Research Development of an Orthotopic Model of Invasive ... · Cancer Therapy: Preclinical Development of an Orthotopic Model of Invasive Pancreatic Cancer in an Immunocompetent Murine

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Published OnlineFirst June 9, 2010; DOI: 10.1158/1078-0432.CCR-09-2384

Cancer Therapy: Preclinical Clinical
Cancer
Research

Development of an Orthotopic Model of Invasive PancreaticCancer in an Immunocompetent Murine Host

William W. Tseng1,2, Daniel Winer1, Justin A. Kenkel1, Okmi Choi1, Alan H. Shain1, Jonathan R. Pollack1,Randy French3, Andrew M. Lowy3, and Edgar G. Engleman1

Abstract

Authors' APalo Alto, CSan FranciDivision ofMoores Ca

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Purpose: The most common preclinical models of pancreatic adenocarcinoma utilize human cells ortissues that are xenografted into immunodeficient hosts. Several immunocompetent, genetically engi-neered mouse models of pancreatic cancer exist; however, tumor latency and disease progression in thesemodels are highly variable. We sought to develop an immunocompetent, orthotopic mouse model ofpancreatic cancer with rapid and predictable growth kinetics.Experimental Design: Cell lines with epithelial morphology were derived from liver metastases ob-

tained from KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice. Tumor cells were implanted in the pancreas ofimmunocompetent, histocompatible B6/129 mice, and the mice were monitored for disease progression.Relevant tissues were harvested for histologic, genomic, and immunophenotypic analysis.Results: All mice developed pancreatic tumors by two weeks. Invasive disease and liver metastases were

noted by six to eight weeks. Histologic examination of tumors showed cytokeratin-19–positive adenocar-cinoma with regions of desmoplasia. Genomic analysis revealed broad chromosomal changes along withfocal gains and losses. Pancreatic tumors were infiltrated with dendritic cells, myeloid-derived suppressorcells, macrophages, and T lymphocytes. Survival was decreased in RAG−/− mice, which are deficient inT cells, suggesting that an adaptive immune response alters the course of disease in wild-type mice.Conclusions: We have developed a rapid, predictable orthotopic model of pancreatic adenocarcinoma

in immunocompetent mice that mimics human pancreatic cancer with regard to genetic mutations, his-tologic appearance, and pattern of disease progression. This model highlights both the complexity andrelevance of the immune response to invasive pancreatic cancer and may be useful for the preclinicalevaluation of new therapeutic agents. Clin Cancer Res; 16(14); 3684–95. ©2010 AACR.

Ductal adenocarcinoma of the pancreas has the worstprognosis of all common epithelial malignancies, withan overall 5-year survival rate of <5% (1). Surgical resec-tion offers the only hope for cure; however, <20% of pa-tients are eligible for surgery and most patients eventuallydevelop recurrence following resection (2). Gemcitabineand, more recently, erlotinib, an epidermal growth factorreceptor tyrosine kinase inhibitor, are the only Food andDrug Administration–approved agents used in the treat-ment of advanced pancreatic cancer. Unfortunately, thesetherapies typically prolong survival by only a few months,and all patients ultimately succumb to progressive disease

ffiliations: 1Department of Pathology, Stanford University,alifornia; 2Department of Surgery, University of California at

sco, San Francisco, California; and 3Department of Surgery,Surgical Oncology, University of California at San Diego,ncer Center, La Jolla, California

. Tseng and D. Winer contributed equally to this work.

ding Author: Edgar G. Engleman, Stanford Blood Center,w Avenue, Palo Alto, CA 94304. Phone: 650-723-7960; Fax:70; E-mail: [email protected].

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(2). The availability of appropriate animal models is there-fore essential for the preclinical evaluation of novel thera-peutic agents.Most preclinical mouse models of pancreatic adeno-

carcinoma utilize immunodeficient hosts into whichresected human tissues or established human cell lines(e.g., MiaPaCa2, PANC-1, BxPC3) are xenografted (3, 4).A major drawback of these models is the lack of an intactimmune system in the host animal, which alters the tumormicroenvironment and creates potentially significant dif-ferences from the human disease. This may in part explainthe poor track record of immunodeficient models in pre-dicting response to therapy in patients with pancreaticcancer (2). Immune cells that infiltrate tumors significant-ly impact the course of tumor growth and progression (5).Infiltrating immune cells have the potential to mount aneffector response to limit tumor growth, but this responseis often overcome by a combination of multiple factors,including the release of immunosuppressive cytokines bythe tumor (e.g., interleukin-10) and the recruitment of im-munosuppressive cell types (e.g., Gr-1+, CD11b+ myeloid-derived suppressor cells, tumor-associated macrophages,and FoxP3+ regulatory T cells; ref. 5). Immune cells can

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Translational Relevance

The most common preclinical models of pancreaticadenocarcinoma utilize human cells or tissues that arexenografted into immunodeficient hosts. In thesemodels, the tumor microenvironment bears little re-semblance to the natural milieu in which tumors typ-ically develop and metastasize in humans. Severalimmunocompetent, genetically engineered mousemodels of pancreatic cancer exist; however, tumor la-tency and disease progression in these models arehighly variable. Using tumor cells obtained from micewith targeted Kras and p53 mutations, we have devel-oped a rapid and predictable orthotopic model of in-vasive pancreatic cancer in immunocompetent mice.The tumors that develop in this model mimic the hu-man disease histologically and recapitulate importantclinical features of disease progression. This modelmay further our understanding of the biology of pan-creatic cancer and prove useful for the preclinical eval-uation of new therapeutic agents.

