Single Cell-Derived Clonal Analysis of Human Glioblastoma Links Functional and Genomic Heterogeneity

Single cell-derived clonal analysis of human glioblastoma links functional and genomic heterogeneity

Mona Meyera,1, Jüri Reimandb,c,1, Xiaoyang Lana,b, Renee Heada, Xueming Zhua, Michelle Kushidaa, Jane Bayanid, Jessica C. Presseye, Anath C. Lionelb,f, Ian D. Clarkea,g, Michael Cusimanoh, Jeremy A. Squirei, Stephen W. Schererb,f, Mark Bernsteinj, Melanie A. Woodine, Gary D. Baderb,c,2, and Peter B. Dirksa,b,k,2

aDivision of Neurosurgery, Program in Developmental and Stem Cell Biology, Arthur and Sonia Labatt Brain Tumour Research Centre, Hospital for Sick Children, Toronto, ON, Canada M5G 1X8; bDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; cThe Donnelly Centre, University of Toronto, Toronto, ON, Canada M5S 3E1 Canada; dDepartment of Transformative Pathology at the Ontario Institute for Cancer Research, Ontario Institute for Cancer Research, Toronto, ON, Canada M5G 0A3; eDepartment of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada M5S 3G5; fThe Centre for Applied Genomics, The Hospital for Sick Children, Toronto, ON, Canada M5G 1L7; gOntario College of Art and Design, Toronto, ON, Canada M5T 1W1; hDivision of Neurosurgery, St. Michael’s Hospital, Toronto, ON, Canada M5B 1W8; iDepartment of Pathology, Queen’s University, Kingston, ON, Canada K7L 3N6; jDivision of Neurosurgery, Toronto Western Hospital, Toronto, ON, Canada M5T 2S8; and kDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada M5S 1A8

Edited by Robert A. Weinberg, Whitehead Institute for Biomedical Research; Department of Biology, Massachusetts Institute of Technology, and Ludwig MIT Center for Molecular Oncology, Cambridge, Cambridge, MA, and approved November 24, 2014 (received for review November 7, 2013) Glioblastoma (GBM) is a cancer comprised of morphologically, Understanding the links between genetic and functional genetically, and phenotypically diverse cells. However, an un- behavior of individual GBM clones, derived from single pa- derstanding of the functional significance of intratumoral hetero- tient samples, will be essential to decipher patient-specific geneity is lacking. We devised a method to isolate and functionally molecular mechanisms of GBM progression and therapeutic profile tumorigenic clones from patient glioblastoma samples. resistance. The study of bulk tumors provides a mixture of Individual clones demonstrated unique proliferation and differen- different signals from a heterogeneous set of clones and cur-

tiation abilities. Importantly, naïve patient tumors included clones rent single cell approaches are amenable to genomics but not MEDICAL SCIENCES that were temozolomide resistant, indicating that resistance to functional studies. Here, we provide a detailed analysis of single conventional GBM therapy can preexist in untreated tumors at cell derived clones from patient GBM samples using a method a clonal level. Further, candidate therapies for resistant clones were that directly links clonal molecular changes with functional detected with clone-specific drug screening. Genomic analyses properties. Parallel phenotypic and genomic analysis of these revealed genes and pathways that associate with specific functional clones reveals a diverse landscape of functional and genetic behavior of single clones. Our results suggest that functional clonal heterogeneity, as well as insights into drug sensitivity pathways profiling used to identify tumorigenic and drug-resistant tumor in glioblastoma. clones will lead to the discovery of new GBM clone-specific treatment strategies. Significance

