A172 CCF-SSTG1 15 - - - 1 1 1 2 2 3 4 6 7 8 10101112131617192022-1 1 1 2 2 3 4 6 7 8 10 10 11 12 13 16 17 19 20 22 T98G U373MG - - - 1 1 1 2 2 3 4 6 7 8 10 10 11 12 13 16 17 19 20 22-1 1 1 2 2 3 4 6 7 8 10 10 11 12 13 16 17 19 20 22 U178MG TP365MG - - - - 1 1 1 2 2 3 4 6 7 8 10 10 11 12 13 16 17 19 20 22 1 1 1 2 2 3 4 6 7 8 10 10 11 12 13 16 17 19 20 22 U118MG U251MG - U118MG - - 1 1 1 2 2 3 4 6 7 8 10101112131617192022-1 1 1 2 2 3 4 6 7 8 10101112131617192022 Supplementary Figure 1
Supplementary Table 1. Patient characteristics of astrocytic glioma samples on the tissue microarrays (TMAs) TMA AJAP1 TMA EMP3 TMA PDPN Age (y) Mean 54.2 53.9 57.7 Minimum 21 15 26 Maximum 80 80 78 Gender Male 128 (64%) 131 (66%) 36 (57%) Female 72 (36%) 69 (34%) 27 (43%) WHO grade II 20 (10%) 21 (1%) 9 (14.3%) III 8 (4%) 10 (5%) 11 (17.5%) IV 172 (86%) 169 (84.5%) 42 (66.7%) unknown 1(%) Chemotherapy Yes 11 (6%) 10 (5%) 25 (39.7%) No 189 (94%) 190 (95%) 15 (23.8%) Unknown 23 (36.5%) Radiotherapy Yes 158 (79%) 158 (79%) 44 (69.8%) No 42 (21%) 42 (21%) 1 (1.6%) Unknown 18 (28.6%) Total 200 200 63
Supplementary Table 2. Primers used for quantitative RT-PCR Target gene Forward primer Reverse primer METTL7B 5 -GCTCCATGGATGTGGTGGTC-3 5 -AAAGAGCACACCTCCCGGTC-3 CDKN1A 5 -GGCAGACCAGCATGACAGATT-3 5 -GCGGATTAGGGCTTCCTCTT-3 EMP3 5 -TGCACGTGGAACAACGACAC-3 5 -CTCGTCGCATGGTGTAGAGC-3 PDPN 5 -ACGATGTGGAAGGTGTCAGCT-3 5 -AGCCAGACTTATAGCGGTCTTCG-3 CENPF 5 -ACCAGAAGCGGGCGAATTGG-3 5 -GAGCTCTTGTAGGCAGCCCT-3 UHRF1 5 -GACTCGCTGTCCAGGCTGAC-3 5 -GTGTCATTCAGGCGGACCTC-3 NUP107 5 -GCAGCCAACTTTGGTTGTGTG-3 5 -CTGTTTCCGTGCAGTCCGTG-3 CCNB2 5 -AGTTGGCTCCAAAGGGTCCT-3 5 -CGTAGTCACTGCAGAGCTGAGG-3 AJAP1 5 -TCCCTCATCATGGTCATAGCTGC- 5 -TGCAGGGTCTCGTTATAGGCCGTG-3 RTN1 5 -GCATCGTGTTTGGGAGTTTCCTGC- 5 -AGGAAGAGCCTCCTCAGTTCC-3 GABBR1 5 -TCCACAACCCTACCCGCGTGAA-3 5 -TTTGACGGGCACAGCTGGATCTG-3
Supplementary Table 3. Amplifications and putative homozygous deletions detected by array-cgh in 20 GBM spheroid cultures High-level amplifications Localization Start clone End clone Size (Mb) Candidate gene (s) (%) 1p21.1 RP5-936J12 RP5-936J12 0.17 COL11A1 5 1p34.3-1p34.2 RP4-615P17 RP11-72N2 2.22 PABPC4, ZMPSTE24 5 1q31.1 RP11-201A3 RP11-201A3 0.15 No annotated gene 5 1q32.1 RP11-203F10 RP11-430C7 0.44 MDM4 10 2p23.3 RP11-558C24 RP11-558C24 0.18 RAB10 5 4q12 RP11-317M11 RP11-463H12 3 PDGFRA 20 5q35.1 RP11-117L6 RP11-779O18 1.49 NPM1 5 6p22.3 RP1-177P22 RP11-33I5 2.22 E2F3 5 7p11.2 RP11-449G3 RP11-339F13 0.74 EGFR 15 7q31.1-7q31.32 RP11-45K3 RP11-384A20 3.88 PTPRZ1 5 8q24.21 RP11-351C8 RP1-80K22 1.12 MYC 20 11p13 RP5-1083G3 RP11-48O9 1.26 WT1 5 12p13.32 RP11-543P15 RP11-264f23 1.24 CCND2 5 12q14.1 RP11-571M6 RP11-155I23 0.22 CDK4 30 12q15 RP11-611O2 RP11-611O2 0.15 MDM2 35 Putative homozygous deletions 1p36 RP5-1096P7 RP3-410I8 10.18 AJAP1 5 1p35.2 RP4-742P4 RP11-336O20 6.45 5 1p34 RP11-100H21 RP5-848E13 5.08 5 1p33 RP4-697E16 RP11-20F20 11.88 5 4q35.1 RP11-13N11 CTC-963K6 1.36 5 5p15.2 CTC-485I21 RP3-502L6 8 5 8p21.2 RP11-299D14 RP11-263C6 3.19 5 8q21.13 RP11-86O7 RP11-216N21 5.74 5 9p21.3 RP11-15P13 RP11-495L19 3,38 CDKN2A. CDKN2B 35 10q23.31 RP11-165M8 RP11-186O14 1.03 PTEN 5