Immunocompetent Mouse Model of Pancreatic Cancer


also influence the formation and maintenance of tumor-supporting stroma, which in the case of pancreatic cancercan make up the bulk of the tumor and affect tumor resis-tance to chemotherapy and radiation therapy (6). Immu-nocompetent rat and hamster models of pancreatic cancerhave been reported (7, 8); however, compared with themouse, few reagents are available for identifying and iso-lating immune cells from these species.In 2003, Hingorani et al. described a genetically engi-

neered mouse model of pancreatic cancer in which expres-sion of oncogenic Kras-G12D was limited to pancreatictissue (9). The authors utilized a pancreas-specific promot-er (Pdx-1) to drive expression of Cre-recombinase, whichrecognizes and excises a Lox-flanked stop repressor ele-ment upstream of the mutant Kras-G12D allele. As a re-sult, these mice express mutant Kras-G12D driven by itsnative promoter resulting in the gradual development ofpancreatic intraepithelial neoplasia (PanIN) that slowlyprogresses to invasive ductal adenocarcinoma (9). In2005, the authors reported that progression to pancreaticcancer could be accelerated in mice by using the same Pdx-1-driven, Cre-Lox system to simultaneously induce expres-sion of Kras-G12D and a mutant form of the p53 tumorsuppressor gene (R172H; ref. 10). Of thesemice, designatedLSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre, 96% devel-oped pancreatic cancer and 63% were noted to have livermetastases (10); however, the time to the development ofinvasive disease varied widely, from 47 to 355 days (10).Tumor cells derived from liver metastases found in thesemice were initially used to develop cell lines for mutationalanalysis (10). These cells manifested mixed morphology inculture: some cells were predominantly epithelioid whereasothers seemedmesenchymal. Utilizing subclones with pure

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epithelial morphology (hereafter referred to as LM-P), wehave developed and characterized an immunocompetent,orthotopic mouse model of pancreatic cancer in which dis-ease develops consistently and with rapid and predictablegrowth kinetics.

Materials and Methods

AnimalsEight- to ten-week-old immunocompetent female B6/

129 mice, histocompatible (H-2b) with the tumor cellsused in this study, were obtained from Jackson Laborato-ries. Age-matched, immunodeficient female RAG−/− miceon the identical hybrid background were also obtainedfrom Jackson. All procedures were approved by the Insti-tutional Animal Care and Use Committee of StanfordUniversity.

Isolation of LM-P cells from liver metastases andin vitro cultureMetastatic tumors were isolated from the livers of

KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice and cell lineswere derived as previously described (10). Cells weregrown in DMEM (high glucose 4.5 g/L) supplementedwith 10% fetal bovine serum, 1 mol/L sodium pyruvate,2 mmol/L L-glutamine, 1× nonessential amino acids(Gibco), and 1× penicillin/streptomycin (100 U/mL pen-icillin, 100 ug/mL streptomycin). Cells were plated at lowdensity, specifically, ∼50,000 cells/100-mm tissue culturedish (Fisher), in the above growth medium. Individualcolonies displaying uniform epithelial morphology werecloned and subcultured. Individual colonies were then re-cloned for two more rounds. Purified cells (LM-P) wereamplified in T75 flasks and vials of cells were stored at−125°C. In preparation for in vivo implantation, frozencells were thawed and cultured in growth medium at37°C in a humidified, 10% CO2 incubator. Subconfluentcells were detached with 0.25% trypsin and cell viabilitywas assessed by trypan blue exclusion. LM-P cells weretested every six months for microbial contamination (in-cluding mycoplasma) using a PCR-based analysis andwere found to be negative.

