SUPPLEMENTARY INFORMATION
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1 SUPPLEMEARY IFORMAIO doi:./nture correction to Supplementry Informtion Adenom-linked rrier defects nd microil products drive IL-/IL-7-medited tumour growth Sergei I. Grivennikov, Kepeng Wng, Dniel Mucid, C. Andrew Stewrt, Bernd Schnl, Dominik Juch, Koji niguchi, Gunn-Yi Yu, Christoph H. Österreicher, Kenneth E. Hung, Christin Dtz, Ying Feng, Eric R. Feron, Mohmed Oukk, Lino essrollo, Vincenzo Coppol, Felix Yrovinsky, Hilde Cheroutre, Lrs Eckmnn, Giorgio rinchieri & Michel Krin ture doi:./nture (Pulished online Octoer ) In the version of the Supplementry Informtion originlly posted online, the scle rs in Supplementry Figs e, nd were incorrect. hese hve een corrected in the revised version of the Supplementry Informtion; see Supplementry Informtion le of Contents for detils.
2 SUPPLEMEARY IFORMAIO doi:./nture Supplementry le : List of primers used for Q-R-PCR. Humn Q-R-PCR Cludin -AACGCCAACCGCCCC- -AAAAGCCAGCCGAGCG- Cludin -GCAGCCAACACAGAGCC- -AGGGGCACCGAACGG- Cludin 7 -GCAAAAGACGACCGGG- -CACAAACAGGCCAGGAAG- IL-7A -ACCAACCCAAAAGGCCC- -CACGCCCCCAGACAC- IL- p9 -AGAAGCCGCACACGGC- -CCACACGGAAGGGGAAC- JAM-A -CCCCAAGGCGACC- -CCAGGGCAAGAAGGCAC- MUC -GACACCACACCCACCCG- -GAGGCACGCCCCA- Mouse Q-R-PCR Cludin -GGCGAAGGCAAGAGAGC- -CCGGAGGGAGCCCA- EBI -GAGAAGAGCCGGGAAGG- -CCAGCCGGGCGAG- IL- -ACCAGAGGAAACAAAGGC- -GAGCACGCAGAAAACA- IL- p -GAGGACGAAGAGACCAG- -CACGGGAGGAGGGC- IL- p -GACCCGCCCAGAACGGC- -CAACGGCACCAGGACG- IL-7A -GCCCCAGACACCCAACC- -ACACCCACCAGCACCC- IL-7F -AACCAGAACCGCCCAG- -GAGCAGCCGAGGC- IL- -CAGGAGGGGACCCCG- -CGGCGCACCGCGA- IL- p9 -CCAGCGGGACAAGAAC- -AGGCCCCCGAAGAG- IL-7 p -CGGCAAGGACAGGCGA- -CAGGGACAGGAGACCGG- JAM-C -CGCCGACCCCGC- -AGACCACGGGCGG- MUC -GAGCCGGGAACAGG- -AGCCCACCCCCAAAGAC-
3 RESEARCH SUPPLEMEARY IFORMAIO d X - X -... IL- p umour (size) CPC-APC CRC X - IL- p9 e IL- p X - W CRC IL-7 p c X - 9 f W X - IL- p9 K-Rs B-Rf ML EBI APC IEC umour IL- F/, IL--GFP GFP (IL-) CD Supplementry Figure. Expression of IL- fmily memers in norml nd tumour tissue of CPC-APC mice. -c: R-qPCR nlysis of cytokine mra expression in norml colon (), tumours () nd mesenteric lymph nodes (ML) of months old CPC-APC or W mice (, ) or in tumours of Apc IEC mice without or in comintion with Krs GD (K-Rs) or Brf VE (B-Rf) lleles, ctivted y intr-colonic dministrtion of Adenovirus-Cre into Apc F/F, Apc F/F /K-rs-LSL GD or Apc F/F / Brf VE mice (c). d,e: Immunostining for IL- (d) or IL--driven GFP in CRC, nd co-stining of F/ + cells (e). Arrows indicte positive cells. f: Flow cytometric nlysis of single cell suspensions isolted from mesenteric lymph nodes (ML) or colorectl tumours of Il -/- CPC- APC mice stined with nti-cd ntiody. Live/Ded - CD + re shown. Dt represent verges ± s.e.m., n= for nd, n= for c. p=. for IL-p in. Differences etween the different tumour models in c re not significnt. p<.. Scle rs, μm.
