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Tumor Microenvironment

Inhibition of Discoidin Domain Receptor 1Prevents Stroma-Induced PeritonealMetastasis in Gastric CarcinomaHyejin Jin1, In-Hye Ham1,2, Hye Jeong Oh1, Cheong A Bae1,2, Dakeun Lee3,Young-Bae Kim3, Sang-Yong Son1, Yong-Joon Chwae4, Sang-Uk Han1,Rolf A. Brekken5, and Hoon Hur1,2

Abstract

Discoidin domain receptor 1 (DDR1) is activated byfibrillar (triple-helical) collagens and collagen IV, whichare major components of tumor stroma; thus, DDR1 mightbe a critical mediator of communication between cancercells and stroma. The aim of this study was to investigatethe effect of DDR1 inhibition on stroma-induced perito-neal metastasis in gastric carcinoma. We analyzed byimmunohistochemistry the correlation between DDR1expression and the pattern of recurrence in gastric carci-noma tissues from a previously characterized and estab-lished gastric carcinoma patient cohort. We also coculturedhuman gastric carcinoma cell lines with gastric cancer–associated fibroblasts (CAF) and investigated DDR1expression and activation. We evaluated CAF-inducedtumorigenic properties of gastric carcinoma cell lines andthe effect of a DDR1-specific inhibitor in organotypiccultures and in a peritoneal seeding xenograft animal

model. The expression of DDR1 in gastric cancer tissueswas positively associated with early recurrence (P ¼ 0.043)and a high incidence of peritoneal recurrence (P ¼ 0.036).We confirmed that coculturing with CAFs elevated DDR1protein expression in gastric carcinoma cell lines andenhanced gastric carcinoma cell line spheroid formationin organotypic cultures in a tumor cell DDR1-dependentmanner. Coimplantation of CAFs with gastric carcinomacells enhanced peritoneal tumor formation in vivo, an effectthat was sensitive to pharmacologic inhibition of DDR1.

Implications: This study highlights that CAF-induced ele-vation of DDR1 expression in gastric carcinoma cellsenhances peritoneal tumorigenesis, and that inhibitionof DDR1 is an attractive strategy for the treatment ofgastric carcinoma peritoneal metastasis. Mol Cancer Res; 16(10);1590–600. �2018 AACR.

IntroductionGastric carcinoma is one of the most common malignant

tumors and the third leading cause of cancer-related deathsworldwide (1). Although mortality from gastric carcinomahas gradually decreased in recent years, patients with late-stage disease still have poor prognosis due to tumor non-resectability or recurrence after resection. Large-scale clinicaltrials showed that adjuvant chemotherapy following curativeresection could reduce the recurrence rate of stage II or IIIgastric carcinoma. However, approximately one fourth of all

patients enrolled in those trials suffered recurrence during thefollow-up period, and the peritoneum is one of the mostcommon sites of recurrence (2, 3). To date, various studieshave reported that patients with peritoneal metastasis haveworse response to treatments than patients with hematoge-nous or lymphatic metastasis (4–6). However, specificmodalities to prevent gastric carcinoma peritoneal metastaseshave not been established. Thus, new strategies basedon specific molecular biomarkers related to peritoneal metas-tasis must be developed to prevent gastric carcinoma perito-neal recurrence.

Several histologic features of primary gastric carcinomas haveimplicated the tumor stroma in the peritoneal metastasis ofgastric carcinoma. First, it is well known that the likelihood ofperitoneal recurrence increases when primary gastric carcino-mas invade the gastric wall deeply (6). During invasion, cancercells that originate from the mucosal layer of the gastrointes-tinal tract are exposed to stromal tissues and activate variousnoncancerous cells, such as fibroblasts and inflammatory cells(7). Second, the subtypes of gastric carcinomas with highproportions of tumor stroma preferentially metastasize to theperitoneum (6). Moreover, several studies have reported thatcancer-associated fibroblasts (CAF) contribute to the peritonealdissemination of gastric carcinoma (8, 9). Taken together, thesefindings indicate that the interaction between cancer cells andstromal cells could be a therapeutic target to prevent peritonealmetastasis of gastric carcinoma. However, drugs targeting the

1Department of Surgery, Ajou University School of Medicine, Suwon, Korea.2Brain Korea 21 Plus Research Center for Biomedical Sciences, Ajou University,Suwon, Korea. 3Department of Pathology, Ajou University School of Medicine,Suwon, Korea. 4Department of Microbiology, Ajou University School ofMedicine, Suwon, Korea. 5Division of Surgical Oncology, Department of Surgery,Hamon Center for Therapeutic Oncology Research, University of TexasSouthwestern Medical Center, Dallas, Texas.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Hoon Hur, Department of Surgery, Ajou UniversitySchool of Medicine, 164 Worldcup-ro, Yeongtong-gu, Suwon-si, Gyeonggi-do16499 Republic of Korea. Phone: 82-31-219-5200; Fax: 82-31-219-5575; E-mail:[email protected], [email protected]

doi: 10.1158/1541-7786.MCR-17-0710

�2018 American Association for Cancer Research.

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tumor stroma have not been applied to treat gastric carcinomain a clinical setting.