cancer | glioblastoma | clonal heterogeneity | genomic analysis | functional analysis Glioblastoma is an incurable brain tumor. It is characterized by intratumoral phenotypic and genetic heterogeneity, but the functional significance of this heterogeneity is unclear. We lioblastoma (GBM) is the most common and most aggres- devised an integrated functional and genomic strategy to ob- Gsive primary malignant brain tumor in adults. Despite major tain single cell-derived tumor clones directly from patient efforts to improve GBM survival, radiation therapy with concurrent tumors to identify mechanisms of aggressive clone behavior temozolomide (TMZ) chemotherapy achieves only a median sur- and drug resistance. Genomic analysis of single clones iden- vival of 15 months with few long-term survivors. Many patients fail tified genes associated with clonal phenotypes. We predict to respond to TMZ, and treatment of all patients at disease pro- that integration of functional and genomic analysis at a clonal gression uniformly fails. level will be essential for understanding evolution and ther- Glioblastoma is one of the first cancer types systematically apeutic resistance of human cancer, and will lead to the dis- – studied at a genomic and transcriptomic level (1 3). Tran- covery of novel driver mechanisms and clone-specific cancer scriptional profiling of GBM samples has revealed a landscape treatment. of intertumoral heterogeneity with distinct molecular tumor subtypes, although only slight prognostic differences are ap- Author contributions: M.M., J.R., X.L., I.D.C., J.A.S., M.A.W., G.D.B., and P.B.D. designed parent in patients except for a clearly better prognosis in the research; M.M., J.R., X.L., R.H., X.Z., M.K., J.B., J.C.P., I.D.C., M.A.W., and G.D.B. performed CIMP+/IDH1 mutant subgroup (1, 4–6). Increasing evidence research; M.M., J.R., X.L., M.C., M.B., and G.D.B. contributed new reagents/analytic tools; M.M., J.R., X.L., M.K., J.B., J.C.P., A.C.L., I.D.C., J.A.S., S.W.S., M.A.W., G.D.B., and P.B.D. suggests that cancer tissues are more complex than previously analyzed data; M.M., J.R., X.L., G.D.B., and P.B.D. wrote the paper; and P.B.D. originated thought, as tumors comprise considerable intratumoral het- and supervised the study at all stages. erogeneity with mixtures of genetically distinct subclones that The authors declare no conflict of interest. – likely escape therapy and cause disease progression (4, 7 10). This article is a PNAS Direct Submission. In particular, GBM is a cancer type comprised of morpholog- Freely available online through the PNAS open access option. ically and phenotypically diverse cells (11). Recent studies have Data deposition: Gene expression profiles and copy number data are available at ArrayEx- also uncovered genetic diversity apparent in subsamples of in- press, www.ebi.ac.uk/arrayexpress/ (accession nos. E-MTAB-2693 and E-MTAB-2694). – dividual patient GBMs (12 15), and more recently in single 1M.M. and J.R. contributed equally to this work. GBM cells (9). Disease recurrence is associated with muta- 2To whom correspondence may be addressed. Email: [email protected] or peter. tional events that are not shared with the primary tumor sug- [email protected]. gesting evolution from minority populations present at time of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. initial diagnosis (10). 1073/pnas.1320611111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1320611111 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 Results independent experiments with clones of different passages, the Single Cell-Derived Clones of Human Glioblastoma Exhibit Functional clones exhibited wide and independent variation in proliferation B SI Appendix Heterogeneity. To enable functional characterization of hetero- and differentiation abilities (Fig. 2 and , Figs. S3 and A–C geneous clones in glioblastoma, we devised a method to isolate S4 ), demonstrating intrinsic distinct functional properties. and grow individual clones from patient tumors using flow We then assessed clonal population response to the conven- cytometry sorting (FACS) and single cell plating in stem cell tional GBM chemotherapeutic agent TMZ (17), in at least three culture conditions (Fig. 1A). We generated 44 individual single independent experiments with clones of different passages. Two tumors contained clones with heterogeneous TMZ responses: 2 cell derived clones from four patient tumors. All tested clones of 8 tested populations in the naïve tumor GBM-472 showed established typical GBM tumors after orthotopic injection in significantly increased resistance to TMZ (P = 0.0011, t test with mice, indicating their tumorigenic potential (SI Appendix, Table Welch correction; clones 472_8, 472_18), whereas 1 clone of 10 in A–D B − S1 and Fig. S1 and Fig. 1 ). the recurrent tumor GBM-482 was relatively sensitive (P < 10 4; We next functionally characterized individual clones. We 482_9; Fig. 2C). We observed only TMZ-resistant clones from identified heterogeneous expression of key GBM proteins phos- the two additional naïve and recurrent tumors. TMZ response phatase and tensin homolog (PTEN), epidermal growth factor and proliferation ability was not significantly correlated (SI Ap- receptor (EGFR), and the constitutively active EGFR deletion pendix, Figs. S5 and S6). Clonal analysis of O6-methylguanin- mutant, EGFRvIII (2, 3, 10, 16), in three tumors, indicating that DNA methyltransferase (MGMT), a common biomarker of TMZ distinct known molecular GBM drivers vary at a clonal level (Fig. resistance in GBM (18), showed variable promoter methylation 2A and SI Appendix,Fig.S2) and suggesting limitations to targeted and protein expression levels that were inconsistent with TMZ therapies based solely on bulk GBM analysis. In identical cul- response (Fig. 2 D and E and SI Appendix,Fig.S7). These data ture conditions and with each resultconsistentinatleasttwo show that TMZ-resistant clones are present ab initio and stably in untreated patient tumors and are not necessarily acquired under therapeutic selection pressure. Given that many GBM clones were TMZ resistant, we sought A FACS to identify potential alternative chemotherapeutic agents. We screened six to eight individual clones from each of three tumors with the National Cancer Institute (NCI) oncology library of 98 drugs and found that every tested tumor showed clone by clone Tumor sample variability in responses to multiple drugs (Fig. 2F and SI Ap- pendix, Table S2 and Figs. S8 and S9). These data readily suggest Expand in reasons why this disease is so refractory to chemotherapies stem cell media and suggest that clone-specific therapies could be developed for GBM treatment.

