Table S1. Molecular Classification of Low-Grade and Anaplastic Gliomas

WHO Grade 1p/19q codel IDH1 mut Histological Diagnosis Low-grade #1 II (-) (+) Oligoastrocytoma Low-grade #2 II (-) (+) Oligoastrocytoma Low-grade #3 I (-) (-) Pilocytic astrocytoma Low-grade #4 II (-) (-) Diffuse astrocytoma Anaplastic glioma #1 III (-) (+) Anaplastic astrocytoma Anaplastic glioma #2 III (-) (+) Anaplastic oligoastrocytoma Anaplastic glioma #3 III (-) (-) Anaplastic astrocytoma Anaplastic glioma #4 III (+) (+) Anaplastic oligodendroglioma Anaplastic glioma #5 III (-) (-) Anaplastic astrocytoma Anaplastic glioma #6 III (-) (+) Anaplastic oligoastrocytoma Anaplastic glioma #7 III (+) (+) Anaplastic oligodendroglioma

Table S2. WNT5A and FRIZZLED5 gene copy number in glioma tissues and hGBM TPCs

Samples/CN status GENE Chromosomal Low Grade Anaplastic Glioma hGBM tissues hGBM TPCs position Tissues Tissues Mesenchymal Classical Proneural Mesenchymal Classical Proneural

Low Grade #1-normal Anaplastic glioma #1-deletion GBM #19-normal GBM #1-normal GBM #15-normal TPCs #20-normal TPCs #26-normal TPCs #18-duplication Low Grade #2-normal Anaplastic glioma #2-normal GBM #20-normal GBM #2-normal GBM #16-duplication TPCs #24-normal TPCs #27-normal TPCs #30-normal Low Grade #3-normal Anaplastic glioma #3-normal GBM #21-normal GBM #3-normal GBM #17-normal TPCs #22-normal TPCs #28-normal TPCs #31-normal Low Grade #4-normal Anaplastic glioma #4-normal GBM #22-normal GBM #4-normal GBM #38-normal TPCs #25-normal TPCs #1-normal TPCs #32-deletion Anaplastic glioma #5-normal GBM #23-normal GBM #5-normal TPCs #33-normal TPCs #2-normal TPCs #41-normal Anaplastic glioma #6-normal GBM #24-normal GBM #6-normal TPCs #39-normal TPCs #34-normal TPCs #38-normal

WNT5A 3p14.3 Anaplastic glioma #7-normal GBM #25-normal GBM #7-normal TPCs #40-normal TPCs #35-normal Anaplastic glioma #8-normal GBM #36-normal GBM #8-normal TPCs #29-normal GBM #39-normal GBM #9-normal GBM #40-normal GBM #10-normal GBM #11-normal GBM #12-normal GBM #13-normal GBM #14-normal

Low Grade #1-normal Anaplastic glioma #1-normal GBM #19-normal GBM #1-normal GBM #15-normal TPCs #20-normal TPCs #26-normal TPCs #18-normal Low Grade #2-normal Anaplastic glioma #2-normal GBM #20-normal GBM #2-normal GBM #16-normal TPCs #24-normal TPCs #27-normal TPCs #30-normal Low Grade #3-normal Anaplastic glioma #3-normal GBM #21-normal GBM #3-duplication GBM #17-normal TPCs #22-normal TPCs #28-duplication TPCs #31-normal Low Grade #4-normal Anaplastic glioma #4-normal GBM #22-normal GBM #4-normal GBM #38-normal TPCs #25-normal TPCs #1-normal TPCs #32-normal Anaplastic glioma #5-normal GBM #23-normal GBM #5-normal TPCs #33-normal TPCs #2-normal TPCs #41-normal Anaplastic glioma #6-normal GBM #24-normal GBM #6-normal TPCs #39-normal TPCs #34-duplication TPCs #38-normal

FZD5 2q33.3 Anaplastic glioma #7-normal GBM #25-normal GBM #7-normal TPCs #40-normal TPCs #35-duplication Anaplastic glioma #8-normal GBM #36-normal GBM #8-normal TPCs #29-normal GBM #39-normal GBM #9-normal GBM #40-normal GBM #10-normal GBM #11-normal GBM #12-normal GBM #13-normal GBM #14-normal