Tumor implantation and monitoringTo establish s.c. tumors, 106 LM-P cells were resus-

pended in HBSS and injected under the skin overlyingthe right flank. To establish orthotopic tumors, mice werefirst anesthetized with a single i.p. injection of ketamine(100 mg/kg) and xylzine (10 mg/kg), and then the pan-creas was exposed through an abdominal incision (lapa-rotomy). Two techniques were used for orthotopic tumorimplantation: (a) direct injection of a single cell suspen-sion of LM-P cells into the pancreas or (b) transplantationof a s.c. LM-P tumor fragment onto the pancreas. For di-rect injection, 106 cells were resuspended in 50 uL HBSSand injected into the pancreas using a 30G needle (BD).Leakage occurred in <5% of mice, and only mice withoutleakage during injection were included in experiments. For

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s.c. tumor transplantation, 1-cm tumors were harvestedand minced into 3- to 4-mm fragments. One tumor frag-ment was secured to the pancreas using 7-0 Prolene su-ture. After tumor implantation (with either technique),the pancreas was carefully returned to the peritoneal cavityand the abdomen was closed with 4-0 Vicryl suture. Aftertumor implantation, all mice were monitored at leasttwice weekly for disease progression by abdominal palpa-tion and for overall signs of morbidity such as ruffled fur,hunched posture, and immobility. Moribund mice wereeuthanized by CO2 inhalation. For survival studies, micewere followed until death or euthanized by a blinded ob-server when signs of morbidity were evident.

Tumor progressionA separate cohort of mice that had undergone either LM-P

cell injection (n = 3) or LM-P tumor fragment transplantation(n = 3) underwent repeat laparotomy to directly measure tu-mor size at two, four, and six weeks after implantation. Tu-mor volume was calculated based on the formula forellipsoid volume, V = 4/3 × π × (a/2)(b/2)(c/2) where a, b,and c represent the maximal length, width, and height, inmillimeters, respectively.

Tumor implantation using PDA cell linesPDA1-1 and PDA3-5 cells were derived from primary tu-

mors found in the pancreas of KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice, using a similar protocol for isolation,purification, and microbial testing as described above forLM-P cells. Orthotopic tumor implantation into immuno-competent B6/129 mice was done, and disease progres-sion and tumor histology were assessed.

Histologic analysisPancreatic tumors, livers, and lungs were harvested from

mice at three and six weeks after orthotopic tumor implan-tation. Tissues were placed in 10% phosphate-buffered for-malin and 24 to 72 hours later were embedded in paraffin.Sections (4-5 μm) were dewaxed in three changes of xyleneand brought to water through graded alcohols. Representa-tive sections were stained with H&E. For immunohisto-chemistry, sections were antigen-retrieved in rodentdecloaker antigen retrieval solution (Biocare Medical) in-side of a pressure cooker. Peroxidase activity was blockedusing 3% hydrogen peroxide (Lab Vision). Sections weretreated for 20 minutes with serum-free protein blocker(Dako), then incubated for 2 hours with polyclonal rabbitanti-CK19 (AbCam) at 1/200, followed by detection usinga Rabbit onRodent Polymer detection kit (BiocareMedical),which consists of an antirabbit horseradish peroxidase–conjugated polymer, for 30 minutes. Color was developedwith 3,3 ‘-diaminobenzidine solution (Vector Labs), andsections were mounted with Faramount aqueous mountingmedium (Dako). Microscopic images were taken with aV2.4 Nuance Multispectral Imaging VIS Flex Camera system(CRI) using an Olympus BX51 microscope (OlympusAmerica Inc.). Magnification for all images was recordedat the eyepiece with a ×10 ocular objective.

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Comparative genomic hybridizationTumor genomic DNA was isolated using a Qiagen DNA/

RNA Allprep Mini kit. Array comparative genomic hybrid-ization (CGH) was done according to the manufacturer'sprotocol on catalog Agilent 44 K murine whole genomearrays. Briefly, 3 μg of genomic DNA from each test sam-ple were random primer-labeled with Cy5 and cohybri-dized to the microarray along with 3 μg of Cy3-labelednormal pancreas reference DNA from syngeneic mice. Tu-mor/normal log2 ratios were extracted using Agilent soft-ware (CGH Analytics), and gains and losses were calledusing the circular binary segmentation algorithm (11).

Immune cell isolation and surface marker stainingPancreatic tumors were minced using a sterile scalpel

blade, and single cell suspensions were generated using acombination of enzymatic digestion (600 U/mL collage-nase IV, Worthington) and mechanical dissociation (Gen-tleMACS Tissue Homogenizer, Miltenyi Biotec). Tumordebris was removed using 70- and 100-μm filters (BD Fal-con). Peripancreatic draining lymph nodes were harvestedand processed into single cell suspensions by dissociationover filters. Pancreas and draining lymph nodes from tu-mor-naïve mice were used as controls for comparison withtissue obtained from tumor-bearing mice. Cells from alltissues were washed with PBS containing 2% fetal bovineserum, Fc blocked (1:50 dilution, BioLegend), and thenstained using a cocktail of antimouse antibodies againstCD45.2 (PacBlue or Alexa700); lineage markers CD19,NK1.1, TCR (PE); B220 (PETxR); CD11c (PECy7); CD11b(PacBlue); Gr-1 (APCCy7); I-Ab (APC); CD3 (FITC); CD4(PECy7); CD8 (Alexa700). All antibodies were purchasedfrom BD Biosciences or BioLegend. Isotype-matched anti-bodies were used as negative controls when appropriate.Surface marker expression was determined by multicolorflow cytometry on an LSR II flow cytometer, and data wereanalyzed using FlowJo 8.7.1 software.

