A Novel Platform for Detection of CK⁺ and CK⁻ CTCs

Capture of Carcinoma Cells Independent of EpCAM Expression

We have developed a microfluidic-based system for capture and analysis of rare cells in circulation, including CTCs (Supplementary Fig. S1A). The platform is capable of capturing rare cells from blood for subsequent molecular characterization directly within a uniquely designed microchannel (Supplementary Fig. S1B and S1C). Each microchannel consists of a roughly rectangular chamber (40 mm × 12 mm × 55 μm) in which approximately 9,000 variable diameter posts are randomly placed to disrupt laminar flow and maximize the probability of contact between target cells and the streptavidin-derivatized posts, resulting in their capture (15). The platform is versatile, permitting assay optimization with flexibility in the number of antibodies selected for capture and detection as well as allowing immediate and direct single-cell microscopic analyses through the transparent microchannel without the need to manipulate cells onto glass slides. This approach utilizes an antibody cocktail that is added directly to cells prior to capture, enabling recovery of variable CTC phenotypes. Briefly, this mixture contains antibodies directed toward a variety of epithelial cell surface antigens (EpCAM, HER2, MUC1, epidermal growth factor receptor, folate-binding protein receptor, TROP-2) and mesenchymal or stem cell antigens (c-MET, N-cadherin, CD318, and mesenchymal stem cell antigen). Each of these antibodies was tested by flow cytometry for reactivity to several well-characterized cancer cell lines (e.g., SKOV3, LNCaP, SKBR3) and shown to be additive in binding to cells (Supplementary Fig. S2). Antibodies were tested to have minimal cross-reactivity to nucleated cells in healthy control blood. Analytical control samples, namely blood from healthy donors spiked with cancer (SKOV3) cells, were run using this antibody mixture to ensure optimal performance in detecting tumor cells (based on CK staining).

Analytical validation of the platform to show precision, reliability, and reproducibility in recovery and detection of CTCs based on CK⁺/CD45⁻/DAPI⁺ (4′,6-diamidino-2-phenylindole) staining was done with ex vivo spiking experiments of carcinoma cells into whole blood. Tumor cell capture efficiency was validated through a series of cell-spiking experiments. We show analytic precision in recovery and detection of low- to high-EpCAM-expressing target cells, independent of the number of cells spiked (Fig. 1A). We further show >95% reproducibility in tumor cell capture with several cell lines (Fig. 1B). Capture efficiency using the antibody cocktail was shown with low (T24) and medium (SKOV3) EpCAM-expressing cell lines (16). While SKOV3 cells had high capture efficiency with EpCAM alone as well as with cocktail (>80%), T24 capture efficiency was markedly improved from <10% capture with EpCAM alone to >80% with antibody cocktail (P = 0.0001; Fig. 1C).

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Figure 1.

Efficiency and reproducibility of cell capture and comparison to CellSearch technology. A, median number of captured carcinoma cells after ex vivo spiking of approximately 10, 25, or 50 cells into 10 mL of human blood when using antibody cocktail against cells of varying EpCAM expression. Each spike was done in triplicate. B, percent reproducibility of cell capture after 10 separate ex vivo spikes into blood (pink: minimum outlier; red: maximum outlier). C, percentages of captured SKOV3 and T24 carcinoma cells after ex vivo spiking of approximately 150 cells into 10 mL of human blood when using only EpCAM only, antibody cocktail, or antibody cocktail without EpCAM antibody. D, comparison with CellSearch platform for CK⁺ CTCs captured from patients with breast, colorectal, lung, or prostate carcinoma. For samples from breast, colorectal, and lung cancer patients, the antibody cocktail (AC15) composed of 10 monoclonal antibodies was used. For samples from prostate cancer patients, the antibody cocktail (AC16) composed of 11 monoclonal antibodies was used. *P < 0.05; **P = 0.001; ‡ P = 0.0001.

Next, clinical verification of capture efficiency was tested by examining CTCs in patient samples. In the first cohort of 21 patients with advanced-stage disease (colon, breast, lung, and prostate cancers), we compared CTC detection with our platform using either EpCAM alone or antibody cocktail (Supplementary Table S1). Improved recovery was observed with higher CTC numbers in 14 of 21 samples with the antibody cocktail (P = 0.07). In 100 normal blood controls, only 1 CK⁺/CD45⁻/DAPI⁺ cell was detected, demonstrating high specificity of the staining procedure. In the second, larger cohort of 93 patients with advanced-stage lung, breast, colorectal, or prostate cancer, we compared CellSearch® and our platform using the antibody cocktail for capture. Based on standard CK⁺/CD45⁻/DAPI⁺ stain criteria, our platform was found to be significantly more sensitive for CTC enumeration in colorectal, prostate, and lung cancers (Fig. 1D).