Table S3. Ingenuity analysis of the LVWnt5a classical TPCs genes demonstrating the associated cancer genes and molecular and cellular functions (5% FDR)

n° of Category Function P value Gene names Genes

Cell Morphology Polarization 4 7.79E-03 CD47,CXCR4,ENPP2,mir-10,DLRL Cellular Movement Migration 10 3.96E-03 A2M,AGT,CD47,CXCR4,ENPP2,mir-10,mir-193,mir-515,mir-7,SERPINF1 Cancer, Neurological Disease, Organismal Injury and Metastasis 10 6.81E-03 ABCC3,CD248,CD47,CXCR4,ENPP2,KCNJ2,mir-10,mir-196,mir-7,SERPINF1 Abnormalities B4GALT1,C3,CCL3,CCL3L3,CCL5,CD274,CD44,CD47,CD5L,CSF2,CTSC,CXCL1,CX CL10,CXCL2,CXCL9,CYR61,DEFB4A/DEFB4B,EDIL3,EGR1,FOS,HCAR2,HGF,ICAM1 Invasion 11 6.92E-04 ,IFNG,IL1A,IL1RN,KDM6B,KITLG,MMP1,MMP2,NINJ1,PIK3R5,PTGS2,TGFB1,THBS1, Cellular Movement TNFRSF1B Cellular Movement Invasion 13 6.91E-04 A2M,AGT,CNR1,CXCR4,ENPP2,FXYD5,mir-10,mir-193,mir-515,mir-7,mir-9 Differentiation of Cellular Development 8 3.89E-03 CNR1,CXCR4,ELAVL3,ENPP2,HES1,mir-219,mir-9,SERPINF1 Nervous System Cancer, Neurological Disease, Organismal Injury and Astrocytoma 9 7.53E-03 ABCC3,CT47A6,CXCR4,ENPP2,mir-10,mir-128,mir-7,NFE2L3,PDP1 Abnormalities Activation of Cell-To-Cell Signaling and Tumor Cell 3 6.74E-03 AGT,CXCR4,mir-515 Interaction Lines

Table S4. Ingenuity analysis of the LVWnt5a U87 and U87 vector genes demonstrating the associated cancer genes and molecular and cellular functions related to Cellular Movement (5% FDR)

n° of Category Function P value Gene names genes

AKT3,CCL3,CCL5,CD44,CD47,CD82,COL4A3BP,CSF2,CXCL1,CXCL10,FOXL1,HGF,IL20,ITGA3,ITGA5,MMP2,PTGS migration of tumor cells 25 2.17E-07 2,RELA,SERPINB2,SPRY2,TGFB1,THBS1,TM4SF1,TUSC3,VCAN

AKT3,ATF3,BMP6,CA9,CCL3,CCL5,CD44,CD47,CD82,CD9,CREB3L1,CSF2,CTSL,CUL4A,CXCL10,CXCL2,CXCL9,C migration of tumor cell YR61,DAB2,DEFB4A/DEFB4B,EGR1,FAT1,GDF15,GJA1,GRN,HAS2,HGF,ICOS,IFNG,IGFBP3,IL1A,ITGA5,ITPR1,JA 63 6.08E-11 lines K1,KITLG,LAMB3,LOX,MME,MMP1,MMP19,MMP2,NDRG1,NINJ1,NR4A2,NRP2,PIK3R5,PLD1,PPIF,PRKG1,PTGS2, RELA,SDCBP,SERPINB3,SPRY2,STAT1,TBX3,TCF4,TGFB1,THBS1,VCAN,WASL,WBP2,XIAP

