Fig. 4

Expression of the metastatic gene twist in breast cancer cells induces Cx43 expression, tumor cell extravasation and microtumor formation in the brain. (A) Representative images show 3D reconstructions of confocal z-stacks (60×, top views) of HMLE or HMLEtwist cell monolayers co-immunostained with Cx43 (green) and the cell–cell junction marker ZO-1 (red). (B) Western blots of the indicated proteins in HMLE cells transduced with the empty lentiviral vector (HLME), transduced with lenti-twist (HMLEtwist) or expressing twist and shRNAs to Cx43 (HMLEtwistKNCx43). HLMEtwist cells showed a 2.3-fold increase in Cx43 compared with HLME cells and HLMEtwistKNCx43 cells showed a 2.0-fold decrease compared with HLMEtwist cells, as measured by densitometry. (C) HMLE, HMLEtwist or HMLEtwistKNcx43 cells expressing dsRed were injected into the circulation of Tg(fli1:egfp) transgenic zebrafish and allowed to extravasate in the brain for 24hours. Shown are representative 3D confocal reconstructions (20×) of the brain vasculature (green) and tumor cells (red) in the head region. The left-most panel shows 3D reconstruction of the head of a control, non-injected embryo. Insets show higher magnification (60×) of areas delineated by the dashed squares. (D) Brain microtumor formation of HMLE, HMLEtwist and HMLEtwistKNCx43 cells was assessed using the chicken metastasis assay. Representative 3D reconstructions (10×, top views) show confocal z-stacks of chicken embryo brains 3days after intravenous injection of HMLE, HMLEtwist or HMLEtwistKNCx43 cells. (E) Average percentage of HMLE, HMLEtwist or HMLEtwistKNcx43 cells that extravasated in the zebrafish embryo brain 24hours after injection. (F) Average number of microtumors per zebrafish brain induced by HMLE, HMLEtwist and HMLEtwistKNCx43 cells 24hours after injection. (G) Average number of microtumors per chicken brain induced by HMLE, HMLEtwist and HMLEtwistKNCx43 cells 3days after injection. Data indicate means + s.e.m. Scale bars: 20μm (A) or 200μm (C,D).