Integrated Analysis of Functional and Genomic Clonal Heterogeneity Predicts Pathways of Drug Resistance. Copy number (CN) profiling Functional analysis Genomic analysis of all samples with Affymetrix SNP6.0 microarrays reveals a Tumorigenicity Proliferation Copy number analysis landscape of clonal genetic heterogeneity, consistent with pub- TMZ response Drug response Gene expression analysis lished observations (2, 3, 9) (Fig. 3A). All tumors carried hallmark Differentiation Pathway analysis GBM alterations such as chr7 gains and chr10 losses, validating our genetic data. Phylogenetic analysis of CN profiles confirms 498_3 hu-Nestin hu-GFAP DAPI B the heterogeneous genetic composition of GBMs and shows that clones of common origin are genetically closer to each other than to those from other tumor samples (Fig. 3B and SI Appendix, Fig. S10). We did not find single clones from one patient crossing into the genomic space of another. These results suggest that the impact of our stem cell culturing on genetic stability is negligible compared with clonal genetic heterogeneity. They also suggest that unsorted populations have one or more dominant clones: two 100 μm 100 μm unsorted populations (GBM-489, GBM-498) most resemble single clones (489_13, 498_5) and thus likely have single dominant clones, whereas the unsorted population of tumor GBM-472 is similar to multiple clones or the primary tumor sample and thus is likely polyclonal. CN analysis reveals clonal chromosomal and focal genomic alterations (Fig. 3C and SI Appendix,Fig.S11A–C). For example, gain of chr7 appears in a subset of clones from tumor GBM-482, whereas chr3 gain is only observed in clone 482_3 (Fig. 3D and SI 100 μm 100 μm Appendix,Fig.S12). Fluorescence in situ hybridization (FISH) validates these amplifications and highlights gain of chr3 in clone Fig. 1. Single cell-derived clonal cultures from primary GBMs show a neural 482_3 as a minor cell population of the primary tissue, as well as precursor phenotype and are tumorigenic. (A) Functional characterization of the genetic homogeneity of derived clones (Fig. 3D and SI Ap- single cell-derived clones. Glioblastoma samples were dissociated into single pendix,Fig.S13). These data confirm genetic polyclonality in cell suspensions. Cells were then sorted using stem cell markers to enrich for sample GBM-482 and provide evidence that our derived clones clonogenic activity. Single live cells were then expanded in EGF/FGF media and analyzed functionally and genomically. (B) Immunostaining of a repre- are homogeneous and originate from the primary tumor sample. sentative xenograft shows tumor formation with an infiltrative phenotype Clonal genetic heterogeneity is also apparent at the gene level reminiscent of human GBM cell behavior in situ (green, human nestinl; red, (Fig. 3E and SI Appendix, Table S3 and Fig. S14). The most human GFAP; blue, DAPI). frequent clonal variation comprises hallmark GBM events such

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1320611111 Meyer et al. Downloaded by guest on September 26, 2021 A GBM-472 GBM-482 B - + U 13 4 7 9 1417188 +P134891113- 7 EGFR Differentiation EGFRvIII hu-Nestin EGFRvIII hu-GFAPDAPI Map2 EdU βIII-tubulinDAPI PTEN βAct - - negative control P - primary tissue

+ - positive control U - unsorted sample EGF/FGF C Clone 489_5 NAïVE uns RECURRENT differentiating 13 GBM-472 GBM-482 9 4 EGF/FGF

11 withdrawal 7 1.0 1 1.0 14 2 8 0.5 *** 16 7 0.5 18 3 14 ** 17 ] 4 8

0 0 EGF/FGF GBM-489 uns GBM-498 1 uns Clone 489_8 Cell survival 1.0 3 1.0 6 5 7 non-differentiating EGF/FGF 0.5 10 0.5 withdrawal 6 1 Bars=50 μm 7 12 2 0 8 13 0 -6.5 -5.5 -4.5 -6.5 -5.5 -4.5 3 log (c) TMZ (M) F GBM−482 Dasatinib Cytarabine HCL D Bleomycin GBM-482, MGMT promoter methylation Tretinoin Cladribine Paclitaxel

100bp Valrubicine U MU MUU MU M U M U M U M U M U M U M U M Mitomycin C Ixabepilone -13478911+ H2O Clofarabine * Topotecan HCL MEDICAL SCIENCES U/M - (un)methylated primer + - positive control Gemcitabine HCL * - TMZ sensitive cells - - negative control Teniposide Doxorubicin HCL Mitoxantrone Docetaxel Vinorelbine tartrate E Plicamycin GBM-482, MGMT protein expression Dactinomycin Vinblastine sulfate MGMT Vincristine sulfate Cabazitaxel Actβ Daunorubicin HCL 134789 11 Romidepsin % growth Bortezomib Clone * inhibition 11 4321613 7 9 075 Clone