AKT3,AQP9,ATF3,B4GALT1,BCAT1,BMP6,C3,CA9,CADPS2,CBLB,CCL3,CCL3L1,CCL3L3,CCL5,CD274,CD44,CD47, CD55,CD5L,CD82,CD9,CFB,COL4A3BP,CREB3L1,CSF2,CTSC,CTSL,CUL4A,CXCL1,CXCL10,CXCL2,CXCL9,CYLD, CYR61,DAB2,DDIT4,DEFB4A/DEFB4B,DPYSL3,EDIL3,EGR1,ELMO2,EPS8,FAT1,FNDC3B,FOS,FOXL1,FURIN,FZD3 ,GDF15,GJA1,GPR183,GRN,HAS2,HCAR2,HGF,HLAA,HSPA5ICAM1,ICOS,ID2,IDO1,IFNG,IGF2R,IGFBP3,IL1A,IL1R N,IL20,IL24,ITGA1,ITGA3,ITGA5,ITK,ITPR1,JAG1,JAK1,KDM6B,KITLG,KLF2,LAMB3,LGMN,LMNB1,LOX,MARCKS,M migration of cells 145 2.70E-15 ME,MMP1,MMP10,MMP12,MMP19,MMP2,NAMPT,NDEL1,NDRG1,NFIA,NFKBIZ,NINJ1,NPC1,NR1D1,NR4A2,NRP2, P2RX4,P2RX7,P4HA2,PDE4DIP,PIK3R5,PLD1,PLTP,PPIF,PRDM1,PRKG1,PTGES,PTGS2,QPCT,REL,RELA,RND3, RRAS,RSPO3,SCPEP1,SDCBP,SERPINB2,SERPINB3,SLC1A3,SLIT3,SMPD1,SPAG9,SPRY2,STAT1,TBX3,TCF4,T GFB1,THBS1,TM4SF1,TNFAIP3,TNFAIP6,TNFRSF1B,TNFSF14,TNFSF9,TUSC3,UNC5B,VCAN, VLDLR,WARS,WASL,WBP2,XIAP

AKT3,CCL3,CCL5,CD44,CD47,CD82,COL4A3BP,CXCL10,HGF,IL20,ITGA3,ITGA5,MMP2,PTGS2,SPRY2,TGFB1,THB migration of cancer cells 20 7.32E-06 S1,TM4SF1,TUSC3,VCAN

ABI1,AKT3,ARHGEF26,ATF3,BMP6,CD44,CD82,CD9,CSF2,CTSD,DAB2,DRAM1,EGR1,EPS8,FAT1,FURIN,GBP1,G invasion of tumor cell DF15,GRN,HAS2,HBP1,HGF,ID2,IFNG,IL13RA2,ITGA3,ITGA5,KDM6B,LAMB3,LOX,MARCKS,MMP1,MMP16,MMP2, 53 4.92E-08 lines NAMPT,NDRG1,NR4A2,NRP2,PPIF,PTGS2,REL,RELA,RND3,RORA,SDCBP,SMPD1,SPRY2,STAT1,TGFB1,UNC5B, VCAN,WBP2,XIAP Cellular Movement ABI1,AKT3,ANG,ARHGEF26,ATF3,B4GALT5,BCAT1,BMP6,CA9,CD274,CD44,CD82,CD9,CSF2,CTSD,CTSL,CXCL1, CXCL10,CYR61,DAB2,DRAM1,EGR1,EPS8,FAT1,FOS,FOXL1,FURIN,GBP1,GDF15,GJA1,GRN,HAS2,HBP1,HGF,HS invasion of cells 75 1.18E-11 PA5,ID2,IER3,IFNG,IL13RA2,ITGA1,ITGA3,ITGA5,JMY,KDM6B,KITLG,LAMB3,LGMN,LOX,MARCKS,MFGE8,MMP1, MMP16,MMP2,NAMPT,NDRG1,NR4A2,NRP2,PLD1,PPIF,PTGS2,REL,RELA,RND3,RORA,SDCBP,SMPD1,SPRY2,S TAT1,TBX3,TGFB1,THBS1,UNC5B,VCAN,WBP2,XIAP B4GALT1,C3,CBLB,CCL3,CCL3L3,CCL5,CD274,CD44,CD47,CD55,CFB,CSF2,CXCL10,CXCL2,CXCL9,CYR61,EDIL3 ,GBP1,HGF,HLAA,ICAM1,ICOS,IDO1,IFNG,IL1A,IL1RN,ITGA1,ITK,KITLG,MMP12,MMP2,NDRG1,NFKBIZ,NINJ1,NPC infiltration of cells 50 4.37E-14 1,P2RX4,P2RX7,PLTP,PRDM1,PRKG1,PTGES,PTGS2,RELA,RSPO3,STAT1,TGFB1,THBS1,TNFAIP3,TNFRSF1B,U NC5B

