Fig 1: Related to Fig 1. CAF-induced breast carcinoma cell clusters with the Ehi and E/M states, collective invasion, and metastasis.(A) Appearance of 21-d-old tdTomato+ DCIS tumor xenografts subcutaneously (s.c.) developed with no fibroblasts (No fibro.), control fibroblasts (+cont. fibro.) or CAFs (+CAFs) in mice. Note the decreased number of intact acini (arrows) in tumors admixed with CAFs. (B) Detection of tdTomato+ DCIS cells and GFP+ fibroblasts (arrowheads) in 21-d-old subcutaneous tumor xenografts. (C). Immunohistochemistry of sections prepared from subcutaneous DCIS tumors developed with no fibroblasts, control fibroblasts, or CAFs using human-specific antivimentin (Vim) antibody. Note the presence of the injected human vimentin-positive control fibroblasts (arrowheads, middle) and CAFs (arrowheads, right) in 21-d-old tumor xenografts. (D) Immunohistochemistry of sections prepared from 21-d-old DCIS tumors subcutaneously developed with no fibroblasts, control fibroblasts, or CAFs using antibodies for human-specific antivimentin (Vim) and antifibronectin (FN). The positively stained tumor cells (arrows) and stromal cells (arrowheads) are shown. (E) Immunofluorescence of sections prepared from 21-d-old subcutaneous DCIS tumors developed with control fibroblasts or CAFs using anti–E-cadherin (E-cad) (Cat. No. ab40772; Abcam) and anti-ZEB1 (Cat. No. sc-515797; Santa Cruz) antibodies. The Ehi (simple arrow) and E/M (triangular arrow) cancer cells as well as nuclear ZEB1+ stromal cells (arrowheads) are shown. (F) Immunofluorescence of sections prepared from DCIS tumors admixed with CAFs using the indicated antibodies. E-cad+FN+ and E-cad+Vim+ tumor cells (arrows) and FN+ stromal cells (arrowheads) are shown. (G) Flow cytometry of the cell suspension dissociated from 30-d-old tumor xenografts raised by tdTomato+ DCIS cells admixed with CAFs using anti–E-cad and anti-ZEB1 antibodies. Ehi, E/M, and E-cadhiZEB1hi cell populations are marked. (H) Tumor weight measured at 30 d after subcutaneous injection of the indicated cells into mice. (I) Lung metastatic indices evaluated by nodule volume at 60 d after subcutaneous injection of the indicated cells into mice. (J) Immunohistochemistry of sections prepared from the lungs at 60 d after subcutaneous injection of DCIS cells admixed with CAFs into mice, using antibodies for human-specific antivimentin (Vim). Note the presence of vimentin-positive DCIS cells (arrows), but not CAFs. (K, L) Appearance of tdTomato+ metastatic nodules in the indicated organs dissected from mice at 60 d after subcutaneous injection of tdTomato+ DCIS cells with CAFs (K) or control fibroblasts (L). Note the tdTomato+ metastatic nodule (arrow) in the liver of the animal from the CAF group, whereas there are no tdTomato+ cells in the indicated organs in the control fibroblast group. The image (L-lung) is also shown, as in Fig 1G. Data information: Star indicates intact acinar structure of DCIS cells (B, D). Scale bars, 1 mm (A, K, L), 30 µm (B, C, D, J), and 10 µm (E, F). Asterisk indicates a significant difference relative to No fibro. and +cont. fibro. groups (H, I). t test (H) and Wilcoxon rank sum test (I). The horizontal line represents the mean value (H, I).