Fig. 2. 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.

as EGFR amplification (2). In total, we found copy number (Fig. 4A). Most clones from the same tumor were more similar to alterations in 456 genes, including 31 of 100 published frequently each other than to clones from a different tumor at the level of − CN altered genes in GBM (2) (P = 1.2 × 10 25; OR, 19; Fisher’s transcription, generally confirming our observed pattern of genetic exact test; SI Appendix, Fig. S15). Clonally altered genes are heterogeneity. However, a few clones were more similar to clones enriched in functions such as regulation of fibroblast proliferation from another tumor, in agreement with previous observations from [6 genes; false discovery rate (FDR), P = 0.022 from g:Profiler single cell RNA sequencing (9). In particular, the TMZ-sensitive (19)], and signaling pathways such as PI3K/AKT (2) (13 genes; clone 482_9 is clearly distinct from other clones of GBM-482. FDR, P = 0.014), TGFβ (20) (3 genes; FDR, P = 0.0080), and Transcriptional profiling of bulk tumors shows that GBM mTOR (21) (5 genes; FDR, P = 0.020), suggesting that genetic comprises several distinct subtypes, generally agreed to be clas- alterations contribute to functional clonal heterogeneity (SI Ap- sical, proneural, and mesenchymal (1, 4, 6, 22). We sought to pendix, Table S4 and Fig. S16). However, we did not observe analyze the relationship of clonal subtypes and TMZ sensitivity. statistically significant correlations between genetic pathway Using the previously defined subtype signatures, we calculated alterations and clonal chemoresistance. subtype scores for 29 GBM clones from all four tumors and two Clonal heterogeneity is also evident at the level of gene ex- unsorted populations (GBM-472 and GBM-489) to distinguish pression (Fig. 4). We performed microarray analysis of 32 GBM between the three subtypes. Consistent with recent RNA-seq based clones from all four tumors and two unsorted populations (GBM- subtyping of single GBM cells (9), these clones show a hybrid 472 and GBM-489). Principal component analysis of clonal ex- cellular state with high scores in more than one subtype (Fig. 4B pression profiles shows extensive heterogeneity within each tumor and SI Appendix, Fig. S17). The TMZ-sensitive clone 482_9

Meyer et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 A Chromosomes 19 21 B 482_16 C 482_3 123456789101112131415161718 20 2222 482_8 GBM-482 22 1 482_2 482_13 21 Z-score 482_11 482_4 20 482_7 2 482_1 19 482_12 1 2 GBM-472 482_14 482_9 18 0 472_14 17 472_pr 482_pr 489_6 -1 16 472_10 489_pr 3 -2

GBM-482 489_12

472_uns 489_8 15 489_uns 14 472_8 489_13

489_1 4 tumor samples 489_7 13 472_4 489_3 GBM-489 472_17 489_10489_5 12 472_13

498_uns 5

GBM-498 472_18 11 472_7 498_6 498_pr 498_5 Relative copy 498_4 498_7 number, log2 10 6

498_3 -1 0.50 498_2 498_1 7 D GBM-482 primary tissue 9 8

- cells with 3 copies of Chr3 * - cells with 4 copies of Chr7 - cells with 3 copies of Chr7

E 482_pr. 489_5 489_13 489_1 489_8 489_6 472_4 472_pr. 472_7 472_9 472_8 472_uns. 498_pr. 498_5 498_1 472_18 472_17 498_uns. 498_7 498_6 498_4 498_3 498_2 472_13 472_14 489_uns. 489_7 489_3 489_pr. 489_12 489_10 SAMPLE ALTERATION 482_4 482_1 ORIGIN 482_11 balanced 482_9 482_14 472 gain 482_7 482 amplification 482_3 482_16 489 loss 482_2 482_13 498 deletion 482_12 482_8 MKX SVIL NFIA WAC IFN # FZD8 ZEB1 GJD4 CUL2 EPC1 NRP1 MPP7 MTAP KIF5B CCNY ITGB1 CREM INADL ZNF # KLHL9 BAMBI LRP1B RAB18 LYZL # PARD3 VOPP1 CCDC7 MTPAP ARMC4 C9orf53 SEPT7L LANCL2 C10orf # MAP3K8 SEC61G * MYC PTCHD3 VSTM2A NAMPTL TMEM75 CDKN2B KIAA1462 KIAA1217 POU5F1B RHGAP12 * EGFR MTRNR2L7 * CDKN2A ANKRD30A A