AKT3,ATF3,BMP6,C3,CA9,CCL3,CCL3L1,CCL5,CD44,CD47,CD82,CD9,COL4A3BP,CREB3L1,CSF2,CTSL,CUL4A,C XCL10,CXCL2,CXCL9,CYR61,DAB2,DEFB4A/DEFB4B,DRAM1,EGR1,EPS8,FAT1,FOS,FURIN,GDF15,GJA1,GRN,H cell movement of tumor 72 8.63E-11 AS2,HGF,ICOS,IFNG,IGFBP3,IL1A,ITGA1,ITGA5,ITPR1,JAK1,KITLG,LAMB3,LOX,MME,MMP1,MMP19,MMP2,NDRG cell lines 1,NINJ1,NR4A2,NRP2,PIK3R5,PLD1,PPIF,PRKG1,PTGS2,RELA,SDCBP,SERPINB3,SHC3,SPRY2,STAT1,TBX3,TCF 4,TGFB1,THBS1,VCAN,WASL,WBP2, XIAP

AKT3,AQP9,ATF3,B4GALT1,BCAT1,BMP6,C3,CA9,CADPS2,CBLB,CCL3,CCL3L1,CCL3L3,CCL5,CD274,CD44,CD47, CD55,CD59,CD5L,CD82,CD9,CFB,COL4A3BP,CREB3L1,CSF2,CTH,CTSC,CTSL,CUL4A,CXCL1,CXCL10,CXCL2,CX CL9,CYLD,CYR61,DAB2,DDIT4,DEFB4A/DEFB4B,DPYSL3,DRAM1,EDIL3,EGR1,ELMO2,EPS8,FAT1,FNDC3B,FOS, FOXL1,FURIN,FZD3,GBP1,GDF15,GJA1,GPR183,GRN,HAS2,HCAR2,HGF,HLAA,HSPA5,ICAM1,ICOS,ID2,IDO1,IFN G,IGF2R,IGFBP3,IL13RA2,IL1A,IL1RN,IL20,IL24,ITGA1,ITGA3,ITGA5,ITK,ITPR1,JAG1,JAK1,KDM6B,KITLG,KLF2,LA cell movement 157 3.88E-15 MB3,LGMN,LIMS1,LMNB1,LOX,MARCKS,MME,MMP1,MMP10,MMP12,MMP19,MMP2,MTSS1,NAMPT,NDEL1,NDRG 1,NFIA,NFKBIZ,NINJ1,NPC1,NR1D1,NR4A2,NRP2,P2RX4,P2RX7,P4HA2,P4HB,PDE4DIP,PDIA3,PIK3R5,PLD1,PLTP ,PPIF,PPP3CC,PRDM1,PRKG1,PTGES,PTGS2,QPCT,REL,RELA,RND3,RRAS,RSPO3,SCPEP1,SDCBP,SERPINB2, SERPINB3,SHC3,SLC1A3,SLIT3,SMPD1,SPAG9,SPRY2,STAT1,STX3,TBX3,TCF4,TGFB1,THBS1,TM4SF1,TNFAIP3 ,TNFAIP6,TNFRSF1B,TNFSF14,TNFSF9,TUSC3,UNC5B,VCAN,VLDLR,WARS,WASL,WBP2,XIAP

Table S5. Ingenuity Analisys of the Wnt5aHigh Genes Demostrating The Associated Cancer Genes Molecular And Cellular Function (5% FDR)

