Fig 2: AAT increases the tumor burden, upregulates the IL-8 levels, and increases the numbers of CXCR2+ tumor cells that line the VM structures in GBM animal models. Quantitative data are expressed in mean ± SEM. *P < .05, **P < .01, and ***P < .001. (A) Representative MR images from vehicle (n = 12), vatalanib (n = 16), and avastin (n = 5) animal groups. The quantification of the tumor volume for these treatment groups is presented on the right. (B) Human cytokine array data showing upregulation of IL-8 levels in vivo and (C) in vitro. Representative immunofluorescence images showing (D) increased expression of CXCR2 (red) in vatalanib-treated tumors. (E) CXCR2+ GBM cells in the formation of VM structures in vatalanib-treated tumors. Scale = 50 μm. (F) Laminin and CXCR2 staining in vehicle- and avastin-treated groups and (G) CXCR2 and PAS staining in a rat model of U251 GBM showing increased numbers of CXCR2+ GBM cells lining PAS-positive VM structures in the early treatment vatalanib 0-21 and delayed treatment vatalanib 8-21 groups compared to vehicle-treated group (n = 9 per group); Scale = 50 μm. (H) BFP+ U251GBM (blue) cells as indicated by the white arrows incorporating into the host endothelial vascular structures (green) injected with Br1-GFP AAV; scale = 50 μm.

Fig 3: CXCR2-KD cells show significant overall decrease in tumor volume and form lesser laminin-positive VM structures. (A) Western blot with densitometry quantification and (B) immunofluorescence imaging showing the decreased expression CXCR2 in the CXCR2 knockdown cells (CXCR2 Cw#5) compared to scramble cells (n = 2). Representative images of (C) CXCR2-KD tumors showing significant reduction in the volume (five-fold) compared to scrambled tumors (n = 4). Immunofluorescence staining showing (D) reduction in the laminin-positive VM structures in the CXCR2-KD tumors compared to the scrambled tumors; scale = 100 µm. Quantitative data are expressed in mean ± SEM. **P < .01. (E) Absence of CXCR2+ GBM cells lining the laminin-positive structures in CXCR2-KD tumors; scale = 50 µm. (F) Scramble U251 and CXCR2 knockdown (CXCR2 DKD) U251 GBM cells were seeded on Matrigel for 6 hours in hypoxia. Quantitative data are expressed in mean ± SEM. *P < .05 and ***P < .001. (G) Differential expression patterns of phosphoproteins and proposed pathway schematic of IL8-CXCR2–mediated VM in GBM tumor cells.

Fig 4: SB225002 therapy decreases the tumor burden and CXCR2+ endothelial cell-like and stem cell–like populations in GBM tumors. Quantitative data are expressed in mean ± SEM. *P < .05, **P < .01, and ***P < .001. n = 3. (A) Representative MR images from vehicle, SB225002, and vatalanib + SB225002 animal groups. The quantification of the tumor volume for these treatment groups is presented on the right. Flow cytometry data in vivo (B) showing different CXCR2 GBM subpopulations and (C) endothelial-like GBM subpopulations in vehicle, SB225002, and vatalanib + SB225002 animal groups. (D) U251 GBM cells treated with vehicle (control) and two different concentrations of SB225002 (1 µM and 2 µM) and cultured in both normoxic (upper panel) and hypoxic (lower panel) conditions for 6 hours. Quantitative data are expressed in mean ± SEM. *P < .05 and ***P < .001. (E) Representative images of the areas within the tumor showing decreased expression of CXCR2+ GBM cells and vascular laminin coverage compared to the vehicle-treated groups, n = 3.

Fig 5: U87 GBM, HF2303, and GBM811 PDX models were developed in athymic nude mice. (A) MRI data show significantly increased tumor burden in vatalanib-treated groups compared to the vehicle- and avastin-treated groups. The quantification of the tumor volume for these treatment groups is presented on the right (n=3). (B) CXCR2 and PAS staining is represented with quantification of the CXCR2+ GBM cells lining the PAS-positive structures after treatment with vehicle (control) and HET0016 in the HF2303 neurospheres (upper panel) and GBM811 PDX models. Scale: 100 μm. Quantitative data are expressed in mean ± SEM. ***P < .001 and ****P < .0001.