Figure 5. In vivo effect of Bevacizumab in combination with glucose analogue 2-DG in U251 induced tumors.

(A) In ovo pictures at 13^th day of embryo development (4 days of tumor growth) and at 17^th day (4 days of treatment, in ovo and ex ovo); representative pictures were taken at 20x in a Zeiss stereomicroscope. (B) Graphical representation of tumors perimeter (in relation to day 13 that was considered as 1 in each group) at the upper panel and of blood vessels (in relation to control without drugs, considered as 1) in the lower panel, showing a slight decrease on tumor size and significantly decrease in blood vessels on tumors treated with both Beva (2 mg/ml), 2-DG (1 mM) alone and 2-DG plus Beva. The results are representative for a media of 8 eggs per group; ****p<0.001 treated group vs control for blood vessels counting. (C) H&E and IHC expression of hypoxia and metabolic markers after 4 days of treatment with Beva, 2-DG and 2-DG+Beva. It is observed in the H&E a higher number of necrotic cells in the tumors that were treated with 2-DG and with Beva+2-DG. The IHC shows a decrease in CAIX expression in the combination group; GLUT1 increased in all groups; MCT1 expression increased after Beva treatment and decreased in combination group; MCT4 expression increased only in 2-DG conditions. The H&E pictures were taken with 4 and 20X objective, while and the ones from IHC were taken with 200X amplification in an Olympus microscope. The yellow line defines the necrotic area of U251 micro-tumors.