4 SUPPLEMEARY IFORMAIO RESEARCH umour numer APC x IL-R umour size (mm) umour lod c AU Apoptotic index +/+ -/- APC x IL- AU Prolifertion index +/+ -/- APC x IL- +/+ APC x IL- -/- d P-SA Cspse E-cdherin AU Ki7 +/+ -/- APC x IL- Supplementry Figure. IL- signling promotes colorectl tumour development nd growth. : Colonic tumours from. months old Ilr -/- /CPC-APC nd control CPC-APC mice were enumerted nd mesured. n=, p=.,.,., respectively.,c: Prffin sections of colons from Il -/- /CPC-APC or control CPC-APC mice were stined with ctive cspse nd E- cdherin ntiodies, UEL kit or Ki7 ntiody nd UEL-positive poptotic or Ki7 positive proliferting cells were quntified. n=, p=. for prolifertion index (c). d: Quntifiction of phospho-sa high cells in colonic tumours of Il -/- /CPC-APC or control CPC-APC mice (see Fig. g). fields from different sections of independent tumours were counted. p=.. Dt represent verges ± s.e.m. p<.. Scle rs, μm.
5 RESEARCH SUPPLEMEARY IFORMAIO IL- -/- BM W BM BM W IL- -/- c umer Size (mm) Lod d umer Size (mm) Lod e X IL- BM IL- IL- +/- IL- -/- X -.. IL-7A X - +/+ gfp/gfp IL-R-GFP BM IL-7F X - +/+ gfp/gfp +/+ gfp/gfp IL- Supplementry Figure. he pro-tumourigenic function of IL- nd IL-R is exerted within hemtopoietic cells. Six weeks old CPC-APC mice were lethlly irrdited ( x rd) nd trnsplnted with one mrrow from Il -/-, Ilr gfp/gfp or W donors nd nlyzed -. months lter.,: Representtive histology () nd gross ppernce of tumour-ering colons () from chimeric CPC-APC mice reconstituted with W or Il -/- one mrrow. c,d: Quntifiction of tumour development in chimeric CPC-APC mice reconstituted with Il -/- (c) or Ilr gfp/gfp (d) one mrrow. n=, p=.,. for size nd lod of Il -/- one mrrow trnsfer, nd.,. for Ilr GFP one mrrow trnsfer, respectively. e: R-qPCR nlysis of cytokine mras in tumours () nd djcent norml colon () of chimeric CPC-APC mice reconstituted with control or Il -/- one mrrow. n=, p=.,.x -,. nd., respectively. Dt represent verges ± s.e.m. p<.. Scle rs, μm.
6 SUPPLEMEARY IFORMAIO RESEARCH umer Size (mm) Lod APC x IL-7R +/- APC x IL-7R -/-... APC x IL-7R c % of ll tumours 7 Size distriution IL-7R +/- <mm -mm >mm IL-7R -/- Supplementry Figure. IL-7R signling promotes growth nd progression of colorectl tumours. umours from months old Il7r -/- /CPC-APC nd control mice were nlyzed. : umour numer, size nd lod. n=7, p=.,.,., respectively. : H&E stining of colon sections from CPC- APC mice of the indicted genotypes. c: Distriution of tumour sizes mong ll detectle tumours. n=7, p=. for tumours igger thn mm. Dt represent verges ± s.e.m. p<.. Scle rs, μm.
7 RESEARCH SUPPLEMEARY IFORMAIO Size (mm) Lod BM W LR,,9 -/- W LR,,9 -/- Supplementry Figure. Signling through LRs is importnt for CRC tumour growth. Six to seven weeks old CPC-APC mice were lethlly irrdited ( x rd) nd trnsplnted with one mrrow from lr,,9 -/- or W donors nd nlyzed. months lter. Quntifiction of tumour size nd lod in chimeric CPC-APC mice is presented. n=, p=.,.9 respectively. Dt represent verges ± s.e.m. p<..
8 SUPPLEMEARY IFORMAIO RESEARCH X - IL- p9 W IL-R -/- umer Size (mm) Lod ML +/+ -/- +/+ -/- +/+ -/- IL-R BM Supplementry Figure. IL-R signling in hemtopoietic cells is not required for IL- induction nd growth of colorectl tumours. Six weeks old CPC-APC mice were lethlly irrdited nd trnsplnted with Ilr -/- or W one mrrow nd scrificed for nlysis months lter. : Single cell suspensions from norml colons, tumours nd ML were prepred nd CD + cells were sorted y positive selection on mgnetic eds. IL-p9 mra expression ws mesured y R-qPCR. : umour numer, size nd lod were determined. n=. one of the differences (numer, size nd lod) re significnt. Dt represent verges ± s.e.m. 7
9 RESEARCH SUPPLEMEARY IFORMAIO X - JAM-A X - JAM-B Cludin- X - Cludin-7 Cludin- MUC X - X - X - X - JAM-C X - Cludin- X - MUC Supplementry Figure 7. Humn CRC nd mouse colorectl tumours disply dysregultion of mras encoding rrier nd junctionl proteins.,: otl RA ws isolted from frozen tumour () nd tumour-free () colon tissue of CRC ptients () or tumour ering CPC-APC mice () nd sujected to R-qPCR nlysis of indicted mras. Humn: n=7, p=.7,.,.,.,. for JAM-A, JAM-B, cludin-, cludin-7, nd Muc, respectively; Mouse: n=, p=.,. nd.7 for JAM-C, cludin- nd Muc, respectively. Dt represent verges ± s.e.m. p<..
