Figure 2
CXCR7 regulates breast cancer tumor growth in vivo by regulating angiogenic, proliferative, and signaling pathways. Female BALB/c mice (6 to 8 weeks old, n = 5) were anesthetized and injected with 2.5 x 105 breast cancer cells 4T1 Vec and 4T1 sh-CXCR7 (downregulated for CXCR7). (A) Tumors were measured by digital calipers weekly for 36 days (mice model inset). (B) Representative photographs of tumors, 36 days after the injection of cells. (C) Tumor weight after 36 days. Female BALB/c mice (6 to 8 weeks old) were anesthetized and injected with 1 × 105 viable 4T1.2 cells into the fourth mammary fat pad. Mice were divided into three groups of five mice each and were injected intraperitoneally with either DMSO or CXCR7 inhibitor (CCX771) or STAT3 inhibitor (S31-201) at 5 mg/kg body weight 3 times a week for 21 days (inhibitors were injected in mice after tumors were palpable), (D, G) tumors were measured with digital calipers (mice model inset) (E, H) Representative photographs of tumors, 31 days after the injection of cells. (F, I) Tumor weight after 31 days. (J) 4T1 Vec, 4T1 sh-CXCR7, and 4T1.2 cell line-derived tumors from CCX771 or S31-201 treated or untreated group were subjected to immunohistochemical (40×) staining with anti-CD31 or Ki67. (K, L) The stained cells were counted in four different fields by using bright-field microscope in each experimental group and the average was calculated. Bars represent the mean ± SD of number of CD31-positive blood vessels and Ki67-positive cells to that of total cells. (M) 4T1 Vec and 4T1 sh-CXCR7 cell line-derived tumors were lysed and analyzed for Phospho STAT3, STAT3, Phospho-ERK (p-ERK), ERK, cyclin D1, and GAPDH with immunoblotting. Data represent the mean ± SD per experimental group. Scale bars, 0.03 mm. *P < 0.05, **P < 0.01, ***P < 0.001 versus control.