Harris Supplementary Table 1. In vivo injection summary of tumorspheres and neurospheres. Intracranial injections are in bold and flank injections are italicized.

passage latency # mice with sample ID genotype # # cells injected (days) tumors 3447 VerbBp53+/- 5,6 250000 22 4/4

2670 VerbBp53-/- 8 10000 21-25 3/3

3447 VerbBp53+/- 10 Single sphere 28 4/4 3447 VerbBp53+/- 4 100 49-62 3/3 3447 VerbBp53+/- 4 500 35-62 3/3 3447 VerbBp53+/- 4 1000 25-42 3/3 3447 VerbBp53+/- 5 200000 21 4/4

4346 VerbBp53-/- 7 Single sphere 42-119+ 2/4 4346 VerbBp53-/- 2 350000 21 3/3

2670 VerbBp53-/- 3 100000 30 3/3

932-1 (3447 clone) VerbBp53+/- 1 10000 33-43 2/2 932-2 (3447 clone) VerbBp53+/- 1 10000 33 2/2 932-3 (3447 clone) VerbBp53+/- 1 10000 33-43 2/2 932-4 (3447 clone) VerbBp53+/- 1 10000 33 2/2 932-5 (3447 clone) VerbBp53+/- 1 10000 33 2/2 932-6 (3447 clone) VerbBp53+/- 1 10000 31 2/2 932-7 (3447 clone) VerbBp53+/- 1 10000 31 2/2 932-8 (3447 clone) VerbBp53+/- 1 10000 31-43 2/2

NSC svz VerbBp53-/- 7 Single sphere 139+ 0/4 NSC svz -/- 9 100000 117+ 0/2

Harris Supplementary Table 2. In vitro limiting dilution assay.

Tumor 1 Tumor 2

# Cells Average # # Cells Average # Plated spheres Frequency Plated spheres Frequency

1500 4.5 1/333 1500 3 1/500

150 0 0 150 0 0

15 0 0 15 0 0

Freshly dissociated primary tumor cells were plated in 12 well plates in 1.5ml of medium/well indicated doses of cells. 3 replicate wells were plated per cell dose. Number of spheres containing more than 10 cells were counted 6 days after plating. Harris Supplementary Table 3. Injection summary of SP and non-SP cells from 4346 and 3447 tumorspheres.

Tumor Non-SP Tumor SP # cells Tumors Percent tumor # cells Tumors Percent tumor injected formed tumors onset injected formed tumors onset 50 0/3 0% 125+ days 50 4/12 33% 37-77+ days 100 0/2 0% 125+ days 100 2/2 100% 35-48 days 500 1/3 33% 42+ days 500 3/3 100% 55-70 days 1000 2/4 50% 83+ days 1000 5/5 100% 10-53 days

Summary of injections from 4 independent Hoechst staining and FACS sorts from two tumorsphere lines, 4346 and 3447. SP cells are more tumorigenic than non-SP cells (p<0.0285). Harris Supplementary Table 4

Ingenuity networks generated by 345 over-expressed in cancer SP (using q-value 0.05 and 1.5 log2 fold change) (A) and by 193 genes under-expressed in cancer SP (using q-value

0.05 and 1.5 log2 fold change) (B). Genes in bold are on our list.

A.

