Intracardiac and Intraprostatic Metastasis Assays

PC3-MM2 sh-control, talin1-silenced with GFP, GFP-talin1^WT and GFP-talin1 mutant cells were transduced with a plasmid directing luciferase expression (pLenti-PGK-Blast-V5-LUC,^{45 (link)} Addgene plasmid #19166) as described above. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC). For intracardiac injection (experimental metastasis assays), 1 × 10⁶ cells were injected into left ventricle of male nude mice. Bioluminescence images were acquired immediately after intracardiac injection to proper ensure distribution of cells in mice, and then were used to monitor tumor growth by a Xenogen IVIS 200 system (PerkinElmer). X-ray and matched bioluminescence images were acquired and superimposed using a Kodak In-Vivo Multispectral Imaging System FX (Carestream, Rochester, NY). For intraprostatic injection (spontaneous metastasis assays), 5 × 10⁵ cells were injected into nude mice. Tumor growth was monitor by bioluminescence imaging. Mice with similar sized primary tumors were sacrificed, tumors were removed and lymph node metastases were identified by bioluminescence imaging.