Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. $EHUUDQW JO\FRV\ODWLRQ SURPRWHV OXQJ FDQFHU PHWDVWDVLV WKURXJK DGKHVLRQ WR JDOHFWLQV LQ WKHPHWDVWDWLFQLFKH 1DWKDQ(5HWLFNHU)O\QQDQG6DQJHHWD1%KDWLD 'DYLG+.RFK,QVWLWXWHIRU,QWHJUDWLYH&DQFHU5HVHDUFK0DVVDFKXVHWWV,QVWLWXWHRI 7HFKQRORJ\&DPEULGJH0DVVDFKXVHWWV86$ +DUYDUG0,7'LYLVLRQRI+HDOWK6FLHQFHVDQG7HFKQRORJ\0DVVDFKXVHWWV,QVWLWXWHRI 7HFKQRORJ\&DPEULGJH0DVVDFKXVHWWV86$ +RZDUG+XJKHV0HGLFDO,QVWLWXWH&DPEULGJH0DVVDFKXVHWWV86$ 'LYLVLRQRI0HGLFLQH%ULJKDPDQG:RPHQ¶V+RVSLWDO%RVWRQ0DVVDFKXVHWWV 86$ 'HSDUWPHQWRI(OHFWULFDO(QJLQHHULQJDQG&RPSXWHU6FLHQFH0DVVDFKXVHWWV,QVWLWXWHRI 7HFKQRORJ\&DPEULGJH0DVVDFKXVHWWV86$ 7RZKRPFRUUHVSRQGHQFHVKRXOGEHVHQWVEKDWLD#PLWHGX 5XQQLQJ7LWOHSt6galnac4Gcnt3DQGJDOHFWLQVDVGULYHUVRI16&/&PHWDVWDVLV .H\:RUGV16&/&PHWDVWDVLVJO\FRV\ODWLRQJO\FRV\OWUDQVIHUDVHJDOHFWLQ,/7KRPVHQ )ULHGHQUHLFK$QWLJHQSt6galnac4Gcnt3WXPRUPLFURHQYLURQPHQW 7KLVZRUNZDVVXSSRUWHGLQSDUWE\WKH.RFK,QVWLWXWH6XSSRUWFRUH*UDQW3&$IURP WKH1DWLRQDO&DQFHU,QVWLWXWH15HWLFNHU)O\QQLVVXSSRUWHGE\D/XGZLJ&HQWHUIRU0ROHFXODU 2QFRORJ\*UDGXDWH)HOORZVKLS6%KDWLDLVDQ++0,LQYHVWLJDWRU7KLVZRUNZDVVXSSRUWHGE\ D6WDQG8S7R&DQFHU'UHDP7HDP7UDQVODWLRQDO5HVHDUFK*UDQW*UDQW1XPEHU68&$$&5 '76WDQG8S7R&DQFHULVDSURJUDPRIWKH(QWHUWDLQPHQW,QGXVWU\)RXQGDWLRQ DGPLQLVWHUHGE\WKH$PHULFDQ$VVRFLDWLRQIRU&DQFHU5HVHDUFKDQGWKH.RFK&7&3URMHFW´ 6%KDWLDFRQWDFWLQIRUPDWLRQ 0DVVDFKXVHWWV$YH %XLOGLQJ &DPEULGJH0$ VEKDWLD#PLWHGX SKRQH ID[ Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 7KHDXWKRUVKDYHQRFRQIOLFWVRILQWHUHVWWRGLVFORVH :RUG&RXQWLQFOXGLQJ0HWKRGV :RUG&RXQWH[FOXGLQJ0HWKRGV )LJXUH&RXQW Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. *DOHFWLQVDUHDIDPLO\RIȕ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ȕ*DO1$FĮ 6HU7KUH[SRVXUHRIWKHGLVDFFKDULGHIRUPLVJHQHUDOO\RFFOXGHGE\IXUWKHUJO\FRV\ODWLRQ,WLV HVWLPDWHG KRZHYHU WKDW WKH FHOOV LQ DV PDQ\ DV RI FDUFLQRPDV H[SUHVV WKLV GLVDFFKDULGH ZLWKRXWIXUWKHUJO\FRV\ODWLRQ1RQHWKHOHVVWKHPHFKDQLVPVE\ZKLFKWKLVFRQVHUYHGJO\FDQ PRWLILVUHJXODWHGE\FDQFHUFHOOVDORQJZLWKWKHSUHFLVHUROHWKDWLWSOD\VLQWXPRULJHQHVLVDQG PHWDVWDVLVDUHSRRUO\XQGHUVWRRG 5HFHQWO\ WKH UROH WKDW G\QDPLF UHPRGHOLQJ RI WKH WXPRU PLFURHQYLURQPHQW SOD\V LQ WKH SURPRWLRQ RI PHWDVWDVLV KDV EHHQ SDUWLDOO\ HOXFLGDWHG 6ROXEOH IDFWRUV UHOHDVHG IURP WXPRUVDFWWRPRELOL]HERQHPDUURZGHULYHGFHOOV%0'&VDQGLQSDUWLFXODUP\HORLGFHOOVLQWR WKHSHULSKHUDOEORRGDQGUHFUXLWWKHPWRWKHHDUO\PHWDVWDWLFQLFKHDVLWHZKHUHPHWDVWDVHVKDYH Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. GRZQUHJXODWLRQ RI WKH FRUH NDFHW\OJOXFRVDPLQ\OWUDQVIHUDVH &*Q7 Gcnt3 DUH NH\ PHGLDWRUV RI WKH FKDQJHV LQ JO\FRV\ODWLRQ WKDW SRWHQWLDWH PHWDVWDWLF SURFOLYLW\ E\ SUHVHUYLQJ SUHVHQWDWLRQRIWKH7$QWLJHQ 5HVXOWV 7XPRUEHDULQJPLFHH[KLELWHOHYDWHGOHYHOVRIJDOHFWLQLQWKHHDUO\PHWDVWDWLFQLFKH 7R GHWHUPLQH WKH UROH RI JDOHFWLQ LQ PHWDVWDVLV ZH XVHG D JHQHWLF PRXVH PRGHO RI OXQJ DGHQRFDUFLQRPDPHWDVWDVLVKrasLSL-G12D/+;p53flox/floxIURPZKLFKFHOOOLQHVUHSUHVHQWLQJGLVWLQFW VWDJHV RI PHWDVWDWLF SURJUHVVLRQ ZHUH GHULYHG )ROORZLQJ &UHPHGLDWHG UHFRPELQDWLRQ DQG WKH GHYHORSPHQW RI DXWRFKWKRQRXV OXQJ WXPRUV DQG PHWDVWDVHV