Network Genes # genes Top functions

id

1 ACSL1, ADAMTS5, AGC1, ASPN, CAV1, CCND3, 32 Cellular Assembly

CDKN1A, COL11A1, COL11A2, COL2A1, CTF1, and Organization,

FBXO7, FXYD1, GJB2, GNAO1, HOXA10, IAPP, Cellular Function

MMP17, NKX2-2, P53CP, PDGFRA, PPFIBP1, and Maintenance,

RECK, S100A1, S100A4, S100A6, S100B, SNAI2, Connective Tissue

SREBF1, STAT5A, TFPI, TIMP2, TIMP3, TUBB3, Development and

UCP2 Function

2 ABLIM3, ACLY, ARFGAP3, CAV1, CCND3, CD2, 20 Cancer, Cellular

CDKN1A, CDKN2A, CXCL14, DECR1, EHD3, FGF2, Growth and

FGFR3, GPNMB, GRIA1, GRIA3, HLA-A, HMGB2, Proliferation,

IFNG, ITGB3, KCNK1, KIAA1276, MDM2 (includes Cardiovascular

EG:246362), MLANA, NFYB, PCSK2, PDGFRA, System

RAB3C, SILV, SLIT3, STAT5A, TCFL5, TENC1, Development and

TIMP2, TIMP3 Function

3 AP1S2, AP2B1, CAPG, CCND3, CCT5, CD82, 20 Cellular Assembly

CD1D, CGI-38, CHI3L1, CHST6, CSPG4, EMP3, and Organization,

ENPP1, FABP5, GP5, HSPA1B, IL3, IL4, IL1B, Cell-To-Cell

LGALS2, MBP, MIA, MMP16, MYO1C, P2RX7, Signaling and

PCSK2, PLB1, PLCD1, PRKCA, SCG5, SLC1A1, Interaction, Cellular

SNCA, SPI1, TGM2, TIMP2 Growth and

Proliferation 4 ADAM28, ANXA6, ARHGEF6, BGN, CAV1, CCND3, 20 Cell Morphology,

CNTN1, CPXM2, DAG1, DDC, ELA1, ELN, ENO3, Nervous System

FDPS, FGF19 (includes EG:9965), FLOT2, FOXP3, Development and

FYN, ITM2A, KRAS, MBP, MCAM, MMP10, NRK, Function,

PAK3, PLP1, SCN1B, SGCA, SGCB, SGCD, SIM1, Developmental

SREBF1, SYT9, THRB, UGT8 Disorder

5 AURKB, BAG1, BIRC5, CASP3, CASP4, CAV1, 19 Cancer, Cell

CCND3, CD82, CDC42, CDKN1A, CYFIP2, DOCK9, Death, Neurological

ELL, FBLN1, FMOD, FOXM1, HS3ST1, LAMA4, MET, Disease

P2RX4, PHLDA3, PKN1, PLXNB3, POU4F1,

RACGAP1, ROBO1, SLIT2, SNCA, SNCB, SREBF1,

TP53, UBE2C, UNC5C, WASL, WASPIP

6 ABCG1, ACVRL1, AGC1, AXL, BYSL, CAV1, 18 Cell Morphology,

CDKN1A, COL2A1, COL4A2, CRK, CTSK, CXCL12, Connective Tissue

EFNA1, EPHA4, HOXA2, HSPG2 (includes EG:3339), Development and

IRF6, KRT8, KRT18, KRT19, MMP11, NR1H2, Function, Cellular

NR4A1, PGCP, PKD1, PKN1, PRELP, RHOA, Assembly and

ROCK1, STARD13, TGFB1, TGM2, TRO, TROAP, Organization

UGDH

7 ADRA2A, ADRB3, AKT1, ARRB1, ATP1A2, CAV1, 17 Lipid Metabolism,

CAV2, CCND3, CEBPA, CFD, CYP3A4, CYP3A5, Small Molecule

CYP3A7, FOXA3, FOXC2, FXYD5, INS1, MBTPS1, Biochemistry,

MICAL1, MYO5A, MYRIP, PDGFRA, PLIN, PSCD3, Cellular

PTGER4, RAB27A, RAB27B, SEPT5 (includes Development

EG:5413), SNCA, SRC, SREBF1, STAT5A, STX4,

SYT4, SYTL2

8 ADIPOQ, AFP, ATBF1 (includes EG:463), CAPN3, 16 Cancer, Cellular

CAPN6, CAV1, EGF, EMB, FOS, FOXD1, GDF2, Growth and

GIT1, GLI1, HAS2, HHIP, MYH10, NANOG, NDRG2, Proliferation, PALM2-AKAP2, POU5F1, PRKACA, PRKAR1A, Tissue