Intracellular staining for FoxP3Tumor-infiltrating T cells were identified with labeled

antibodies to T-cell surface markers and then permeabi-lized and fixed using the Mouse Regulatory T cell StainingKit (eBioscience). Intracellular staining was done usingantimouse FoxP3 (APC) versus isotype control antibody,as part of the Staining Kit.

Survival of immunocompetent versusimmunodeficient miceAfter establishing growth kinetics with 106 and lower cell

numbers, B6/129 (n = 6) and immunodeficient RAG−/−mice (n = 5) were orthotopically injected with 105 LM-Pcells and then monitored for survival.

Treatment of LM-P tumors with gemcitabineand LY364947For evaluation of in vitro cytotoxicity, LM-P cells were

first plated in quadruplicate (2.5 × 103 cells/well) onto96-well plates and allowed to grow for 24 hours. Escalating

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concentrations of gemcitabine (courtesy of Clinical Phar-macy, Stanford Hospital) or a transforming growth factor(TGF)-β receptor-1 kinase inhibitor (LY364947, Calbio-chem) were then added and MTT assay (R&D Systems)was done 48 hours later, as per the manufacturer's proto-col. For in vivo studies, orthotopic tumor implantation wasdone using later passaged (P8), more aggressive LM-Pcells. At two weeks, mice with similarly sized pancreatictumors (determined by palpation) were randomized toreceive PBS, gemcitabine alone, or gemcitabine plus theTGF-β receptor-1 kinase inhibitor. Gemcitabine was givenat 100 mg/kg by i.p. injection on days 0, 3, 6, and 9 afterrandomization. The TGF-β receptor-1 kinase inhibitor wasgiven at 1 mg/kg i.p. on days 0, 2, 4, 6, and 8.

Statistical analysisTumor volume comparisons were analyzed using the

paired Student's t-test. The frequency of tumor uptakeand metastases between groups was analyzed usingFischer's exact test. Survival differences between groupswere assessed with the log-rank test using GraphPadPrism 5.02 software. P < 0.05 was considered statisticallysignificant.

Results

Pancreatic tumor uptake is uniform and diseaseprogression occurs predictablyAfter either (a) injection of suspended LM-P cells into

the pancreas or (b) implantation of a s.c. LM-P tumor frag-ment onto the pancreas, tumors were noted as early as twoweeks and progressed predictably by four weeks (Fig. 1A).Comparison of average tumor volumes at two and fourweeks showed no statistically significant differences, sug-gesting similar growth kinetics with either technique(Fig. 1B). By six weeks, tumors in some mice became dif-ficult to measure owing to their extensive, locally invasivenature and poorly defined borders (Fig. 1C, left). At thisstage in disease progression, liver metastases were also fre-quently noted (Fig. 1C, right) and the mice appeared ill.Of note, some mice also developed biliary and gastric out-let obstruction during the course of disease progression(Fig. 1D). By eight weeks, mortality was consistently100% (data not shown).In total, pancreatic tumors developed in 100% of mice

following orthotopic implantation of LM-P cells (Table 1).Of these mice, 90% developed liver metastases whereas nomice developed peritoneal carcinomatosis or hemorrhagicascites. No significant differences were noted in the fre-quencies of uptake and metastases with either technique.Lung metastases were noted in some mice, particularlythose with more advanced disease; however, the frequencyof metastasis to this organ was not systematically evaluat-ed. Pancreatic tumors and liver metastases also developedwith <106 cells injected, but tumor volumes were initiallysmaller and disease progression occurred more slowly; incontrast, the use of cells passaged more frequently in vitro



generated pancreatic tumors with more rapid disease pro-gression (data not shown).

Pancreatic and metastatic tumor histology resembleshuman diseaseH&E staining of representative LM-P pancreatic tumors

showed adenocarcinoma with high-grade nuclear features,areas of necrosis and hemorrhage, and regions of desmo-plasia (Fig. 2A). Adjacent areas of poorly differentiated,solid sheets of cells were also noted, particularly in moreadvanced tumors. Cytokeratin-19, a marker expressed inmost human pancreatic ductal adenocarcinomas, was ex-pressed in tumors as shown by immunostaining (Fig. 2B).Glandular areas showed more consistent cytoplasmic CK-19 staining than poorly differentiated areas of the tumor.In many pancreatic tumors, perineural invasion was pres-ent (Fig. 2C) and peripancreatic lymph nodes were oftenheavily infiltrated by tumor (Fig. 2D). Histologic examina-tion of liver and lung metastases (Fig. 2E and F, respectively)confirmed the presence of adenocarcinoma.