Capture and Detection of CK⁻ CTCs in Patients with Breast, Ovarian, or Colorectal Cancer

Given that CK expression levels can often be variable among epithelial cells and absent among other nonepithelial cell types, we examined CK staining efficiency within the microchannel using a third cohort of HER2-neu-positive breast cancer samples (n = 19) for which FISH analysis of the HER2 locus enabled confirmation of CTC recovery independent of CK/CD45 staining (Fig. 2A). CK⁺ cells were located in each of the 19 cases and used as target cells for analysis of HER2 by FISH. In addition, all CK⁻/CD45⁻ cells were classified as “possible” CTCs for subsequent analysis of HER2 signals. In 18 of 19 cases (94.7%), a range of 0.04 to 2.4 HER2-amplified CTCs/mL of blood were detected among the cells classified as CK⁺/CD45⁻ and CK⁻/CD45⁻, suggesting that the CK⁻ HER2-amplified CTCs originated from the primary tumor. When excluding CK⁻ HER2-amplified CTCs, only 12 (63.2%) of the patient samples were found to have CK⁺ HER2-amplified cells, consistent with the findings of other similar reports (17). Interestingly, only 24.3% of all CK⁺ CTCs were found to have HER2-neu amplification (P = 0.007; Fig. 2B). Surprisingly, 49.7% of the HER2-amplified cells were CK⁻ (Fig. 2B). These results show the inefficiency in detection of CTCs based solely on CK⁺/CD45⁻ stain criteria, resulting in failure to detect a significant population of HER2-amplified cells. Thus, given the unique design and amenity to sequential staining and FISH, the platform enabled incorporation of FISH directly within the microchannel as a valuable independent method in confirming recovery of both CK⁺ and CK⁻ CTCs immediately after CK staining.

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Figure 2.

Capture of CK⁺ and CK⁻ complex aneuploid CTCs in breast, ovarian, or colorectal cancer. A, representative images illustrating detection of HER2⁺/CK⁺ and HER2⁺/CK⁻ CTCs. Both cells display a > 2.2 HER2/centromere 17 ratio, confirming positive HER2 amplification. B, comparison of total CK⁺/CD45⁻, CK⁺/CD45⁻/HER2⁺ and CK⁻/CD45⁻/HER2⁺ cells from advanced-stage breast cancer patients. C, capture of circulating ovarian (top) and colorectal (bottom) carcinoma cells that stain for CK. Subsequent FISH shows an ovarian cancer cell with trisomy in chromosome 8 (blue, arrows) and monosomy in region 20Q11 (red, arrows), whereas the colorectal cancer cell has trisomy in chromosome 8 and tetrasomy in chromosome 17 (orange, arrows). D, capture of CK⁻ circulating ovarian (top) and colorectal (bottom) carcinoma cells. FISH of an ovarian cancer cell with trisomy in chromosome 8 (blue), monosomy in chromosome 11 (green), and tetrasomy in region 20Q11 (orange), whereas the colorectal cancer cell has trisomy in chromosomes 8 (blue) and 11 (green) and monosomy in chromosome 17. The average number of total CK, complex aneuploid CK⁺, and CK⁻ circulating tumor cells per milliliter of blood is shown for (E) ovarian and (F) colorectal cancer patients. *P < 0.05; **P = 0.007.

To confirm further the use of the FISH-based strategy, we applied the same antibody cocktail capture approach and CTC detection (CK⁺/CD45⁻/DAPI⁺ staining) with samples from patients with advanced ovarian or colorectal cancer. To verify detection of CTCs, we again used FISH, but with the aim to detect aneuploidy (unbalanced genomic copy number changes). We rarely found cells stained with CK⁺/CD45⁻; roughly 0.1 cells/mL of blood from ovarian cancer patients regardless of stage, tumor grade, or CA-125 levels were CK⁺/CD45⁻ (Supplementary Table S2). FISH probes targeted to frequently gained genomic regions in ovarian and colorectal carcinoma were chosen based on array comparative genomic hybridization data (18, 19). Each probe was first tested on a total of 2,500 normal peripheral blood cells from five donors and in CK⁺ cultured SKOV3 cells to determine hybridization efficiency and scoring accuracy in interphase cells captured within the microchannel (see Methods section). Cells that displayed only a monosomic signal for any one of the three probes were excluded (potential signal overlap). Therefore, only cells classified as either trisomic or complex aneuploid (at least one locus gain and another gain or loss at a second locus) were enumerated. As observed with the HER2-positive breast cancer cohort, for both ovarian and colorectal cancer, we found that not only captured CK⁺/CD45⁻ cells (Fig. 2C), but also co-captured CK⁻/CD45⁻ staining cells (Fig. 2D; Supplementary Tables S3 and S4) had complex aneuploidy. Complex aneuploidy was not observed among normal control blood samples. Similar to breast cancer patients, when assessing for frequency of complex aneuploidy, colorectal and ovarian cancer patients had nearly the same number of CK⁻ as CK⁺ CTCs (Fig. 2E, F). Thus, the presence of CK⁻ complex aneuploid cells further shows the inefficiency of CK as a marker to detect all candidate CTCs.

