Supplementary Methods, Figures 1 - 19, Tables 1 - 6 from Contributions to Drug Resistance in Glioblastoma Derived from Malignant Cells in the Sub-Ependymal Zone
Supplementary experimental procedures: this file contains an expanded materials and methods section on 5-ALA administration and sample collection, cell lines propagation, immunofluorescence and in vivo assays, genomic and molecular clock analysis, fluorescence in situ hybridization, drug concentration used in the proliferation assay and MGMT promoter methylation analysis. Supplementary Figure S1: this figure illustrates the identification of the sub-ependymal zone (SEZ) in two GB patients included in this study. Supplementary Figure S2: this figure shows the sampling and morphology of the SEZ tissue. Supplementary Figure S3: this figure summarizes the results of real time analysis for markers of glial, precursor, stem cells and proliferation in paired T and SEZ from three GB patients. Supplementary Figure S4: this figure shows an example of common copy number aberration breakpoints between the SEZ and the corresponding T in each of the GB patients included in this study. Supplementary Figure S5: this figure summarizes the fluorescence in situ hybridization (FISH) results for EGFR, MET and PTEN in three GB patients. Supplementary Figure S6: this figure shows the copy number aberration breakpoints between T and SEZ and the corresponding cell lines in patient sp14. Supplementary Figure S7: this figure shows the results of growth curve analysis of tumor-initiating cells (TICs) derived from matched T and SEZ of five GBs. Supplementary Figure S8: this figure summarizes the results of clonogenic index between three paired T and SEZ cells and multipotency of SEZ cells. Supplementary Figure S9: this figure shows the immunofluorescence results for stem cell markers on T and SEZ cells in two GBs. Supplementary Figure S10: this figure summarizes the results of cumulative Kaplan-Meier survival analysis from five additional GBs. Supplementary Figure S11: this figure shows an analysis of the in vivo properties of TICs isolated from the SEZ of three GBs. Supplementary Figure S12: this figure shows the results of the cell proliferation assay of 7 paired TICs using 50μM of Temozolomide (TMZ). Supplementary Figure S13: this figure summarizes the cell proliferation data of additional 20 TICs treated with TMZ for dose escalation analysis. Supplementary Figure S14: this figure shows the results of cell proliferation assay of additional 4 paired TICs treated with TMZ, Cisplatin and Cediranib. Supplementary Figure S15: this figure shows a gel image of MGMT promoter status analysis for T and SEZ of the analyzed 7 paired TICs. Supplementary Figure S16: this figure summarizes the immunofluorescence results for vascular endothelial growth factor receptor 2 (Vegfr2) on T and SEZ cells from three GBs. Supplementary Figure S17: this figure summarizes the cell proliferation data of three control lines. Supplementary Figure S18: this figure illustrates a model of residual disease in human GB and summarizes the cardinal features of T and SEZ. Supplementary Figure S19: this figure shows the results of growth curve analysis of two SEZ in absence of fluorescence. Supplementary Table S1: this table summarizes the results of the histological features of T and SEZ tissues. Supplementary Table S2: this table provides a summary of the samples used for the genomic analysis and of the clinical information available for the eleven GB patients included in this study. Supplementary Table S3: this table shows the gene ontology (GO) terms whose genes are differentially expressed between SEZ and T. Supplementary Table S4: this table provides a summary of the results of MGMT methylation analysis by pyrosequencing in sixteen GBs. Supplementary Table S5: this table summarizes the overall survival of the eight GB patients whose TICs were used for the cell proliferation assay and includes the MGMT methylation by pyrosequencing. Supplementary Table S6: this table shows the gene expression data of PDGF and VEGF receptors in T and SEZ.