OLQHV ZHUH JHQHUDWHG IURP SULPDU\WXPRUVWKDWGLGQRWJLYHULVHWRPHWDVWDVHV7QRQ0HWGLGJLYHULVHWRPHWDVWDVHV70HW O\PSK QRGH PHWDVWDVHV 1 DQG OLYHU PHWDVWDVHV 0 3UHYLRXVO\ ZH IRXQG WKDW LQFUHDVLQJO\ PHWDVWDWLF OLQHV H[KLELWHG LQFUHDVHG DGKHVLRQ WR JDOHFWLQ in vitro )LJ $ $V (&0 PROHFXOHV DQG WKHLU PRGLILFDWLRQ E\ %0'&V SOD\ LPSRUWDQW UROHV LQ WKH GHYHORSPHQW RI WKH PHWDVWDWLF QLFKH ZH DVNHG ZKHWKHU JDOHFWLQ KDV D UROH LQ LWV HVWDEOLVKPHQW /LYHUV ZHUH KDUYHVWHGIURPWXPRUEHDULQJPLFHSULRUWRWKHGHWHFWLRQRIRYHUWPHWDVWDVHVDQGH[DPLQHGIRU DFFXPXODWLRQRIJDOHFWLQ:HVWHUQEORWDQDO\VLVRIOLYHUSURWHLQO\VDWHVUHYHDOHGDQHOHYDWLRQLQ JDOHFWLQLQWKHOLYHUVRIWXPRUEHDULQJPLFHFRPSDUHGWRQDwYHPLFH)LJ%7RLGHQWLI\WKH VRXUFH RI WKLV JDOHFWLQ ZH H[DPLQHG ERWK WKH WXPRU FHOOV DQG UHFUXLWHG OHXNRF\WHV *HQH H[SUHVVLRQ PLFURDUUD\ DQG ZHVWHUQ EORW DQDO\VHV RI JDOHFWLQ LQ WKH WXPRU FHOO OLQHV UHYHDOHG HTXLYDOHQWH[SUHVVLRQRIWKHPROHFXOHVDFURVVDOOIRXUFODVVHVRIOLQHVLQGLFDWLQJWKDWJDOHFWLQ H[SUHVVLRQ E\ WXPRUV LV QRW GLIIHUHQWLDOO\ UHJXODWHG GXULQJ WXPRU SURJUHVVLRQ 6XSSOHPHQWDU\ )LJ $ % ,Q OLJKW RI UHFHQW UHSRUWV RI P\HORLGGHULYHG SRSXODWLRQV VXSSRUWLQJ RXWJURZWK RI Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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wYHPLFHRUPLFHEHDULQJWXPRUVJHQHUDWHGE\HLWKHU7QRQ0HWRU0OLQHV 6XSSOHPHQWDU\)LJ$&DQGDQDO\]HGLWIRUWKHSUHVHQFHRIJDOHFWLQDQG&'EP\HORLG FHOOV $QDO\VLV RI SHULSKHUDO EORRG FHOOV E\ IORZ F\WRPHWU\ UHYHDOHG D PDUNHG LQFUHDVH LQ JDOHFWLQ DQG &'E OHXNRF\WHV LQ WKH FLUFXODWLRQ RI PLFH EHDULQJ ERWK WKH 0 DQG 7QRQ0HW WXPRUV )LJ $& DQG 6XSSOHPHQWDU\ )LJ ') &RQVLVWHQW ZLWK WKH ILQGLQJV RI JDOHFWLQ PDFURSKDJHV LQ WKH OLYHU WKHUH ZDV DQ HQULFKPHQW LQ JDOHFWLQ&'E GRXEOHSRVLWLYH SRSXODWLRQVLQWKHEORRGRIWXPRUEHDULQJPLFH)LJ$',QDJUHHPHQWZLWKSUHYLRXVUHSRUWV RI LQFUHDVHV LQ LQIODPPDWRU\ P\HORLG FHOOV LQ WXPRUEHDULQJ PLFH WKHUH ZDV D VLJQLILFDQW LQFUHDVH LQ WKH IUHTXHQF\ RI &'E*U DQG &'E/\&KL SRSXODWLRQV )LJ () DQG Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. FHOOPRELOL]DWLRQZHH[DPLQHGIl6JHQHH[SUHVVLRQOHYHOVLQDOORIWKHFHOOOLQHVIURPHDFKRIWKH IRXUJURXSV)LJ'$QDO\VLVRIJHQHH[SUHVVLRQPLFURDUUD\GDWDUHYHDOHGFRQVWDQWH[SUHVVLRQ RI Il6 DFURVV DOO IRXU FODVVHV RI FHOO OLQHV VXJJHVWLQJ WKDW H[SUHVVLRQ RI ,/ E\ WXPRUV FRPPHQFHVHDUO\LQWXPRULJHQHVLV 7RGHWHUPLQHWKHUHOHYDQFHRIWXPRUGHULYHG,/WRWKHKXPDQGLVHDVHZHH[DPLQHGJHQRPLF DPSOLILFDWLRQRIWKHIL6JHQHLQSDWLHQWVZLWKOXQJDGHQRFDUFLQRPDV&RPSDUHGWRQRUPDOEORRG RUOXQJOXQJDGHQRFDUFLQRPDVKDGVLJQLILFDQWO\HOHYDWHGFRS\QXPEHUVRIWKHIL6JHQH)LJ( P î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sf3r *&6)5 NQRFNRXW PLFH ZKLFK H[KLELW LPSDLUHG JUDQXORF\WHSURGXFWLRQDQGPDWXUDWLRQ:KLOH,/ZDVVWLOOFDSDEOHRILQGXFLQJ&'E/\&KL FHOO PRELOL]DWLRQ Csf3r-/- PLFH H[KLELWHG D SURIRXQG UHGXFWLRQ LQ &'EJDOHFWLQ DQG &'E*U OHXNRF\WHV FRPSDUHG WR ZLOGW\SH PLFH 6XSSOHPHQWDU\ )LJ $' 7KHVH ILQGLQJV VXJJHVW WKDW WXPRUGHULYHG ,/ LV VXIILFLHQW WR LQGXFH PRELOL]DWLRQ RI JDOHFWLQ OHXNRF\WHV 7R GHWHUPLQH ZKHWKHU JDOHFWLQ H[SUHVVLRQ E\ WKH P\HORLG FHOOV RU DQRWKHU QRQSDUHQFK\PDO SRSXODWLRQLVDSUHUHTXLVLWHWRWKHLUPRELOL]DWLRQLQWRFLUFXODWLRQZHUHSHDWHGWKH,/LQMHFWLRQV