Figure 1.
Human breast cancer specimen displays morphological and biochemical evidence suggestive of EMT and MET.
Images of the patient’s primary tumor focus #3 (top row), focus #6 (second row), chest wall recurrence (third row), and bone marrow biopsy (bottom row) showing H&E staining and immunohistochemistry for CK5, CK8/18, vimentin, and E-cadherin.
Table 1.
Characterization of human primary tumor, chest wall recurrence and bone marrow metastasis.
Figure 2.
A representative mitotic DKAT cell from sideline 1 is shown. A. Metaphase cell with chromosomes shown in SKY display colors. B. Inverted and contrast-enhanced DAPI image of the same metaphase cell. C. The same metaphase cell with chromosomes shown in spectra-based classification colors. D. Spectral karyotype of the same metaphase cell shown in SKY display colors.
Figure 3.
DKAT cells express markers of basal epithelial cells. A.
Immunofluorescence for CK5, CK17, p63 and CK18 in DKAT, immortalized HMEC, MCF-7, and MDA-MB-231 cells. B. Western blotting of total cell lysates from HMEC, MCF-7, MDA-MB-231, and DKAT cells grown in MEGM. C. FACS analysis of DKAT-MEGM, MCF-7 and MDA-MB-231 cells stained for CD44 and CD24. D. Results from DKAT, MDA-MB-231, and HMEC Affymetrix HG-U133 2+ arrays were analyzed by unsupervised hierarchical clustering with a published data set [28]. Cell lines are labeled by breast cancer subtype; luminal-like (blue); basal-like (red); mesenchymal-like (green); unknown subtype (black). Displayed is an expanded view of selected gene cluster containing basal-specific gene expression. The full gene cluster is provided as Figure S1.
Table 2.
Summary of DKAT marker expression (MEGM/SCGM).
Figure 4.
Evidence for morphologic and phenotypic changes consistent with in vitro EMT.
A. Phase contrast or immunofluorescence images of DKAT cells cultured in either MEGM (upper) or SCGM for 14 days (lower). Phase contrast images were acquired with a 20 × objective, scale bar = 40 µm. IF images were acquired with a 40 × objective, scale bar = 20 µm. Cells were stained simultaneously for vimentin (green) and E-cadherin (red), or CK18 (green) and CK5 (red). B. E-cadherin immunofluorescence images of DKAT cells cultured in MEGM or SCGM for 2 hours. C. Total cell lysates from DKAT cells cultured in MEGM or SCGM for 1 h, 24 h, or 14 days were analyzed by western blot for vimentin, E-cadherin and actin. D. Scratch wound healing assay comparing DKAT cells grown in MEGM or SCGM (14 days) and MDA-MB-231 cells. Images (upper panel) were taken at the time of the scratch (T = 0) and 12 hours later with a 5 × objective. Scale bar = 200 µm. Results (lower panel) are expressed as the percentage of the scratch area closed at 12 hrs as measured by ImageJ software, and are an average of 12 scratches per sample. E. Total cell lysates from a clonal DKAT cell line cultured in MEGM or SCGM (14 days) were analyzed by western blot for vimentin, E-cadherin and actin. F. Total cell lysate from passage matched cultures of DKAT cells grown in MEGM, SCGM, or SCGM for 10 passages and then returned to MEGM for 5 passages were analyzed by western blot for epithelial and mesenchymal markers. G. Immunofluorescence (IF) images of subcloned DKAT-SCGM cells cultured in SCGM or MEGM for 14 days. Cells were stained simultaneously for vimentin (green) and E-cadherin (red), and images were acquired with a 40 × objective. H. Total cell lysates from the indicated cell lines cultured in their normal media or switched to MEGM or SCGM for 14 days were analyzed by western blot for vimentin, E-cadherin, and actin expression. N = Normal, M = MEGM, S = SCGM.
Table 3.
Differential gene expression (MEGM/SCGM).
Figure 5.
Zeb1 regulates DKAT EMT/MET. A.
Total cell lysate was prepared from DKAT-MEGM or DKAT-SCGM cells and subjected to western blot analysis for markers of EMT. B. Total cell lysate was prepared from DKAT-MEGM cells stably transfected with a control vector (V) or vector containing Zeb1 transcript (Zeb1) and subjected to western blot analysis for markers of EMT. C. Scratch wound healing assay comparing DKAT cells grown in MEGM or SCGM as indicated, stably expressing either a non-targeting shRNA sequence or one of two Zeb1-targeting sequences. Results are expressed as the percentage of the scratch area closed at 16 hrs as measured by ImageJ software, and are an average of 12 scratches per sample.
Figure 6.
Comparison of H&E and IHC patterns from DKAT xenografts.
A. IHC showing H&E (i and ii) or CK5 staining (iii) illustrating the three recurring morphological patterns observed in DKAT xenografts transplanted without Matrigel. B. IHC showing H&E, CK8/18, CK5, and SMA staining of DKAT xenografts transplanted with Matrigel. The left column shows the center of a transplant and the right column shows the leading edge of the tumor.
Figure 7.
Basal/myoepithelial differentiation of DKAT xenografts. A.
DKAT xenograft tumor stained with DAPI (blue) nuclear counter stain, CK14 (green) showing basal/myoepithelial differentiation of tumor cells, and human-specific p53 (red) confirming that both the CK14-positive cells and the more luminal, CK14 weak/negative cells are human DKAT tumor cells. 100 micron scale bar in the DAPI channel applies to all four images. B. DKAT tumor from a second mouse at higher magnification shows an area of vague glandular architecture with somewhat elongated CK14-positive basal/myoepithelial cells (arrows) surrounding CK14 weak/negative cells which surround a poorly formed glandular lumen (asterix). 25 micron scale bar show in in DAPI channel.
Figure 8.
Immunofluorescence of DKAT tumorspheres stained for E-cadherin, β-catenin, CK17 and CK18 (upper and middle panels). Tumorspheres were also dual stained for E-cadherin (red) and vimentin (green), or CK5 (red) and CK18 (green) (lower panel). Images were acquired with a 63 × objective. Scale bar = 40 µm.