Carcinomatosis and more aggressive diseaseprogression occur when pancreatic tumors aregenerated from primary tumor cell linesOrthotopic implantation of PDA1-1 and PDA3-5 cell

lines, which were derived from primary pancreatic tumors(as opposed to liver metastases for LM-P) in the KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice, led to pancreatic tumordevelopment in 100% of the mice (data not shown). Miceimplantedwith PDA1-1 cells had significantly worse surviv-al compared with mice implanted with PDA3-5 or LM-P(Fig. 1E). In contrast to LM-P–implanted mice (Table 1),all of the PDA1-1– and PDA3-5–implantedmice developedextensive peritoneal carcinomatosis (Fig. 1F) and onlyoccasionally, small liver metastases. On histologic analy-sis, PDA1-1 pancreatic tumors were poorly differentiated(Fig. 2G) compared with PDA3-5 (Fig. 2H) and LM-P(Fig. 2A), which correlated with the survival data.

CGH on tumor cells from different sourcesTo characterize our tumor model at the genomic level,

we carried out array CGH on in vitro propagated LM-P cells,a s.c. tumor derived from LM-P cells, and an orthotopic tu-mor derived from a transplanted s.c. tumor (Fig. 3). TheLM-P cells displayed several broad low-level gains andlosses along with one focal deletion on cytoband 4D1. Itis possible that these genomic aberrations might cooperatewith the Kras and p53mutations to transform cells. The s.c.tumor mirrored the LM-P cells with the exception of mod-est gains on chromosomes 5A1, 5G3, and 6A1. The ortho-topic tumor genome shared some features with the LM-Pcells and the s.c. tumor but also contained many new ge-nomic aberrations, including further gains on the ends ofchromosomes 5 and 6. Interestingly, among many othergenes, hepatocyte growth factor and its receptor, Met, residein these regions on chromosomes 5 and 6, respectively,which were further amplified with progression from in vitrocells to orthotopic tumors.




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Fig. 1. Early and late disease in our orthotopic model. At 2 weeks, LM-P tumor uptake is noted, with progressive growth at 4 weeks. Comparison ofthe two techniques for implantation (see Materials and Methods) reveals no difference in tumor appearance (A) or total tumor volume (B) at these early timepoints. C, by 6 to 8 weeks, mice develop extensive local growth into adjacent organs (dotted line, tumor invading into stomach) and liver metastases(open arrows). D, in some mice, clinically relevant disease manifestations, such as biliary obstruction (arrowheads, dilated common bile duct) producingjaundice (swab tip of bilious peritoneal fluid), and gastric outlet obstruction (*, massively dilated stomach) are seen. In comparison with LM-P, orthotopicimplantation of PDA cells results in more aggressive disease progression with decreased survival (for PDA1-1, E; *, P < 0.05) and development ofcarcinomatosis (for both PDA1-1 and PDA3-5, F).

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Immunophenotyping of tumor-infiltratingmononuclear cells reveals a compleximmune responseSingle cell suspensions obtained from processed LM-P

pancreatic tumor and draining lymph nodes three weeksafter orthotopic tumor implantation were analyzed byflow cytometry for surface marker expression. After gatingin CD45+ hematopoietic cells and gating out lineage-positive (CD19, NK1.1, TCR) cells, multiple expandedpopulations of CD11c- and CD11b-expressing cells werenoted in pancreatic tumors compared with the pancreasof tumor-naïve normal mice (Fig. 4A). Population 1(CD11c-high, CD11b-negative) and population 2(CD11c-high, CD11b-positive) were characterized as den-dritic cells as both populations also expressed high levelsof MHC Class II (I-Ab). A small proportion of cells inthese two dendritic cell populations also coexpressedCD86, an activation marker and T-cell costimulatoryfactor; however, the majority (80-90%) lacked CD86 ex-pression. Population 3 (CD11c-negative/low, CD11b-positive) did not express either MHC Class II or CD86(data not shown), but could be further subdivided intoGr-1–positive myeloid-derived suppressor cells (MDSC)and Gr-1–negative macrophages. Each of these three my-eloid cell populations was also expanded, but to a lesserextent, in the adjacent tumor-draining lymph nodes. Inboth the pancreatic tumors and draining lymph nodes,over two thirds of the T cells (CD45+ CD3+) wereCD4+ (Fig. 4B). A proportion (>20%) of these cellsexpressed CD25 and intracellular FoxP3, the phenotypeof regulatory T cells. A similar immune cell infiltrateconsisting of dendritic cells, MDSCs, macrophages, andregulatory T cells was noted in pancreatic tumor anddraining lymph nodes as early as 1.5 weeks after ortho-topic tumor implantation (data not shown). When com-pared with normal control tissues, no consistent changesin natural killer (NK) or B-cell populations were foundin either tumor or lymph nodes at 1.5 or 3 weeks (datanot shown).

Impact of an adaptive immune response on survivalWhen LM-P tumor cells were orthotopically implanted

into RAG−/− mice, which lack T and B cells, these miceshowed more rapid disease progression and, overall, sig-nificantly decreased survival compared with fully immu-nocompetent B6/129 mice (Fig. 4C).



