The cell growth was evaluated at different time points using cell number counting. 1B). We found the prevalent expression of VASH1 in endothelial cells in both cancer stroma and paracancerous normal tissues (Figure ?(Figure1A).1A). However, in the paracancerous normal tissues, the numbers of VASH1+ vessels are very low (mean numbers of 3.1), whereas significantly increased numbers of VASH1 expression in vascular endothelial cells were detected in colon cancer BMS-906024 stroma (mean numbers of 4.7) (Figure ?(Figure1B).1B). The result strongly suggested the activated angiogenesis in colon cancer patients. In addition, we investigated the expression levels of the other well-known angiogenic molecules CD34 and VEGF-A, as well as lymphoangiogenenic molecules D2-40 and VEGF-C in colon cancer tissues and paracancerous normal tissues (Figure 1C & 1D). CD34 expression was mainly localized in the cytoplasm and membrane of the blood endothelial cells, while D2-40 expression was observed in the cytoplasm and cellular membrane of lymph endothelial cells (Figure ?(Figure1C).1C). BMS-906024 Furthermore, VEGF-A and VEGF-C were found expression in the cytoplasm both in cancer cells and in paracancerous normal tissues (Figure ?(Figure1C).1C). In addition, expression TNFSF10 levels of CD34, D2-40, VEGF-A and VEGF-C in colon cancer tissues were significantly higher than those in paracancerous normal tissues (Figure ?(Figure1D).1D). Our results collectively suggest that both active angiogenesis and lymphoangiogenesis exist in colon cancer patients, and that VASH1 is prevalent in the cancer stroma of cancer tissues. Open in a separate window Figure 1 Expression of VASH1 in cancer stroma of BMS-906024 colon cancer patients(A) & (B) Significantly increased VASH1 expression density in endothelial cells of blood vessels was detected in colon cancer stroma, BMS-906024 compared with that expressed in paracancerous normal tissues. Numbers of VASH1+ vessels in 75 colon cancer tissues and 59 paracancerous normal tissues were detected and summarized using the immunohistochemical staining. (C) & (D) Expression levels of angiogenic molecules CD34 and VEGF-A, as well as lymphoangiogenenic molecules D2-40 and VEGF-C in colon cancer tissues (n=75) and paracancerous normal tissues (n=59) were determined using the immunohistochemical staining. Expression level of each dot shown in (B) and (D) is the average numbers (VASH1, CD34 and D2-40) or scores (VEGF-A and VEGF-C) per high field (400 x) in each tissue sample. The mean number of each molecule in each group is shown as a horizontal line. Significance was determined by unpaired (cancer tissue vs paracancerous tissue) T test. Stroma VASH1 is an important cancer angiogenic molecule in human colon cancer Given that high density of VASH1 expression in blood endothelial cells in cancer stroma, and that active angiogenesis and lymphoangiogenesis were observed in colon cancer tissues, we next determined whether cancer stroma VASH1 is associated with colon cancer lymphangiogenesis and angiogenesis. The correlations between cancer stroma VASH1 expression level and expressions of CD34, D2-40, VEGF-A, VEGF-C in cancer tissues were analyzed. We found that cancer stroma VASH1 was positively correlated with its expression in paracancerous normal tissues (Figure ?(Figure2A).2A). Furthermore, box plot and linear correlation analyses demonstrated that there was a significant correlation between stroma VASH1 and CD34, a key microvessel density (MVD) marker, in colon cancer tissues (Figure 2B and 2C). However, there were no correlations between cancer stroma VASH1 expression and VEGF-A expression in cancer cells, and lymphoangiogenenic molecules D2-40 (a lymphatic vessel density marker) and VEGF-C in cancer tissues (Figure 2D, 2E and 2F). Open in a separate window Figure 2 Correlations between cancer stroma VASH1 expression and levels of other angiogenic and lymphoangiogenenic BMS-906024 molecules in colon cancer tissues(A) Scatter diagram showing a positive correlation between cancer stroma VASH1 and paracancerous tissue VASH1. (B) and (C) Scatter diagram (B) and box plot (C) analyses showing positive correlations between expression levels of cancer stroma VASH1 and cancer tissue CD34. The mean number of VASH1 in each group is shown as.