Figure 4.

Characterization and capture of cytokeratin-negative cells after induction of EMT. (a) SKOV3 cells were either grown in regular culture media (Pre-EMT) or in serum-free media with 10 ng/mL 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 cytokeratin expression. Phase contrast images of the same cells (bottom) demonstrate 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) Following 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 media (Pre-EMT) or serum-free media with 10 ng/mL 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 cytokeratin 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.