Discoidin domain receptor 1 (DDR1) is a member of thetransmembrane receptor tyrosine kinase family with a discoi-din homology domain in its extracellular region (10–12).Distinct from other receptor tyrosine kinases, DDR1 is activatedby collagens, including types I, II, III, IV, V, VIII, and XI, whichare major components of the extracellular matrix (ECM) insolid tumors. Various studies have reported that increasedDDR1 expression is associated with a poor prognosis in malig-nant tumors (13–16). Moreover, our previous report describedthat approximately half of the gastric carcinomas exhibiteda positive expression of DDR1, which was related to the depthof gastric carcinoma invasion. In addition, gastric carcinomapatients with DDR1 expression showed worse overall survivalrates than gastric carcinoma patients without DDR1 expres-sion. We also demonstrated that type I collagen could activateDDR1 signaling and enhance the aggressive phenotype ofgastric carcinoma cells (17). However, the significance ofDDR1 expression on the pattern of recurrence after resectionof primary gastric carcinoma was not evaluated. If the tumorstroma contributes to the peritoneal recurrence of gastric car-cinomas, DDR1 could be a key regulator that links peritonealmetastasis and tumor stroma in gastric carcinoma.

Here, we found that DDR1 expression correlated with gastriccarcinoma peritoneal recurrence. The aim of this study was toevaluate if tumor stromal cells enhance the peritoneal metastasisof gastric carcinoma through the upregulation of DDR1, and ifinhibition of DDR1 expression and activity could be appliedtherapeutically to prevent peritoneal recurrence.

Materials and MethodsHuman samples and immunohistochemical staining

We analyzed the features of gastric carcinoma recurrence in202 patients who had undergone curative gastric resectionswith proper lymphadenectomy at the Department of Surgery,Ajou University Hospital, Korea, from May 2003 to December2005. We examined the incidence of tumor recurrence and itsfeatures in that cohort during the follow-up period. The sites ofrecurrence were classified into three groups: the peritoneum,hematogenous sites, and distant lymph nodes. The definitionof DDR1 positivity was described in our previous report (17).We analyzed the association of DDR1 expression in gastriccarcinomas with recurrence-free survival rates and the recur-rence rates at the specific sites.

Cell lines and chemicalsWe purchased gastric carcinoma cell lines MKN74 (KCLB

No. 80104) and MKN45 (KCLB No. 80103) from the KoreanCell Line Bank. These cells were cultured in RPMI-1640 medi-um (Invitrogen) supplemented with 10% fetal bovine serum(FBS; Equitech-Bio), 1% penicillin/streptomycin (Invitrogen),and 1% amphotericin B (Sigma-Aldrich). The cells were incu-bated at 37�C in a humidified atmosphere containing 5% CO2.

Fibroblasts were isolated from fresh gastric carcinoma andpaired normal gastric specimens, as described in our previousstudy (18). In the present study, we used fibroblasts isolated fromsurgical specimens of a gastric carcinomapatient diagnosedwith a7.5-cm-sized moderately differentiated tumor adenocarcinoma.Stomach cancer specimens were obtained from patients under-

going surgery at Ajou University Hospital (Suwon, Korea) whosepreoperative pathologic diagnosis was gastric carcinoma. Thisstudy was conducted in accordance with the ethics code of theWorld Medical Association (Declaration of Helsinki) and wasapproved by the Institutional Review Board of Ajou UniversityHospital (AJIRB-BMR-SMP-14-155).

For cocultures, fibroblasts were seeded into the upper cham-bers of 6-well transwells, and gastric carcinoma cell lines wereseeded in 6-well tissue culture dishes.

CRISPR/Cas9-mediated knockout (KO) of DDR1We established DDR1-deficient gastric carcinoma lines using

the CRISPR-Cas9 system in order to investigate the role ofDDR1 in interactions between CAFs and gastric carcinoma celllines (Supplementary Fig. S1A). The target primer sequences forhuman DDR1 were #1 (forward, 50-CACCGGTGGAATGTCG-CTTCCGGCG-30; reverse, 50-AAACCGCCGGAAGCGACATTC-CACC-30) or #2 (forward: 50-CACCGCCCCCTAGGTTGTGGC-GCAT-30; reverse, 50-AAACATGCGCCACAACCTAGGGGGC-30). Oligonucleotides with BsmB1 restriction sites for guideRNAs were synthesized at Xenotech, then phosphorylated usingT4 kinase (NEB). The phosphorylated oligonucleotides werecloned into LentiCRISPR v2 and the sequences of the clonedplasmids that were extracted from several selected colonieswere confirmed at Cosmo Genetech.

MKN74 cells were transfected with pLentiCRISPR-sgRNADDR1 using Lipofectamine 2000 (Invitrogen), according to themanufacturer's instructions. When the cells were confluent indishes, they were treated with 1 to 2 mg/mL puromycin forapproximately 2 weeks. The silencing of DDR1 in the survivingcells after puromycin selection was validated byWestern blotting.

