Talin promotes mammary tumor cell invasion, intravasation, and lung metastasis. (A) Inverted 3D ECM invasion assay. MDA-MB-231 cells were treated with control or talin siRNA and plated on the bottom of transwell filters and allowed to migrate into the 3D collagen/Matrigel matrix over the course of 4 d. The relative invasion index was quantified as the number of cells migrating above 60 µm/the number of cells below 30 µm. n > 30 cells; three independent experiments. **, P < 0.029. (B) Western blot analysis of MTLn3 cells stably expressing control or talin shRNA. Blots were stained for talin and GAPDH (loading control). (C and D) Spontaneous lung metastasis assay. (C) Representative fields of lungs from severe combined immunodeficiency mice harboring MTLn3-Dendra2 tumors expressing either control or talin shRNA. Bar, 200 µm. (bottom) Maximum intensity projection multiphoton image of control and talin shRNA metastatic MTLn3 tumor cells (green) that have extravasated into the lung parenchyma (second harmonic collagen; blue). Bars, 40 µm. (D) Quantification of the number of metastatic tumor cells per lung lobe in control or talin shRNA mice. n = 5 mice; ***, P = 6.4 × 10−5. (E) Quantification of the number of motile cells and protrusions per 100-µm field (see Videos 3 and 4). *, P < 0.036. (F) Quantification of the number of circulating tumor cells per millimeter of blood in control or talin shRNA mice. n = 5 mice; *, P = 0.045. Error bars represent the SEM.
