Fig. 4: TRF2 knockdown reduces formation of spontaneous metastases in vivo.

A Schematic representation of spontaneous metastasis experiment. Luminescent MDA-MB-231 shSCR and shTRF2 N1 were injected in the mammary gland of female NSG mice. Primary tumor was resected at day 19 and spontaneous lung metastases formation was monitored by bioluminescence imaging followed by ex vivo analyses. B Left panel, representative images of all mice ten days post primary tumor resection. Right panel, Box-and-whisker plot representing lung metastases bioluminescence intensity; shSCR (N = 7), shTRF2 (N = 7). Two-tailed t student test was used to calculate P value. C Representative images of Cytokeratin AE1/AE3 immunostained sections of lungs from spontaneous metastasis experiment. Scale bars, 100 μm. D Quantification of the number of single breast cancer cells and metastatic foci per 25X field. E Schematic representation of intravital experiment. MDA-MB-231 shSCR and shTRF2 N1 cells stably expressing H2B-Dendra2 were injected into the mammary gland of female NSG mice. After formation of primary tumors, animals have been subjected to intravital imaging. F Left panel, representative images of shSCR and shTRF2 N1 H2B-Dendra2 positive cells in their in vivo setting at the end point (6 h). Right panel, Box-and-whisker plots (10–90 percentile) of cell velocity, displacement/hour, and persistence; n = 900 cells from three mice per condition were analyzed. Two-tailed Mann Whitney test was used to calculate P value. G Schematic representation of experimental metastasis experiment. Luminescent MDA-MB-231 shSCR and shTRF2 N1 were injected in the tail vein of NSG mice and colonization of the lungs was monitored by bioluminescence imaging followed by ex vivo analyses. H Left panel, representative images of all mice ten days post cell injection. Right panel, Box-and-whisker plots representing lung metastases bioluminescence intensity; shSCR (N = 5), shTRF2 (N = 5). I Representative images of Citokeratin AE1/AE3 immunostained sections of lungs from experimental metastasis experiment. Scale bars, 100 μm. J Quantification of the number of single breast cancer cells and metastatic foci per 25X field. K Representative images of TRF2 immunostained sections of primary tumor, spontaneous and experimental lung metastases. Scale bars, 30 μm. L Quantification of the percentage of TRF2 positive cells in primary tumors and lung metastasis sections. For (D, J, L), twelve fields derived from three animals for condition were analyzed. Data are the mean ± SD, two-tailed t student test was used to calculate statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.