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Published OnlineFirst October 19, 2010; DOI: 10.1158/0008-5472.CAN-10-1506

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cular and Cellular Pathobiology

s Mutation Targeted to Gastric Tissue Progenitor Cellsults inChronic Inflammation, anAlteredMicroenvironment,

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Progression to Intraepithelial Neoplasia

yuki Okumura1, Russell E. Ericksen1, Shigeo Takaishi1, Sophie S.W. Wang1, Zinaida Dubeykovskiy1,
u Shibata1, Kelly S. Betz1, Sureshkuma Muthupalani2, Arlin B. Rogers3, James G. Fox2, . Rustgi4, and Timothy C. Wang1
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onic infectious diseases, such as Helicobacter pylori infection, can promote cancer in a large part throughion of chronic inflammation. Oncogenic K-ras mutation in epithelial cells activates inflammatory path-which could compensate for a lack of infectious stimulus. Gastric histopathology and putative progenitorrs [doublecortin and calcium/calmodulin-dependent protein kinase-like 1 (Dcamkl1) and keratin 19 (K19)]-K-ras-V12 (K19-kras) transgenic mice were assessed at 3, 6, 12, and 18 months of age, in comparison withbacter felis–infected wild-type littermates. Inflammation was evaluated by reverse transcription–PCR oflammatory cytokines, and K19-kras mice were transplanted with green fluorescent protein (GFP)–labeledarrow. Both H. felis infection and K-ras mutation induced upregulation of proinflammatory cytokines,

sion of Dcamkl1+ cells, and progression to oxyntic atrophy, metaplasia, hyperplasia, and high-grade dyspla-9-kras transgenic mice uniquely displayedmucousmetaplasia as early as 3months and progressed to high-dysplasia and invasive intramucosal carcinoma by 20months. In bonemarrow–transplanted K19-krasmicerogressed to dysplasia, a large proportion of stromal cells were GFP+ and bone marrow–derived, but onlyFP+ epithelial cells were observed. GFP+ bonemarrow–derived cells included leukocytes andCD45− stromalat expressed vimentin or α smooth muscle actin and were often found surrounding clusters of Dcamkl1+
cells th
cells at the base of gastric glands. In conclusion, the expression of mutant K-ras in K19+ gastric epithelial cells caninduce chronic inflammation and promote the development of dysplasia. Cancer Res; 70(21); 8435–45. ©2010 AACR.

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cobacter-related gastric cancer arises through a multi-rogression that involves the conversion of normal gas-ithelium to regions of hyperplasia, dysplasia, and,, invasive adenocarcinoma. This multistep process isated with the accumulation of genetic and/or epigenet-rations in the target cell population that leads to theirant transformation. Helicobacter infection contributespathogenesis of gastric cancer in a large part throughduction of chronic inflammation (1, 2), which can in-roliferation and thus opportunity for mitotic error. In

bacter infection can promote the develop-through mobilization and recruitment of

(8). Eximmu

ions: 1Division of Digestive and Liver Diseases,y Medical School, New York, New York; 2Division ofdicine, Massachusetts Institute of Technology,sachusetts; 3Lineberger Comprehensive Cancerof North Carolina at Chapel Hill, Chapel Hill, Northpartments of Medicine and Genetics, University ofcal School, Philadelphia, Pennsylvania

tary data for this article are available at Cancerttp://cancerres.aacrjournals.org/).

on: T. Okumura: Conception and design, experiment,. R.E. Ericksen: Conception, experiment, manuscript: Conception, experiment. S.S.W. Wang: Conception,

experimW. ShiS. Mutnosis. Jof stud

T. Okum

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Research. on August 30, 20cancerres.aacrjournals.org ded from

marrow–derived cells (BMDC) that contribute to can-velopment in diverse ways (3–5).ever, whereas chronic inflammation can likely initiateromote oncogenic mutation and transformation, accu-ing data suggest that, in many cases, the converse is alsond that oncogenic mutations can also lead to the devel-t of chronic inflammation (6). Cytokines and solubletors produced by oncogene mutations in cancer cells re-nd activate inflammatory cells to develop a tumor micro-nment, which further stimulates tumor progression (6, 7).oncogene that has been strongly linked to the devel-t of chronic inflammation has been the Ras oncogene
pression of a mutant K-ras oncogene induces strongne responses through activation of cytokines and
ent. Z. Dubeykovskiy: Conception, provision of study materials.bata: Conception, experiment. K.S. Betz: Conception, experiment.hupalani: Pathological diagnosis. A.B. Rogers: Pathological diag-.G. Fox: Pathological diagnosis. A.K. Rustgi: Conception, provisionymaterials. T.C. Wang: Conception and design, manuscript writing.

ura and R.E. Ericksen contributed equally to this work.

ponding Author: Timothy C. Wang, 1130 St. Nicholas Avenue,925, Columbia University Medical Center, New York, NY 10032.212-851-4581; Fax: 212-851-4590; E-mail: [email protected].

.1158/0008-5472.CAN-10-1506

American Association for Cancer Research.