Effect of gemcitabine and TGF-β receptor-1 kinaseinhibition on LM-P tumor growthTo evaluate the sensitivity of LM-P tumors to an agent

with known activity against human pancreatic cancer, wecultured these cells in the presence of gemcitabine. We alsostudied the effect of a TGF-β receptor-1 kinase inhibitor,LY364947, based on recent studies indicating a role forTGF-β in the growth of pancreatic tumors and developmentof metastases (12, 13). In vitro, gemcitabine resulted in sig-nificant cytotoxicity against LM-P cells at all concentrationstested. In contrast, some cytotoxicity was noted withLY364947 only at higher concentrations tested (Fig. 5A).In vivo, gemcitabine alone significantly improved survivalin mice with pancreatic tumors, an effect that was slightlyenhanced by coadministration of LY364947 (Fig. 5B).

Discussion

The development of a genetically engineered mousemodel of pancreatic cancer marks a significant turningpoint in preclinical model development in pancreatic can-cer (14). Other groups have shown that mutant Kras incombination with other targeted genetic mutations, includ-ing Ink4a/Arf and MUC1, can also accelerate the develop-ment and progression of pancreatic cancer (15, 16). Thesespontaneous mouse models of pancreatic cancer in immu-nocompetent mice have significant advantages over tradi-tional, immunodeficient xenograft models, in which boththe host environment and the local tumor microenviron-ment bear little resemblance to the natural milieu in whichtumors typically develop and metastasize in humans.Although our orthotopic implantation model is not a

spontaneous model, it has several advantages over geneti-cally engineered models. The most obvious advantage isthat the model is rapidly established without the needfor time-consuming and expensive breeding. Furthermore,the disease develops rapidly and predictably. In LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice, diseaselatency is highly variable, ranging from weeks to almostone year of age (10). With either direct tumor cell injec-tion or s.c. tumor fragment transplantation, all mice inour study developed palpable pancreatic tumors by twoweeks that progressed to advanced disease by six to eightweeks (Fig. 1). The rapidity and consistency of disease de-velopment in our model makes it possible to carry out

Table 1. Frequency of pancreatic tumor uptake, liver metastases, and peritoneal carcinomatosis six toeight weeks following orthotopic implantation of LM-P cells

Pancreatic tumor

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15 of 15 13 of 15 0 of 15 s.c. LM-P tumor fragment transplantation 15 of 15 14 of 15 0 of 15 Total (%) 30 of 30 (100%) 27 of 30 (90%) 0 of 30 (0%)
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Fig. 2. Histology of pancreatic tumors andmetastases. A. LM-P tumors form glandsand regions of desmoplasia (arrows).B, immunohistochemistry shows thatpancreatic tumors stain for cytokeratin-19,a ductal epithelial marker (brown,cytokeratin-19; blue, hematoxylincounterstain). Areas of perineural invasion(C; arrowheads, nerve; dotted line, tumorinvasion) and peripancreatic lymph nodeinfiltration (D; T, tumor; GC, germinalcenter) are also noted. Liver (E) and lung(F) metastases confirm adenocarcinoma.PDA pancreatic tumors show similarhistology to LM-P but are predominantlypoorly (1-1; G) or well differentiated (3-5;H). H&E, original magnification, × 100 forA and D to F, × 200 for B, C, G and H.


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time-dependent and large-scale studies in a resource- andcost-effective manner.Our orthotopic mouse model of pancreatic cancer is es-

tablished by surgical implantation of tumor cells into thepancreas of an immunocompetent host. Although othercell line–based immunocompetent mouse models of pan-creatic cancer exist (17, 18), the cells used in those modelsare toxin and virally induced. Close examination of reportsusing these models reveals that the tumor histology is pre-dominantly sarcomatoid, with tumors mainly composedof solid sheets of cells (19). In contrast, the cells used toestablish our model were “induced” by mutations in Krasand p53, mutations found in over 90% and 60% of hu-man pancreatic cancers, respectively (2). Histologic exam-ination of the tumors in our model showed regions ofclear glandular differentiation and some regions of desmo-plasia (Fig. 2A), which are characteristic of human pancre-atic cancer. Moreover, pancreatic tumors in our modelexpressed cytokeratin-19 (Fig. 2B), a marker expressed bymost human pancreatic ductal adenocarcinomas. Recently,Olive et al. also reported on the use of a mouse pancreaticcancer model generated by implantation of tumor cell linesderived from LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cremice (20); however, most mice had s.c. tumors, and onlyhuman cell lines (e.g., MiaPaCa2) were used to establishorthotopic xenograft tumors.With regard to initial disease development, when LSL-

KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice develop tu-mors, they often occur throughout the pancreas, as allductal epithelial cells within the organ harbor Kras andp53 mutations and are susceptible to malignant transfor-mation (10). In contrast, in orthotopic implantationmodels such as ours, the host pancreas cells are wild typeand tumors eventually invade into this otherwise normalparenchyma, similar to what occurs in human pancreaticcancer. A disadvantage of orthotopic implantation, how-ever, is that tumor development does not proceedthrough the typical stepwise progression of preinvasivestages (PanIN); thus, carcinogenesis may be better studiedusing the original LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice.



