Fig. 4
Patient-derived primary epithelial tumor cells cultured under hydrodynamic stress (SS) demonstrate significantly elevated in vivo tumorigenicity. a NOD/SCID mice were injected subcutaneously with increasing numbers of breast cancer cells derived from chemotherapy-treated patients CT-PCs or CT + SS, and tumor volumes (middle panel) and weights (right panel) were measured at 4 weeks after tumor cell inoculation. Representative images of tumors from the mice injected with the indicated number of cells (102–104 cells per site) are shown on the right (n = 3, *p < 0.05). Tumor tissues from the CT + SS-injected mice were analyzed by H&E staining or immunohistochemical analysis with the indicated antibody. Each indicated region (small squares) is magnified in the insets. b Tumor volumes and weights of successive passages of xenografts harboring CT + SS. The secondary xenograft was generated from injection of 50 cells isolated from the tumors formed from the preceding passage. Representative images of tumors harvested from mice are shown on the right (n = 4, *p < 0.05). c Protein expression of E-CADHERIN, N-CADHERIN, and TWIST was analyzed by western blot using the tumor tissues harvested from the 1st and 3rd passaged mice. Numbers refer to the densitometry analysis of each signal normalized against the corresponding anti-ACTIN values. d In vivo tumorigenicity of NOD/SCID mice injected orthotopically into the mammary fat pads with CT-PCs or CT + SS. Left panel, representative BFI images of mice at 4 and 8 weeks post-injection of the indicated cells (n = 3). Right panel, the percentage of green fluorescent protein (GFP)+ cells in the blood at 4 and 8 weeks post-injection was analyzed by flow cytometry. e The ratio of ionized calcium levels in whole blood from the CT-PCs- or CT + SS-injected mice were measured by the ABL800 analyzer. Error bars correspond to mean ± SD. Statistically significant differences were tested at p < 0.05