LQ Lgals3 JDOHFWLQ NQRFNRXW PLFH DQG REVHUYHG QR GLIIHUHQFHV LQ &'E P\HORLG FHOO PRELOL]DWLRQFRPSDUHGWRZLOGW\SHPLFH)LJ+DQG6XSSOHPHQWDU\)LJ$(7KHUHOHYDQW Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ȕJDODFWRVLGH JO\FDQV WKURXJK WKHLU FDUERK\GUDWH UHFRJQLWLRQ GRPDLQ &5' ZH DVNHG ZKHWKHU FKDQJHV LQ JO\FDQ SUHVHQWDWLRQ E\ WXPRU FHOOV FRQIHU WKHLU DGKHVLRQ WR JDOHFWLQ RQ UHFUXLWHG OHXNRF\WHV GXULQJ PHWDVWDVLV $QDO\VLV RI JO\FDQ PLFURDUUD\ GDWDVHWV DQG H[LVWLQJ OLWHUDWXUH UHYHDO D YDULHW\ RI FDUERK\GUDWH OLJDQGV IRU WKHVH JDOHFWLQV )LJ $ :KLOH DOO JDOHFWLQV ELQG ODFWRVDPLQHV JDOHFWLQH[KLELWVVSHFLILFDIILQLWLHVIRUWKH7KRPVHQ)ULHGHQUHLFK$QWLJHQ7$QWLJHQ*DOȕ *DO1$FĮ6HU7KUDSDQFDUFLQRPDPDUNHUH[SUHVVHGRQPDQ\JO\FRSURWHLQV)LJ% *DOHFWLQ LV XQDEOH WR LQWHUDFW ZLWK WKH 7$QWLJHQ LI WKH GLVDFFKDULGH LV IXUWKHU JO\FRV\ODWHG 7KXVZHDVNHGZKHWKHUPHWDVWDWLFFHOOVH[KLELWHOHYDWHG7$QWLJHQOHYHOVRQWKHLUVXUIDFHVWKDW PLJKW PHGLDWH LQWHUDFWLRQV ZLWK JDOHFWLQ 6WDLQLQJ RI 7QRQ0HW 70HW DQG 0 OLQHV ZLWK D 7 $QWLJHQVSHFLILF OHFWLQSHDQXW DJJOXWLQLQ 31$ UHYHDOHG WKDW WKH OHYHOV RI 7$QWLJHQ VXUIDFH SUHVHQWDWLRQaIROGLQFUHDVHFRUUHODWHZLWKPHWDVWDWLFSRWHQWLDO)LJ&DQG6XSSOHPHQWDU\ )LJ $ 7R GHWHUPLQH ZKHWKHU PHWDVWDWLF FHOO DGKHVLRQ WR JDOHFWLQ LV LQGHHG FDUERK\GUDWH PHGLDWHG ZH LQFXEDWHG WKH FHOOV ZLWK IOXRUHVFHQW JDOHFWLQ LQ WKH SUHVHQFH RI D FRPSHWLWLYH Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ELQGHU N$FHW\OODFWRVDPLQH /DF1$F )ORZ F\WRPHWU\ UHYHDOHG D UHGXFWLRQ LQ ELQGLQJ LQ WKH SUHVHQFH RI /DF1$F FRPSDUHG WR FRQWURO VXFURVH VXJJHVWLQJ WKDW JDOHFWLQ DGKHVLRQ WR WKH PHWDVWDWLFFHOOVLVFDUERK\GUDWHPHGLDWHG)LJ' 7R LQYHVWLJDWH ZKHWKHU WKHVH DOWHUDWLRQV DUH REVHUYHG LQ WKH KXPDQ GLVHDVH ZH VWDLQHG KXPDQ 16&/& WLVVXH PLFURDUUD\V RI WLVVXH VDPSOHV ZLWK 31$ WR DVVHVV WKH SUHVHQFH RI WKH 7 $QWLJHQ 7KLV VWDLQLQJ UHYHDOHG HOHYDWHG OHYHOV RI FHOOVXUIDFH 7$QWLJHQ SUHVHQWDWLRQ LQ FDQFHURXVWLVVXHFRPSDUHGWRQRQFDQFHURXVWLVVXHZLWKDQRWDEOHHQULFKPHQWLQWKHO\PSKQRGH PHWDVWDVHV )LJ () DQG 6XSSOHPHQWDU\ )LJ % $GGLWLRQDOO\ ZH H[DPLQHG IRXU FRPPRQ 16&/&FHOOOLQHVIRUWKHLUUHODWLYHH[SUHVVLRQRIWKH7$QWLJHQ)ORZF\WRPHWU\UHYHDOHGWKDW LQGHHG WKUHH RI WKH IRXU OLQHV ERXQG 31$ DW FRPSDUDEOH OHYHOV WR WKH 0 OLQH ZLWK WKH PRVW LQYDVLYH RI WKH OLQHV $ H[KLELWLQJ WKH KLJKHVW GHJUHH RI VWDLQLQJ )LJ * 7DNHQ WRJHWKHUWKHVHGDWDVXJJHVWWKDWOXQJWXPRUVELQGJDOHFWLQWKURXJKFDUERK\GUDWHHSLWRSHVVXFK DVWKH7$QWLJHQDQGWKDWWKLVHSLWRSHLVH[SUHVVHGPRUHKLJKO\RQPHWDVWDWLFFHOOV 'LIIHUHQWLDO J\FRV\OWUDQVIHUDVH H[SUHVVLRQ SURPRWHV LQFUHDVHG 7$QWLJHQ SUHVHQWDWLRQ WKURXJKUHGXFWLRQRIJO\FDQFKDLQHORQJDWLRQDQGLQFUHDVHGFDSSLQJ $ YDULHW\ RI OOLQNHG JO\FRSURWHLQV VXFK DV 08& FDQ H[KLELW SURIRXQG GHJUHHV RI O JO\FRV\ODWLRQDQGGLIIHUHQWLDOUHJXODWLRQRIVXFKJO\FRSURWHLQVFDQVLJQLILFDQWO\DOWHUWKHRYHUDOO JO\FDQSUHVHQWDWLRQRIFHOOV7KXVZHDVNHGZKHWKHULQFUHDVHG7$QWLJHQH[SUHVVLRQLVDUHVXOW RI XSUHJXODWLRQ RI LQGLYLGXDO JO\FRSURWHLQV RU DOWHUHG JO\FRV\OWUDQVIHUDVH DFWLYLW\ :H LVRODWHG FHOOPHPEUDQHSURWHLQVIURP7QRQ0HW70HWDQG0OLQHVDQGSHUIRUPHG31$OHFWLQEORWV7KHVH