8435

21. © 2010 American Association for Cancer

http://cancerres.aacrjournals.org/

T cellcancernificancanceroncogresultsfiltratithat mmokintransfdent oIn a

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s (9–11). In transgenic mice modeling pancreatic, based in part on K-ras mutations in PDX1+ cells, sig-t inflammatory and stromal responses correlate withprogression (12). Expression of the mutant K-ras-V12

ene in keratin 19 (K19)–expressing pancreatic cells alsoin pancreatic ductal hyperplasia with lymphocytic in-on (13). Studies from Sparmann and Bar-Sagi showedany of the key downstream targets of K-ras were che-es and cytokines (10, 14). Further studies showed thatormed cell survival and tumor progression are depen-n these induced cytokines (15).previous report, K-ras-V12 was expressed in theepithelium using the K19 promoter and resulted ins metaplasia and parietal cell loss (13). However, ad investigation of the inflammatory responses and de-ment of dysplasia in this model has not yet beened. K19 is a member of the family of intermediate fila-that is highly expressed in the proliferative zone of theintestinal tract (13, 16–18). In the stomach, K19 is ex-d in the neck/isthmus region of the glandular unit, inme area where stem/progenitor cells reside (13, 16). In, induction of cancer is most likely to occur when onco-mutations accumulate in long-lived stem or progenitorWhereas an authentic stem or progenitor cell for thec glands of the stomach has not yet been identified,ndidate progenitor marker that has emerged is double-and calcium/calmodulin-dependent protein kinase-likemkl1), a gene shown to be upregulated in an enrichedepithelial progenitor cell population from the murine

s (19). Dcamkl1+ cells reside in the neck/isthmus regionstomach glands and do not express biomarkers associ-ith differentiated lineages (19, 20).aim of the current study was to compare the effects

tant K-ras expression to chronic Helicobacter felis infec-n the gastric epithelium. We show that mutant K-rasbstitute for Helicobacter infection in the induction of
c inflammation, leading to expansion of putative gastric
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rials and Methods

K19-K-ras-V12 (K19-kras) transgenic mice were gener-reviously (13), using a 2.1-kb genomic fragment of theking region/promoter of the mouse K19 gene linked toll-length mutant human K-ras (Val 12) gene. K19-krasenic mice were backcrossed to C57/BL6 mice for at leastenerations under the Columbia University Institutionall Care and Use Committee. Chicken β-actin enhancedfluorescent protein (EGFP) transgenic mice (8–10 weeksere purchased from The Jackson Laboratory.

s infection, histopathology, andnohistochemistryelis infection was carried out by gavage as previously
bed with H. felis (American Type Culture Collection; ref. 4). Stomachs were fixed in 10% neutral buffered
muno(1:300

r Res; 70(21) November 1, 2010

Research. on August 30, 20cancerres.aacrjournals.org Downloaded from

ldehyde, and routine H&E sections were reviewedard-certified veterinary pathologists and gradeding to previously described criteria (21, 22). Immuno-hemistry was performed on 4-μm sections with avidin--peroxidase complex kits (Vector Laboratories) anderstained with Mayer's hematoxylin. For primary anti-, mouse anti-hydrogen/potassium ATPase (H/K-ATPase)inity BioReagents), rabbit anti-Ki67 (Abcam), rabbit anti-bcam), rabbit anti-Dcamkl1 (Abagent), rabbit anti--TFF2 (established in our laboratory), rabbit anti-TFF1m), and mouse anti-Muc5AC (Abcam) or rabbit anti-nvitrogen) were used.

nd quantitative real-time-PCR analysisl-time PCR reactions with QuantiTect SYBR green PCRIAGEN) and primers (Supplementary Table S1) werean ABI Prism 7300 (Applied Biosystems). mRNA quan-were analyzed in duplicate and normalized againstaldehyde-3-phosphate dehydrogenase as an internall. Results are expressed as relative gene expression us-e ΔΔCt method.

marrow transplantatione marrow transplantation was carried out as previouslybed with 5 million cells per animal (3). Mice were eutha-at 3, 6, and 12 months posttransplantation.

cytometry and mesenchymal stem cell cultureshly isolated bone marrow cells were processed for flowetry as previously described (4, 6) and after incubationhycoerythrin (PE)–conjugated antimouse CD45 anti-BD Pharmingen) or PE-conjugated rat IgG2a antibodye control; Jackson ImmunoResearch) and 4′,6-diamidino-ylindole (DAPI) were analyzed using BD LSRII (Becton,son). Mesenchymal stem cells (MSC) were cultured asusly described (23).

omosome and K19 in situ hybridizationitu hybridization for Y chromosome or K19 were per-d using a Texas Red–labeled Y-chromosome paintFISH, Cambio) or a DIG-labeled antisense riboprobee) following the manufacturer's protocols, as previouslybed (24).

nofluorescenceunofluorescence was performed following in vitro

n-perfusionwith 4%paraformaldehyde followed by freez-d embedding in OCT, frozen 4-μm sections were pre-and blocked with 1% fetal bovine serum (FBS) in PBSour at room temperature, and then rat anti-E-cadherindy (Zymed), rat anti-CD45 (eBioscience), rabbit anti-αhmuscle actin (αSMA; Abcam), rabbit anti-K19 (Abcam),anti-Dcamkl1 (Abagent), and mouse anti-vimentin

m), diluted in PBS with 1% FBS (1:100 dilution), wered. Following overnight incubation at 4°C, Texas Red–ated antirat, rabbit, ormouse IgG antibodies (Jackson Im-
Research) were applied, respectively, with 1% FBS in PBSdilution) and incubated for 1 hour at room temperature.
Cancer Research



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K-ras Mutation in Gastric Progenitors Induces Neoplasia

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i were stained with DAPI and mounted using Vecta-(Vector Laboratories) for microscopy.

tical analysisistical analysis was performed using Student's t test,values of <0.05 were considered significant. Data aresed as mean ± SEM.

lts

ras mice developed gastric hyperplasia andogic inflammationassess the effects that the 2.1-kb K19 promoter-driven