CK⁻ CTCs Found within the Primary Tumor

We hypothesized that if the isolated CK⁻ CTCs are the consequence of EMT, then similar cells should be present within the primary tumor. We sought to verify that the circulating aneuploid cells identified in patients with ovarian cancer had molecular features reflective of the primary tumor and, thus, are a useful population of CTCs. Blood was collected from 7 patients just before cytoreductive surgery. Multiple regions in the matched tumor and CTCs shared similar complex aneuploid patterns (Fig. 3A). Interestingly, about 20% of these aneuploid regions within the tumor were CK⁻ and heterogeneous in distribution (Fig. 3B). Although similar findings using FISH in CTCs and circulating endothelial cells compared to primary tumors have been described (20), these were based on circulating CK⁺ cells.

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Figure 3.

Matched CK⁺ and CK⁻ cells in circulation and primary tumor. A, CK⁻ ovarian cancer cells identified in circulation (top) at the time of surgical resection have similar aneuploidy as regions in the tumor (bottom). Represented are cells with trisomy of chromosome 8. B, CK staining of ovarian carcinoma samples reveals CK⁻ cells with aneuploidy (arrows) similar to those detected in circulation. Top: left panel (merged), middle panel (DAPI), right panel (CK staining). Bottom: FISH signals in a CK⁻ tumor cell for chromosome 8 (left) and chromosome 11 (right). Approximately 20% of the tumor had such CK⁻ cells.

Linking CK⁻ CTCs to an EMT

One possible mechanism giving rise to the presence of CK⁻ CTCs is EMT, a biologic process reported to play a significant role in tumor progression and metastasis. Thus, there is growing interest in methods that enable capture and analysis of EMT-derived CTCs. To determine whether our assay captures cells that have undergone EMT, we studied the effect of TGF-β (an inducer of EMT) treatment on SKOV3 ovarian carcinoma cells. After 72 hours of treatment, CK staining was lost in about 20% of cells, correlating with an EMT morphologic change (Fig. 4A). Quantitative PCR analysis of these cells before and after TGF-β treatment showed an increase in expression of mesenchymal markers (Fig. 4B). Cells, before and after treatment, were spiked ex vivo into mouse blood and run through the microchannel. All untreated cells captured were CK⁺; however, after TGF-β treatment, 16% of the cells captured were CK⁻ and had complex aneuploidy (Fig. 4C). Direct visualization of the FISH staining within the microchannel shows these CK⁺ and CK⁻ cells had nearly identical complex aneuploid patterns (Fig. 4D).

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Figure 4.

Characterization and capture of CK⁻ cells after induction of EMT. A, SKOV3 cells were grown in either regular culture medium (pre-EMT) or serum-free medium with 10 ng/mL of TGF-β (post-EMT) for 72 hours. Pictured are representative immunofluorescent images (top) of pre-EMT cells demonstrating 100% CK expression and areas of post-EMT cells with absent CK expression. Approximately 20% of post-EMT cells were found to have complete loss of CK expression. Phase contrast images of the same cells (bottom) show a morphologic change characteristic of EMT. B, quantitative real-time PCR for markers of EMT of SKOV3 cells with and without TGF-β treatment for 72 hours. C, after 72 hours, pre- and post-EMT cells were spiked ex vivo into mouse blood and run through the CEE microchannel. All pre-EMT cells that were captured were CK⁺ and had complex aneuploidy, while 16% of post-EMT cells were CK⁻ and had similar complex aneuploidy. The bar graph represents ratios of CK⁺ and CK⁻ complex aneuploid captured cells in each group. D, representative images of CK⁺ and CK⁻ complex aneuploid SKOV3 cells are shown from within the microchannel. E, HeyA8 cells were cultured in regular medium (pre-EMT) or serum-free medium with 10 ng/mL of TGF-β (post-EMT) for 72 hours. Representative immunofluorescent images of Pre-EMT cells demonstrating nearly 100% CK expression and TGF-β-treated cells with absent CK expression are shown. Approximately 60% of TGF-β–treated cells were found to have complete loss of CK expression. F, HeyA8 cells were injected into 10 mice to establish a metastatic ovarian model. Once moribund, blood was collected from each mouse by cardiac puncture. Pictured are a CK⁺ and CK⁻ CTC within the microchannel demonstrating hyperploidy of chromosomes 11 and 17. G, correlation of total aggregate tumor burden with enumeration of complex aneuploid CK⁻ CTCs by mouse.

Next, to examine the utility of our CTC detection system for capturing tumor cells with EMT features in an in vivo setting, we studied the effect of TGF-β treatment on HeyA8 ovarian carcinoma cells. After 72 hours of TGF-β treatment, approximately 50% to 60% of cells lost their CK staining (Fig. 4E). To determine whether these CK⁻ cells can be captured in circulation, we established a metastatic orthotopic model with HeyA8 cells. Ten tumor-bearing mice were monitored for signs of morbidity, at which point approximately 350 μL of blood was obtained per mouse by cardiac puncture prior to sacrifice. All sites of metastatic tumor were carefully removed and weighed. Both CK⁺ and CK⁻ complex aneuploid CTCs were isolated from circulation within the microchannel (Fig. 4F). Enumeration of complex aneuploid CK⁻ CTCs correlated with aggregate tumor burden (Fig. 4G).