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Supplementary Data SUPPLEMENTAL INFORMATION A study restricted to chemokine receptors as well as a genome-wide transcript analysis uncovered CXCR4 as preferentially expressed in Ewing's sarcoma (Ewing's sarcoma) cells of metastatic origin (Figure 4). Transcriptome analyses showed that in addition to CXCR4, genes known to support cell motility and invasion topped the list of genes preferentially expressed in metastasis-derived cells (Figure 4D). These included kynurenine 3-monooxygenase (KMO), galectin-1 (LGALS1), gastrin-releasing peptide (GRP), procollagen C-endopeptidase enhancer (PCOLCE), and ephrin receptor B (EPHB3). KMO, a key enzyme of tryptophan catabolism, has not been linked to metastasis. Tryptophan and its catabolites, however, are involved in immune evasion by tumors, a process that can assist in tumor progression and metastasis (1). LGALS1, GRP, PCOLCE and EPHB3 have been linked to tumor progression and metastasis of several cancers (2-4). Top genes preferentially expressed in L-EDCL included genes that suppress cell motility and/or potentiate cell adhesion such as plakophilin 1 (PKP1), neuropeptide Y (NPY), or the metastasis suppressor TXNIP (5-7) (Figure 4D). Overall, L-EDCL were enriched in gene sets geared at optimizing nutrient transport and usage (Figure 4D; Supplementary Table 3), a state that may support the early stages of tumor growth. Once tumor growth outpaces nutrient and oxygen supplies, gene expression programs are usually switched to hypoxic response and neoangiogenesis, which ultimately lead to tumor egress and metastasis. Accordingly, gene sets involved in extracellular matrix remodeling, MAPK signaling, and response to hypoxia were up-regulated in M-EDCL (Figure 4D; Supplementary Table 4), consistent with their association to metastasis in other cancers (8, 9). Together, these data show that EDCL offer a relatively reasonable model to investigate gene expression patterns associated with localized and metastatic ES, and further support the notion that CXCR4 associates with metastatic ES, as shown in both cell lines and primary tumors. References 1. Ino K, Yamamoto E, Shibata K, et al. Inverse correlation between tumoral indoleamine 2,3-dioxygenase expression and tumor-infiltrating lymphocytes in endometrial cancer: its association with disease progression and survival. Clin Cancer Res 2008;14: 2310- 7. 2. Chao C, Ives K, Hellmich HL, Townsend CM, Jr., Hellmich MR. Gastrin-releasing peptide receptor in breast cancer mediates cellular migration and interleukin-8 expression. J Surg Res 2009;156: 26-31. 3. Holmberg J, Genander M, Halford MM, et al. EphB receptors coordinate migration and proliferation in the intestinal stem cell niche. Cell 2006;125: 1151-63. 4. Wu MH, Hong TM, Cheng HW, et al. Galectin-1-mediated tumor invasion and metastasis, up-regulated matrix metalloproteinase expression, and reorganized actin cytoskeletons. Mol Cancer Res 2009;7: 311-8. 5. Sobolik-Delmaire T, Katafiasz D, Keim SA, Mahoney MG, Wahl JK, 3rd. Decreased plakophilin-1 expression promotes increased motility in head and neck squamous cell carcinoma cells. Cell Commun Adhes 2007;14: 99-109. 