n° of Category Function P value Gene names genes

AIF1,AKR1B1,AMICA1,ANGPT2,AQP9,BID,C3,C3AR1,C5AR1,CCL1,CCL18,CCL20,CCL3,CCL3L1,CCL3L3,C CL4,CCL5,CCR1,CCR4,CCRL2,CD14,CD2,CD247,CD28,CD3E,CD69,CD74,CD86,CD93,CES1,CLEC4E,CLE C5A,CLEC7A,CSF1R,CSF2RA,CTSB,CTSS,CXCL1,CXCL16,CXCL2,CXCL5,CXCL8,CXCR3,CYBB,CYP1B1,D EFA1,ENG,FABP4,FAM65B,FCAR,FCER1G,FCGR2A,FERMT3,FGR,FN1,FYB,GATA3,GNLY,GPR183,GREM Cell Movement 128 2.66E-66 1,GRN,HCK,ICOS,IL10,IL1A,IL1B,IL1RN,IL2RB,ITGA1,ITGA2,ITGA4,ITGAL,ITGAX,ITGB2,ITK,JAK1,KIT,KLRC 4KLRK1/KLRK1,LCK,LCP1,LGALS9,LPAR3,LTB,LY96,LYN,LYZ,MAP3K8,MAP4K1,mir54,MMP1,MMP14,MMP 7,MMP9,MPP1,MSR1,NCF1,NPS,OCLN,OLR1,PECAM1,PIK3R5,PLA2G7,PLAUR,PPARG,PPBP,PRF1,PRKC Q,PTPN6,PTPRC,RASGRP1,SERPINA1,SH2D1A,SIGLEC9,SIRPA,SKAP1,SLFN12L,SPI1,SPP1,SYK,THBD,T LR2,TLR4,TNF,TNFRSF21,TPSAB1/TPSB2,TREM2,TYROBP,XCL1 AIF1,AMICA1,ANGPT2,ANPEP,AQP9,AREG,BID,C3,C3AR1,C5AR1,CAMK4,CCL1,CCL18,CCL20,CCL3,CCL3 L1,CCL3L3,CCL4,CCL5,CCR1,CCR4,CCRL2,CD14,CD2,CD247,CD28,CD3E,CD69,CD74,CD86,CD93,CDCP1 ,CES1,CLEC4E,CLEC5A,CLEC7A,CSF1R,CSF2RA,CTSB,CTSH,CTSL,CTSS,CXCL1,CXCL16,CXCL2,CXCL5 ,CXCL8,CXCR3,CYBB,CYP1B1,DAB2,DEFA1,DUSP6,ENG,EOMES,EREG,FABP4,FAM65B,FCAR,FCER1G,F CGR2A,FERMT3,FGR,FN1,FYB,GATA3,GNLY,GPR183,GREM1,GRN,GZMB,HAVCR2,HCK,HTR2B,ICOS,IL1 Migration 156 8.53E-46 0,IL1A,IL1B,IL1RN,IL24,IL2RB,ITGA1,ITGA2,ITGA4,ITGAL,ITGAX,ITGB2,ITK,JAK1,KIT,KLRC4KLRK1/KLRK1, LCK,LCP1,LGALS9,LPAR3,LTB,LY96,LYN,LYZ,MAP3K8,MAP4K1,mir154,mir21,MITF,MMP1,MMP10,MMP14, MMP19,MMP7,MMP9,MPP1,MSR1,NCF1,NCF2,NPS,NRP1,OCLN,OLR1,PDCD4,PDGFRB,PECAM1,PIK3R5, PLA2G7,PLAUR,PPARG,PPBP,PPIF,PPP2R5C,PRF1,PRKCQ,PTPN6,PTPRC,RASGRP1,SERPINA1,SERPIN B2,SH2D1A,SIGLEC9,SIRPA,SKAP1,SLFN12L,SPI1,SPP1,SYK,SYNE2,TGFBI,THBD,TLR2,TLR4,TNF,TNFR SF21,TNFSF15,TPSAB1/TPSB2,TREM2,TYROBP,XCL1,XCL2 AIF1,AKR1B1,AMICA1,ANGPT2,ANPEP,AQP9,AREG,BID,C3,C3AR1,C5AR1,CAMK4,CCL1,CCL18,CCL20,C CL3,CCL3L1,CCL3L3,CCL4,CCL5,CCR1,CCR4,CCRL2,CD14,CD2,CD247,CD28,CD3E,CD69,CD74,CD86,CD 93,CDCP1,CES1,CLEC4E,CLEC5A,CLEC7A,CSF1R,CSF2RA,CTSB,CTSH,CTSL,CTSS,CXCL1,CXCL16,CXC L2,CXCL5,CXCL8,CXCR3,CXCR6,CYBB,CYP1B1,DAB2,DEFA1,DGKA,DRAM1,DUSP6,ENG,EOMES,EREG, FABP4,FAM65B,FCAR,FCER1G,FCGR2A,FERMT3,FGL2,FGR,FN1,FYB,GATA3,GNLY,GPR183,GREM1,GR Cellular Movement N,GZMB,HAVCR2,HCK,HTR2B,ICOS,IGHM,IL10,IL1A,IL1B,IL1RN,IL24,IL2RB,IRF8,ITGA1,ITGA2,ITGA4,ITGA Cell Movement 165 1.41E-45 L,ITGAX,ITGB2,ITK,JAK1,KIT,KLRC4KLRK1/KLRK1,LCK,LCP1,LGALS9,LPAR3,LTB,LY96,LYN,LYZ,MAP3K8, MAP4K1,mir154,mir21,MITF,MMP1,MMP10,MMP14,MMP19,MMP7,MMP9,MPP1,MSR1,NCF1,NCF2,NPS,NP TX1,NRP1,OCLN,OLR1,PDCD4,PDGFRB,PECAM1,PIK3R5,PLA2G7,PLAUR,PPARG,PPBP,PPIF,PPP2R5C,P RF1,PRKCQ,PTPN6,PTPRC,RASGRP1,SERPINA1,SERPINB2,SH2D1A,SIGLEC9,SIRPA,SKAP1,SLC11A1,S LFN12L,SPI1,SPP1,SYK,SYNE2,TGFBI,THBD,TLR2,TLR4,TNF,TNFRSF21,TNFSF15,TPSAB1/TPSB2,TREM 2,TYROBP,XCL1,XCL2 AKR1B1,ANGPT2,BID,C3,C3AR1,CCL18,CCL3,CCL3L3,CCL4,CCL5,CCR1,CD14,CD2,CD28,CD86,CD93,CE S1,CLEC5A,CSF1R,CTSB,CTSS,CXCL16,CXCL2,CXCL5,CXCL8,CYBB,ENG,FABP4,FCAR,FCER1G,FGL2,F Cellular Infiltration 68 3.09E-37 N1,HCK,ICOS,IGHM,IL10,IL1A,IL1B,IL1RN,IL2RB,IRF8,ITGA1,ITGA4,ITGAL,ITGB2,ITK,LTB,MAP3K8,mir21,M MP14,MMP9,MSR1,NCF1,NRP1,PLA2G7,PPARG,PPBP,PRF1,PRKCQ,SERPINA1,SPI1,SPP1,THBD,TLR2,T LR4,TNF,TNFRSF21,XCL1

