Figure 1.

Full-length ITIH5 suppressed tumor cell growth in vivo. A tumorigenesis model was established to analyze the tumor growth of breast cancer cells either overexpressing ITIH5 or lacking ITIH5 expression and transplanted into the mammary fat pad. Tumor volume was determined by both using a caliper (A) and µCT imaging (B,C (see arrows)), showing the significantly decreased tumor growth of MDA-MB-231 breast cancer cells transfected with ITIH5. (D) ITIH5 overexpression in tumors grown in the mouse model was confirmed by qPCR analysis. In order to assess the putative effects of ITIH5 on the mechanisms of neovascularization, which may impact growth rates in vivo, we visualized the vascular system by contrast-enhanced µCT imaging (E,F) and calculated the relative blood volume (rBV) (G) between both groups. (H) Immunofluorescence staining of the markers involved in blood vessel maturation, i.e., CD31 (a specific marker for vascular structures) and αSMA (a specific marker for vascular differentiation). Hoechst dye was used to stain the nuclei. (I) The staining intensity of CD31 (H) was analyzed for tumors derived from either ΔpBK-mock (dark grey) or ΔpBK-ITIH5 clones (grey), shown as the area fraction. (J) The correlation analysis between relative blood volume (rBV) determined by µCT and the CD31 area fractions showed a significant association in the tumors of both groups. * p ≤ 0.05; ** p < 0.01; *** p < 0.001.