3, 6, and 12 mo after H. felis infection (*, P < 0.01; n = 3 for each group)

t K-ras-V12 oncogene has on the induction of chronicmation, we first investigated the gross appearance,

sectiolished

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histopathology, and inflammatory responses in K19-ransgenic mice compared with H. felis–infected wild-WT) mice over time. Colonization of H. felis organismsoric and fundic glands of WT mice was confirmed bynation of H&E-stained sections and PCR amplificationfelis–specific DNA sequence (Supplementary Fig. S1).tomach weight and the ratio of wet stomach weighty weight of K19-kras mice were significantly higherhose of control mice, regardless of H. felis infectionA). This was consistent with the gross appearance ofomach, where severe mucosal thickness and enlargedere consistently observed in K19-kras mice (Fig. 1B).

topathologic scoring of H&E-stained gastric tissue

1. Gastric histology of WT and K19-kras mice. A–C, from mice in 12-mo cohort: body and stomach weights (A), representative gross presentation (B),E-stained and Alcian blue-stained gastric tissue sections (C). Representative Alcian blue+ cells in WT H. felis–infected mice are indicated bymagnified insets are of boxed regions (original magnification, 100× and 150×, respectively). D, histopathology scores for gastric lesions of the

ns from mice in each group using previously pub-criteria (21, 22) showed that H. felis–infected WT

Cancer Res; 70(21) November 1, 2010 8437



mice eatrophprogremodeInflamof WToped oand dthe enterizeblue swas obin WTwith tshowelymphthoseFig. S2cells isthe ga

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Okumura et al.

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xhibited much greater chronic active gastritis, oxynticy, intestinal metaplasia, hyperplasia, and dysplasia thatssed over time (Fig. 1C), consistent with the multistagel of Helicobacter-related gastric carcinogenesis (22).mation scores in K19-kras mice were similar to thosemice with H. felis infection. K19-kras mice also devel-xyntic atrophy, pseudopyloric metaplasia, hyperplasia,ysplasia by 3 months, and the lesions persisted untild of the study (Fig. 1D). Mucous metaplasia, charac-d by replacement of parietal cells with foamy Alciantaining H/K-ATPase− cells without glandular dysplasia,served in the oxyntic mucosa of K19-kras mice, but notmice, even with H. felis infection (Fig. 1C), consistenthe previous reports (13, 22). The K19-kras mice alsod a marked lymphocytic infiltrate with hyperplasticoid follicles, similar in appearance and frequency tofound in H. felis–infected WT mice (SupplementaryA). These results show that K-ras mutation in epithelialsufficient to induce chronic inflammatory responses instric epithelium.

enic K-ras mutation in gastric epithelial cellsulates cytokine and chemokine expressionfinding of chronic inflammation in K19-kras mice sug-that K-ras mutation in gastric epithelial cells triggeredtivation of specific immune responses that may be sim-H. felis–related chronic gastritis. Reverse transcrip-CR (RT-PCR) analysis of mice 6 to 12 months of age
ed increased gastric mRNA expression of interleukin-) and IL-1β in both H. felis–infected WT and K19-kras
lesiontory e

2. Expression of chemokines/cytokines/growth factors. A, B, and D, quantitativeeach group). C, relationship between dysplasia score and mRNA expression of



ompared with WT uninfected controls (Fig. 2A). Addi-ly, CXCL1 was upregulated in both these groups,gh the expression of CXCL5 was upregulated only inras mice (Fig. 2B). Interestingly, CXCL1 and IL-6 ex-on retained the highest degree of correlation with dys-scores in K19-kras mice (Fig. 2C). Similar trends wereed for IL-1β and CXCL5, albeit with smaller R2 values.were no differences among the three cohorts in thesion of other detected cytokines and chemokines, in-g IFNγ, tumor necrosis factor α, CCL2, CCL3, and2 (data not shown). In younger (3 months) mice, expan-f pit cells was moderate (Supplementary Fig. S2B).quently, at this point, the aforementioned inflammato-okines were minimally elevated, whereas the chemo-XCL1 was significantly upregulated (SupplementaryB), supporting an important role for CXCL1 in pheno-development. Although K-ras is known to inducexygenase-2 (COX-2) expression, which can drive tu-enesis (25), we were unable to detect any upregulationX-2 expression in younger mice (≤12 months); in con-expression was nearly 3-fold higher in K19-kras versusice over 12 months of age, correlating with progressionplasia (Supplementary Fig. S3).ression of growth factors heparin-binding epidermalh factor (HB-EGF) and amphiregulin was significantlyulated in the stomachs of K19-kras mice when com-with WT mice, regardless of H. felis infection (Fig. 2D).results suggest that progression of gastric preneoplastic
s in K19-kras mice occurs in the setting of an inflamma-nvironment similar to that found in H. felis–dependent
RT-PCR analysis of 12-mo cohort (*, P < 0.1; §, P < 0.05;CXCL1 and IL-6 in K19-kras mice at 12 mo.

Cancer Research



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c gastritis, characterized by expression of CXCL1, IL-1β,-6. However, the unique phenotype of the K19-krasis associated with the expression of additional inflam-y chemokines and growth factors that are relativelyic to the K-ras-V12 mutation, which include CXCL5,F, and amphiregulin.

