Figure 3.

Pazopanib inhibited PDGFRβ phosphorylation from human immortalized astrocytes and tumor-activated primary cultured human glial in vitro. A: Microfluorimetric image analysis of pazopanib effect on p751-PDGFRβ immunostaining (in green) by immortalized human astrocytes cultured in basal conditions overnight and treated with 5 μmol/L pazopanib (added every 24 hours) or DMSO for 48 hours. p751-PDGFRβ staining intensity was quantified and normalized to DAPI staining. Data represent average values from five or seven images per well from three independent experiments (n = 17 per group). Box-and-whisker plots of the raw data are shown, but the statistical analysis was performed on transformed data. ^∗P < 0.0001. B and C: Representative images of p751-PDGFRβ staining after DMSO (B) or pazopanib (C) treatments. D–G: Identification of PDGFRβ phosphorylation in GFAP-expressing cells from a primary culture of astrocyte-enriched human glial cells. Glial cells were isolated by mechanical cell separation, enzyme digestion, and differential plating, from a tumor-unaffected area in human brain tissue obtained by neurosurgery from a patient with a metastatic brain tumor. Same antibodies were used as above. DAPI staining in blue (D). Some p751-PDGFRβ–expressing cells in green (E) and GFAP-expressing cells in red (F) are colocalized in yellow (G). H: Same enriched glial cell cultures were exposed to basal medium (untreated astrocytes) or the conditioned medium from serum-free and basal condition-cultured 231-BR-HER2 cells (tumor-activated astrocytes) for 18 hours in the presence of 5 μmol/L pazopanib or DMSO. The number of cells expressing p751-PDGFRβ was quantified by flow cytometry after immunostaining with the same antibody as above. Three independent experiments, using primary culture from three different patients (n = 3 per group), are shown in the dot plots. The bars represent the median. Scale bars: 100 μm (B and C); 10 μm (D–G).