Loss of Rb expression promotes metastatic behavior in human breast cancer cell lines.

<p>(A) Phase contrast images and quantification of mammosphere-forming potency of MCF7ras and T47D cells with Rb knockdown. Arrows indicate protrusions formed by invading cells and cell clusters. Scale bar, 50 μm; unequal variance Student's t-test, * p<0.05, ** p<0.01, *** p<0.001. (B) Morphology of primary tumors and lymphovascular (LV) or mammary fat pad (MFP) invasion. MCF7ras cells stably expressing shRNA against Rb or scrambled sequence were injected into the MFP of NOD/SCID mice. Primary tumors and other tissues were harvested after 8 weeks and H&E stained. Bar graph depicts primary tumor weight in each group expressed as mean ± SD where <i>n</i> is the number of animals in each group. Arrows indicate sites of lymphovascular invasion in Rb knockdown experiment. Scale bar, 50 μm (left panel) and 25 μm (middle panel). (C) Fluorescent and H&E stained images of metastatic tumor growth in lungs. Metastases containing EGFP-expressing Rb knockdown or control cells originated from orthotopic site. Images from <i>n</i> number of animals were quantified using ImageJ software (NIH). Data are presented as mean ± SD. Scale bar, 5 mm or 100 μm; equal variance Student's t-test, ** p<0.01, *** p<0.001. (D) Scheme showing isolation of circulating cancer cells (CCC) from mice bearing primary tumors. CCC were co-separated with the mononuclear fraction of blood and adhered to Poly-L-lysine-coated plates. (E) Fluorescent images and quantification of isolated EGFP-expressing circulating cancer cells and cell clusters. Scale bar, 200 μm; unequal variance Student's t-test, * p<0.05, ** p<0.01. (F) Western blot analysis of cell lysates from in vitro cultures of MCF7ras cells expressing Rb or control shRNA. β-tubulin staining was used as a loading control. (G) Western blot analysis of lysates from primary tumors. Total Akt was used as a loading control.