Fig. 2.

Inducing c-Met/β1 integrin binding in breast cancer cells increases tumor cell migration and extravasation out of circulation. (A) MDA-MB-231 cells were engineered to express HA-tagged β1 integrin and FLAG-tagged c-Met fused to FRB (DmrC) and FKBP (DmrA), respectively, and treated with AP21967 (A/C heterodimerizer) to induce c-Met/β1 binding. (B) MDA-MB-231-iDimerize-c-Met-β1 cells were treated with AP21967 or vehicle (control), after which HA and FLAG IP confirmed that AP21967 induces c-Met/β1 binding. (C) MDA-MB-231-iDimerize-c-Met-β1 cells were assessed for morphology via a form factor plugin on ImageJ, revealing decreased circularity shape factor after AP21967 treatment (P = 6 × 10⁻⁷; n = 12 cells per group). (D) MDA-MB-231-iDimerize-c-Met-β1 cells were scratched at time = 0 and assessed after 5 and 24 h via mosaic imaging and analyzed using T-Scratch software to confirm that AP21967 induced migration (P = 0.001 at 5 h, P = 0.02 at 24 h) (n = 8 per group). (E) AP21967 also increased MDA-MB-231-iDimerize-c-Met-β1 invasion in Matrigel (n = 6 per group; P = 0.007). (F) MDA-MB-231-iDimerize-c-Met-β1 cells were pretreated for 2 h with or without AP21967, followed by tail vein injection into NSG mice (n = 6 mice per group). After 2 h, mice receiving AP21967-pretreated cells exhibited more human vimentin staining by immunohistochemistry versus those pretreated with vehicle, as shown in representative images here and quantified in SI Appendix, Fig. S9 (P = 0.02), with qPCR revealing the FKBP–c-Met fusion sequence in lungs of all mice receiving AP21967-pretreated cells, with the lack of PCR signal in all vehicle-treated mice precluding statistical comparison. (G) Analysis of lungs from the same experiment as F at 7 d postinjection revealed further increased human vimentin staining after tail vein injection of cells pretreated with AP21967 compared with no staining when injecting vehicle-pretreated cells (lack of staining in control group prevented statistical comparison). Similarly, qPCR at 7 d revealed increased tumor-specific FKBP–c-Met fusion sequence in mice receiving AP21967-treated cells, with lack of signal in most mice receiving vehicle-treated cells precluding statistical comparison. *P < 0.05; **P < 0.01; ***P < 0.001.