Fig. 3. Heterogeneous clonal genetics as mapped by copy number (CN) analysis. (A) Probe-level CN heat map shows genetic variation in clonal populations. Samples are grouped vertically by patient tumor (left vertical color bar; right label: asterisk, primary sample; diamond, unsorted population). (B) Phylogenetic analysis confirms the common origin of derived clones (colors) and shows that primary samples (asterisks) are relatively distant from derived clones. (C) Circos plot of segmented CN alterations in GBM-482. Inner circle represents baseline (no CN change), and lines expanding toward the center or the periphery ofthe diagram represent CN losses or gains, respectively (primary, black; clonal, colored). (D) FISH confirms chr3 gain in primary tumor tissue of GBM-482. (E) Heat map of frequently altered genes with clonal heterogeneity (at least five alterations).

scores lower in mesenchymal and higher in proneural and clas- Fig. 4C and SI Appendix,TableS5). This list includes a signif- − sical subtypes compared with resistant clones of tumor GBM- icant enrichment of 28 known cancer genes (P = 2.5 × 10 4; 482, suggesting that functional heterogeneity of drug response is OR, 2.2), suggesting that known cancer pathways are involved driven by transcriptional heterogeneity and that a mixture of in differential drug response of clones of GBM-482. For ex- clones of different subtypes is present in the bulk tumor. ample, the well-defined GBM oncogenes hepatocyte growth To further explore variable TMZ sensitivity within single pa- factor receptor (MET) and EGFR show reduced expression in tient tumors, we compared gene expression profiles of GBM-482 the TMZ-sensitive clone of GBM-482. qRT-PCR assays con- clones. We ranked 777 genes with the most pronounced differ- firm the results of microarray analysis and validate dramatic ential expression in the TMZ-sensitive clone relative to mean up-regulation of EGFR and MET in TMZ-resistant clones expression in six TMZ-resistant clones (absolute z-score > 2; (>10-fold; Fig. 4D).

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1320611111 Meyer et al. Downloaded by guest on September 26, 2021 A B CL MES PN D E ● ● Hemostasis ● ● ● Cell migration ● ● ● ● ● ● ● 4 MET ● 10 - 0.3 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 3 ● ● ● ● ● ● ● ● ● ● ● 10 - ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.2 TGFβ ● ● ● ● 472 ● ● 100- ● ● ● ● ● ● ● ● 482 ● ● ● Score ● ● ● ● ● ● ● ● 489 ● ● ● ● ● ● ● 10- Angiogenesis Extracellular matrix Protein ● ● ● 0.1 ●

● fold induction ● 498 ● ● Focal adhesion glycosylation 482_sens. MET, EGFR, MPP1, EGF, WNT7B

Principal component 2 1- ● TMZ 489_uns. ● ● ● ● ● resistant ● ● ● ● * 0.0 ● ● 472_uns. ● sensitive ● ● Ion channels C Principal component 1 100- EGFR 1.0-

0.5- 10- Neurotransmitters OLIG2 MYCNGAS7 MAP3K1 MAFHIP1 ANCA PTCH1RPS6KA1 ARID2 ANCGWASF BCL7ANUP214LMO1 0.0- RANBP2 RB1MUTYH LASP1F PRDM1MYH9CYLD

CHCHD7EGFR * fold induction COL1A2MALT1 −0.5- PL 1- Synaptic Membrane Gliogenesis AG1 membrane potential MET * 0.5 91117432 3 −1.0- 0.0 GRM3, GRIA2, GRIK3, GRIN2D, Log2 fold change −0.5 * GBM-482 −1.0 KCNH4, CACNG5, PTK2B, ERBB3 −1.5-

Fig. 4. Clonal function is correlated with gene expression and pathway function. (A) Principal component analysis of gene expression profiles of 32 GBM clones and unsorted populations highlights divergence of the TMZ-sensitive clone 482_9. (B) Transcriptional analysis of GBM-482 predicts that the TMZ- sensitive clone (orange, 482_9) belongs to a different GBM subtype than the six TMZ-resistant clones (gray). Three transcriptional subtypes of GBM (classical, mesenchymal, proneural) were analyzed with bootstrapping and permutation tests. (C) Barplot shows differentially expressed genes in the TMZ-sensitive clone 482_9 compared with mean expression in six TMZ-resistant clones of tumor GBM-482 (absolute z-score > 2). Genes are ranked according to fold change. (D) qRT-PCR analysis confirms significantly lower gene expression of MET and EGFR in the TMZ-sensitive clone 482_9 compared with six TMZ-resistant clones of

tumor GBM-482. (E) Pathway analysis illustrates differentially expressed pathways in the TMZ-sensitive clone with increased (red) and decreased (blue) gene MEDICAL SCIENCES expression. Selected genes associated with pathways are shown below network diagrams.

