SUPPLEMENTARY INFORMATION: Single cell derived clonal analysis of human glioblastoma links functional and genomic heterogeneity ! Mona Meyer*, Jüri Reimand*, Xiaoyang Lan, Renee Head, Xueming Zhu, Michelle Kushida, Jane Bayani, Jessica C. Pressey, Anath Lionel, Ian D. Clarke, Michael Cusimano, Jeremy Squire, Stephen Scherer, Mark Bernstein, Melanie A. Woodin, Gary D. Bader**, and Peter B. Dirks**! ! * These authors contributed equally to this work.! ** Correspondence: [email protected] or [email protected]! ! Supplementary information - Meyer, Reimand et al. Supplementary methods" 4" Patient samples and fluorescence activated cell sorting (FACS)! 4! Differentiation! 4! Immunocytochemistry and EdU Imaging! 4! Proliferation! 5! Western blotting ! 5! Temozolomide treatment! 5! NCI drug library screen! 6! Orthotopic injections! 6! Immunohistochemistry on tumor sections! 6! Promoter methylation of MGMT! 6! Fluorescence in situ Hybridization (FISH)! 7! SNP6 microarray analysis and genome segmentation! 7! Calling copy number alterations! 8! Mapping altered genome segments to genes! 8! Recurrently altered genes with clonal variability! 9! Global analyses of copy number alterations! 9! Phylogenetic analysis of copy number alterations! 10! Microarray analysis! 10! Gene expression differences of TMZ resistant and sensitive clones of GBM-482! 10! Reverse transcription-PCR analyses! 11! Tumor subtype analysis of TMZ-sensitive and resistant clones! 11! Pathway analysis of gene expression in the TMZ-sensitive clone of GBM-482! 11! Supplementary figures and tables" 13" "2 Supplementary information - Meyer, Reimand et al. Table S1: Individual clones from all patient tumors are tumorigenic. ! 14! Fig. S1: clonal tumorigenicity.! 15! Fig. S2: clonal heterogeneity of EGFR and PTEN expression.! 20! Fig. S3: clonal heterogeneity of proliferation.! 21! Fig. S4: clonal differentiation potential.! 22! Fig. S5: correlation of TMZ resistance and proliferation. ! 27! Fig. S6: doubling time of clones from tumor GBM-472. ! 28! Fig. S7: clonal MGMT status.! 29! Fig. S8: clonal drug response in NCI library.! 30! Table S2: clonal drug response in NCI library.! 31! Fig. S9: clonal dose response of bortezomib, daunorubicin, and romidepsin.! 37! Fig. S10: PCA clustering of clonal copy number alterations.! 38! Fig. S11: circos plots of clonal copy number alterations.! 39! Fig. S12: Copy number of chromosome 3 in tumor GBM-482. ! 42! Fig. S13: FISH validation of clonal chromosomal alterations. ! 43! Table S3: genes with clonal copy number alterations.! 44! Fig. S14: genes with consistent copy number alterations.! 49! Fig S15: clonal copy number alterations observed in TCGA.! 50! Fig. S16: pathway analysis of clonal copy number alterations.! 51! Table S4: pathway analysis of clonal copy number alterations.! 52! Fig S17: Subtype scoring of clones from tumor GBM-472, GBM-489, and GBM-498.! 57! Table S5: genes with differential expression in TMZ sensitivity. ! 58! Table S6: pathway analysis of TMZ expression profile. ! 67! Bibliography! 69 "3 Supplementary information - Meyer, Reimand et al. Supplementary methods" ! Patient samples and fluorescence activated cell sorting (FACS)" Tumor samples were obtained from consenting patients, as approved by the Research Ethics Boards at The Hospital for Sick Children and Toronto Western Hospital (Toronto). Within 1 hour of surgical resection tumors were processed and acutely dissociated in oxygenated artificial cerebrospinal fluid (CSF) and subjected to enzymatic dissociation. Following dissociation tumor cells were stained for the cell surface markers CD15 (#347423, BD Bioscience) and human specific CD133/1 (AC133/1-PE, Miltenyi Biotech) and single cells from each population (CD negative, CD15 positive, CD133 positive and CD15/CD133 double positive) were sorted into a 96 well plate (BD FACSAria III, BD FACSAria II, Beckman Coulter MoFlo-XDP). Single cells were cultured in EGF/FGF conditions as previously described (1, 2) and grown as adherent monolayer on poly-L- ornithine (Sigma) and laminin (Sigma) coated plates. Cells were grown in serum-free Neurocult NS-A Basal media (Stemcell Technologies), supplemented with 2 mM L- glutamine, N2 and B27, 75 µg/mL BSA, 10 ng/mL rhEGF, 10 ng/mL bFGF and 2 µg/mL !heparin. Cells were treated with Accutase (Sigma) for expansion or harvesting. ! Differentiation" 10,000 cells from each clone were seeded onto poly-L-ornithine/laminin (Sigma) coated coverslips in 24-well plates. Clones were grown under EGF/FGF conditions. In parallel, clones were also grown under growth factor withdrawal conditions to promote neuronal differentiation. Briefly, cells were cultured overnight in NS media with B27 supplement, 10 ng/mL rhEGF, 10 ng/mL bFGF and 2 µg/mL heparin. To promote differentiation, the