Figure 4. The E^hi state required for invasive and metastatic abilities in DCIS^CAF2cy.

(A–C) Lung metastatic indices (left) evaluated at 60 d after subcutaneous injection of the indicated cells into mice. Lung metastatic volume (right) was evaluated at 30 d after intravenous injection of the indicated cells into mice. (D) Appearance of GFP⁺DCIS^CAF2cy (arrows) attached to the top layer of the indicated cells (left). The relative numbers of GFP⁺DCIS^CAF2cy attached to the indicated cells (n = 4) are shown (right). (E) Appearance of larger cell aggregates (arrows) formed by DCIS^CAF2cy compared to DCIS^alone2cy and DCIS^cnt2cy on low attachment culture dishes (left). The relative volume of cell aggregates is shown in the indicated cells (n = 5) (right). (F) The ECM (collagen)–cell adhesion measured in the indicated cells (n = 4–9). (G) Cell apoptosis measured in the indicated cells (n = 5) on low attachment culture dishes. (H) The relative apoptotic (left) and proliferating (right) tumor cell proportions in experimental lung metastases generated by DCIS^CAF2cy expressing the indicated shRNA (n = 4) by immunostaining using anti-cPARP and anti–Ki-67 antibodies, respectively. Data information: Asterisk indicates a significant difference relative to GFP-shRNA–expressing DCIS^CAF2cy (A–H). t test (D–H) and Mann–Whitney U test (A–C). Error bars, SE. The horizontal line represents the mean value (A–C). Scale bars, 30 μm (E) and 300 μm (D). See also Fig S4.