PRKG2, RARG, RIMS1, SLC8A1, SNAP25, SNIP, Development

SOX9, STAT5A, TIMP3, VIL2, WASF1, WIF1, WWP2

9 ANKH, CAV1, CCND3, CDH11, CRABP2, CRYL1, 15 Organismal

CSNK1E, CTNNB1, DKK3, FGF1, FREM2, GRIA4, Development,

GRIP1, GRIP2, HAPLN1, HOXA3 (includes Cancer, Cell Death

EG:3200), JARID1A, MGP, NCOA5, PPP2CA,

PPP2CB, PPP2CBP, PPP2R4, PPP2R1A, PPP2R1B

(includes EG:5519), PPP2R2A, PPP2R2B, PPP2R2C,

PPP2R3A (includes EG:5523), PPP2R5B, RARA,

S100A13, SPP1, VDR, WISP1

10 ADAM9, ADAM10, ADAM12, ADAM17, ALDOA, 15 Cell Death,

ANKS1B, APP, CDKN1A, CLDN1, CLDN2, CYP2J2, Cellular Movement,

ENPP2, EPHB1, EYA2, G6PD, HERPUD1, HSPG2 Skeletal and

(includes EG:3339), IFI35, IL15, IL7R, JUN, M6PR, Muscular System

MST1, MSX1, MYOD1, PAX3, PTPN3 (includes Development and

EG:5774), S100B, SH3D19, SH3GL3, SIX1, Function

SLC12A2, SNCA, TIMP3, WNK4

11 ARNT, C1QL1, CAV1, CCNA1, CCND3, CDKN1A, 15 Tissue

COL9A1, COL9A2, COL9A3, DGKA, , ETV4, Development,

F2R, FLT1, GDF2, GJB1, HES1, HEY2, LPPR4, Cardiovascular

MMP12, NOTCH1, NR4A1, NRG2, NRP1, NRP2, System

PLAG1, PLG, RBPSUH, RLBP1, SEMA3A, SEMA3D, Development and

SEMA3E, STARD8, STK23, VEGF Function, Cellular

Movement

12 ACHE, ALDH1A7, APOE, BCHE, CARD6, CDKN1A, 14 Hematological

CLDN11, COL15A1, COLQ, CPM, CTSG, DHRS3, Disease, Cellular

FRZB, GP1BA, GP1BB, IRF5, KDR, MAP4K4, Movement,

MAPK11, MAPK12, MAPK13, MMP1, MMP3, Immunological NFE2L2, PDRG1, PF4, POU2F1, PROC, RIPK1, Disease

RIPK2, SERPINA3, ST3GAL5, TDRD7, TNF, TRADD

13 ACTA1, CD200, CD200R1, CDKN1A, DAP, DOK1, 13 Cancer, Cellular

DOK2, ERBB2, EREG, F2R, FLJ36748, GALNT3, Development,

GDPD3, GRB7, GSN, HNRPC, KLK3, MMP1, Cellular Growth

MMP14, NUP214, NXF3, NXT1, P4HA2, PDE8A, and Proliferation

RET, SDK1, SOX10, STUB1, TERT, TPD52,

TPD52L1, USF2, VIL2, WNT5A, XPO1

14 ANXA1, ANXA2, BGN, BIRC5, CALD1, CDH11, 13 Genetic Disorder,

CDKN1A, CHI3L1, COL6A1, COL6A2, COL6A3, Skeletal and

CTSB, DRD1, DRD2, DYSF, FMR1, GPRASP1, HD Muscular

(includes EG:3064), HRAS, IL2RB, LECT1, Disorders, Cancer

M6PRBP1, MAP2K6, MUC2, ODZ3, PCYT1A,

RAD9A, S100A4, SERPIND1, SMAD7, SP3, STK10,

TAGLN, TIMP1, TNC

B.

