Individual clones within the same tumor show phenotypic heterogeneity. (A) Differential expression of EGFR, EGFRvIII, and PTEN in individual clones from two patient tumors. EGFRvIII-transfected human fetal brain cell line HF7450NS was used as control. (B) Individual clones of tumor GBM-489 vary in their ability to express markers of differentiation. Staining for human Nestin (green), human GFAP (red), Map2 (green), βIII-tubulin (red), and EdU (red). (C) Variable temozolomide (TMZ) drug responses of individual clones from four different tumors. Clones were treated once with TMZ (0.39–100 µM) and measured for viability. Significant differences in TMZ response were observed among clones from two tumors, GBM-472 (P = 0.0011), and GBM-482 (P < 10−4). (D) Heterogeneous promoter methylation of MGMT in GBM-482. Asterisks indicate sensitive cell populations. (E) Heterogeneous protein expression of MGMT in GBM-482. (F) NCI drug library screen reveals clones with variable drug sensitivity in GBM-482. Drugs with at least 50% growth inhibition on individual samples are shown and are ranked by response variability.