media was changed to Neurocult NS-A Basal media supplemented with FGF2 (5ng/mL) and 1X B27 supplement only for 7 days, then to a 1:1 mix of Neurocult NS-A Basal media with Neurobasal media (Invitrogen) with 1X B27 supplement for 14 days. Immunocytochemistry (IHC) was performed using antibodies against the epitopes Nestin (AB5922, rabbit polyclonal, Millipore), #III-tubulin (MAB1637, mouse monoclonal, Millipore), GFAP (MAB360, mouse monoclonal, Millipore), MAP2 (MAB3418, mouse monoclonal, Millipore). Slides were visualized and collected using a Leica STP 6000 microscope. Representative images were taken at 20x magnification as merged images !and their individual filter images.! Immunocytochemistry and EdU Imaging" Clones were grown on poly-L-ornithine/laminin–coated glass coverslips. Cells were fixed in 4% paraformaldehyde for 30 min at room temperature, washed with PBS, and permeabilized in 0.3% Triton X for 30min. Cells were stained with antibodies against human-specific Nestin (1:1,000; AB 5922; Millipore), human-specific GFAP (1:1,000; MAB 360, Millipore), and Map2 (1:MAB 3418, mouse monoclonal, Millipore). Secondary antibodies used include Alexa Fluor 488 Goat anti-Rabbit IgG (1:500; A11034, Invitrogen), Alexa Fluor 488 Goat anti-Mouse IgG (1:500, A21042, Invitrogen), and Alexa Fluor 568 Goat anti-Mouse IgG (1:500, A11004, Invitrogen). DNA staining was !performed using DAPI (Sigma).! "4 Supplementary information - Meyer, Reimand et al. Proliferation" All clones were seeded onto poly-L-ornithine/laminin (Sigma) coated 96-well plates at an initial density of 2000 cells per well. Relative cell proliferation was measured for each clone at days 0, 2, 4, 6, and 8 using an AlamarBlue assay (Life Technologies), as per manufacturer's protocol. Briefly, 10 $L of Alamar blue was added to 90 $L of culture medium in each well. After 6 hours, plates were read on an Spectramax Gemini EM microplate reader (Molecular Devices), with an excitation wavelength of 544 nm and emission wavelength of 595 nm. Data were acquired using SOFTmaxPRO and analysed using GraphPad Prism 5 software. Each data point is an average of at least 3 replicates, and error bars represent standard deviation. ! The population doubling level (PDL) for each clone from tumor 472 was calculated using the formular: PDL = X + 3.322 (log Y – log I), where, X = initial PDL; I = cell inoculum (number of cells plated); Y = final cell yield (number of cells at the end of the !growth period). Doubling time assay for each clone was performed in triplicates." Western blotting " Clonal protein expression of EGFR, EGFRvIII, and PTEN was analyzed with western blots using EGFRvIII-transfected human fetal brain cells (HF7450NS) as positive control and eGFP transfected cells as negative control. Western blotting was performed using the following primary antibodies: anti-EGFR (1:1,000; ab2430; Abcam), anti-EGFRvIII (1:1,000; Zymed; now discontinued), anti-PTEN (1:200; sc7974; Santa Cruz Biotechnology), anti-Act# (1:5,000; A5441; Sigma-Aldrich), anti-GABRA3 (1:1000; HPA000839; Sigma-Aldrich), anti-phospho-H2A.X (Ser139) (1:1000; #2577; Cell Signaling). Secondary antibodies included goat anti-mouse IgG, HRP conjugate (1:100,000; A9044; Sigma-Aldrich), and goat anti-Rabbit IgG, HRP conjugate (1:40,000; A0545; Sigma-Aldrich). The human EGFR variant III cDNA [3] was cloned into the pIRES2-EGFP vector (#6029-1, Clontech Laboratories). The EGFRvIII construct was a gift from Dr. Abhijit Guha (The Hospital for Sick Children, Toronto). 5$g of pIRES2- EGFP and pIRES2-EGFRvIII were used to transfect approximately 1 million cells of the human fetal derived neural stem cell line HF7450. The transfection was performed using the Amaxa Nucleofector Kit (VPG-1004; Amaxa Biosystems) and the Nucleofector II Electroporator (Amaxa Biosystems) as per the manufacturer's instructions. Protein !lysates were harvested for analysis 48 hours following transfection. ! Temozolomide treatment" 3,000 cells of each clone were seeded into a well of a coated 96-well plate. All cells were treated once with temozolomide (TMZ) at a dose range of 0.39-100 µM. Cell viability was measured when DMSO treated control cells reached 70% confluence, using the AlamarBlue assay (Life Technologies), as per manufacturer's protocol, and read 6 hours later on an Spectramax Gemini EM microplate reader (Molecular Devices). Data were analysed with GraphPad Prism 5 software. Data points represent an average of three replicates, and error bars represent standard deviation. Unpaired T-test with Welch’s correction was used to calculate p-values of cell survival between relatively !sensitive and resistant clones.! "5 Supplementary information - Meyer, Reimand et al. NCI drug library screen" Clones were seeded onto poly-L-ornithine/laminin