Western blot analysisThe cells were washed with phosphate buffered saline and

lysed in phospho-specific lysis buffer. Lysates were incubatedon ice for 20 minutes, then centrifuged at 13,000 � g for20 minutes at 4�C. Protein concentrations were determinedby Bradford assay (Bio-Rad). Equal amounts of protein fromeach sample were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a poly-vinylidene difluoride membrane (Millipore. The immunoblotswere blocked by incubation in 5% skim milk, 250 mmol/L tris(hydroxymethyl) aminomethane–HCl (pH 8.0), 1500 mmol/LNaCl, and 0.1% Tween 20 for 1 hour at 25�C. Membraneswere incubated with the following primary antibodies: DDR1(1:1,000 dilution, #5583, Cell Signaling Technology), phos-phorylated-DDR1 (1:1,000 dilution, #11994, Cell SignalingTechnology), and b-actin (1:5,000 dilution, sc-47778, SantaCruz Biotechnology), followed by the corresponding HRP-conjugated secondary antibodies (Jackson ImmunoResearchLabs). Protein detection was performed with an enhancedchemiluminescence kit (Abclon).

Cell viability testCells were seeded at 1� 104 cells per well in 96-well plates, and

cell viability was measured using the highly sensitive water-soluble tetrazolium salt (WST)-based cell viability, cytotoxicityassay kit (DoGen). The EZ-Cytox solution (10 mL) was added toeach well, cells were incubated for 1.5 hours, and the absorbancewas measured by spectrophotometry at 450 nm. To evaluatethe effect of 7rh benzamide (provided by Dr. Ke Ding, Jinan

DDR1 in Peritoneal Metastasis of Gastric Carcinoma

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University) on cell viability, the cells were treated with differentconcentrations of 7rh benzamide over 72 hours.

Reverse transcription PCRTotal RNA was prepared from gastric cancer cell lines and

fibroblasts and converted to cDNA using 1 mg of each RNA in afinal volume of 20 mL. The cDNA mixture (3 mL) was used for 35cycles of PCR amplification (denatured at 94�C for 5 minutes,annealed at 62�C (DDR1), or 64�C (COL1A) for 30 seconds, andextended at 72�C for 5 minutes). The primers were as follows:DDR1 (forward, 50-CGAGCAGGTCA TCGAGAACG-30; reverse,50-CCCACCTGCAGTCTCACTG-30), COL1A (forward, 50-GTCA-CCCACCGACCAAGAAACC-30; reverse, 50-AAGTCCAGGCTGTC-CAGGGA TG-30), b-actin (ACTB) (forward, 50-TCAAGATCATT-GCTCCTCCTGAGC-30; reverse, 50-TGCTGTCACCTTCACCGTTC-CAGT-30).

Organotypic culture and immunocytochemical stainingFor organotypic culture experiments, the artificial ECM con-

sisted of a mixture of Matrigel (5 mg/mL, BD Biosciences) andcollagen I (1.5 mg/mL, BD Biosciences) with or without3,000 CAFs. Gastric cancer cell lines (3,000 cells/well) wereseeded on the artificial ECM. To measure the outcomes, wecounted the number of spheroids 200 mm or more in size andthe mean size of the 5 largest spheroids 7 days after seeding thecells under 40� magnification. Immunocytochemical stainingwas conducted after measuring outcomes approximately 1 weekafter the plating of the gastric carcinoma cell lines. Additionalmethodologic details for organotypic cultures and immunocy-tochemical staining were previously published by Dang andcolleagues (19).

Secretome and transcriptome analysisWe performed secrotome analysis using Proteome Profiler

Human Cytokine Array Kit (R&D Systems Inc.) in order toidentify upregulated secretory factors in culture supernatantsof cocultured MKN74 cells with CAFs relative to MKN74without CAFs. Conditioned medium from MKN74 cultureswas collected after 48 hours in serum-free medium and incu-bated with arrays containing 36 human cytokine-specific anti-bodies according to the manufacturer's instructions.

Using RNeasy (Qiagen), RNA was extracted from MKN74cells cultured alone or together with CAFs. RNA quality wasassessed using a 2100 Bioanalyzer (Agilent), and cDNA wassynthesized using the GeneChip WT (Whole Transcript) Ampli-fication kit. Approximately 5.5 mg of labeled DNA target washybridized to Affymetrix GeneChip Human Gene 2.0 ST Arrays.Array data processing and analysis were performed using Affy-metrix GeneChip Command Console Software. Gene setenrichment analysis (GSEA) was used to identify upregulatedpathways in MKN74 cells cocultured with CAFs relative toMKN74 alone. The pathway categories were obtained fromMSigDB (https://software.broadinstitute.org). The cutoff valuefor statistical significance used in GSEA is a false discovery rate(FDR) of 25%. In order to reduce the likelihood of false positiveresults, this study used an FDR value of 5% as the cutoff levelfor enriched gene sets.

Peritoneal xenograft mouse modelsAnimal care and handling procedures were performed in

accordance with the Ajou University School of Medicine

Institutional Animal Care and Use Committee guidelines,and all animal experiments described were approved by theAnimal Research Committee of the institution. The peritonealseeding animal model was established using 6- to 8-week-oldmale athymic nude mice (Orient Bio), weighing 16 to 18 g.We performed 3 types of experiments. In experiment 1, micewere randomly assigned to 3 groups. Mice in the first groupwere intraperitoneally injected with 1 � 107 cancer cells alone(experiment 1, group 1), or with 1 � 106 (experiment 1, group2) or 4 � 106 (experiment 1, group 3) CAFs. In experiment 2,mice were randomly assigned to 2 groups. Mice were intraper-itoneally injected with DDR1 KO cancer cell line only (exper-iment 2, group 1) or with 1� 106 CAFs (experiment 2, group 2).In experiment 3, mice were first assigned to 2 groups. Micewere intraperitoneally injected with 1 � 107 cancer cells only(experiment 3, group 1), or with 1 � 106 CAFs (experiment3 group 2). Each group was randomly divided into 2 sub-groups, and mice were orally administered the DDR1 inhibitor7rh (25 mg/kg) or vehicle every 2 days over 3 weeks, startingon day 1 after cell injection. The mice were sacrificed 3 weeksafter injection, and the numbers and sizes of the peritonealnodules were assessed.