UHYHDOHGDJOREDOLQFUHDVHLQ7$QWLJHQH[SUHVVLRQDFURVVPDQ\JO\FRSURWHLQVLQWKHLQFUHDVLQJO\ PHWDVWDWLFFHOOVDVRSSRVHGWR31$ODEHOLQJRIDVSHFLILFJO\FRSURWHLQ)LJ$ Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ,QOLJKWRIWKLVEURDGGLVWULEXWLRQLQJO\FRV\ODWLRQZHLQYHVWLJDWHGDOWHUDWLRQVLQWKHH[SUHVVLRQ DQG DFWLYLW\ RI JO\FRV\OWUDQVIHUDVHV $QDO\VLV RI JHQH H[SUHVVLRQ PLFURDUUD\V IRU JO\FRV\OWUDQVIHUDVH JHQHV GLG QRW GHPRQVWUDWH ZLGHVSUHDG VKLIWV LQ H[SUHVVLRQ QRU ZHUH WKHUH VLJQLILFDQWFKDQJHVLQWKHVXEVHWRIJHQHVWKDWHQFRGHWUDQVIHUDVHVVSHFLILFIRUJHQHUDWLRQRIWKH JDOHFWLQ OLJDQGV 6XSSOHPHQWDU\ )LJ $ % &RPSDULVRQV EHWZHHQ WKH DYHUDJHV IRU WKH SULPDU\ WXPRU DQG PHWDVWDVLVGHULYHG FHOO OLQHV KRZHYHU LGHQWLILHG Gcnt3 DQG St6galnac4 DV EHLQJXQGHUDQGRYHUH[SUHVVHGLQWKHPHWDVWDWLFOLQHVUHVSHFWLYHO\)LJ%WKHVHDOWHUDWLRQV ZHUH FRQILUPHG E\ T573&5 LQ WKH UHSUHVHQWDWLYH FHOO OLQHV )LJ & WRS &*Q7 Gcnt3 LQGXFHVWKHDGGLWLRQRIDȕ*OF1$FWRWKH7$QWLJHQZKHUHDV6W*DO1$F,9DGGVDQĮ 1HX$F WR WKH VLDO\O7$QWLJHQ )LJ & ERWWRP $V IXUWKHU HORQJDWLRQ RI WKH FRUH GLVDFFKDULGHSUHYHQWVELQGLQJE\JDOHFWLQ)LJ%FKDQJHVLQWUDQVIHUDVHDFWLYLW\FRQVLVWHQW ZLWK WKH H[SUHVVLRQ FKDQJHV LQ Gcnt3 DQG St6galnac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î$GGLWLRQDOO\ZHH[DPLQHGJHQHH[SUHVVLRQRIWKHUHOHYDQW Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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cnt3DQGSt6galnac4PHGLDWHJDOHFWLQDGKHVLRQin vitroDQGPHWDVWDVLVin vivo 7R WHVW WKH IXQFWLRQDO UROH RI WKHVH WUDQVIHUDVHV RQ JDOHFWLQ ELQGLQJ DQG PHWDVWDVLV ZH WUDQVIHFWHG WKH PHWDVWDWLF FHOOV ZLWK Gcnt3 RU NQRFNHG GRZQ St6galnac4 ZLWK VKRUW KDLUSLQV 7UDQVIHFWLRQ RI Gcnt3 UHVXOWHG LQ GHFUHDVHG 7$QWLJHQ SUHVHQWDWLRQ DQG GHFUHDVHG JDOHFWLQ ELQGLQJ DQG LQ D FRPSOHPHQWDU\ IDVKLRQ NQRFNGRZQ RI St6galnac4 \LHOGHG D UHGXFWLRQ LQ JDOHFWLQ ELQGLQJ ZKLOH KDYLQJ QR HIIHFW RQ QRQVLDO\ODWHG 7$QWLJHQ SUHVHQWDWLRQ DV 31$ GRHV QRW UHFRJQL]H VLDO\ODWHG IRUPV RI WKH DQWLJHQ )LJ $ DQG 6XSSOHPHQWDU\ )LJ $( $ % 7R GHWHUPLQH WKH IXQFWLRQDO UROH RI 6W*DO1$F,9 DFWLYLW\ RQ PHWDVWDVLV in vivo ZH SHUIRUPHGH[SHULPHQWDOPHWDVWDVLVDVVD\VZLWKWKHSt6galnac4NQRFNGRZQOLQH&HOOVH[SUHVVLQJ DKDLUSLQIRUWKHVLDO\OWUDQVIHUDVHRUDFRQWUROKDLUSLQDJDLQVWILUHIO\OXFLIHUDVHZHUHLQMHFWHGLQWR WKH VSOHHQV RI PLFH DQG PRQLWRUHG IRU OLYHU WXPRU IRUPDWLRQ IROORZLQJ KHPDWRJHQRXV GLVVHPLQDWLRQ:HIRXQGWKDWPLFHLQMHFWHGZLWKWKH0VKSt6galnac4FHOOVH[KLELWHGJUHDWHU WKDQ IHZHU OLYHU PHWDVWDVHV WKDQ FRQWURO PLFH WZR ZHHNV IROORZLQJ WXPRU FHOO LQMHFWLRQ )LJ %& DQG 6XSSOHPHQWDU\ )LJ :H REVHUYHG QR GLIIHUHQFHV LQ SUROLIHUDWLRQ UDWHV EHWZHHQ WKH NQRFNGRZQ DQG FRQWURO KDLUSLQ FHOO OLQHV in vitro 6XSSOHPHQWDU\ )LJ 7DNHQ Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ĮVLDO\OWUDQVIHUDVH6W*DO1$F,97KHVHFKDQJHVLQ WUDQVIHUDVH DFWLYLW\ DQG 7$QWLJHQ SUHVHQWDWLRQ UHVXOW LQ LQFUHDVHG FDUERK\GUDWHGHSHQGHQW ELQGLQJ RI WKH WXPRU FHOOV WR JDOHFWLQ DQG LQFUHDVHG PHWDVWDVLV in vivo $V WKH SUHVHQFH RI WXPRUV LQGXFHV HOHYDWHG QXPEHUV RI JDOHFWLQ P\HORLG FHOOV LQ WKH FLUFXODWLRQ DQG