kl1+ putative tissue stem/progenitor cells areded in the preneoplastic gastric lesions ofras miceprogression to metaplasia and dysplasia in the sto-of K19-kras mice was associated with changes in

lial differentiation and proliferation. In the stomachmal WT mice, K19 staining was observed primarilycells, which comprised the upper one sixth of the gas-ands (Fig. 3A). Ki67+ proliferating cells reside in thes and upper neck region (Fig. 3C), in close proximitye Dcamkl1+ cells found in this region (Fig. 3D).ghout and below the neck region are H/K-ATPase+ pa-cells, which make up the majority of glands (Fig. 3B).gastric epithelium of K19-kras mice at 12 months of
splayed obvious parietal cell loss, with only a smaller of H/K-ATPase+ cells remaining at the base of the
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acrjournals.org


c glands (Fig. 3F). Excluding these rare parietal cells,ority of the remaining cells were mucus-producingpit cells (Fig. 3E), which had expanded to comprisetwo thirds of the entire gland, consistent with previ-ports (13). Expression of TFF1 and Muc5AC in the K19-ouse stomach indicated that the majority of K19+ cellsupper middle region of glands were expanded surfacells (Supplementary Fig. S4A and B). Ki67+ proliferatinghifted over time from the neck region to the bottomtric glands, slightly above the remaining parietal cells.stingly, the number of Dcamkl1+ cells increased mark-many cells in each glandular unit, located just belowoliferative zone and just above the residual parietalFig. 3G and H). It should be noted that Dcamkl1 isy a putative progenitor marker based on its homeo-location and frequency throughout the gastric epithe-gr5 can give rise to gastric antral glands in the murinebut Lgr5 is not expressed in the adult gastric corpuse confirmed that Lgr5-EGFP is expressed at the basetric antral glands, whereas Dcamkl1+ cells residey above, never colocalizing (Supplementary Fig. S4C).

Figure 3. Immunohistochemicalstaining in gastric epithelium ofuninfected WT and K19-kras mice at12 mo. Expression of K19 (A and E),H/K ATPase (B and F), Ki67 (C and G),and Dcamkl1 (D and H) in the gastricepithelium of WT (A–D) and K19-kras(E–H) mice (arrows indicate Dcamkl1+

cells; original magnification, 150×).

fore, these results suggest that the Dcamkl1+ putativelial progenitor pool expands independent of Lgr5




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Okumura et al.

Cance8440


sion, under inflammatory responses induced by mutantexpression.

ras mice develop high-grade dysplasia bynths of agefollowed a cohort of aged mice, combining data fromhat survived 16 to 21 months of age, to investigate pos-progression of the gastric preneoplastic lesions. Ased, K19-kras mice and H. felis–infected WT mice con-to exhibit higher inflammation scores when comparedT mice without H. felis infection, becoming progres-

more severe with longer observation periods (Supple-ry Fig. S5; see also Fig. 1D). Histology scores forsia were also elevated after a long-term observation,allel with the severity of inflammation (Fig. 4A; see also). Among these mice, three of four (75.0%) H. felis-d WT mice and three of eight (37.5%) K19-kras miceped gross papillary tumors in the stomach (Fig. 4B).sions of three K19-kras mice were histologically de-as high-grade dysplasia or intramucosal carcinoma,ome areas invading into submucosal layers (Fig. 4C).unohistochemistry on serial tissue sections revealede lower portions of dysplastic glands contained a mix-f TFF2+ and Dcamkl1+ cells (outlined in red in Fig. 4D;mentary Figs. S6B and C and S7B and D). More apicals of these glands typically contained strongly stainingcells, consistent with a surface epithelial phenotype
lementary Figs. S6A and S7C). K19 expression wasally strong in glands with squamous metaplasia (Sup-
proteiin par

cosal carcinoma (original magnification, 200×). D, expression of Dcamkl1, K19, Ks mouse (original magnification, 300×).



ntary Fig. S6A, bottom right). Infrequently, weaklyng K19+ regions also contained expanded Dcamkl1+

outlined in black with arrowheads and as magnified in-ig. 4D; arrowheads in Supplementary Fig. S7A and B).ver, TFF2 and K19 expression were clearly distinct fromkl1, and colocalization of these markers was never ob-in these serial sections or double immunofluorescencelementary Fig. S7E). It appears that K19 mRNA issed early on as the pit cell migrates upward throughand and before K19 protein expression. Therefore,ikely that K-ras is expressed in a few cells that areactive to the K19 antibody. Whereas the transgene-d mutant human K-ras could not be directly detectedhigh homology with murine K-ras, we observed thatRNA expression as assessed by in situ hybridization ist in a few cells below K19 protein+ cells, but distinctlythe region where Dcamkl1 is typically expressed (Sup-ntary Fig. S7F).ilar immunohistochemical observations were made ins of high-grade dysplasia, where Dcamkl1+ cells wereded but never coexpressed as TFF2 or K19 (arrows4D). Proliferating Ki67+ cells typically resided betweenorementioned cell types, although they were occasion-ound to be TFF2+ (Fig. 4D). Although we cannotetely exclude the possibility that the observed expan-f Dcamkl1+ cells is due to the expression of K-ras incells, the lack of colocalization with K19 mRNA and
n would suggest that it is more likely due to alterationsacrine signals and/or their niche.
4. Aged K19-kras mice develop high-grade dysplasia. A, histopathology scores for dysplasia in gastric lesions of mice between 16 and 19 mo.s presentation of the stomachs with papillary tumors. Stomachs from a WT mouse at 19 mo with H. felis infection (7445) and K19-kras mice at 16 to(9003 and 7456, respectively). C, H&E-stained gastric tissue section of a K19-kras mouse at 16 mo displaying submucosal penetration of

i67, and TFF2 in low-grade and high-grade dysplasia of a

Cancer Research



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ras transgenic mice display recruitment ofs to the tumor microenvironmentnvestigate the role of mutated K-ras in the recruitmentDCs to the tumor microenvironment, we performedmarrow transplantation (BMT) studies. Bone marrowdonor chicken β-actin-EGFP mice (WT/GFP) waslanted into K19-kras or uninfected WT recipients. Sixs after BMT, half of the bone marrow cells from recip-ice expressed GFP, similar to donor mice, with mostGFP+ cells CD45+ hematopoietic cells (SupplementaryA). Successful engraftment was confirmed by demon-n of GFP expression by MSC cultures (Supplementary8B), quantitative detection of transgene DNA se-es in BMT recipient mice (Supplementary Fig. S8C),etection of GFP+ cells in MSC cultures establishedperipheral blood of all BMT recipient K19-kras micelementary Fig. S8B). Twelve to 18 months after BMT,tissues from K19-kras BMT recipient mice displayed

e phenotypic changes of nontransplanted K19-krasincluding mucosal thickness, histologic pathologies,f the Ki67+ proliferation zone, and dramatic expansionmkl1+ cells (Supplementary Fig. S8B and D).he BMT recipient mice, only rare GFP+ cells were ob-in the gastric mucosa of uninfected WT mice, whereasas mice had a >10-fold increase in the number of re-d cells (Fig. 5A–C). E-cadherin, an epithelial-specific

histochemistry (original magnification, 200×). F, Y-FISH (red) in a gastricl magnification, 400×).

