Increased microvessel density in PC3 holoclone-derived tumors. Tumor angiogenesis was significantly increased in holoclone-derived tumors compared to tumors derived from parental PC3 cells and paraclone 2B6 cells. A, qPCR analysis of the endothelial cell marker CD31 using mouse-specific primers (mean ± SE, n = 3-4 tumors/group). B, quantification of immunohistochemical staining of mouse CD31-positive vascular area (NIH ImageJ quantitation, mean ± SE for n = 6-7 parental PC3 tumors and holoclone 2G7-derived tumors/group, based on three separate regions for each tumor). C, CD31 staining of PC3 tumors seeded with parental PC3 cells, which give rise to poorly vascularized tumors (PC3), as did tumors seeded from paraclones, such as 2B6. In contrast, all 5 holoclones tested ('h') showed a significant increase in vascularity compared to parental PC3 cell-derived tumors.