We chose cells derived from liver metastases found inthe LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre mice basedon the rationale that cells that have already metastasizedto the liver have a higher propensity to metastasize again,as shown in other mouse models of cancer (21, 22). In-deed, 90% of our mice implanted with LM-P cells devel-oped liver metastases (Table 1). In contrast, we observedthat mice with pancreatic tumors generated from PDA(primary tumor) cell lines had fewer liver metastases anduniformly developed carcinomatosis. Our findings suggestthat, depending on which cell line is used, different aspectsof pancreatic cancer disease progression may be highlight-ed for closer study, with the advantage of more rapid andpredictable kinetics compared with the original geneticallyengineered mice.The genome of human pancreatic cancer exhibits sub-

stantial complexity, and CGH array analysis of the pancre-atic tumors in our model indicates similar complexity. Inaddition to Kras and p53, it is well accepted that manymore genetic alterations are involved in pancreatic carci-nogenesis (23). Additionally, these genomic abnormalitiesincrease with disease progression (23). This progressive ge-nomic instability is mimicked in our model as revealed byCGH data obtained from in vitro cultured cells, s.c. tumors,and invasive pancreatic tumors (Fig. 3). The data point tointeresting genomic regions that may harbor genes in-volved in disease progression; however, due to the com-plex nature of the genome of pancreatic cancer, analysisof additional samples together with mechanistic studieson candidate genes will be required to confirm the identi-ties and roles of involved genes.Characterization of the immune cells found within the

tumors and draining lymph nodes in our model revealedthat the immune response to invasive pancreatic cancer ishighly complex and involves an infiltration of tumors bymultiple immune cell types (Fig. 4A, B). Some popula-tions, such as MDSCs, tumor-associated macrophages,and regulatory T cells, are known to inhibit effector T celland NK activity, allowing for tumor escape (5, 24, 25). Incontrast, dendritic cells, defined by their high expression ofMHC Class II and activation markers/T-cell costimulatory

Fig. 3. Comparative genomic hybridization. Shown are genomic profiles of copy number alteration for LM-P cells (bottom, green), a s.c. tumor (middle, blue),and an orthotopic tumor (top, red). Tumor/normal CGH log2 ratios (moving average 0.2 megabases) are plotted by chromosome position; gains and lossesappear as peaks and valleys, respectively. Selected sites of gain and loss (see text) are indicated.




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molecules (e.g., CD86), are the most potent antigen-presenting cells and are capable of orchestrating anadaptive antitumor immune response (26, 27). How-ever, poorly activated “tolerogenic” dendritic cells have



been shown to induce T-cell anergy, allowing for contin-ued tumor growth and progression (28, 29). Immuno-phenotypic analysis of the tumor-infiltrating dendriticcells in our model would suggest that, within the tumor

Fig. 4. LM-P tumors are infiltrated with distinct mononuclear cell types. A, three expanded populations are seen in pancreatic tumors and, to a lesser extent,in draining lymph nodes (dLN), based on surface marker expression: population 1, CD11c-high, CD11b-negative; population 2, CD11c-high, CD11b-positive; and population 3, CD11c-negative/low, CD11b-positve cells. The two CD11c-high populations (1 and 2) express MHC Class II, and a smallproportion of the cells also express CD86. The CD11c-negative/low CD11b-positive cells (population 3) are composed of Gr-1+ MDSCs and Gr-1− tumor-associated macrophages. B, the T-cell response in pancreatic tumors and draining lymph nodes is shifted toward CD4+ cells. Tumor-infiltrating CD4+T cells (+) include a distinct population of CD25+ FoxP3+ regulatory T cells. Numerical values represent CD4:CD8 ratios. Isotype controls (for CD86, FoxP3)are shown in gray. C, lymphocyte-deficient RAG−/− pancreatic tumor-bearing mice have decreased survival compared with fully immunocompetent mice(*, P < 0.05); however, both ultimately succumb to disease.






microenvironment, the majority of dendritic cells aretolerogenic; however, a small proportion of the dendriticcells present may be appropriately activated, based onCD86 expression (Fig. 4A).The results from the survival study (Fig. 4C) suggest

that, in the fully immunocompetent B6/129 mice, at leastsome degree of effective dendritic cell priming of T cellsagainst tumor likely occurs, leading to improved survivalcompared with T lymphocyte–deficient RAG−/− mice. Thisis supported by our finding of activated dendritic cells inthe tumor-draining lymph nodes (Fig. 4A and data notshown), which suggests that at least some of these cellssuccessfully trafficked to sites where priming of naïveT cells can occur. In time, however, all mice succumbed toprogressive disease, indicating that the adaptive immuneresponse is ultimately ineffective, perhaps thwarted bythe simultaneous presence of tolerogenic dendritic cells,MDSCs, macrophages, and regulatory T cells. In fact, theseparticular cell types have been identified as key elementshindering the efficacy of vaccine-based immunotherapyfor human pancreatic cancer (30).Clark et al . analyzed the immune response in