11q24.3 RP11-122H4 RP11-469N6 6 10 12q12 RP11-210N13 RP11-474P2 2.69 5 14q13.2 RP11-330O19 RP11-561B11 9.48 5
Supplementary Table 4. Top ranked highly expressed genes in spheroid cultures (n=10) Gene symbol FC to nonneoplastic brain Description FC to nonneoplastic spheroid culture* ACTG1 3.22 Actin, gamma 1 11.00 HNRPA1 3.18 Heterogeneous nuclear ribonucleoprotein A1 4.15 CDK4 3.14 Cyclin-dependent kinase 4-2.97 IGFBP2 3.06 Insulin-like growth factor binding protein 2, 2.31 NUSAP1 3.05 Nucleolar and spindle associated protein 1 0.30 VIM 3.04 Vimentin 0.06 PTPRZ1 2.92 Protein tyrosine phosphatase, receptor-type, Z polypeptide 1 4.63 UBE2C 2.88 Ubiquitin-conjugating enzyme E2C 1.16 CDKN1A 2.82 Cyclin-dependent kinase inhibitor 1A 1.34 PDGFRA 2.81 Platelet-derived growth factor receptor, alpha polypeptide -0.40 CCNB2 2.76 Cyclin B2 0.44 TSPAN3 2.7 Tetraspanin 3 6.28 ANXA1 2.68 Annexin A1-0.81 CDK6 2.67 Cyclin-dependent kinase 6 2.33 NUP107 2.65 Nucleoporin Nup107 0.21 KIAA0101 2.64 KIAA0101 0.05 KCNQ2 9 Potassium voltage-gated channel, KQT-like subfamily, member 2 6.21 FAM119B 8 Family with sequence similarity 119, member B 0.31 HNRPK 8 Heterogeneous nuclear ribonucleoprotein K 5.14 UHRF1 6 Ubiquitin-like, containing PHD and RING finger domains, 1 0.20 TUBA6 4 Tubulin, alpha 1c 1.63 NES 4 Nestin 2.25 METTL7B 3 Methyltransferase like 7B 2.34 TMSL8 2 Thymosin-like 8-2.23 CBX2 1 Chromobox homolog 2 1.99 TNC Tenascin C 4.77 CDCA7 2.49 Cell division cycle associated 7 2.29 GAS1 2.44 Growth arrest-specific 1-0.07 TYMS 2.41 Thymidylate synthetase 1.32
PBK 2.4 PDZ binding kinase 1.11 FAM60A 2.37 Family with sequence similarity 60, member A 1.76 TNFRSF19 2.36 Tumor necrosis factor receptor superfamily, member 19 6 PTTG1 2.33 Pituitary tumor-transforming 1 0.80 SOX11 2.33 SRY (sex determining region Y)-box 11 2.22 H19 2.32 H19, imprinted maternally expressed transcript -0.46 HOXA5 2.31 Homeobox A5 3.93 CENPF 2.3 Centromere protein F, 350/400ka (mitosin) 0.85 MYC 2.29 V-myc myelocytomatosis viral oncogene homolog (avian) 1.73 EMP3 2.29 Epithelial membrane protein 3 19 RPL6 2.27 Ribosomal protein L6 4.66 NPM1 2.27 Nucleophosmin -1.91 S100A10 2.24 S100 calcium binding protein A10 5.27 CD44 2.22 CD44 molecule -0.45 PDPN 2.22 Podoplanin 9.02 median fold change compared to non-neoplastic brain (n = 5, pooled) expressed as log-2 ratio * median fold change compared to spheroid culture isolated from non-neoplastic brain expressed as log-2 ratio
Supplementary Table 5. Relationship between expression of candidate genes and WHO grade WHO grades sgbm pgbm II-III low AJAP1 expression 8 (28%) 2 (100%) 93 (55%) high AJAP1 expression 20 (72%) 0 (0%) 77 (45%) low EMP3 expression 26 (84%) 2 (67%) 73 (44%) high EMP3 expression 5 (16%) 1 (33%) 93 (56%) low PDPN expression 17 (85%) 3 (100%) 6 (15%) high PDPN expression 3 (15%) 0 (0%) 33 (85%) Abbreviations: sgbm, secondary glioblastoma; pgbm, primary glioblastoma