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. 2019 Jul 22;2(4):e201900425. doi: 10.26508/lsa.201900425

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© 2019 Matsumura et al.

This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

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Figure 2. — (A) Schematic representation of isolation of CAF-primed highly invasive and metastatic breast cancer cells. See text for details. (B) Scratch wound assay in the indicated cells. Error bars, SE. (C) Appearance of DCIS^cnt2cy and DCIS^CAF2cy organoids generated on Matrigel gel for 5 d. (D) H&E staining and immunostaining of sections prepared from 21-d-old subcutaneous tumors generated by the indicated cells using the described antibodies. p63⁺ or E-cadherin (E-cad)⁺ cancer cells (simple arrows), nuclear ZEB1⁺ cancer cells (triangular arrows), and nuclear ZEB1⁺ stromal cells (arrowheads) are also shown. (E) Immunofluorescence of 21-d-old tumor sections from DCIS^CAF2cy and DCIS^cnt2cy using anti–E-cad and anti-ZEB1 antibodies. The Ehi (simple arrow) and E/M (triangular arrow) tumor cells, and nuclear ZEB1⁺ stromal cells (arrowheads) are shown. (F) Representation of metastatic nodules (arrows) in whole lungs dissected from mice subcutaneously injected with the indicated cancer cells (upper). The size (d: diameter) and number of metastatic nodules are shown for the indicated groups (n = 6) (left). The lung metastatic indices were also evaluated in each group at 60 d after injection (right). (G) Lung metastasis volumes evaluated at 30 d after intravenous injection of the indicated cells into mice. (H) Metastases evaluated by tdTomato fluorescent intensity in the lungs at 30 d after intravenous injection of the indicated tdTomato⁺ tumor cells into mice. (I) Detection of GFP fluorescence (arrow) in bone (upper) and liver (lower) of mice at 60 d after intracardiac injection of the indicated GFP⁺ cancer cells. H&E staining of bone metastasis (middle) is also shown for the described groups. Quantification of GFP intensity of the indicated cells colonizing bone and liver (graphs). Data information: Star indicates intact acinar structure of DCIS cells (D, E). Asterisk indicates a significant difference relative to others (F–I). Mann–Whitney U test (F, H, I) and Wilcoxon rank sum test (G). The horizontal line represents the mean value (F–I). Scale bars, 10 μm (E), 30 μm (C, D and I-H&E), 3 mm (I-GFP image), and 5 mm (F). See also Fig S2.

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