The size of PGCC nucleus was three times and up to 10–20 times larger than that of the regular diploid cancer cell. The shape of PGCCs nuclei was irregular. Ki-67 IHC staining data showed that Ki-67 expressed in all the glioma tissues and the positive ratio increased with the grade of gliomas. Most of PGCCs were positive for Ki-67 staining (Figure 1B).

Based on these morphologic characteristics and Ki-67 staining, Vorinostat in vitro 76 cases of glioma were graded into 28 cases of low grade glioma (4 cases of grade I and 24 cases of grade II) and 48 cases of high grade (28 cases of grade III and 20 cases of grade IV). PGCCs can be observed in all these glioma tissues (Figure 1A), but there were more PGCCs in high grade tumors than those in low grade tumors and the difference was find more statistically significant (χ 2 = 4.781, P = 0.015) (Figure 1C). Figure 1 Identification of PGCCs in glioma tissues. A. PGCCs present in human VS-4718 purchase gliomas. a) PGCCs in grade I gliomas (Black arrow points) (×200). b) PGCCs in grade II gliomas (Black arrows point) (×200). c) PGCCs in grade III gliomas (Black arrows point) (×200). d) PGCCs in grade IV gliomas (Black arrows point) (×200). B. Ki-67 IHC staining in gliomas and black arrows indicate the PGCCs. a) Ki-67 expression in grade I gliomas (×200). b) Ki-67 expression in

grade II gliomas (×200). c) Ki-67 expression in grade III gliomas (×200). d) Ki-67 expression in grades IV gliomas (×200). C. Association of PGCCs number with the grades of human gliomas. Erythrocyte generation by PGCCs Zhang et al. reported that PGCCs of breast cancer cell line BT-549 was able to generate erythrocytes in vitro and in vivo [20]. To determine whether glioma PGCCs can directly generate erythrocytes, H&E and anti-hemoglobin-β/γ/ϵ/δ chain IHC staining were performed on glioma tissue sections and the results showed that there were many red bodies budding from PGCCs. These red bodies located in the cytoplasm or adhered

to the surface of PGCCs (Figure 2A -a). Figure 2A-b showed that some red bodies located in the cytoplasm of PGCC. An interesting phenomenon indicated that some PGCCs generating mafosfamide erythrocytes form the wall of VM and MVs. Figure 2A-c showed that PGCCs and their generating erythrocytes can form VM structure and PGCCs lined in the basement membrane of VM. Hemoglobin-β/γ/ϵ/Δ IHC staining confirmed that these red bodies generated by PGCCs were erythrocytes (Figure 2A -d). Figure 2 Human high grade glioma cells generated erythrocytes. a) H&E staining showed that there were many red bodies adhered to the surface of PGCCs (Black arrows point) (×200). b) Red bodies located in the cytoplasm of PGCC (Black arrows point) (×200). c) PGCCs and their budding erythrocytes form vessel-like structure with basement membrane (Black arrows point) (×200). d) IHC staining of hemoglobin-β/γ/ϵ/δ confirmed that the red bodies generated by PGCCs were erythrocytes (Red arrows point) (×200).