Statistical analysisRelapse-free survival rates were evaluated using log-rank tests,

and survival curves were generated using the Kaplan–Meier meth-od. The differences between the expression of DDR1 and thepatterns of recurrence were evaluated using the c2 test. All exper-imental studies were performed independently 3 times. Statisticalanalysis was performed using IBM SPSS statistics (version 21 forMac OS X, IBM) and GraphPad Prism (version 6.0 for Mac OS X,GraphPad) software.

ResultsDDR1 expression in primary gastric carcinoma tissuescorrelates with peritoneal recurrence

In a previous report, DDR1 staining was observed at the cellmembrane or in the cytoplasm of tumor cells, and 50.5% ofgastric carcinoma tissues showed positive DDR1 expression(17). Gastric carcinoma tissues with DDR1 expression wereshown to be associated with deeper invasion and poorer overallsurvival, compared with tissues that did not express DDR1 (17).When we performed additional analyses with follow-up dataincluding recurrence, we found that patients with DDR1 expres-sion showed significantly shorter relapse-free survival timesthan DDR1-negative patients (P ¼ 0.043; Fig. 1A). During thefollow-up period, 51 patients (25.2%) presented with recur-rence, and 74.5% of all patients with recurrence had metastaticlesions in the peritoneum, the most common site of recurrence(Fig. 1B). Patients with DDR1 expression had significantlyhigher rates of peritoneal recurrence compared with thosenegative for DDR1 (P ¼ 0.036). However, hematogenous(P ¼ 0.101) or distant lymph node (P ¼ 0.285) recurrenceswere not significantly related to DDR1 expression (Table 1).

CAFs enhance the peritoneal tumorigenic potential of gastriccarcinoma

At first, we screened for the expression of DDR1 and mesen-chymal markers, such as a-SMA and Vimentin in gastric carcino-ma cell lines and fibroblasts, and we identified that DDR1 was

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only expressed in gastric carcinoma cell lines (SupplementaryFig. S1B).

To identify if CAFs enhance the tumorigenesis of gastriccarcinoma cell lines, we conducted organotypic cultures ofgastric carcinoma cell lines (Fig. 2A and B). We found that thenumber of spheroids and the mean size of the 5 largest spher-oids in MKN74 culture were significantly higher in the presenceof CAFs relative to cells cultured on ECM only (Fig. 2C). Inaddition, we established the peritoneal seeding xenograft mousemodel to assess the effects of CAFs on the peritoneal tumori-genesis of gastric carcinoma cell lines in vivo (Fig. 2D). Asexpected, the number of tumors and mean weight of the largestnodules were significantly increased in groups injected withCAFs, and these factors depended on the number of CAFs(Fig. 2E and F). These results indicated that CAFs enhance theperitoneal tumorigenesis of gastric carcinoma cell lines.

CAFs induced upregulation of DDR1 in gastric carcinomacell lines

We evaluated the phosphorylation of DDR1 in organotypiccultures of MKN74 through immunocytochemistry (Fig. 3A).Moreover, we identified, through immunohistochemistry, theupregulation of DDR1 in the peritoneal seeding xenograftmouse model, in the groups that received coinjection ofMKN74 and CAFs (Fig. 3B).

We used a transwell coculture system to investigate CAF-induced changes in DDR1 expression. In gastric carcinoma celllines, MKN74 and MKN45 cells that were cocultured with CAFsusing the transwell coculture system showed increased expressionof DDR1 relative to cultures without CAFs by Western blottinganalysis (Fig. 3C). However, the coculture with fibroblasts iso-lated from normal gastric tissues (normal tissue-associated fibro-blasts: NAFs) did not enhance the expression of DDR1 in gastriccarcinoma cells (Supplementary Fig. S2A). In addition, DDR1

gene expression in MKN74 cells was not increased despite cocul-ture with CAFs (Supplementary Fig. S2B). These results indicatedthat CAF upregulated DDR1 at the posttranscriptional level andCAF-induced upregulation of DDR1 was involved in the perito-neal spread of gastric carcinoma.

CAF-induced STAT3 activation may be involved in DDR1upregulation

We investigated the release of 36 different cytokines, growthfactors and other secreted proteins in conditioned culture mediaof MKN74 with/without CAFs (Supplementary Fig. S3A).Results after 48 hours of culture are shown in SupplementaryFig. S3B. IL6, IL8, and C–C motif chemokine ligand 2 (CCL2)were readily detected in conditioned medium when coculturedwith CAFs. When we analyzed signal transduction pathwaysbased on the KEGG pathway database (https://www.genome.jp/kegg/pathway.html), they were all related to the Janus kinase(JAK1)-signal transducer and activator of transcription (STAT3)signaling pathway (Supplementary Fig. S3B).