PHWDVWDWLF Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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³SUH´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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ĮȕSURPRWHVPHWDVWDVLVWKURXJKELQGLQJWRFRPELQDWLRQVRIILEURQHFWLQZLWKHLWKHU JDOHFWLQ RU ,QWHJULQV FRQWDLQ D YDULHW\ RI JO\FRV\ODWLRQ VLWHV DQG WKHVH GDWD PD\ Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. VXJJHVWWKDWJO\FRV\ODWLRQRILQWHJULQVFRXOGSURPRWHPHWDVWDVLVWKURXJKV\QHUJLVWLFLQWHUDFWLRQV EHWZHHQ JDOHFWLQV DQG RWKHU (&0 PROHFXOHV ZLWKLQ WKH PHWDVWDWLF QLFKH 5HJDUGOHVV RI WKH SDUWLFXODU SURWHLQV H[KLELWLQJ WKHVH JO\FRV\ODWLRQ SDWWHUQV RXU REVHUYDWLRQV RI DOWHUDWLRQV LQ WUDQVIHUDVHH[SUHVVLRQDQG7$QWLJHQSUHVHQWDWLRQLQKXPDQVZLWKWKHGLVHDVHVXJJHVWVDUROHIRU D KLJKO\ FRQVHUYHG PHFKDQLVP RI JO\FDQPHGLDWHG OXQJ FDQFHU PHWDVWDVLV WKDW ZDUUDQWV FRQVLGHUDWLRQIRUWKHUDSHXWLFLQWHUYHQWLRQ Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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î FHOOV UHVXVSHQGHG LQ / RI 3%6 LQWR WKH VXEFXWDQHRXV UHJLRQ RI IODQNV RI PLFH ZKLOH DQHVWKHWL]HG ZLWK LVRIOXRUDQH 7XPRUV DQG SHULSKHUDO EORRG ZHUH KDUYHVWHG ZHHNV IROORZLQJ LQMHFWLRQV $SSUR[LPDWHO\ P/ RI SHULSKHUDO EORRG ZDV KDUYHVWHG E\ FDUGLDF SXQFWXUH ZKLOH PLFH ZHUH DQHVWKHWL]HG ZLWK Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. LVRIOXRUDQH DQG XVHG IRU DQDO\VLV E\ IORZ F\WRPHWU\ 0LFH ZHUH LPPHGLDWHO\ HXWKDQL]HG IROORZLQJUHFRYHU\RIWKHEORRGDQGWKHLUWXPRUVZHUHKDUYHVWHG ,QWUDVSOHQLF WUDQVSODQWDWLRQ DVVD\V ZHUH SHUIRUPHG DV SUHYLRXVO\ GHVFULEHG %ULHIO\ î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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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ĮWXEXOLQ &HOO 6LJQDOLQJ /HFWLQ EORWWLQJ IRU JO\FDQV ZDV SHUIRUPHG XVLQJ 31$%LRWLQ 6LJPD IROORZHG E\ GHWHFWLRQ XVLQJ WKH $%& (OLWH .LW 9HFWRU /DEV 3. RU 31$3HUR[LGDVH FRQMXJDWHV 6LJPD 3RQFHDX 6 VROXWLRQ 7KHUPRZDVXVHGIRUWRWDOSURWHLQGHWHFWLRQIROORZLQJWUDQVIHUWR39')PHPEUDQHV Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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¶VLQVWUXFWLRQV &HOO6XUIDFH3URWHLQ,VRODWLRQ &HOO VXUIDFH SURWHLQV ZHUH LVRODWHG SULRU WR :HVWHUQ EORW DQDO\VLV XVLQJ 7KHUPR 6FLHQWLILF NLW IROORZLQJ WKH PDQXIDFWXUHU¶V LQVWUXFWLRQV %ULHIO\ FHOOV DUH ZDVKHG IROORZHG E\ ELRWLQ\ODWLRQRIWKHVXUIDFHSURWHLQVE\D6XOIR1+6%LRWLQWKURXJKFRQMXJDWLRQWRIUHHDPLQHV 7KHUHDFWLRQLVTXHQFKHGDQGFHOOVDUHKDUYHVWHGDQGO\VHG/\VDWHLVZDVKHGWKURXJKDFROXPQ FRQWDLQLQJDQHXWUDYLGLQUHVLQWRFDSWXUHWKHELRWLQ\ODWHGVXUIDFHSURWHLQV)LQDOO\WKHGLVXOILGH OLQNDJHLVUHGXFHGWKURXJKLQFXEDWLRQZLWKP0'77DQGWKHSURWHLQVDUHFDSWXUHG )ORZ&\WRPHWULF$QDO\VLV Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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¶V LQVWUXFWLRQV&'EFHOOVZHUHLVRODWHGXVLQJ0$&606&ROOHFWLRQ&ROXPQV0LOWHQ\L 3HDQXW DJJOXWLQLQ VWDLQLQJ ZDV SHUIRUPHG E\ LQFXEDWLQJ î FHOOV LQ 3%6 ZLWK / 31$ $OH[D)OXRU LQYLWURJHQ RQ LFH IRU PLQXWHV )OXRUHVFHQW JDOHFWLQ ZDV SURGXFHG E\ UHDFWLQJUHFRPELQDQWPXULQHJDOHFWLQ5'6\VWHPVZLWK'\/LJKW1+6(VWHU7KHUPR 3LHUFH &RQMXJDWHG SURWHLQ ZDV SXULILHG E\ )3/& DQG FRQFHQWUDWHG WR PJP/ XVLQJ 