Network Genes # gene Top functions

id

1 A2M, ADM, AGT, BTG1, CCL13, CD53, CDH22, 22 Cellular Growth

CEBPD, CENTD1, CREM, CYP2J2, FZD9, and

GABARAPL2, GJA1, GLDC, HRASLS3, IFNG, Proliferation,

IL15, ITGA5, JUN, KIR2DL3, KLRB1C, LAMB1, Cell Death,

LMO1, LYN, MAPK10, MCC (includes EG:4163), Cancer

NFKBIB, PEA15, PPP1R1A, PRKCA, PRKCB1,

TNFRSF12A, WNT7A, ZFP36

2 AOX1, ARL4C, C9ORF26, CCL13, CEBPD, CMA1, 20 Cell-To-Cell

CXCL6, DCAMKL1, EMX2, FAM19A2, FLJ20701, Signaling and

GADD45G, GJA1, HLA-DRA, HRAS, IL6, IL13, Interaction,

KITLG (includes EG:4254), KRAS, MBP, NFKBIZ, Cellular Growth

NFYB, OXTR, PDPN, RFX2, RFX3, RFX4, RPL30, and

SORT1, TFF3, THRSP, TNFSF4, TPM1, TSLP, Proliferation,

WNT5A Hematological

System

Development

and Function

3 ADAM17, AGTR1B, ANGPT2, C5ORF13, CREM, 17 Cellular

CSPG2, DLL1, EFNB2, EGFR, EMP2, FGF1, FUT8, Movement,

GJB1, GPC1, GPD2, GRB10, GRM5, HMGA2, Drug

HOXB7, HTATIP2, IGFBP2, ITGA5, LRIG1, Metabolism,

MGAT3, NOTCH3, NTS (includes EG:4922), Small Molecule

NTSR2, PPAP2B, PTGS1, SNAI2, STC1, SULF2, Biochemistry

TNC, VAV3, VEGF

4 A2M, ALOX5AP, APOE, BIK, C6, C7, C9, CA2, 17 Hematological

CCL13, CEBPD, CTSE, CXCL6, EIF2S3Y, FGF19 System (includes EG:9965), GABRA1, GABRB3, GABRG1, Development