We examined the gene expression changes in MKN74 cellsinduced by coculture with CAFs (Supplementary Fig. S3A).GSEA was used to determine potentially altered signalingpathways and the results identified that the IL6/JAK/STAT3signaling-related genes were enriched in MKN74 cells cocul-tured with CAFs [enrichment score: 1.83, nominal P < 0.001,FDR Q ¼ 0.001, family wise error rare (FWER) P ¼ 0.006;Supplementary Fig. S3C]. To validate these results, we perform-ed Western blotting analysis for the MKN74 cell line coculturedwith/without CAFs. Along with the upregulation and phos-phorylation of DDR1, phosphorylation of STAT3 was signifi-cantly upregulated in MKN74 cocultured with CAFs relativeto MKN74 cells cultured alone (Supplementary Fig. S3D).Meanwhile, the phosphorylation status of AKT was notaltered in coculture with CAFs. These results suggested that

Figure 1.

Pattern of recurrence in gastriccarcinoma patients according to DDR1expression. A, DDR1 expression inprimary gastric carcinoma tissuescorrelates with early recurrence in theKaplan–Meier curve, analyzedwith thelog-rank test (P ¼ 0.043). B, Venndiagram of the sites of recurrence ingastric carcinoma patients,demonstrating that the peritoneum isthe most common site of recurrence.

Table 1. Pattern of recurrence according to DDR1 expression in GCs

n DDR1 negative (n ¼ 100) DDR1 positive (n ¼ 102) PTotal recurrence 51 19 (19.0%) 32 (31.4%) 0.043Peritoneal recurrence 38 13 (13.0%) 25 (24.5%) 0.036Hematogenous recurrence 19 6 (6.0%) 13 (12.7%) 0.101Distant lymph node recurrence 14 5 (5.0%) 9 (8.8%) 0.285





https://www.genome.jp/kegg/pathway.htmlhttps://www.genome.jp/kegg/pathway.htmlhttp://mcr.aacrjournals.org/

CAFs-induced cytokines such as IL6 activated the JAK/STAT3pathway in gastric carcinoma cells, and this may be related tothe upregulation of DDR1. However, the exact mechanisms ofCAF-induced upregulation of DDR1 need to be clarified throughfuture studies.

DDR1 inhibition using the CRISPR/Cas9 system suppressesthe effects of CAFs

We conducted organotypic cultures for gastric carcinomacell lines using DDR1-knockout MKN74. We found that theDDR1-KO MKN74 cells generated equivalent numbers ofspheroids with a mean size similar to the 5 largest spheroidsin the ECM culture only and ECM with CAF coculture condi-tions (Fig. 4A and B). Moreover, we investigated the activationof DDR1 in the DDR1-KO MKN74 cells from the organotypiccultures through immunocytochemistry. As expected, the ECMwith CAFs condition did not enhance the activation of DDR1in the DDR1-KO MKN74 cells (Fig. 4C). When we establishedthe peritoneal seeding xenograft using DDR1-KO MKN74, thenumber of tumors in the groups with DDR1-KO cell line alonewas not significantly different from DDR1-KO cells coinjectedwith CAFs (P ¼ 0.751). There was no expression of DDR1 inthe xenograft tumors of the DDR1-KO cell line (Fig. 4D). Thesedata suggested that the genetic inhibition of DDR1 in gastriccarcinoma cells suppressed the tumor promoting effectsof CAFs.

Blocking DDR1 signaling through 7rh represses the effects ofCAFs on gastric carcinomas

We next evaluated the effect of 7rh, a specific inhibitor ofDDR1 (20), which may be a new candidate therapeutic agent.First, we demonstrated that low-dose 7rh does not affect theviability of gastric carcinoma cell lines or fibroblasts (Fig. 5A),and that it significantly reduces DDR1 expression in MKN74cells compared with vehicle treatment (Fig. 5B). In addition, weconfirmed that the COL1A gene encoding COL1A, a ligand ofDDR1, was only expressed in fibroblasts (Supplementary FigS4A). Western blotting analysis for MKN74 cocultured withCAFs showed that low-dose 7rh can reduce the phosphoryla-tion of DDR1 as well as upregulation of DDR1 (SupplementaryFig S4B).

Then, we conducted organotypic cultures of gastric carcinomacell lines with 7rh. The organotypic cultures revealed that 7rhreduced the number of spheroids and the mean size of the 5largest colonies despite coculture with CAFs (Fig. 5C and D).Immunocytochemistry for the organotypic cultures showed that7rh suppressed the phosphorylation of DDR1 in MKN74 cells(Fig. 5E).

Moreover, to further clarify the effects of 7rh, we performedin vivo experiments using the peritoneal seeding xenograftmouse model of gastric carcinoma (Fig. 6A). As expected,7rh significantly reduced the number of tumor nodules in theCAF coinjection group (Fig. 6B). Immunohistochemistry on

Figure 2.

