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 p53-/- 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 genes 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 gene 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, E2F1, 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