0:&2$PLFRQFHQWULIXJDOILOWHUXQLWV0LOOLSRUH&HOOVZHUHVWDLQHGE\LQFXEDWLQJîFHOOV LQ3%6ZLWK/RIWKHIOXRUHVFHQWJDOHFWLQRQLFHIRUPLQXWHV )RUJDOHFWLQELQGLQJLQKLELWLRQH[SHULPHQWVRQWXPRUFHOOVFHOOVZHUHLQFXEDWHGZLWKJDOHFWLQ DVGHVFULEHGDERYHLQWKHSUHVHQFHRIP0N$FHW\O'ODFWRVDPLQH&DUERV\QWKRUVXFURVH 6LJPD )RU FRPSHWLWLRQ RI JDOHFWLQ SUHVHQWDWLRQ RQ OHXNRF\WHV SHULSKHUDO EORRG ZDV KDUYHVWHGIURPPLFHEHDULQJ0IODQNWXPRUVDVGHVFULEHGDERYH&HOOVZHUHLQFXEDWHGZLWK Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. P0ȕ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¶V SURWRFRO ,/ ,)1J ,/ 71)D ,/D ,/ ,/ ,/ /XPLQH[ DVVD\V ZHUH DQDO\]HG XVLQJ%LRSOH[ZLWK%LRSOH[0DQDJHU 51$,VRODWLRQDQG([SUHVVLRQ3URILOLQJ 0XULQH JHQH H[SUHVVLRQ PLFURDUUD\ DQDO\VLV ZDV GHVFULEHG SUHYLRXVO\ DQG LV DYDLODEOH IURP 1&%, XQGHU DFFHVVLRQ QXPEHU *6( DQG *6( +XPDQ 16&/& H[SUHVVLRQ PLFURDUUD\GDWDZDVDFFHVVHGIURP1&%,DFFHVVLRQQXPEHU*6(51$ZDVLVRODWHGXVLQJ Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. 51HDV\ PLQL NLWV 4LDJHQ DFFRUGLQJ WR WKH PDQXIDFWXUHU¶V LQVWUXFWLRQV F'1$ UHDFWLRQV ZHUH SHUIRUPHG XVLQJ L6FULSW F'1$ V\QWKHVLV NLW %LR5DG T3&5 UHDFWLRQV ZHUH SHUIRUPHG XVLQJ /RIF'1$/RI,46<%5*UHHQ6XSHUPL[%LR5DGDQGPRORIHDFKSULPHU)ROG FKDQJH LV UHSRUWHG IROORZLQJ DQDO\VLV XVLQJ WKHǻǻ&W PHWKRG ZKHUH JHQHV ZHUH QRUPDOL]HG WR Hprt KRXVHNHHSLQJ FRQWURO 3ULPHUV ZHUH St6galnac4 IRUZDUG ¶ **77**77&$&&$7*$77&7*¶ St6galnac4 UHYHUVH ¶**$*&****$&7&77&7&¶ Gcnt3 IRUZDUG ¶*&$*&&$$*$$**7$&&$$$¶ Gcnt3 UHYHUVH ¶ $&$**&*$**$&&$7&$$¶ RU IRUZDUG ¶$7*$*$*&&$77*&*$*$&7¶ UHYHUVH ¶&77*77**&&$$*$**7*$7¶ Hprt IRUZDUG ¶*7&$$&*****$&$7$$$$*¶ Hprt UHYHUVH ¶&$$&$$7&$$*$&$77&777$&$¶ *HQH H[SUHVVLRQ DQDO\VLV DQG YLVXDOL]DWLRQVZHUHSHUIRUPHGXVLQJ6SRWILUH7LEFRDQG0$7/$%0DWKZRUNV +XPDQ&RS\1XPEHU$QDO\VLV &RS\QXPEHUGHWHUPLQDWLRQVRIGCNT3DQGIL6ZHUHSHUIRUPHGXVLQJ2QFRPLQHEDVHGRQ SXEOLFO\DYDLODEOHGDWDIURPWKH&DQFHU*HQRPH$WODV +DLUSLQVDQG3ODVPLGV .QRFNGRZQ RI 6WJDOQDF ZDV SHUIRUPHG XVLQJ VKRUWKDLUSLQ 51$ ¶ 77&7*&7&&7&$&$&7*7*&$7&77*$&$¶ RU D FRQWURO KDLUSLQ WDUJHWLQJ ILUHIO\ OXFLIHUDVH LQ WKH S*)3956 YHFWRU 2ULJHQH 7* DQG 75 3ODVPLGV ZHUH SDFNDJHG LQWR UHWURYLUXVHV XVLQJ WKH 3KRHQL[(FR V\VWHP 9LUDO VXSHUQDWDQW ZDV ILOWHUHG XVLQJ P V\ULQJH ILOWHUV DQG FXOWXUHG ZLWK 0 FHOOV DW QR GLOXWLRQ 7UDQVGXFHG FHOOV ZHUH VHOHFWHGZLWKSXURP\FLQ,QYLWURJHQDQGNQRFNGRZQHIILFLHQF\ZDVGHWHUPLQHGE\T573&5 IROORZLQJ IRXU ZHHNV RI FXOWXUH 7DQVIHFWLRQ RI Gcnt3 ZDV SHUIRUPHG XVLQJ /LSRIHFWDPLQH Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. ,QYLWURJHQDFFRUGLQJWRWKHPDQXIDFWXUHU¶VLQVWUXFWLRQVZLWKSODVPLGVSXUFKDVHGIURP2ULJHQH 0& .QRFNGRZQ RI /JDOV ZDV SHUIRUPHG XVLQJ WZR VKRUWKDLUSLQ 51$V 6LJPD VK*DO ¶ &&***&$*7$&$$&&$7&**$7*$$&7&*$*77&$7&&*$7**77*7$&7*&77777 *¶ VK*DO ¶ &&**&&*&$7*&7*$7&$&$$7&$7&7&*$*$7*$77*7*$7&$*&$7*&**77777 *¶ RU D FRQWURO KDLUSLQ WDUJHWLQJ ILUHIO\ OXFLIHUDVH LQ 6LJPD 6+&9 $GGLWLRQDOO\ D VHFRQG KDLUSLQ V\VWHP ZDV XVHG WR NQRFNGRZQ St6galnac4 IURP 6LJPD VK6WJDOQDF &&***7$&$&&77&$&7*$$&*&$7&7&*$*$7*&*77&$*7*$$**7*7$&77777 * In vitro 3UROLIHUDWLRQ$VVD\V &HOOVH[SUHVVLQJKDLUSLQVDJDLQVWSt6galnac4RUFRQWUROKDLUSLQVZHUHVHHGHGLQWRZHOOSODWHV 