Gliomas are fatal and refractory to treatment. Targeting angiogenesis may offer a promising therapeutic strategy 1. In this study, we characterized the role of angiogenesis in glioma pathogenesis using the C6 model in Wistar and SD rats 2,3. Angiogenesis was assessed using DCE MRI, and factors contributing to angiogenesis, including VEGF, HIF1, and the polarization of M2 macrophages, were examined by immunohistochemistry.Rat C6 glioma cell line was from the Bioresource Collection and Research Center (ATCC # CCL-107, Taipei, Taiwan). A total of 0.9×105 C6 cells (in 0.6 μl PBS) was injected to 9-week-old Wistar or SD rats at the right striatum [Bregma: 0.2, 3.0, 5.0 mm]. The animals were subjected to MRI examinations on 20 days after implantation, using a horizontal 7.0-T spectrometer (PharmaScan 70/16, Bruker, Germany). Rats were anesthetized, and T2WIs were acquired using a fast spin-echo sequence with FOV = 2.56 cm, slice thickness = 1 mm, 8 slices, TR/TE = 3000/70 ms, echo train length = 8, NEX = 12, and matrix size = 256×128 (zero filled to 256×256). DWIs were obtained with the Stejskal-Tanner spin-echo sequence (TR/TE= 2600/35 ms, NEX = 1, diffusion gradient duration = 5 ms, diffusion gradient separation = 12 ms, four b values = 0, and 1100 mm2/s applied along the X, Y, and Z directions) to generate an averaged ADC maps by customized MATLAB codes. The DCE MRI was using a dynamic series of 80 T1-weighted gradient-echo images with TR/TE = 130.2/4.1 ms, filp angle = 30°, 8 slices, 2D acquisition, FOV = 2.56 cm×2.56 cm, matrix size = 256×128 (zero filled to 256×256), slice thickness = 1 mm, and NEX = 1. An intravenous bolus injection of 0.2 mmol/kg Gd-DTPA (Gadoevist, AG, Germany) was administered during acquisition of the eighth image in total 80 images. ANOVA was used to identify group differences using STATVIEW. Immunohistochemistry of Iba1, VEGF, HIF-1, CD11c, and CD163 was performed with brain sections.Fig. 1 shows the workflow of this study. The T2WIs, DWIs, and ADC maps of C6 tumors developed in Wistar and SD rats, and animal survival were shown in Fig. 2. As measured by T2WIs, significantly increased tumor volume was demonstrated in SD rats, and tumor-bearing SD rats displayed a shorter survival than the Wistar rats. The C6/SD tumors were characterized with well-circumscribed margin, whereas tumors formed in the Wistar rats exhibited blurred boundaries with necrotic lesion in the center. We further examined the functionality of BBB and degree of angiogenesis using DCE MRI (Fig. 3). A significant increase in vascular permeability (Ktrans) was noted in tumors in SD rats as comparison to that in Wistar rats, indicating a higher degree of angiogenesis in C6/SD tumors. In support, immunohistochemistry of the brain sections revealed higher levels of expression of VEGF as well as of its upstream regulator HIF-1 (Fig. 3C and 3D). The M2 type of tumor-associated macrophages (TAMs) have been shown to shape the tumor microenvironment and facilitate angiogenesis. We further examined the polarization of the TAMs in C6 gliomas (Fig. 4). Both C6 tumors in the Wistar and SD rats were stained positive for Iba1, a marker for microglial cells (the resident macrophages) and recruited macrophages. Importantly, the TAMs in SD tumors were predominantly stained positive for M2 marker CD163, whereas M1 type of TAMs were readily detected in the Wistar tumors.The present study clearly demonstrates an important role of angiogenesis in glioma pathogenesis. Using the C6 glioma model, we show that C6/SD gliomas display a more malignant phenotype than the C6/Wistar tumors. The enlarged tumor volume was associated with an increased degree of angiogenesis, accompanied with increased expression of VEGF and HIF-1, as well as with the presence of M2 type of TAMs. These findings suggest that therapeutic approach coupling tailored therapy targeting angiogenesis may represent an attractive strategy for treating glioma patients.