Endothelial

dependent vessels (EVs) counting standard: According to the standard introduced by Weidner et al. [23, 24], capillary VX-689 purchase vessels and microvessels in the tumor stained with CD31 were counted. A single positively stained endothelial cell can be counted as one EV. VM counting standard: The wall of VM is lined with tumor cells, and red cells can be found in the VM, without NVP-AUY922 mw inflammation cells or red cell leakage around the VM [25]. MVs counting standard: The vessel wall was lined with both tumor and endothelial cells [14, 25]. PGCCs counting and definition Five microscopic fields in each tissue section were reviewed and scored under microscopy with × 400 magnification and the average was summarized. The size of PGCCs nuclei was measured by a micrometer using H&E section. We defined the PGCC as a cancer cell that the nucleus of PGCC is at least three times larger than that of diploid cancer cell according Napabucasin in vitro to Zhang et al. description [11]. Tumor xenografts in chicken embryonating eggs Fresh fertilized eggs (less than 5 days after fertilization) (Tianjin Shengchi Inc.) were kept under 75% humidity and 37°C. At day 3 after incubation, the egg shell was cleaned with 75% ethanol. A square window (1 × 1 mm2)

was opened in the end of air cell. The shell was removed and 0.1 ml PBS with 5 × 106 glioma C6 cells was injected into the chorioallantoic membrane (CAM) of each egg. The opening was then closed with a cellophane tape and the eggs were incubated until the 20th day. All these operations were performed in the sterile environment. These fertilized eggs were rotated with 45 degrees every day and the

air cell end was always kept upright. At day 20 after incubation, the fertilized eggs were put into the -20°C freezer to kill the chicken embryos and then the tumor mass were Suplatast tosilate dissociated. The tumor tissues were fixed with formalin and embedded with paraffin for H&E staining to observe the structure of different blood supply patterns and erythrocytes generated by PGCCs. Statistical analysis The statistical analysis was performed using SPSS statistical analysis software (SPSS, Chicago, IL). An unpaired t-test was performed to analyze the differences in the number of VM, MVs and EVs. The χ 2 test was used for the PGCCs number comparison among different grades of gliomas. A P-value less than 0.05 was considered statistically significance. Results Number of PGCCs associated with histologic grade of gliomas To grade all these 76 cases of glioma, new sections were cut from 76 paraffin-embedded glioma samples and stained with H&E and immunohistochemistry for further analysis. These tumors were graded by two pathologists according to the morphologic characteristics and Ki-67 IHC staining.