1HZZHOOVZHUHWU\SVLQL]HGDQGFRXQWHGXVLQJDKHPRF\WRPHWHUZLWK7U\SDQ%OXHH[FOXVLRQDW KRXULQWHUYDOV Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. $XWKRUV¶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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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±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p(HWDO+,)Į,QGXFHVWKH5HFUXLWPHQWRI %RQH0DUURZ'HULYHG9DVFXODU0RGXODWRU\&HOOVWR5HJXODWH7XPRU$QJLRJHQHVLVDQG,QYDVLRQ&DQFHU &HOO Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. .LP 0< 2VNDUVVRQ 7 $FKDU\\D 6 1JX\HQ '; =KDQJ ;+) 1RUWRQ / HW DO 7XPRU 6HOI 6HHGLQJE\&LUFXODWLQJ&DQFHU&HOOV&HOO .DQJ < 6LHJHO 30 6KX : 'UREQMDN 0 .DNRQHQ 60 &RUGy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±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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited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±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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. )LJXUH/HJHQGV )LJXUHMice bearing tumors have accumulation of the adhesion ligand, galectin-3, in the early metastatic niche $ &HOO OLQHV DUH JHQHUDWHG IURP DXWRFKWKRQRXV WXPRUV LQ KrasLSLG12D/+ ;p53flox/flox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¶V 0XOWLSOH &RPSDULVRQ7HVWPP )LJXUHTumor-derived soluble factors induce the mobilization of galectin-3+ myeloid cells into peripheral circulation early in tumorigenesis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¶V0XOWLSOH&RPSDULVRQ7HVW PYDOXHVLQ*ZHUHGHWHUPLQHGE\7ZRZD\$129$ZLWK%RQIHUURQLSRVWWHVWP PP )LJXUH Secretion of IL-6 by tumors induces the rapid mobilization of CD11b+galectin-3+ leukocytes $ $QDO\VLV RI &'EJDOHFWLQ P\HORLG FHOO PRELOL]DWLRQ WR SHULSKHUDO EORRG IROORZLQJ LQMHFWLRQV RI FRQWURO PHGLXP ZKLWH 0 OLQH FRQGLWLRQHG PHGLXP UHG RU UHFRPELQDQW PXULQH JDOHFWLQ VXSSOHPHQWHG FRQWURO PHGLXP JUD\ % $QDO\VLV RI &'EJDOHFWLQP\HORLGFHOOPRELOL]DWLRQIROORZLQJLQMHFWLRQVRIFRQGLWLRQHGPHGLDIURP0 OLQHVFRQWDLQLQJHLWKHUDFRQWUROKDLUSLQVK/XFEODFNRUJDOHFWLQKDLUSLQVUHG &/XPLQH[ F\WRNLQH OHYHOV RI WKH FRQGLWLRQHG PHGLXP ' *HQH H[SUHVVLRQ PLFURDUUD\ DQDO\VLV RI Il6 H[SUHVVLRQ LQ DOO FHOO OLQHV IURP WKH IRXU FODVVHV ( IL6 H[KLELWV D JDLQ RI FRS\ QXPEHU LQ KXPDQOXQJDGHQRFDUFLQRPDVFRPSDUHGWRQRUPDOOXQJWLVVXHRUEORRGP îLQWKH ³/XQJ DGHQRFDUFLQRPD´ GDWD VHW DYDLODEOH IURP 7KH &DQFHU *HQRPH $WODV ZHEVLWH VHH 0HWKRGV)*$QDO\VLVRI&'EJDOHFWLQFHOOPRELOL]DWLRQWRSHULSKHUDOEORRGIROORZLQJ LQMHFWLRQV RI FRQWURO PHGLXP RU PHGLXP VXSSOHPHQWHG ZLWK UHFRPELQDQW PXULQH ,/ + 0RELOL]DWLRQ RI &'E OHXNRF\WHV LQ ZLOGW\SH DQG JDOHFWLQ NQRFNRXW Lgals3-/- PLFH IROORZLQJ,/LQMHFWLRQV(UURUEDUVLQ&DUHVHPµ1'¶QRWGHWHFWHGPYDOXHVLQ$DQG Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. +ZHUHFDOFXODWHGE\2QHZD\$129$ZLWK7XNH\¶V0XOWLSOH&RPSDULVRQ7HVWPYDOXHLQ *GHWHUPLQHGE\6WXGHQW¶VWWHVWPµQV¶QRWVLJQLILFDQW )LJXUH Elevated Thomsen-Friedenreich Antigen presentation promotes increased galectin-3 adhesion in metastatic populations $ 3RWHQWLDO FDUERK\GUDWH OLJDQGV IRU WKH JDOHFWLQ FDUERK\GUDWHUHFRJQLWLRQGRPDLQ&5'7KH7$QWLJHQ*DOȕ*DO1DFĮ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µ1¶QRQFDQFHURXVWLVVXHµ&¶ FDQFHUWLVVXH*31$ODEHOLQJRIKXPDQ16&/&FHOOOLQHVDVGHWHUPLQHGE\IORZF\WRPHWU\ PYDOXHV LQ ) GHWHUPLQHG E\ )LVKHU¶V ([DFW 7HVW 6FDOH EDUV LQ ( DUH P P IRU LQVHWV )LJXUH Metastatic cells downregulate *FQW and upregulate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¶VWWHVW7KHGDVKHG OLQH UHSUHVHQWV HTXLYDOHQW H[SUHVVLRQ EHWZHHQ WKH PHWDVWDWLF DQG SULPDU\ WXPRUGHULYHG OLQHV & T573&5 DQDO\VLV RI WUDQVIHUDVH JHQH H[SUHVVLRQ 7RS JHQH H[SUHVVLRQ RI Gcnt3 DQG St6galnac4 LQ UHSUHVHQWDWLYH 7QRQ0HW 70HW DQG 0 FHOO OLQHV %RWWRP &*Q7 Gcnt3 FDQ WUDQVIHUD*OF1$FWRWKHFRUH*DO1$FRI7$QWLJHQE\DȕOLQNDJH6WJDOQDFFDQWUDQVIHUD 1HX$FWRWKHFRUH*DO1$FRIWKHVLDO\O7$QWLJHQ'GCNT3H[KLELWVDORVVRIFRS\QXPEHU LQKXPDQOXQJDGHQRFDUFLQRPDVFRPSDUHGWRQRUPDOOXQJWLVVXHRUEORRGP îLQ WKH³/XQJDGHQRFDUFLQRPD´GDWDVHWDYDLODEOHIURP7KH&DQFHU*HQRPH$WODVZHEVLWHP YDOXHVLQ&ZHUHGHWHUPLQHGE\2QHZD\$129$ZLWK7XNH\¶V0XOWLSOH&RPSDULVRQ7HVW PPP )LJXUHKnockdown of 6WJDOQDF reduces galectin-3 binding and prevents metastasis LQYLYR $$QDO\VLVRIJDOHFWLQELQGLQJWRWKH0OLQH0IROORZLQJNQRFNGRZQRISt6galnac4E\ UHWURYLUDO WUDQVGXFWLRQ RI VKRUW KDLUSLQV WDUJHWLQJ St6galnac4 RU D FRQWURO JHQH /XF ILUHIO\ OXFLIHUDVH E\ IORZ F\WRPHWU\ () :LOGW\SH PLFH ZHUH WUDQVSODQWHG ZLWK *)3 0 FHOOV EHDULQJWKHVKRUWKDLUSLQWDUJHWLQJSt6galnac40VK6WJDOQDFRUFRQWUROKDLUSLQ0 Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. VK/XF E\ LQWUDVSOHQLF LQMHFWLRQ 7ZR ZHHNV IROORZLQJ LPSODQWDWLRQ WKH OLYHUV RI PLFH ZHUH H[FLVHG DQG LPDJHG % 5HSUHVHQWDWLYH OLYHUV DV YLVXDOL]HG E\ IOXRUHVFHQFH WRS EULJKWILHOG PLGGOH DQG KHPDWR[\OLQ DQG HRVLQ VWDLQLQJ ERWWRP & 4XDQWLILFDWLRQ RI WKH QXPEHU RI VXUIDFH QRGXOHV RQ WKH OLYHUV 6FDOH EDUV LQ % DUH PP PYDOXH LQ & ZDV GHWHUPLQHG E\ 0DQQ:KLWQH\WHVWP )LJXUHT-Antigen presentation promotes metastasis through interactions with galectins in the metastatic niche. 6FKHPDWLF PRGHO RI JDOHFWLQJO\FDQ LQWHUDFWLRQV LQ OXQJ FDQFHU PHWDVWDVLV 3ULPDU\ OXQJ WXPRUV KDYH KLJK H[SUHVVLRQ RI EUDQFKLQJ JO\FRV\OWUDQVIHUDVHV VXFK DV &*Q7 Gcnt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ownloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research. Author Manuscript Published OnlineFirst on November 24, 2014; DOI: 10.1158/2159-8290.CD-13-0760 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Aberrant glycosylation promotes lung cancer metastasis through adhesion to galectins in the metastatic niche Nathan E Reticker-Flynn and Sangeeta N. Bhatia Cancer Discovery Published OnlineFirst November 24, 2014. Updated version Supplementary Material Author Manuscript E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: doi:10.1158/2159-8290.CD-13-0760 Access the most recent supplemental material at: http://cancerdiscovery.aacrjournals.org/content/suppl/2014/11/22/2159-8290.CD-13-0760.DC1.html Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerdiscovery.aacrjournals.org on December 1, 2014. © 2014 American Association for Cancer Research.
© Copyright 2024 ExpyDoc