6. Ogasawara M, Murata J, Ayukawa K, Saiki I. Differential effect of intestinal neuropeptides on invasion and migration of colon carcinoma cells in vitro. Cancer Lett 1997;119: 125-30. 7. Goldberg SF, Miele ME, Hatta N, et al. Melanoma metastasis suppression by chromosome 6: evidence for a pathway regulated by CRSP3 and TXNIP. Cancer research 2003;63: 432-40. 8. Wu WS, Wu JR, Hu CT. Signal cross talks for sustained MAPK activation and cell migration: the potential role of reactive oxygen species. Cancer metastasis reviews 2008;27: 303-14. 9. Larsen M, Artym VV, Green JA, Yamada KM. The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr Opin Cell Biol 2006;18: 463-71. 10. Su AI, Cooke MP, Ching KA, et al. Large-scale analysis of the human and mouse transcriptomes. Proceedings of the National Academy of Sciences of the United States of America 2002;99: 4465-70. SUPPLEMENTARY FIGURES Supplementary Fig. 1. Flow chart of Ican bioinformatic procedures. Additional details and references are within the Materials and Methods section of the paper. Supplementary Fig. 2. Chemokine receptors other than CXCR4 and CXCR7 are not modulated by EWS-FLI1 knockdown in EDCL. Gene expression of chemokine receptors was assessed by gene expression profiling before and after EWS-FLI1 knockdown in six EDCL. Mean ratio of gene expression in the two experimental setups are shown. The six EDCL were considered as six biological replicates and standard deviations were less than 20% for all genes tested. SUPPLEMENTARY TABLES Supplementary Table 1. Gene Ontology (GO) of gene sets enriched in L-EDCL. Ranking of GO functional groups was based on statistical significance (p values) of LogFD, the Log2 fold difference between M-EDCL and L-EDCL. Only those with a LogFD < -1.5 are listed. Supplementary Table 2. Gene Ontology (GO) of gene sets enriched in M-EDCL. Ranking of GO functional groups was based on statistical significance (p values) of LogFD. Only those with a LogFD > +1.5 are listed. Supplementary Table 3. Chemokine-expressing normal tissues. Gene expression profiles derived from two individuals and comprising 75 normal tissues (10) were retrieved from GEO. Highest expressing NT are shown. Supplementary Table 4. Chemokine receptor-expressing normal tissues. As in Table 1, highest expressing NT are listed herein. Supplementary Table 5. Clinical variables of tumor samples tested. Ewing's sarcoma (Ewing's sarcoma) patient tumor sample characteristics are listed. Abbreviations are as in Figure 5. Name LogFD P. Value Group gene list RESPONSE TO NUTRIENT -4.903 4.617E-05 TP53,SREBF1,CDKN2D,CHMP1A,OGT,NUAK2,ENPP1,STC2,FADS1,GNAI2,ENSA,STC LEVELS 1,ASNS,NPY SECONDARY ACTIVE -4.555 6.132E-05 SLC25A11,SLC9A6,SLC12A2,SLC12A9,SLC20A1,SLC13A2,SLC20A2,SLC12A4,SLC3A TRANSMEMBRANE 2,SLC22A7,SLC17A7,SLC9A1,SLC6A8,SLC6A2,SLC5A3,SLC12A7,SLC5A6,SLC7A11 TRANSPORTER ACTIVITY METAL ION TRANSPORT -4.057 1.687E-04 ATP2A3,KCNH2,CCS,RYR1,KCNN4,KCNQ2,ATP2C1,KCNJ4,SCNN1A,KCNN1,SLC30A 5,KCNK4,STIM1,KCNF1,ATP7A,ATOX1,KCNJ5,CACNB3,SCN10A,TRPC4,SCN1B,SLC1 1A2,KCNJ15,HFE,SLC31A2,CHP,KCNS1,COX17,KCNK1,KCNJ6,SLC31A1,SGK1,NPY RECEPTOR COMPLEX -4.108 2.178E-04 MYH9,ITGB7,ITGB3,GRIN1,BCL10,IL13RA1,CHRNB2,SRP9,CHRNE,TRIP6,SDCBP,CD 79A,SYK,ITGAE,ADRB3,TGFBR2,ITGB4,ITGA5,SRPR,ACVR1,SMAD3 