KrasG12D-expressing, p53 wild-type mice, which manifestprecursor PanIN that occasionally progresses to invasiveadenocarcinoma (31). They found, surprisingly, thatMDSCs and regulatory T cells were already present in the



early PanIN stages and continued to increase with diseaseprogression (31). The dendritic cell response was not ana-lyzed; however, the authors did note that about one thirdof intratumor CD4+ T cells had an expression profile(CD45RB-low and CD44-high) that suggests evidence ofdendritic cell priming and antigen experience (31). In an-other spontaneous, genetically engineered mouse modelof pancreatic cancer (TGF-α/p53 mutant), tumor-specificCD8+ T cells were noted (32); however, the presence ofactivated dendritic cells, which would likely contributeto this response, was also not assessed.To further assess the similarity of our model to human

pancreatic cancer, we evaluated the sensitivity of LM-P tu-mors to gemcitabine, a chemotherapeutic agent widelyused in the treatment of human pancreatic cancer (2).We also evaluated the effect of LY364947, a novel TGF-βreceptor-1 kinase inhibitor. TGF-β is one of several tumor-derived cytokines that are thought to induce extracellularmatrix deposition and fibrosis, creating the desmoplasticstroma characteristic of pancreatic cancer (6). Also, inhibi-tion of TGF-β signaling has been shown in a s.c. xenograft(BxPC3) mouse model of pancreatic cancer to potentiatedrug delivery by altering permeability at the tumor micro-environment level (12). Whereas gemcitabine was highlycytotoxic to LM-P cells in vitro and extended survival of tu-mor-bearing mice in our orthotopic, immunocompetent

Fig. 5. Treatment of LM-P tumors with gemcitabine and LY364947, a TGF-β receptor-1 kinase inhibitor (TGF-βR1 KI). A, in vitro, MTT assays show directcytotoxicity of gemcitabine but not TGF-βR1 KI against LM-P cells. B, in vivo, gemcitabine treatment results in improved survival, an effect slightlyenhanced by addition of TGF-βR1 KI. Gem, gemcitabine; *, P < 0.05 for Gem versus PBS control; **, P < 0.05 for Gem + TGF-βR1 KI versus Gem alone).




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model, LY364947 had little or no antitumor activity as asingle agent in vitro but resulted in modest improvementin survival when used in combination with gemcitabine(Fig. 5). These results are consistent with the concept thatTGF-β inhibition likely acts on surrounding tumor stroma.It will be interesting to determine if optimizing the dose ofthis agent results in greater efficacy.Our preliminary findings with gemcitabine and TGF-β

inhibition, however, should be approached with somecaution. Olive et. al. showed recently that due to differ-ences in tumor-supporting stroma and microvasculature,transplant models of pancreatic cancer were much moresensitive to gemcitabine than the original genetically en-gineered mice (20). Although desmoplasia was noted inpancreatic tumors in our model (Fig. 2A), the extentand pattern of fibrosis may not be as profound as inthe genetically engineered mice or in humans. Our re-sults with gemcitabine and TGF-β inhibition will likelyneed to be further validated in the original geneticallyengineered mice; nonetheless, we envision that ourmodel can serve as a complementary resource- andcost-effective screening tool for preclinical testing ofnew therapeutic agents.In conclusion, we have developed a rapid, predictable

model of pancreatic cancer by orthotopic implantation



of tumor cells into immunocompetent mice. Like thehuman disease, tumor cells have both Kras and p53mutations, and the tumors that develop from these cellshave a histologic appearance and pattern of disease pro-gression similar to the human disease. Genomic analysissuggests that this model may help to reveal new genesinvolved in disease progression. Immunophenotypicanalysis highlights the complexity of the immune re-sponse to pancreatic cancer. Our hope is that this modelwill further the understanding of pancreatic cancerbiology and prove useful for the preclinical evaluationof new therapeutic agents.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Grant Support

NIH grants CA141468-01 (E.G. Engleman), CA112016 (J.R. Pollack),and an SGF and NSF fellowship (A.H. Shain).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

Received 09/16/2009; revised 04/30/2010; accepted 05/27/2010;published OnlineFirst 06/09/2010.

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2010;16:3684-3695. Published OnlineFirst June 9, 2010.Clin Cancer Res William W. Tseng, Daniel Winer, Justin A. Kenkel, et al. Cancer in an Immunocompetent Murine HostDevelopment of an Orthotopic Model of Invasive Pancreatic

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