antibody mixture (see Section 2). Cells on the channels were stained with CE-488 and DAPI. The four WBCs stained blue for DAPI only, while the SKOV (attached to post) can be detected only with CE (green). This higher contrast image shows the outline of the posts in the microchannel. Together these images show that CE can be used to augment weakly staining CK cells or can be used to detect cells without CK stain.

Figure 6

Costaining clinical lung cancer CTCs with anti-CK and CEE-Enhanced. (a–c) A single CTC on the microchannel stained with anti-CK (a), CE-AlexaFluor-546 (orange, (b)), and (c), a composite image. (d–f) shows the same order of staining but with 2 attached CTCs. This demonstrates the costaining of the internal CK antigen and the cell surface antigens with CE.

Figure 7

Clinical breast cancer samples sequentially stained with anti-CK and with CEE-Enhanced. The antibody mixture was used to capture CTCs. The dark bars on the bottom represent the number of CK positive cells detected in a sequential series of stage IV breast cancer samples. The location of these cells was recorded and then the channel was restained with CE. The light bars on top represent the newly detected CTCs after CE stain. All cells designated as positive were CD45 negative and DAPI positive.