rface molecule, was detected in all epithelial cells ofstric epithelium, including dysplastic glands. More de-

Toperfor

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analysis of the glandular areas with confocal microsco-ealed that, although a small number of GFP+ cells wereerin+ epithelial cells, a majority were E-cadherin− stro-lls (Fig. 5D).expression of GFP was confirmed by immunohisto-

ical staining for GFP (Fig. 5E). In addition, becauseas present in only half of bone marrow cells (Supple-ry Fig. S8A), we performed Y-chromosome in situization (Y-FISH) on sections prepared from female re-ts that had received male donor bone marrow. Gastricns showed many Y-chromosome+ cells in lymphoid fol-(Supplementary Fig. S8E) but no Y chromosome+ cellsgastric glands (Fig. 5F). These findings suggest that thepressing dysplastic gastric epithelium does not derivehe engrafted BMDCs.further characterize the GFP+ BMDCs, we performednofluorescent staining against CD45, vimentin, and. In all gastric sections, we detected colocalization ofith CD45 or spindloid cells positive for vimentinr αSMA, indicating the BMDCs had differentiatedrily into leukocytes, fibroblasts, and myofibroblasts,tively (Fig. 6A). By 12 months, an estimated 15% ofcruited GFP+ BMDCs in the gastric epithelium of WTith H. felis and K19-kras mice were myofibroblasts, as

cterized by their morphologic appearance and coex-on of αSMA (Supplementary Fig. S9).

section of a K19-kras female receiving male donor BMT

5. Detection of BMDCs in the gastric epithelium of recipient mice. Endogenous GFP fluorescence of BMDCs in gastric tissue of WT (A) ors (B) BMT recipients (original magnification, 150×), quantified in C (*, P < 0.01; n = 3 per group). D, representative E-cadherin (red) and GFP (green)ic tissue of K19-kras BMT recipients (original magnification, 200×). Magnified inset of a representative double positive cell (bone marrow–derivedal cell) and GFP single positive cells (bone marrow–derived stromal cells). E, representative GFP in K19-kras BMT recipients detected by

further understand the expansion of Dcamkl1+ cells, wemed staining against GFP, αSMA, and F4/80 in K19-kras




BMT rroundstromwere tK-rasan alte

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In tK19-eactivaphy, mparallchroncontricorrelitor ceintramK19-krin themodelthrouggether

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FigureBMDCs and stained red for CD45 (left), vimentin (middle), and αSMA (right) and (B) endogenous GFP (left, from a BMT recipient) or stained forF4/80 (middle, green) or αSMA (right, green) and stained for Dcamkl1 (red; original magnification, 400×).

Okumura et al.

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ecipient mice. A plethora of GFP+ cells were found sur-ing most clusters of Dcamkl1+ cells. Excessive αSMA+

al cells, as well as inflammatory F4/80+ macrophages,ypically seen surrounding these clusters, indicating that
expression in adjacent putative progenitor cells favored oncog
(11, 1reportrepormetapthe cupure iPre

in micoverexesis incancelikelymorsprolifecanceinflammutatwith i

red, inflammatory microenvironment (Fig. 6B).

ssion

his study, we show that oncogenic K-ras mutation inxpressing putative gastric epithelial progenitor cellstes inflammatory pathways and induces gastric atro-etaplasia, and dysplasia in a manner that largely

els H. felis–induced carcinogenesis. K-ras–dependentic inflammation leads to recruitment of BMDCs thatbute primarily to the stromal microenvironment andates with the expansion of Dcamkl1+ putative progen-lls and development of high-grade dysplasia and rarelyucosal carcinoma. Interestingly, H. felis infection ofas mice did not accelerate gastric cancer progressionse mice (data not shown), suggesting that the twos (K-ras mutation and H. felis infection) may progress
h overlapping and/or nonadditive pathways. Taken to-, K-ras mutation is able to compensate for a lack of
modethat a



ious stimulus, such as H. felis infection, to inducemation and carcinogenesis.development of dysplasia in the K19-kras mouse wastirely surprising, given the known role of K-ras as anene for the pancreas and other organs in the mouse2, 27) and the fact that K-ras mutations have beened rarely in gastric cancer (28, 29). Although previousts have noted only gastric hyperplasia and mucouslasia in K19-kras mice up to 18 months of age (13),rrent study is the first to examine the phenotype in anbred (C57BL/6) background.vious studies by our group have modeled gastric cancere using chronic Helicobacter infection or transgenicpression of cytokines that can drive gastric carcinogen-dependent of Helicobacter infection (4). Indeed, manyrs arise in the setting of chronic inflammation, whichplays a role in both the initiation and promotion of tu-(30) and can lead to oncogenic mutations by inducingration and increasing the mutation rate (31). However,rs are also thought to arise in the absence of chronicmation through spontaneous or carcinogen-inducedions in key oncogenes and tumor suppressor genes,nflammation developing later on (32). In this latter

