Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods, Figures 1-2, Tables 1-4 - PDF file - 619K
Volume 1, Issue 7
