Figure 4. RARRES3 impairs metastatic cell adhesion to the lung parenchyma.

• A (Left panel) Percentage of apoptotic cells under anoikis conditions, as determined by FACS measurement of Anexin V staining. Same number of cells of the indicated cell lines were plated, and measurements were taken at the indicated time point. Data are averages of three independent experiments ± SD. (Right panel) Apoptotic human luciferase activity of the indicated cell lines was measured in vivo using a Caspase-3 luciferin pro-substrate and normalized to total luciferase activity at the indicated time points after injection (n = 5 per group). Data are averages ± SEM. PI stands for post-injection.
• B Cell tracker green-labeled LM2-mock and LM2-RARRES3 cell lines were injected via the tail vein and allowed to lodge in the lungs. Two day post-injection, mice were inoculated with rhodamine-lectin and 30 min later were perfused with 5 ml of PBS to remove cells attached to the vasculature. Lungs were extracted, flushed with PBS, and fixed-frozen in OCT, and frozen sections were obtained. Representative confocal images of extravasated cells (green) to the lung parenchyma are shown. In red, vasculature staining using rhodamine-lectin. Right panel: Quantification of extravasated cells in each condition is plotted. n = 5 mice per group. 10 sections per mouse were scored. Data are averages ± SD.
• C, D Adhesion to collagen I and fibronectin, respectively. The MBA-MB-231 shControl, shRARRES3 #1 and #2, LM2-Mock and LM2-RARRES3 cells were labeled with cell tracker green and plated (5 × 10⁴) in triplicate in 24-well inserts coated with Collagen I (C) and Fibronectin (D). One hour post-plating, inserts were washed twice with PBS to remove non-attached cells and fixed in PFA. Images were taken, and the area covered by cells was determined using Image J. The percentage of area covered by cells in Collagen I (C) or Fibronectin (D) inserts is shown. Data are averages of three independent experiments ± SD (n = 3).