6. Characterization of BMDCs in BMT recipient mice. Gastric tissue sections from K19-kras BMT recipients detecting (A) endogenous GFP of

l, transformed cells produce inflammatory mediatorsttract tumor promoting inflammatory cells and set up

Cancer Research



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numbcellulation, wanothgenes(9–11)in K19motechronmiceIL-1β,ic Raswhichtumorthe exenhanCXCL1is a knrecruilatestumoris assolular ccruitmmoststudiegastricK-r

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ne networks. Accordingly, a number of studies have alsothat specific oncogenic mutations lead to a chronicmatory state (6, 7).ogenic K-ras mutation in epithelial cells induces aer of molecular pathways that intrinsically regulater processes such as proliferation, survival, and migra-hich contribute to tumorigenesis (1, 12, 33). However,er consequence of oncogenic Ras signaling in carcino-is is the upregulation of cytokines and chemokines. Indeed, most of the cytokine/chemokines elevated-kras mouse in this study have been reported to pro-tumor development (4, 10, 34, 35). The development ofic inflammation in the stomachs of K19-kras transgenicwas associated with significant increases in CXCL1,and IL-6. It has previously been reported that oncogen-mutations are able to induce the secretion of IL-6,acts in a paracrine and autocrine fashion to promotegrowth (34). K-ras has also been shown to upregulatepression of IL-1β in epithelial cells, propagating andcing inflammatory networks (36). The chemokineis overexpressed in human gastric cancers (37) andown critical downstream mediator of mutant Ras thatts neutrophils (38), supports angiogenesis (39), regu-the microenvironment (35), and directly promotesigenesis (39). Moreover, CXCL1 (also known as Gro1)ciated with other epithelial cancers such as hepatocel-arcinoma, even in the absence of overt neutrophil re-ent (40). The induction of dysplasia by K-ras correlatedtightly in our study with increased CXCL1, but furthers will be required to understand the role of CXCL1 incancer progression.

as is known to induce COX-2 expression largelyh upregulation of COX-2 mRNA stability (41), andenotype of K19-kras mice shows striking similaritiese that overexpress COX-2–derived prostaglandins inells (25); notably, both K19-kras mice and K19-C2mEexhibit expansion of mucous-producing cells andal gastric tumor development. Although we were un-o detect increased COX-2 expression before thepment of moderate dysplasia, it is interesting tolate that it may have a role in phenotype deve-nt. Furthermore, we noted lymphoid aggregates inras mice similar to those found in H. felis–infectedWhereas the antigen responsible for this adaptivene response is unknown, it is possible that mutantpeptides may induce the antigenic response, as pre-y suggested (42, 43), and contribute to phenotypepment.itional inflammatory chemokines and growth factors,s CXCL5, HB-EGF, and amphiregulin, were expressedely specific to the K19-kras mice compared withs–dependent chronic gastritis. CXCL5 is an additionalt neutrophil chemoattractant (44), linked to connec-ssue remodeling, and has been associated with gastricr progression independent of Helicobacter infection6). HB-EGF is expressed by both myeloid cells (47)
astric cancer cells (48) and has been shown to play awound healing and carcinogenesis. Amphiregulin is
the renorma

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member of the EGF family that is expressed by epi-cells and fibroblasts (49) and induced in tumor cells

Ras mutations (50). Given the link between theseh factors and EGF receptor signaling, it seems likelyey contribute to the glandular proliferation and meta-phenotype in the K19-kras mice.of the consequences of K-ras–induced chronic inflam-

n was the recruitment of BMDCs to the stromal micro-nment in gastric dysplasia. Many of the BMDCs inecipient K19-kras mice were infiltrating immune cells,tent with the previous observations of lymphocyte in-ion in the pancreatic periduct and gastric mucosa ofras mice (13). However, BMDCs in the K19-kras mousemprised fibroblasts and myofibroblasts [also known asr-associated fibroblasts (CAF)] that expressed vimentinA, which surrounded the base of the dysplastic gastric. Stromal myofibroblasts are believed to contribute toem cell niche in the gastrointestinal tract (51, 52), but, the effect of depleting these stromal cell populationsnown. Previous studies have reported that many CAFst in tumors are likely to be bone marrow derived (53),idence for the importance of a bone marrow contribu-o tumor stroma has previously been recognized (54).this is the first study to show that K-ras mutation onn can lead to the recruitment of bone marrow–derivedFew epithelial cells were bone marrow derived, in con-o earlier reports in the H. felis murine model (3).restingly, in the K19-kras mouse stomach, there wasual expansion of Dcamkl1+ cells that moved down toase of the gastric glands and clustered in a regionnt to bone marrow–derived myofibroblastic nichesimilar to that observed in the intestinal crypts ofin mice (20). Dcamkl1-expressing cells have been

sed as a candidate progenitor cell fraction in bothomach and intestine (19, 20), raising the possibility-ras overexpression in K19 cells may lead to an ex-on of the gastric progenitor pool in the K19-kras. It is probable that the expansion of Dcamkl1+ cellsue to paracrine signals from stromal cells and/or ad-K19+ putative progenitors rather than a direct effectas within Dcamkl1+ cells, given that K19 and Dcamkl1differentially expressed. However, to date the only re-marker to lineage trace an entire gastric gland is

25). Because Lgr5 lineage tracing is restricted to theand dysplasia in K19-kras mice is found in the cor-

t is unlikely that Lgr5-derived cells are the primarye of the corpus dysplasia. However, definitive datathe source of dysplastic lesions will require additionale tracing studies.onclusion, K-ras mutation in K19+ putative gastricprogenitor cells induced proinflammatory responses,ing in early onset of gastric dysplasia. Dcamkl1+ cellsfound clustered in dysplastic glands, adjacent toded K19+ regions. BMDCs contributed significantly toicroenvironment of dysplastic glands as immune cells,lasts, and myofibroblasts. Further characterization of
lationship between K19+ and Dcamkl1+ cells in thel gastric epithelium and dysplastic lesions may provide