Pathway enrichment analysis (19) of differentially expressed Also, recent studies of GBM geography demonstrate maps of genes in the TMZ-sensitive clone highlights multiple significant regional copy number and gene expression differences that im- pathways and processes (FDR, P < 0.05; Fig. 4E and SI Ap- portantly shed light on mechanisms of intratumoral clonal evo- pendix, Table S6). These include ion channels, neurotransmitter lution in space and time and show that individual tumors can signaling, and synaptic membrane genes with increased expres- contain cells corresponding to different GBM subgroups (14). sion (e.g., KCNH4, GRM3,andCACNG5), whereas genes in- These observations have been extended by a single cell genomic volved in cell migration (MET) and angiogenesis (WNT7B, analysis of a large number of GBM cells from five patient EGF) are down-regulated compared with resistant clones. tumors, which strikingly reveals the genomic complexity of GRM3 encodes a glutamate receptor involved in growth, pro- this tumor (9). liferation, and survival of glioma and melanoma cells (23). Acti- To date, however, these features of genetic heterogeneity have vation of MET enhances GBM cell migration (24) and tumor cell not been directly linked to functional readouts such as drug re- WNT7B EGF resistance in response to DNA damage (25). and sponse, growth, or differentiation potential. Unless we consider have been shown to induce vasculature in the central nervous functional analysis from single cell derived populations, it will be system (26, 27). Interestingly, the TMZ-sensitive clone shows in- difficult to find the precise driver genes and molecular pathways creased expression of several genes involved in neurotransmitter GRIA2 GRIK3 responsible for aggressive behavior of individual clones in a het- signaling such as glutamate receptors ( , ,and erogeneous tumor, particularly if those clones are rare. Direct GRIN2D) (28). Neurotransmitters are involved in brain development functional-genomic correlations at a clonal level will be essential and proliferation of progenitor cells (29); however, their role in to developing new GBM therapies. tumor biology is less established. This protein family includes In this study, we developed a strategy to analyze pro- targets of approved drugs and therefore is an interesting candi- spectively isolated single GBM clones from fresh tumors that date for drug repurposing studies. Our data suggest that clonal links genotypes of individual subclones with tumorigenic, chemo-response in GBM may be altered by modulation of neurotransmitter signaling, and this deserves future exploration. We proliferative, differentiation, and, most importantly, drug re- demonstrate an example where transcriptional clonal heterogene- sponsiveness properties. Our proof-of-principle drug screen ity correlates with variable drug response and points to po- shows that single tumors contain clones that respond differ- tential therapeutic mechanisms that could render resistant ently to known cancer drugs, thus highlighting the opportunity clones as sensitive. to identify resistant clones, define prognostic biomarkers, and develop clone-specific combination therapies. Importantly, a Discussion clonal analysis of the standard GBM biomarker MGMT did The very name of this tumor type, glioblastoma multiforme, has not correlate with TMZ responsiveness, suggesting that new long defined its morphologic heterogeneous nature. There is biomarkers of drug responsiveness are sorely needed, consis- increasing appreciation that intratumoral genetic heterogeneity tent with more recent bulk GBM genomic analyses which is central to GBM biology (9, 14) and therapeutic resistance (10). highlight the subgroup limitations of this marker (1). We A number of recent studies demonstrated intratumoral het- predict that further studies of larger groups of patient tumors and erogeneity in GBM by showing regional variation and cell-to-cell derived clones are likely to yield additional clonal vulnerabilities differences in expression of receptor tyrosine kinases (12, 13, 15). that will have clinical relevance.

