Extended Data Fig. 10: Effects of apoptosis, oxidative stress and TGFβ inhibition on dissemination of E-cad+ or E-cad− MMTV-PyMT organoids.
From: E-cadherin is required for metastasis in multiple models of breast cancer
a, Schema of invasion assay in the presence of a pan-caspase inhibitor (z-VAD-FMK). b, Representative micrographs of adeno-Cre-transduced MMTV-PyMT; Cdh1+/+ or Cdh1fl/fl organoids in the presence of z-VAD-FMK. Scale bar, 50 μm. c, d, There is a dose-dependent increase in invasion (c) and dissemination (d) of Cdh1fl/fl organoids relative to control organoids in the presence of z-VAD-FMK. Box plots show the median, box edges represent the first and third quartiles, and the whiskers extend to 5th and 95th percentiles. ****P < 0.0001 (Kruskal–Wallis test). e, Schema of invasion assay in the presence of NAC, soluble TGFβ or TGFβR1 inhibitor (SB525334). f, Representative images of tamoxifen-treated MMTV-PyMT; Cdh1fl/fl or Cdh1fl/fl; CreER organoids in the presence of these inhibitors. Treatment with soluble TGFβ increases dissemination of E-cad+ and E-cad− organoids while inhibition of TGFβR1 decreases dissemination. Treatment with NAC does not change dissemination of E-cad+ or E-cad− organoids. Scale bar, 50 μm. Box plots show the median, box edges represent the first and third quartiles, and the whiskers extend to 5th and 95th percentiles. ****P < 0.0001 (Kruskal–Wallis test). g, Schema of tail-vein assays using NAC pre-treated E-cad+ or E-cad− cancer cell clusters. MMTV-PyMT; Cdh1fl/fl organoids were treated with adeno-GFP or adeno-Cre, trypsinized into small clusters, incubated with NAC for 24 h, and injected via the tail vein of NSG host mice. Lungs from these mice were collected 48 h or 1 week after injection and number of micro- and macrometastases, respectively, were counted. h, There is no significant difference in the number of micrometastases arising from E-cad+ or E-cad− cancer cells after NAC pre-treatment. Horizontal line shows the median. (Mann–Whitney test, two-sided). i, NAC pretreatment partially rescued the ability of E-cad− cancer cells to form macrometastases relative to E-cad+ cancer cells. Horizontal line shows the median. **P = 0.0051 (Mann–Whitney test, two-sided).