CATION TRANSPORT -3.851 3.250E-04 ATP2A3,KCNH2,SLC39A1,NNT,CCS,RYR1,KCNN4,KCNQ2,ATP6AP1,ATP2C1,KCNJ4, SCNN1A,KCNN1,SLC30A5,UCP2,KCNK4,STIM1,ATP6V1E1,KCNF1,TCIRG1,ATP7A,AT OX1,KCNJ5,PKD2,CACNB3,SCN10A,TRPC4,SCN1B,SLC11A2,KCNJ15,ATP6V0B,ATP 6V1F,HFE,UCP3,SLC31A2,CHP,ATP6V0E1,KCNS1,ATP6V1C1,ATP6V0C,COX17,KCN K1,KCNJ6,SLC31A1,SGK1,NPY AUXILIARY TRANSPORT -4.133 4.326E-04 ARPP-19,SRI,TNNI3,KCNAB2,NNAT,SGK2,CHP,KCNS1,ENSA,SMAD3,NPY PROTEIN ACTIVITY RESPONSE TO -4.005 4.715E-04 TP53,CDKN1A,RPS19,SREBF1,CDKN2D,CHMP1A,OGT,NUAK2,ENPP1,STC2,FADS1, EXTRACELLULAR GNAI2,ENSA,STC1,ASNS,NPY STIMULUS GTPASE ACTIVITY -3.770 4.859E-04 RAC3,RRAS,RAC2,MX2,DNM1,RAB27A,GNA13,RAB1A,RAB33A,ARL4C,RAB5B,RAB35 ,RAB7A,RAB5C,OPA1,EIF2S3,GNAI1,ARFRP1,ARL1,RAP1A,TGM3,SEPT9,RAB5A,RAP 2A,SEPT5,GNAQ,GNB1,ARHGAP5,TRIM23,GNAS,RAB13,ARF6,RAB3A,RAB6C,RAB28 ,RAB6A,RHOD,RHEB,RHOA,RAB2A,RABL2A,RABL2B,GTPBP4,ARF1,GNA11,GNA12, GNB2,ARF5,RAB7L1,RAN,RRAS2,RND2,ARF3,DNM1L,ARL4A,ARL4D,RAB9A,RAB11B, RHOG,GNAI2,RAB4A,GNAI3,GSPT1,RRAGC,GNG11,ARF4,RHOB,EIF5,RAB22A,RAC1, CDC42,RAB31,NUDT1,RAB3B,RND3,GNA14 REGULATION OF -3.594 6.773E-04 CXCR4,HCLS1,PROS1,IFI6,TGFB2,MYC,FOXO3,APOE,EDNRA,C3AR1,COPA,PEX6,C BIOLOGICAL QUALITY ALR,GRIK2,ISOC2,TP53,BNIP3,EIF2B5,IQCB1,CDC42EP1,CAPRIN2,CDKN1A,PABPC4 ,IHPK2,FLI1,RPS19,RHOT1,SERTAD2,DERL1,BARD1,BCL2L1,SMAD4,TFPI,STX12,YW HAH,AURKA,CALCB,APP,BCL6,GCLC,ATP6AP2,MYH9,HPS4,MAFB,BCL2,SRI,NPC2,N DUFA13,SERTAD3,ABCA2,CLN5,CDKN2D,MPV17,CDKN2C,NCK1,DRD1,CCR5,ITGB3, EP300,RB1,FBXO7,HIF1A,GPX1,AKT1,NOTCH2,CENTD2,F2R,NPTN,GPI,NF1,PABPC1 ,SEPT5,NDUFS3,ATP2C1,GNAQ,GP1BA,CHGA,TGFB1,GRM4,SLC30A5,ARF6,EIF2B4, BAX,APBB1,CDC42EP4,ATP1A1,RPS6,LMAN1,PAIP1,RAB3A,NPR2,BAK1,LIMA1,TMP RSS6,ATP7A,PPARD,CLCN3,GCHFR,ATOX1,GLRX2,TSPYL2,BCL10,CDKN2A,CACNA 1A,PLAT,ABCG1,CALCA,ATP1A3,CAV1,SLC4A1,RASA1,NPR1,AVPR1B,CLN3,AGT,FT H1,FGD1,CDKN2AIP,ENTPD1,HPRT1,A1CF,ACVR1B,GTPBP4,EIF2B2,APBB2,TAOK2, PTEN,GNA12,SOD1,FXN,HFE,SLC12A4,NPPA,DYRK3,GGCX,FRAP1,EGLN2,CDKN1B, NOL8,ENO1,CDC2L2,NDUFS1,SLC9A1,ABCA1,DDIT3,CD59,TARBP2,APOA1,GCH1,S YT1,GUCA1B,EGLN1,CAPG,APTX,PML,DERL2,ACIN1,RHOT2,CLN6,ACVR2A,TMSB4Y ,CD24,CCL2,SLC1A3,PTGS1,BIN3,MT2A,GSN,NCK2,FTL,CD55,LYN,CEBPG,AKR1C1, PPT1,TXNDC4,COG7,TPP1,GCLM,NPC1,ALDH9A1,DLC1,SMAD3,STC1,UBB,THY1,CD 9,CXCL12,UTS2,NPY POSITIVE REGULATION OF -3.701 9.330E-04 SCG2,TGFB2,IL8,CD1D,EEF1E1,MAP3K7,CFHR1,FYN,TRAF2,TNFRSF1A,LAT2,IKBKG RESPONSE TO STIMULUS ,CD79A,UBE2N,CADM1,CEBPG,MALT1,SLIT2,THY1,NPY ION TRANSPORT -3.524 9.459E-04 ATP2A3,KCNH2,TRPA1,SLC34A2,SLC39A1,SLC26A2,NNT,TSPO,GLRB,CCS,RYR1,KC NN4,LASP1,KCNQ2,ATP6AP1,P2RX1,CLIC1,ATP2C1,KCNJ4,SCNN1A,KCNN1,SLC30A 5,UCP2,KCNK4,CLIC5,VDAC2,STIM1,ATP6V1E1,KCNF1,TCIRG1,ATP7A,VDAC1,ATOX 1,KCNJ5,SLC4A1,PKD2,CACNB3,SCN10A,TRPC4,FXYD3,PLP2,SCN1B,SLC11A2,KCN J15,ATP6V0B,ATP6V1F,HFE,SLC22A7,UCP3,SLC31A2,CHP,ATP6V0E1,KCNS1,SLC17 A7,ATP6V1C1,ATP6V0C,COX17,KCNK1,AKAP7,KCNJ6,SLC31A1,SLC4A2,SGK1,WNK 1,NPY REGULATION OF -3.553 1.184E-03 SCG2,TGFB2,IL8,CD1D,EEF1E1,APOBEC3G,APOBEC3F,UBE2V2,MAP3K7,CFHR1,FY RESPONSE TO STIMULUS N,TRAF2,UBE2V1,TNFRSF1A,LAT2,IKBKG,PTGDS,CD79A,UBE2N,TARBP2,CADM1,C EBPG,MALT1,SLIT2,THY1,NPY LIPID RAFT -3.668 1.300E-03 FLOT1,STX12,PIP5K3,CAV2,LAT,PRNP,CAV1,CLN3,LAT2,ABCA1,CD79A,PI4K2A,CD2
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