Meyer et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 Understanding the significance of cancer genetic heterogeneity AlamarBlue assay was performed for cell survival analysis after drug treatment and the impact on cancer relapse is enormously challenging and (temozolomide; National Cancer Institute oncology drug library). MGMT will require multiple approaches. The integration of genomics promoter methylation was determined by nested two-stage methylation- techniques with sophisticated bioinformatic analysis and, most specific PCR (18), using CpGenome Universal Methylated DNA (Millipore) importantly, clonal functional assays, provide a direct starting and patient blood DNA as controls. Genetic profiling of GBM samples was point, as it will identify tumor subpopulations that drive growth carried out with Affymetrix Human SNP Array 6.0 microarrays, using and therapeutic resistance. Future developments of this strategy CRMAv2 (31) for preprocessing, the CBS algorithm (32) for genome segmentation, and custom R scripts for filtering and global analyses. Broad would consider deep sequencing of bulk tumors and clones com- genomic segments were filtered. Matched blood reference was used for bined with computational inference of intratumoral clonal struc- three tumors, whereas a median diploid genome of 226 patients (33) com- ture (30). In addition, combining single cell approaches (9) with bined with filtering of polymorphic copy number variants (34) was used on single clone derived functional analysis are likely to give a clearer the unmatched tumor GBM-489. Phylogenetic analysis of copy number picture of GBM heterogeneity and the significance of genomic di- variation was conducted with a Markov Chain Monte Carlo procedure using versity. Although our approach may not capture all relevant clones the MrBayes software (35). To enumerate copy number changes of chro- in the primary patient sample, our study focuses on the critical tu- mosomes 7, 3, and 17, the Urovision probe kit (Abbott Laboratories) was morigenic fraction, as functional assays for the bulk population have used. Transcriptional profiling was performed with Affymetrix Human Gene not been developed. We predict that clone-specific functional pro- 1.0 ST microarrays, using the RMA algorithm for preprocessing and z-scores filing of GBMs will help identify aggressive clones, new cancer driver for differential expression estimation. Pathway enrichment analysis was mechanisms, molecular signatures, and therapeutic vulnerabilities performed with g:Profiler (19) and visualized as Enrichment Maps (36) with emphasizing the potential of cancer treatment at a clone-specific Cytoscape. All experiments were conducted on cell cultures below 20 pas- level. We envisage a similar clonal functional analysis strategy will sages. The identity of all samples was confirmed by genotyping analysis with be applicable to deciphering heterogeneity in other types of cancer. Genotyping Console (Partek), and by short tantem repeats (STR) analysis. One potential application of this approach will be the development Extended description of methods is available in SI Appendix, SI Materials and Methods. of anticipatory therapy, directed at themostaggressiverelapse-initiating clones identified at the time of patient diagnosis. ACKNOWLEDGMENTS. We thank P. A. Penttilä, A. Khandani, S. Zhao, T. Velauthapillai, and L. Jamieson for preparative flow cytometry, Materials and Methods Dr. A. Guha for kindly providing the EGFRvIII plasmid, and the support of Two naïve and two recurrent tumors originated from four individual the Toronto Western Hospital Brain Tumor Bank led by Dr. Gelareh Zadeh. patients. Single cell-derived clonal populations were retrieved by FACS live Flow cytometry was performed in The SickKids-University Health Network sorting and expanded in stem cell conditions. Intracranial cell transplantation Flow Cytometry Facility. P.B.D. is supported by grants from Ontario Institute for Cancer Research with funds from the Government of Ontario, Canadian involved injection of 100,000 cells into immuno-compromised (NSG) mice. Institutes for Health Research (CIHR), Genome Canada, Canadian Cancer Immunohistochemistry was performed on paraffin-embedded tissue. Clonal Society, the Hospital for Sick Children Foundation, Jessica’s Footprint Foun- protein expression of EGFRvIII was analyzed with Western blots using dation, and the Hopeful Minds Foundation. G.D.B. is supported by National EGFRvIII-transfected human fetal brain cells (HF7450NS) as a control. Differ- Resource for Network Biology (US National Institutes of Health Grant P41 entiation assays were carried out in growth factor withdrawal conditions. GM103504), and M.W. is supported by a CIHR grant.