Peritoneal tumorigenesis of gastric carcinoma cells stimulated by CAFs. A to C, Organotypic culture of gastric carcinoma cell lines. A, Schematic figureof organotypic culture and B, representative photos of MKN74 colonies (scale bar, 200 mm). C, The number of colonies larger than 200 mm and themean sizes of the 5 largest MKN74 colonies were significantly increased in the presence of CAFs (� , P < 0.05, independent t test). D to F, Peritonealtumorigenesis of MKN74 gastric cancer cell line in BALB/c nude mice (n ¼ 8, each group). D, Representative photos of peritoneal nodules (arrow) 18 daysafter injection of MKN74 with/without CAFs into the abdominal cavity. E, The number of tumor nodules in each group and F, mean weight of thelargest nodules (� , P < 0.05; �� , P < 0.01, independent t test).

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the harvested tumor nodules revealed that 7rh reduced theexpression of DDR1 in peritoneal tumors (Fig. 6C). However,the weight of mice in each group did not show differencesduring the treatment (Fig. 6D). Thus, 7rh suppresses the CAF-induced peritoneal tumorigenesis of gastric carcinomas with-out side effects through the inhibition of DDR1 signaling.

DiscussionThis study reveals the potential of DDR1 as a novel thera-

peutic target to prevent stroma-induced gastric carcinoma peri-toneal recurrence after resection. In our cohort of patients, whounderwent curative resection for gastric carcinomas, we found

that DDR1 expression in primary tumors could be a biomarkerto predict peritoneal recurrence. The in vitro and in vivo experi-ments demonstrated that DDR1 was upregulated in gastriccarcinoma cells stimulated with CAFs. Furthermore, inhibitionof DDR1 induced significant antitumorigenic effects in CAF-stimulated gastric carcinoma cells in organotypic cultures andin a peritoneal xenograft mouse model. Our findings corrobo-rate the favorable evidence for the use of DDR1 inhibitors toprevent peritoneal recurrence after curative resection for gastriccarcinomas and are summarized in Fig. 6E.

Peritoneal metastasis is a distinctive pattern of recurrence ingastric carcinomas and is associated with poorer outcomes thanhematogenous or lymphatic metastasis (21, 22). In particular,

Figure 3.

CAF-induced upregulation of DDR1 in gastric cancer cell lines. A, Immunocytochemistry revealed that DDR1 phosphorylation was induced to a greaterextent on the ECM in the presence of CAFs than on the ECM alone (scale bar, 200 mm; �, P < 0.05, independent t test). B, Immunohistochemistry ofharvested intraperitoneal nodules showed that MKN74 xenografts mixed with CAFs had greater expression of DDR1 and accumulation of fibroblastsexpressing a-SMA than xenografts with only MKN74 cells (scale bar, 50 mm). C, Western blotting showed that coculture with CAFs enhanced DDR1expression in MKN74 and MKN45 gastric carcinoma cells. The DDR1 and b-actin bands were quantified with densitometry using ImageJ and then DDR1 bandintensities were normalized to b-actin band intensity. Relative expression of DDR1 in coculture with CAFs over 48 hours was significantly highercompared with MKN74 cells cultured alone (� , P < 0.05, independent t test).






small peritoneal seeding nodules in the peritoneal cavity are notadequately detected in imaging studies such as abdominal com-puted tomography, so peritoneal recurrence is diagnosed at latestages and associatedwith poor outcomes. Therefore, a predictionof peritoneal metastasis through the analysis of the resectedprimary tumor is required to suggest additional modalities toprevent metastasis after curative resection for gastric carcinomas.However, reliable markers for site-specific recurrence have onlyrarely been investigated in gastric carcinomas. Recently, a tran-scriptome analysis of curatively resected gastric carcinoma tissuesrevealed that SYT8 could be a candidate to predict peritoneal

recurrence during the follow-up period (23). However, theresearchers obtained transcriptome data from only 16 patients,so the statistical power of the data might be insufficient to con-firm SYT8 as a reliable marker. In addition, therapeutic agentstargeting SYT8 protein are not available. Others reported thatthe infiltration of S100-positive dendritic cells could be areliable marker for peritoneal recurrence of gastric carcinomas(24).However, the studies lacked supportive experimental data todemonstrate a correlation between dendritic cells and the peri-toneal tumorigenesis of gastric carcinoma cells. In the presentstudy, we analyzed the clinical data of 202 gastric carcinoma

Figure 4.

Genetic inhibition of DDR1 suppressesthe effects of CAFs. A to C,Organotypic cultures of DDR1-knockout MKN74 cells. A,Representative photos of colonies(scale bar, 200 mm). B, No differenceswere observed in the number ofcolonies larger than 200 mm or themean size of the 5 largest coloniesfrom DDR1-knockout MKN74 cells inthe presence or absence of CAFs. C,Immunocytochemistry showed thatDDR1-knockout MKN74 cells did nothave enhanced DDR1 phosphorylationon the ECM in the presence of CAFs,unlike DDR1-positive MKN74 cells(scale bar, 200 mm). D, Peritonealtumorigenesis of DDR1-knockoutMKN74 cells mixed with or withoutCAFs inBALB/c nudemice (n¼6, eachgroup). There was no difference in thenumber of intraperitoneal tumors(P ¼ 0.751), and we confirmed noexpression of DDR1 in xenografts withDDR1-knockout MKN74 cells.