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grants 5R01 CA120979-02 (T.C. Wang), R01 DK060694 (A.K. Rustgi),143056 (T.C. Wang and A.K. Rustgi), and P30 DK050306 (Mouse Core)Uehara Memorial Foundation.costs of publication of this article were defrayed in part by thet of page charges. This article must therefore be hereby markedement in accordance with 18 U.S.C. Section 1734 solely to indicatet.

ived 04/28/2010; revised 08/16/2010; accepted 08/31/2010; publishedirst 10/19/2010.
l assistance.
rencesne AN, Carneiro F, O'Morain C, Offerhaus GJ. Nature meets nur-e: molecular genetics of gastric cancer. Hum Genet 2009;126:–28.an AO, Chu KM, Huang C, et al. Association between Helicobacterori infection and interleukin 1β polymorphism predispose to CpGnd methylation in gastric cancer. Gut 2007;56:595–7.ughton J, Stoicov C, Nomura S, et al. Gastric cancer originatingm bone marrow-derived cells. Science 2004;306:1568–71.S, Bhagat G, Cui G, et al. Overexpression of interleukin-1βuces gastric inflammation and cancer and mobilizes myeloid-rived suppressor cells in mice. Cancer Cell 2008;14:408–19.uma K, Oshima H, Aoki M, et al. Activated macrophages promotet signaling through tumor necrosis factor-α in gastric tumor cells.BO J 2008;27:1671–81.WW, Karin M. A cytokine-mediated link between innate immunity,ammation, and cancer. J Clin Invest 2007;117:1175–83.dreu P, Johansson M, Affara NI, et al. FcRγ activation regulateslammation-associated squamous carcinogenesis. Cancer Cell0;17:121–34.me S, Balmain A. Integration of positive and negative growth sig-ls during Ras pathway activation in vivo. Curr Opin Genet Dev0;10:106–13.rams SI, Stanziale SF, Lunin SD, Zaremba S, Schlom J. Identifica-of overlapping epitopes in mutant ras oncogene peptides that

ivate CD4+ andCD8+ T cell responses. Eur J Immunol 1996;:435–43.armann A, Bar-Sagi D. Ras-induced interleukin-8 expression playsritical role in tumor growth and angiogenesis. Cancer Cell 2004;6:–58., Houghton AM, Mariani TJ, et al. K-ras activation generates an

ammatory response in lung tumors. Oncogene 2006;25:2105–12.gorani SR, Petricoin EF, Maitra A, et al. Preinvasive and invasivectal pancreatic cancer and its early detection in the mouse.ncer Cell 2003;4:437–50.mbeck FH, Schreiber FS, Deramaudt TB, et al. The mutant K-rascogene causes pancreatic periductal lymphocytic infiltration andstric mucous neck cell hyperplasia in transgenic mice. Cancers 2003;63:2005–9.armann A, Bar-Sagi D. Ras oncogene and inflammation: partnersrime. Cell Cycle 2005;4:735–6.rrello MG, Degl'Innocenti D, Pierotti MA. Inflammation and cancer:oncogene-driven connection. Cancer Lett 2008;267:262–70.mbeck FH, Moffett J, Wang TC, Rustgi AK. The keratin 19 pro-ter is potent for cell-specific targeting of genes in transgenic mice.stroenterology 2001;120:1720–8.lnek D, Quaroni A. Changes in keratin expression during fetal andstnatal development of intestinal epithelial cells. Biochem J 1992;:939–46.lnek D, Quaroni A. Differential localization by in situ hybridizationdistinct keratin mRNA species during intestinal epithelial cellvelopment and differentiation. Differentiation 1993;53:95–104.

y R, Riehl TE, Hunt C, Sureban SM, Anant S, Houchen CW. Iden-ation of a novel putative gastrointestinal stem cell and adenomam cell marker, doublecortin and CaM kinase-like-1, followingiation injury and in adenomatous polyposis coli/multiple intestinaloplasia mice. Stem Cells 2008;26:630–7.gers AB, Taylor NS, Whary MT, Stefanich ED, Wang TC, Fox JG.licobacter pylori but not high salt induces gastric intraepithelialoplasia in B6129 mice. Cancer Res 2005;65:10709–15.gers AB, Houghton J. Helicobacter-based mouse models ofestive system carcinogenesis. Methods Mol Biol 2009;511:7–95.ng SS, Asfaha S, Okumura T, et al. Fibroblastic colony-formingit bone marrow cells delay progression to gastric dysplasia in alicobacter model of gastric carcinogenesis. Stem Cells 2009;27:01–11.sselot C, Spraggon L, Chia I, et al. Non-cell autonomous retinoidnaling is cruicial for renal development. Development 2010;137:3–92.hima H, Oshima M, Inaba K, Taketo MM. Hyperplastic gastricors induced by activated macrophages in COX-2/mPGES-1