GAS1, HOXC8, KITLG (includes EG:4254), LCAT, and Function,

LPL, LYN, MEIS2, MME, MS4A2, OGG1, PBX1, Tissue

PRKCB1, PROM1, SLC4A1, TEAD1 (includes Development,

EG:7003), THY1, VLDLR, ZNF202 Neurological

Disease

5 AKAP5, AXIN1, BMP2, CAMK2B, CNKSR3, 17 Cell-To-Cell

CRMP1, CTNNB1, DLG4, DMP1, FGF1, FRAT1, Signaling and

FZD4, FZD9, GRASP, GRIN1, GRM3, GSK3B, Interaction,

HAP1, HD (includes EG:3064), HTRA1, KCNJ16, Nervous

LPHN2, MAP3K10, MAPK10, NDP, NPTX1, System

NRCAM (includes EG:4897), OPN3, PEG12, Development

PRKCB1, PURB, SHANK2, SLC6A1, SLC6A2, SRF and Function,

(includes EG:6722) Neurological

Disease

6 ADM, AKR1B1, AKT1, BCL2, CALCRL, CCL13, 17 Cell

CCND2, CDKN2B, CDX1 (includes EG:1044), Morphology,

CHGA, CX3CL1, EGFR, ELAVL2, F2, FOXG1B, Cellular

GCG, HTRA2, IAPP, IER2, ITGA5, KITLG (includes Development,

EG:4254), LYN, MBOAT2, MLLT7, NNAT, POU3F4, Cell-To-Cell

RAB3B, RAMP1, RDH5, RHOB, SCG3, SLC2A1, Signaling and

SNAP23, STX11, TCOF1 (includes EG:6949) Interaction

7 ALOX5, ARHGAP29, CASP4, CCND2, CEBPD, 16 Cell Death,

CHUK, CREM, CX3CL1, DKK1, FGD6, GBP2, Cancer, Cellular

GBP4, HBEGF, HDC, IL3, ING1, ITGA7, LTBP1, Development

MAP3K2, MEN1, MSX1, MYO6, MYST1, NDN,

PDE1B, RBBP5, SFN, SLC7A11, TNFSF13B,

TP53, TP73L, UPP1, YAP1, YWHAG, ZFP36

8 AFP, BTBD11, CTSC, D13BWG1146E, DNER, 15 Immune and DUSP6, EGFR, EREG, GNAI3, GNAZ, GNB5, Lymphatic

GSTA4, JAG2, KITLG (includes EG:4254), MNT, System

MT1A, NBL1, NCAM1, PTGS1, RGS7, RGS20, Development

ROBO1, SLIT1, SLIT2, SNN, TERT, TG, THRSP, and Function,

TM4SF1, TNF, TP73, TP53I11, UGCG, WNT5A, Cellular

YWHAQ Movement,

Cellular

Development

Harris Supplementary Table 5

Supplementary Table 5. Real Time PCR validation using primary and secondary tumors.

Indicated are the fold change values of CSC compared to NSC, normalized to 18s. Genes on the 45 brain cancer stem cell gene signature list are in bold; standard deviations are in parentheses.

CSC 1 CSC 2 CSC 1 CSC 2 CSC 3 secondary secondary

A93001N09Rik (n=1) 4.6 2.7 3.9

Abca13 (n=1) 0.40 0.70 Bgn (n=1) 503 147 Cav1 (n=1) 97.0 158.3 28.4 Ccnd3 (n=1) 2.3 1.2 11.7 Cdkn1a (n=1) 10.17 4.0 16.0 Col6a1 (n=3) 67.44 (± 5.7) 36.90 (± 3.1) 21.34 (± 2.3) Dkk3 (n=1) 897.64 7.41 62.8 82.7 Ephb1 (n=2) 0.21 (± 0.057) 0.15 (± 0.007) 5.2 (± 5.38)

Foxc2 (n=2) 119.43 1.99 43.61 (± 12.83) 19.12 (± 1.46) Foxg1 (n=1) 0.07 0.07 0.0001 0.0195 Frat1 (n=1) 0.31 0.66 0.08 0.06 Gadd45g (n=1) 0.46 0.62 0.83 0.32 Gja1 (n=1) 0.00 0.01 Hey2 (n=1) 68.9 2.5 2.61 7.19 Hrasls3 (n=2) 0.26 (± 0.04) 0.65 (± 0.10) Igfbp2 (n=1) 0.60 0.0035 0.0013 0.0005

Ldoc1l (n=1) 5.04 5.90 3.52 2.38 Mamdc2 (n=2) 0.006 (± 0.001) 0.034 (± 0.015) Mettl7a (n=1) 2.29 1.75 2.08 1.58

Mgp (n=1) 52.2 42.20 Mmp16 (n=1) 3.6 1.2 12.0 Mmp17 (n=2) 2.63 (± 0.14) 0.85 (± 0.38) 2.55 (± 1.83) Prickle1 (n=1) 10.29 13.06 11.9 13.1 Robo1 (n=1) 0.005 0.11 0.32

S100a4 (n=3) 1.58 (0.30) 1.83 (± 0.44) 7.24 (± 0.80) S100a6 (n=2) 0.27 (± 0.04) 4.98 (± 0.67) 6.23 (± 0.24) Sall3 (n=1) 0.30 0.16 0.09 0.11 Scg3 (n=1) 0.02 0.09 0.27 Slit2 (n=2) 1.7 (± 0.76) 1.7 (± 0.43) 17.3 (± 14.64)

Slit3 (n=2) 163.8 (± 37.7) 41.1 (± 27.1) 59 (± 9.6) Snai2 (n=3) 39.7 (± 7.4) 4.1 (± 0.74) 8.7 (± 0.75) Stat5a (n=2) 0.83 (± 0.28) 0.64 (± 0.62) 1.67 1.54 0.95 Susd5 (n=2) 530.9 (± 65.7) 84.9 (± 4.2) 383.1 (± 23.8) Tcfl5 (n=2) 2.39 3.07 1.41 1.07 (± 0.32) 0.35 (± 0.32) Wif1 (n=1) 258.97 7.50 167.7 151.0 Zfp36 (n=2) 1.00 (± 0.02) 0.71 (± 0.092) 1.19 0.21 0.34