CCL3,CCL3L3,CCL4,CCL5,CCR1,CD28,CD86,CTSS,CXCL2,CXCL5,CXCL8,ENG,FERMT3,FN1,IL1B,ITGA4,I Transmigration 28 6.60E-19 TGAL,ITGAX,ITGB2,ITK,MMP14,MMP9,PECAM1,PPARG,PRKCQ,SIRPA,SPP1,TNF

ANGPT2,ANPEP,AREG,CCL1,CCL20,CCL3,CCL5,CDCP1,CTSH,CTSL,CXCL2,CXCL5,CXCL8,DAB2,ENG,E REG,FN1,GATA3,GRN,GZMB,HAVCR2,ICOS,IL1A,IL1B,ITGA2,ITGA4,JAK1,KLRC4KLRK1/KLRK1,LCK,LYN, Migration 54 2.36E-14 MAP3K8,mir21,MITF,MMP1,MMP14,MMP19,MMP7,MMP9,NRP1,PDCD4,PECAM1,PIK3R5,PLAUR,PPIF,PRK CQ,RASGRP1,SERPINA1,SIRPA,SPP1,SYK,TGFBI,TLR2,TLR4,TNF

ANGPT2,ANPEP,AREG,CCL1,CCR1,CD14,CTSB,CTSH,CTSL,CTSS,CXCL1,CXCL5,CXCL8,CYP1B1,DAB2, DGKA,DRAM1,DUSP6,ENG,FN1,GATA3,GRN,GZMB,HAVCR2,HDLBP,IER3,IL1B,ITGA1,ITGA2,ITGA4,ITGAL Invasion 60 1.30E-13 ,ITGAX,ITGB2,LCP1,LYN,MAP4K1,mir103,mir21,MITF,MMP1,MMP14,MMP9,NRP1,OCLN,PDCD4,PECAM1,P LAUR,PPARG,PPIF,PRKCQ,RORA,SERPINA1,SPI1,SPP1,SYK,TGFBI,THBD,TLR2,TNF,TREM2 ANGPT2,CCL3,CCL3L3,CCL4,CCL5,CCR1,CXCL1,CXCR3,IL1B,ITGA1,ITGAL,ITGB2,ITK,MMP10,PECAM1,S Extravasation 18 1.65E-13 YK,TLR2,TNF