nsgenic mice. EMBO J 2004;23:1669–78.rker N, Huch M, et al. Lgr5+ve stem cells drive self-renewal in themach and build long-lived gastric units in vitro. Cell Stem Cell10;6:25–36.bbe N, Shi G, Meguid RA, et al. Spontaneous induction of murinencreatic intraepithelial neoplasia (mPanIN) by acinar cell targetingoncogenic K-ras in adult mice. PNAS 2008;105:18913–8.tari J, Tanaka A, Tanabe H, et al. K-ras mutations and cell kineticsHelicobacter pylori associated gastric intestinal metaplasia:omparison before and after eradication in patients with chronicstritis and gastric cancer. J Clin Pathol 2007;60:921–6.ama T, Haruma K, Kitadai Y, et al. K-ras mutation in helicobacterlori-associated chronic gastritis in patients with and without gas-cancer. Int J Cancer 2002;97:562–6.Visser KE, Eichten A, Coussens LM. Paradoxical roles of themune system during cancer development. Nat Rev Cancer 2006;4–37.x JG, Wang TC. Inflammation, atrophy, and gastric cancer. J Clinest 2007;117:60–9.lkwill F, Charles KA, Mantovani A. Smoldering and polarizedlammation in the initiation and promotion of malignant disease.ncer Cell 200;7:211–7, Review.hubbert S, Shannon K, Bollag G. Hyperactive Ras in developmen-disorders and cancer. Nat Rev Cancer 2007;7:295–308, Review.crile B, Lim KH, Counter CM. Oncogenic Ras-induced secretion ofis required for tumorigenesis. Genes Dev 2007;21:1714–9.

ng G, Rosen DG, Zhang Z, et al. The chemokine growth-regulatedcogene 1 (Gro-1) links RAS signaling to the senescence of stromalroblasts and ovarian tumorigenesis. PNAS 2006;103:16472–7.kahashi M, Mutoh M, Shoji Y, et al. Transfection of K-rasAsp12NA markedly elevates IL-1β- and lipopolysaccharide-mediated in-
cible nitric oxide synthase expression in rat intestinal epitheliallls. Oncogene 2003;22:7667–76.nnila S, Kokkola A, Mizuguchi T, et al. Gene expression analysis
Cancer Research



idegas

38. Mopro199

39. Haeff53–

40. Foingen

41. Zhcyl52

42. Shagcan

43. BritionT cres73–

44. Qiukinob968

45. Palate

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47. BetioRe

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51. DirbuRe

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www.a


ntifies over-expression of CXCL1, SPARC, SPP1, and SULF1 intric cancer. Genes Chromosomes Cancer 2010;49:28–39.ser B, Clark-Lewis I, ZwahlenR, BaggiollniM. Neutrophil-activatingperties of the melanoma growth-stimulatory activity. J Exp Med0;171:1797–802.ghnegahdar H, Du J, Wang D, et al. The tumorigenic and angiogenicects of MGSA/GRO proteins in melanoma. J Leukoc Biol 2000;67:62.

x JG, Feng Y, Theve EJ, et al. Gut microbes define liver cancer riskmice exposed to chemical and viral transgenic hepatocarcino-s. Gut 2010;59:88–97.ang Z, Sheng H, Shao J, et al. Posttranscriptional regulation ofooxygenase-2 in rat intestinal epithelial cells. Neoplasia 2000;2:3–30.ono Y, Tanimura H, Iwahashi M, et al. Specific T-cell immunityainst Ki-ras peptides in patients with pancreatic and colorectalcers. Br J Cancer 2003;88:530–6.stol JA, Orsini C, Lindinger P, Thalhamer J, Abrams SI. Identifica-of a ras oncogene peptide that contains both CD4+ and CD8+

ell epitopes in a nested configuration and elicits both T cell subsetponses by peptide or DNA immunization. Cell Immunol 2000;205:83.Y, Zhu J, Bandi V, et al. Biopsy neutrophilia, neutrophil chemo-

e and receptor gene expression in severe exacerbations of chronicstructive pulmonary disease. Am J Respir Crit Care Med 2003;168:–75.

rk JY,ParkKH,BangS,et al.CXCL5overexpression is associatedwithstage gastric cancer. J Cancer Res Clin Oncol 2007;133:835–40.veking D, Mitchell HM, Day AS. Gastric epithelial cell CXC chemo-

54. GutumCh

acrjournals.org


e secretion following Helicobacter pylori infection in vitro. J Gas-enterol Hepatol 2004;19:982–7.sner G, Higashiyama S, Klagsbrun M. Isolation and characteriza-n of a macrophage-derived heparin binding growth factor. Cellgul 1990;1:811–9.kson JH, Grabowska A, El-Zaatari M, Atherton J, Watson SA.licobacter pylori can induce heparin-binding epidermal growthtor expression via gastrin and its receptor. Cancer Res 2006;66:24–31.oyab M, Plowman GD, McDonald VL, Bradley JG, Todaro GJ.ucture and function of human amphiregulin: a member of theidermal growth factor family. Science 1989;243:1074–6.ulany M, Dittmann K, Krüger M, Baumann M, Rodemann HP.dioresistance of K-ras mutated human tumor cells is mediatedough EGFR-dependent activation of PI3K-AKT pathway. Radio-r Oncol 2005;76:143–50.ekze NC, Hodivala-Dilke K, Jeffery R, et al. Bone marrow contri-tion to tumor-associated myofibroblasts and fibroblasts. Cancers 2004;64:8492–5.deny M, Marini FC, Dembinski JL, et al. Mesenchymal stem

lls: potential precursors for tumor stroma and targeted-deliveryhicles for anticancer agents. J Natl Cancer Inst 2004;96:93–603.ii G, Sangai T, Oda T, et al. Bone-marrow-derived myofibroblastsntribute to the cancer-induced stromal reaction. Biochem Biophyss Commun 2003;309:232–40.o X, Oshima H, Kitmura T, et al. Stromal fibroblasts activated by
or cells promote angiogenesis in mouse gastric cancer. J Biolem 2008;283:19864–71.



2010;70:8435-8445. Published OnlineFirst October 19, 2010.Cancer Res Tomoyuki Okumura, Russell E. Ericksen, Shigeo Takaishi, et al. and Progression to Intraepithelial Neoplasia

Microenvironment,Results in Chronic Inflammation, an Altered K-ras Mutation Targeted to Gastric Tissue Progenitor Cells

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