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patients, whose primary carcinoma tissues were processed forDDR1 expression by immunohistochemistry. These resultsshowed that DDR1 expression in patients was related to perito-neal recurrence (the most common recurrence), but not to hema-togenous or lymphatic metastasis. Moreover, our experimentalstudies indicated that stroma-induced DDR1 upregulation ingastric carcinoma cell lines enhances their peritoneal tumorigen-esis, whereas the inhibition of DDR1 reduces it. Our findingsprovide support for the use of DDR1 expression as a predictivebiomarker for peritoneal recurrence in patients with gastric car-cinomas, and for the application of 7rh, a DDR1 inhibitor, for theprevention of peritoneal recurrence after curative resection.

Our previous report using the same cohort as that of the presentstudy indicated that the proportion of DDR1-positive cells inprimary gastric carcinoma tumors increased among the tumorcells that had more deeply invaded the gastric wall (17). Otherstudies, which evaluated the clinical relevance of DDR1 expres-sion for gastrointestinal malignancies, including gastric andesophageal carcinomas, showed similar results (25, 26). Carci-

noma of the gastrointestinal tract usually originates from epithe-lial cells in the mucosal layer, which subsequently invade thesubmucosa, the muscle, and the serosal layers. The initial step ofcancer invasion is the degradation of the basement membrane,followed by increased cross-talk between the invading carcinomacells and stromal tissue (27). Stromal tissue consists of variouscomponents, including noncancerous cells and ECM. Amongthose components, fibroblasts are essential to contribute to ECMremodeling by secreting proteases such as metalloproteinases,which effectively degrade the ECM (28). In addition, fibroblastscan produce various soluble autocrine and paracrine secretoryproteins that regulate the activity of the surrounding cells as wellas themselves (29). In the present study, we speculated that gastriccarcinoma cells invading the gastric wall upregulate DDR1through interaction with CAFs. Our experimental data supporta role for CAFs in enhancing the upregulation of DDR1 incocultures, organotypic cultures, and peritoneal xenografttumors, whereas DDR1 gene transcription was not altered aftercoculture with CAFs. These findings are consistent with a recent

Figure 5.

Effects of 7rh on gastric cancer cell lines. A, Low-dose 7rh did not affect the viability of gastric carcinoma cell lines or fibroblasts. B, Western blottingrevealed that 7rh reduced DDR1 expression in MKN74 cells. The DDR1 and b-actin bands were quantified with densitometric program ImageJ and thenrelative DDR1 band intensities were normalized to b-actin band intensity. C to E, Organotypic cultures of MKN74 cells with 7rh treatment. C, Representativephotos of colonies (scale bar, 200 mm). D, The number of colonies larger than 200 mm and the mean size of the 5 largest colonies from MKN74 cellswere markedly decreased by 7rh treatment on the ECM with CAFs (� , P < 0.05; �� , P < 0.01, independent t test). E, Immunocytochemistry showed that DDR1phosphorylation in MKN74 was suppressed by 7rh treatment on the ECM in the presence of CAFs (scale bar, 200 mm).






report that insulin-like growth factor 1 (IGF-1) induces upregula-tion of DDR1 by suppressing miR-119 through the PI3K/AKTpathway, andnot by enhancing the transcriptionofDDR1 (30). Inthe present study, we suggested several CAF-produced factors,including IL6, IL8, and CCL2, that act as activators to enhanceupregulation of DDR1 in gastric carcinoma cells through secre-tome analysis. Moreover, those factors can activate the JAK/STAT3signaling pathway, which iswell known to be related to cancer cellsurvival (31, 32). CAF-induced JAK/STAT3 signal activation wasvalidated through transcriptome analysis andWesternblotting forgastric carcinoma cells cocultured with CAFs; however, we couldnot discern a direct relationship between JAK/STAT3 and the

upregulation of DDR1, so the exact mechanisms of CAF-inducedupregulation of DDR1 in gastric carcinomas needs to be morefully explored in the future.

In the present study, we hypothesized that the upregulationof DDR1 is essential for the tumorigenic ability of CAF-stimulated, free-floating cancer cells in the peritoneum. Ourdata support this hypothesis, as DDR1-knockout cells did notundergo CAF-stimulated increases in colony number or clonesize. There are several studies describing the ability of DDRsto promote tumor growth in metastatic sites like the boneand liver (33, 34). However, there had been no proof of therole of DDR1 in the peritoneal metastasis of cancer cells. In

Figure 6.

Effects of 7rh in mouse xenograft model. A to D, Peritoneal tumorigenesis of gastric cancer cell lines in BALB/c nude mice with 7rh treatment (n ¼ 6,each group). Mice were treated with 25 mg/g 7rh 3 times per week for 3 weeks. A, Representative photomicrographs of peritoneal nodules (arrow) frommice that were treated with 7rh for 18 days after MKN74 injection into the abdominal cavity with/without CAFs. B, The number of tumor nodules wasreduced by 7rh treatment in mice injected with MKN74 and CAFs, whereas 7rh treatment did not statistically affect the number of tumor nodules in miceinjected with MKN74 cells only. The effects on the MKN45 tumor nodules were the same as those for MKN74. C, Immunohistochemistry on harvestedintraperitoneal nodules showed that 7rh reduced DDR1 expression in MKN74 xenograft tumors (scale bar, 50 mm). D, Weight of mice showed a similarpattern among each group during treatment. E, Schematic displaying the proposed consequences of peritoneal metastasis of gastric carcinomas,which include invasion of cancer cells from the mucosal layer to serosa, and the activation of cancer cells by CAFs during invasion. Activated cancercells increase DDR1 expression and are exfoliated into the peritoneal cavity as free-floating cells. These cells can form tumor nodules at the peritonealsurface. 7rh can reduce tumorigenesis at the peritoneal surface through inhibition of DDR1.

