Rb/CD44-dependent regulation of initial cell survival and metastatic progression.

<p>(A) Illustration of tail vein injection experiment in mouse. (B) MCF7ras cells were infected with shRNA to Rb or control sequence expressed in an EGFP vector. The resulting cells were injected in tail vein and fluorescent images of whole lungs were taken at indicated time points. Representative images are shown. Total fluorescence of whole lung images was quantified and is plotted as mean ± SD. Scale bar, 500 μm (left and middle panels) and 100 μm (right panel); equal variance Student's t-test, ** p<0.01. (C) Metastatic-like tumor growth in lungs 8 weeks after tail vein injection. Total number of metastases quantified by ImageJ is plotted into the graph on the left. The ratio of macrometastases to the total number of metastases in each pair of lungs is presented in the graph on the right. The number of animals in each group is indicated as <i>n</i>. Data is shown as mean ± SEM. Scale bar, 3 mm; equal variance Student's t-test, ** p<0.01, *** p<0.001. (D) Immunofluorescence of frozen sections from lungs of mice with metastatic growth after tail vein injection. Number of Ki-67 positive cells was quantified by ImageJ and is presented as mean ± SD. Scale bar, 25 μm; equal variance Student's t-test, *** p<0.001. (E) Dissecting microscope fluorescent images and quantification of cells/cell clusters in lungs one hour or 72 hours after tail vein injection of MCF7ras cells with suppressed Rb and/or CD44 expression. Representative pictures are shown. Scale bar, 25 μm; unequal variance Student's t-test, ** p<0.01. (F) Whole lungs fluorescent images from metastatic tumor growth eight weeks after tail vein injection of single/double Rb/CD44 knockdowns. Number of metastatic foci per mouse was quantified by ImageJ and is plotted in the graph. Scale bar for fluorescent images, 1 mm; unequal variance Student's t-test, ** p<0.01.