Figure 3.

Activation of DRD2 signaling increases the self-renewal capacity of GBM cells. A, PDX GBM cells treated with either DMSO or TMZ were sorted based on DRD2 expression and neurosphere assays were performed. Using the extreme limiting dilution assay algorithm, frequency of GICs was calculated (DMSO, DRD2 + 1/458, and DRD2- 1/2091, p < 0.001; TMZ, DRD+ 1/196, and DRD2 − 1/515, p < 0.001). B, In vivo engraftment of 500 cells sorted on DRD2 ± expression demonstrates DRD2+ cells engraft more efficiently. Figure 3-1 includes more images of engrafted tumors from other mice. PDX lines treated with dopamine agonist demonstrate elevation in key stem cell genes via Western blot. Figure 3-1 includes Western blots from other PDX lines demonstrating similar trends. C, PDX GBM cells were treated with either DRD2 agonist (30 nm) or equimolar vehicle control DMSO and plated for neurosphere assays. Using the extreme limiting dilution assay algorithm, frequency of GICs was calculated [GBM6: DMSO stem cells 1/62 and agonist stem cells 1/29.2 (p < 0.01); GBM39: DMSO stem cells 1/181.9 and agonist stem cells 1/87.8 (p < 0.05); GBM5: DMSO stem cells 1/118.9 and agonist stem cells 1/67.4 (p < 0.05)]. Figure 3-1 includes a nonresponding GBM12 neurosphere assay. D, Two PDX lines were treated with either CPM (1 μm) or equimolar DMSO and plated for neurosphere assays as above. Fraction Nonresponding graph is shown for GBM 39. Difference between groups was determined by score test of heterogeneity. Error bars represent upper and lower limit of 95% confidence interval computed for stem-cell frequency. E, Viral knock-out of DRD2 using two different SH-RNA constructs in GBM43 demonstrates reduced stem-cell frequency. Student t tests were performed for each comparison. **p < .001, ****p < 0.0001.