Jin et al.





the early stage of peritoneal metastasis, the floating cells dis-seminated from gastric carcinoma primary tumors must survivein the peritoneal cavity (35). In some tumor cells, DDR1directly enhances prosurvival mechanisms through inter-actions with Notch1 or the activation of NF-kB and the down-stream effector COX-2 (36, 37). Finally, the disseminated cellsaggregate into metastatic nodules on the peritoneal surface.DDR1 is known to play a critical role in supporting cell–celladhesion in a variety of noncancerous cells (38–40) andmalignant cell lines (41). Studies suggest that DDR1 stabilizesE-cadherin at the cell surface and promotes cell–cell aggrega-tion (38, 40). Although we have not proposed an exact mech-anism, our results support the hypothesis that the upregulationof DDR1 in gastric carcinoma cells is a critical step in theformation of tumor nodules from disseminated cancer cellson the peritoneal surface.

The efficacy of adjuvant intraperitoneal chemotherapy hasbeen studied for the prevention of peritoneal recurrence aftercurative resection in patients with locally advanced gastriccarcinomas. Cisplatin and mitomycin C have been used ascytotoxic agents for intraperitoneal administration, but theirsurvival benefits have been controversial (42). A meta-analysisshowed that the risk of postoperative abscess in the abdominalcavity was increased by 137% in patients with intra-abdominalchemotherapy relative to those receiving surgery alone (43).Those findings have resulted in physicians doubting the ben-efits of intraperitoneal chemotherapy for the prevention ofperitoneal recurrence in gastric carcinoma s. Based on thefindings of the present study, we suggest that the novel, orallyavailable drug 7rh may prevent the tumorigenesis of free-floating cancer cells in the abdominal cavity. In experimentsperformed by Gao and colleagues, 7rh potentially inhibited theproliferation, adhesion, and tumorigenicity of cancer cellsexpressing high levels of DDR1 and had a good pharmacoki-netic profile with oral bioavailability of 67.4% (44). Anothergroup described that 7rh could attenuate DDR1 expression innasopharyngeal cancer cells as seen in our data for gastriccarcinoma cells, and the authors suggested JAK1–STAT3 sig-naling pathway as the downstream target of DDR1 (45). In thepresent study, we also confirmed that 7rh potentially attenuatesCAF upregulation and phosphorylation of DDR1 using acoculture system. In addition, our in vivo studies revealed noadverse effects of the oral administration of 7rh, and we also

confirmed that 7rh reduced the expression of DDR1 in xeno-graft tumors. These results suggested that 7rh could be orallyadministered to patients after resection for primary gastriccarcinomas in order to prevent peritoneal recurrence in patientsat high risk through the downregulation of DDR1 levels.

Although this study has several limitations such as thenonvalidation of clinical data with an external cohort and theusage of only two types of gastric carcinoma cell lines, we canconclude that intratumoral stromal cells, particularly CAFs,enhance the upregulation of DDR1 in gastric carcinomas, andthat DDR1 is a key factor in CAF-induced tumorigenesis. Wesuggest that treatment with 7rh, a specific inhibitor of DDR1, inan adjuvant setting after curative resection might prevent peri-toneal recurrence of gastric carcinomas.

Disclosure of Potential Conflicts of InterestR.A. Brekken has ownership interest (including stock, patents, etc.) in and is

a consultant/advisory board member for Tuevol Therapeutics. No potentialconflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: R.A. Brekken, H. HurDevelopment of methodology: H. Jin, I.-H. HamAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): H.J. Oh, C.A. Bae, D. Lee, S.-U. HanAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): H. Jin, S.-Y. Son, H. HurWriting, review, and/or revision of the manuscript: H. Jin, S.-Y. Son,R.A. Brekken, H. HurAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H. Jin, Y.-J. ChwaeStudy supervision: Y.-B. Kim, S.-U. Han, H. Hur

AcknowledgmentsThis research was supported by the Basic Science Research Program

through the National Research Foundation of Korea (NRF), funded by theMinistry of Education (2016R1D1A1B03933083). This research was alsosupported by a grant from the Korea Health Technology R&D Projectthrough the Korea Health Industry Development Institute (KHIDI), fundedby the Ministry of Health and Welfare, Republic of Korea (HI17C0364).

The authors thank Professor Ding Ke at Jinan University provision of 7rhand Dave Primm for help in editing this article.

Received December 13, 2017; revised April 11, 2018; accepted May 30, 2018;published first June 4, 2018.

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2018;16:1590-1600. Published OnlineFirst June 4, 2018.Mol Cancer Res Hyejin Jin, In-Hye Ham, Hye Jeong Oh, et al. Stroma-Induced Peritoneal Metastasis in Gastric CarcinomaInhibition of Discoidin Domain Receptor 1 Prevents

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