1. Brennan CW, et al.; TCGA Research Network (2013) The somatic genomic landscape of 18. Esteller M, et al. (2000) Inactivation of the DNA-repair gene MGMT and the clinical glioblastoma. Cell 155(2):462–477. response of gliomas to alkylating agents. N Engl J Med 343(19):1350–1354. 2. Cancer Genome Atlas Research Network (2008) Comprehensive genomic characterization 19. Reimand J, Arak T, Vilo J (2011) g:Profiler—a web server for functional interpretation defines human glioblastoma genes and core pathways. Nature 455(7216):1061–1068. of gene lists (2011 update). Nucleic Acids Res 39(web server issue):W307-15. 3. Parsons DW, et al. (2008) An integrated genomic analysis of human glioblastoma 20. Ikushima H, et al. (2009) Autocrine TGF-beta signaling maintains tumorigenicity of multiforme. Science 321(5897):1807–1812. glioma-initiating cells through Sry-related HMG-box factors. Cell Stem Cell 5(5):504–514. 4. Verhaak RG, et al.; Cancer Genome Atlas Research Network (2010) Integrated ge- 21. Akhavan D, Cloughesy TF, Mischel PS (2010) mTOR signaling in glioblastoma: Lessons nomic analysis identifies clinically relevant subtypes of glioblastoma characterized by learned from bench to bedside. Neuro-oncol 12(8):882–889. abnormalities in PDGFRA, IDH1, EGFR, and NF1. Cancer Cell 17(1):98–110. 22. Sturm D, et al. (2014) Paediatric and adult glioblastoma: Multiform (epi)genomic – 5. Noushmehr H, et al.; Cancer Genome Atlas Research Network (2010) Identification of culprits emerge. Nat Rev Cancer 14(2):92 107. a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer 23. Prickett TD, Samuels Y (2012) Molecular pathways: Dysregulated glutamatergic sig- – Cell 17(5):510–522. naling pathways in cancer. Clin Cancer Res 18(16):4240 4246. 6. Sturm D, et al. (2012) Hotspot mutations in H3F3A and IDH1 define distinct epigenetic 24. Demuth T, Berens ME (2004) Molecular mechanisms of glioma cell migration and – and biological subgroups of glioblastoma. Cancer Cell 22(4):425–437. invasion. J Neurooncol 70(2):217 228. 7. Shah SP, et al. (2012) The clonal and mutational evolution spectrum of primary triple- 25. Li Y, et al. (2011) c-Met signaling induces a reprogramming network and supports the glioblastoma stem-like phenotype. Proc Natl Acad Sci USA 108(24):9951–9956. negative breast cancers. Nature 486(7403):395–399. 26. Goldman CK, et al. (1993) Epidermal growth factor stimulates vascular endothelial 8. Navin N, et al. (2011) Tumour evolution inferred by single-cell sequencing. Nature growth factor production by human malignant glioma cells: A model of glioblastoma 472(7341):90–94. multiforme pathophysiology. Mol Biol Cell 4(1):121–133. 9. Patel AP, et al. (2014) Single-cell RNA-seq highlights intratumoral heterogeneity in 27. Stenman JM, et al. (2008) Canonical Wnt signaling regulates organ-specific assembly primary glioblastoma. Science 344(6190):1396–1401. and differentiation of CNS vasculature. Science 322(5905):1247–1250. 10. Johnson BE, et al. (2014) Mutational analysis reveals the origin and therapy-driven 28. Chen RS, Deng TC, Garcia T, Sellers ZM, Best PM (2007) Calcium channel gamma evolution of recurrent glioma. Science 343(6167):189–193. subunits: A functionally diverse protein family. Cell Biochem Biophys 47(2):178–186. 11. Singh SK, et al. (2004) Identification of human brain tumour initiating cells. Nature 29. Diamandis P, et al. (2007) Chemical genetics reveals a complex functional ground 432(7015):396–401. state of neural stem cells. Nat Chem Biol 3(5):268–273. 12. Little SE, et al. (2012) Receptor tyrosine kinase genes amplified in glioblastoma ex- 30. Roth A, et al. (2014) PyClone: Statistical inference of clonal population structure in hibit a mutual exclusivity in variable proportions reflective of individual tumor het- cancer. Nat Methods 11(4):396–398. – erogeneity. Cancer Res 72(7):1614 1620. 31. Bengtsson H, Wirapati P, Speed TP (2009) A single-array preprocessing method for 13. Snuderl M, et al. (2011) Mosaic amplification of multiple receptor tyrosine kinase estimating full-resolution raw copy numbers from all Affymetrix genotyping arrays – genes in glioblastoma. Cancer Cell 20(6):810 817. including GenomeWideSNP 5 & 6. Bioinformatics 25(17):2149–2156. 14. Sottoriva A, et al. (2013) Intratumor heterogeneity in human glioblastoma reflects 32. Olshen AB, Venkatraman ES, Lucito R, Wigler M (2004) Circular binary segmentation – cancer evolutionary dynamics. Proc Natl Acad Sci USA 110(10):4009 4014. for the analysis of array-based DNA copy number data. Biostatistics 5(4):557–572. 15. Szerlip NJ, et al. (2012) Intratumoral heterogeneity of receptor tyrosine kinases EGFR 33. Silversides CK, et al. (2012) Rare copy number variations in adults with tetralogy of and PDGFRA amplification in glioblastoma defines subpopulations with distinct Fallot implicate novel risk gene pathways. PLoS Genet 8(8):e1002843. growth factor response. Proc Natl Acad Sci USA 109(8):3041–3046. 34. Iafrate AJ, et al. (2004) Detection of large-scale variation in the human genome. Nat 16. Nishikawa R, et al. (1994) A mutant epidermal growth factor receptor common in hu- Genet 36(9):949–951. man glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA 91(16):7727–7731. 35. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under 17. Stupp R, et al.; European Organisation for Research and Treatment of Cancer Brain mixed models. Bioinformatics 19(12):1572–1574. Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials 36. Merico D, Isserlin R, Stueker O, Emili A, Bader GD (2010) Enrichment map: A network- Group (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glio- based method for gene-set enrichment visualization and interpretation. PLoS ONE blastoma. N Engl J Med 352(10):987–996. 5(11):e13984.

6of6 | www.pnas.org/cgi/doi/10.1073/pnas.1320611111 Meyer et al. Downloaded by guest on September 26, 2021