Quantifying Tumor Cell Proliferation and Angiogenesis

Tumor samples were stained with anti-Ki67 antibody (CST, Danvers, MA, USA). Ki67-positive tumor cells were observed with a Zeiss Axioskop 2 plus microscope. The proliferation index (PI) was determined as the ratio of the number of Ki67-positive cells to the total number of counted tumor cells [34 (link)]. Cluster of differentiation 31 (CD31) is a key biomarker for vascular endothelial cells, and its immunostaining density is represented by the tumor microvessel density (MVD) [35 (link)]. Tumor tissues were stained with CD31 antibody (CST, Danvers, MA, USA). Vessels were observed and counted by using a Zeiss Axioskop 2 plus microscope [31 (link)].