10 SUPPLEMEARY IFORMAIO RESEARCH Cldn Cldn orml umour Cldn Cldn7 Cldn7 Cldn JAM-A JAM-A JAM-B JAM-B Supplementry Figure. Mouse colorectl tumours disply dysregultion of rrier nd junctionl proteins. Prffin-emedded colon sections from CPC-APC mice were stined with ntiodies to cludin, cludin 7, JAM-A or JAM-B nd nlyzed y fluorescent microscopy () or with cludin nd cludin specific ntiodies (). orml () nd umour () res re shown. Scle rs, μm. 9
11 RESEARCH SUPPLEMEARY IFORMAIO IL- p9 IL-7A X - X - X - IL- p9 Con APC X -... MUC Con APC Supplementry Figure 9. Erly humn denoms nd mouse rpidly trnsformed colons disply upregultion of cytokines nd dysregultion of rrier proteins. otl RA ws isolted from frozen humn denom tissue removed during colonoscopy () nd mtching tumour-free () colon tissue () or from Cdx ER-Cre x Apc F/F nd control mice injected times with tmoxifen nd left for dys for rpid colonic trnsformtion (). RA ws sujected to R-qPCR nlysis of the indicted trnscripts. n=, p=.,. for IL-p9 nd IL-7A in. n=, p=.,. for IL-p9 nd MUC in. Dt represent verges ± s.e.m. p<..
12 SUPPLEMEARY IFORMAIO RESEARCH Microil Products Brrier disruption Altered differentition MyD h7-like response IL- IL-7 IL- umour elicited Inflmmtion Myeloid cells SA? SA F-κB orml mucos -ctenin APC prolifertion Adenom K-Rs B-Rf prolifertion Advnced denom p Crcinom Supplementry Figure. Scheme: umour elicited inflmmtion is triggered y the clssicl genetic pthwy tht drives colorectl tumourigenesis nd cn further stimulte tumour progression. Inctivtion of tumour suppressor APC is the first step in the clssicl genetic pthwy of colorectl tumourigenesis, leding to persistent ctivtion of -ctenin signling. We propose tht -ctenin signling in erly neoplstic lesions induces ltered tissue homeostsis nd disruption of the epithelil rrier, including downregultion of mucin expression due to filed golet cell differentition nd disruption of tight junctions. umour-specific deteriortion of rrier function llows commensl microflor nd microil products to penetrte the denoms, ctivte LR-MyD signling nd induce IL- in tumour ssocited mcrophges (AM). IL- regultes tumour elicited inflmmtion nd the h7-like response, prticulrly its effector cytokines tht include IL-7A nd IL-, to ctivte trnscription fctor SA, which together with -ctenin enhnces the prolifertion nd growth of denom cells. umour elicited inflmmtion further contriutes to tumour progression nd mlignnt conversion y multiple mechnisms, some of which mny lso involve SA ctivtion. umour promoting events re in lue, tumour suppressors re in red.
13 RESEARCH SUPPLEMEARY IFORMAIO c Supplementry Figure. Genertion of Ilr F/F nd Ilr -/- mice. : rgeting strtegy. wo LoxP sites were introduced into introns nd of the Ilr gene, such tht Cre-medited deletion results in excision of exon nd exon, which encode the lignd-inding extrcellulr portion of the receptor. he trgeting construct ws introduced into Bruce C7Bl ES cells nd homologous integrnts were used to generte Ilr F/F mice. : Southern lot screening of Bruce ES clones. Representtive clones re shown. c: Selected clones were injected into C7BL/- Alino lstocysts nd trnsferred to pseudopregnnt femles. Chimeric offspring were red with C7BL/-Alino prtners to generte F mice tht were screened for integrtion y Southern nlysis (for more detils see Methods). Resulting mice with the Ilr SA-flox-neo llele were crossed with C7BL/-FLPe mice to fcilitte recomintion etween Frt sequences nd generte the Ilr flox llele. he null llele ws generted y crossing Ilr F/F mice with CPC-Cre trnsgenic mice (for more detils see Methods). Complete ltion of IL-R ws confirmed y Q-R-PCR of spleen nd tumour-derived cells.
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{ OI: 1.138/n31 Srifie n nlyze APs on week 1 s of iet 1 4 6 High-ft iet BrU High-ft iet BrU 4 High-ft iet BrU 6 High-ft iet BrU Lin - Lin - : C34 + : C9 + 1 1 3 1 4 1 5 C45 1 C34 1 1 1 1 3 1 4 1 5 S-1
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