The Pancreas Cancer...

The Pancreas Cancer Microenvironment

Christine Feig1, Aarthi Gopinathan1, Albrecht Neesse1, Derek S. Chan1, Natalie Cook1,2, andDavid A. Tuveson1,2,3

AbstractPancreatic ductal adenocarcinoma (PDA) is a common and lethal malignancy resulting in more than

250,000deaths per yearworldwide.Despite extensive efforts, cytotoxic and targeted therapies have provided

only limited efficacy for patients with PDA to date. One contributing factor to the failure of systemic

therapies may be the abundant tumor stromal content that is the characteristic of PDA. The PDA stroma,

aptly termed the tumor microenvironment, occupies the majority of the tumor mass, and consists of a

dynamic assortment of extracellular matrix components and nonneoplastic cells including fibroblastic,

vascular, and immune cells. Recent work has revealed that the PDA stroma supports tumor growth and

promotes metastasis and simultaneously serves as a physical barrier to drug delivery. Accordingly,

methods that alter stromal composition or function, for instance interference with the vasculature via

Notch/Hedgehog pathway inhibition or relief of vascular compression by hyaluronidase, are under active

investigation. Here, we will review our current understanding of the PDA tumor microenvironment, and

highlight opportunities for further exploration that may benefit patients. Clin Cancer Res; 18(16); 4266–76.

�2012 AACR.

Tumor Microenvironment—Achilles' Heel ofPancreatic Cancer?

Pancreatic ductal adenocarcinoma (PDA) is an aggressivemalignant disease of the exocrine pancreas with a 5-yearsurvival rate of less than 5% (1). In the United States, itrepresents the fourth-leading cause of cancer-related deathswith an estimated 43,920 new cases and 37,390 deaths in2012 (2). The majority of patients initially present withadvanced and metastatic disease with only 10% to 15% ofpatients being candidates for surgical resection. Unfortu-nately, postsurgically, most patients still relapse despiteadjuvant systemic therapies (3). This dismal prognosis isa result of the late diagnosis of the disease, the lack ofbiomarkers allowing early screening, the early metastaticdissemination, and ultimately the resistance to systemictherapies.

Recent years have seen significant advances in the treat-ment for many tumor types, including melanoma, lung,and colorectal cancer based on the rational design of tar-geted therapies directed at molecular alterations arising incancer cells (4). Unfortunately, similar success has notoccurred in PDA, which remains a lethal disease. Gemcita-

bine, the current standard-of-care chemotherapeutic, wasapproved mainly on the basis of patient benefit and pro-duced only a modest increase in survival (5). Even targetedtherapy approaches have had limited success so far.Indeed, the only other drug approved is for the EGF recep-tor (EGFR) tyrosine kinase inhibitor erlotinib (Tarceva;Genentech), which, when combined with gemcitabine,increased overall the survival from 5.91 to 6.24 months(6). A promising classical combination chemotherapyapproach recently reported is FOLFIRINOX (oxaliplatin,irinotecan, leucovorin, and 5-fluorouracil), which achiev-ed a significant survival benefit for patients with metastaticPDA compared with gemcitabine (11.1 vs. 6.8 months;ref. 7). Unfortunately, FOLFIRINOX is only suitable forpatients with a good performance status due to increasedtoxicity. Therefore, new approaches are sorely needed forthe vast majority of patients with PDA.

What are the reasons that most conventional and tar-geted therapies fail to provide substantial response rates inpancreatic cancer? The challenges faced by oncologists inthe treatment of pancreatic cancermay in part be explainedby the diverse influences exerted by the microenvironmenton the cancer cells. Intriguingly, there is a huge discrepancybetween the relative success and effectiveness of therapies,including gemcitabine, reported in preclinical assays (cellculture and xenograft mouse models) and subsequentfailure in human PDA (8). Revealing the underlyingmolecular mechanisms of the microenvironment–tumorcell cross-talk is challenging due to the heterogeneousnature of the PDA stroma. Importantly, the generation ofgenetically engineered mouse models (GEMM) for pan-creatic cancer that faithfully recapitulate the human dis-ease, including resistance to gemcitabine, has enablednew approaches to understand the importance of the

Authors' Affiliations: 1Li Ka Shing Centre, Cambridge Research Institute,Cancer Research UK; 2Department of Oncology, Cambridge UniversityHospitals NHS Foundation Trust, Cambridge, United Kingdom; and 3TheLustgarten Foundation Pancreatic Cancer Laboratory at Cold Spring Har-bor, Cold Spring Harbor, New York

Corresponding Author: David Tuveson, Li Ka Shing Centre, CambridgeResearch Institute, Cancer Research UK, Robinson Way, Cambridge CB20RE, United Kingdom. Phone: 44-1223-404190; Fax: 44-1223-404199;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-3114

�2012 American Association for Cancer Research.

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Clin Cancer Res; 18(16) August 15, 20124266

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tumor microenvironment (TME) in disease pathogenesisand therapeutic response (9–11). These GEMMs arefounded on early genetic analyses that revealed the pres-ence of a single point mutation in the KRAS oncogene inmore than 90% of the human PDA specimens (12).Subsequent genetic manipulation of the orthologousgene in mice showed that this mutation was sufficientto initiate the formation of premalignant ductal transfor-mation (pancreatic intraepithelial neoplasia, PanIN). Fur-ther studies showed that the loss or mutation of tumorsuppressor genes commonly acquired during human dis-ease progression (Tp53 and Ink4a/Arf) cooperates withKras in mice to promote invasive cancer (13–15). Moreinsight into the underlying genetic alterations in pancre-atic cancer is provided by Iacobuzio-Donahue and col-leagues in this CCR Focus section (16).

Pancreatic ductal adenocarcinoma is one of the moststroma-rich cancers. It is not uncommon for stromal com-ponents to outnumber cancer cells, as illustrated in Fig. 1.PDA stroma is very heterogeneous and comprises cellularand acellular components, such as fibroblasts, myofibro-blasts, pancreatic stellate cells, immune cells, blood vessels,extracellular matrix (ECM), and soluble proteins such ascytokines and growth factors. The TME is not a static entity;rather, it is constantly changing in composition, especiallyin the progression from preneoplastic PanIN to invasivePDA. We aim to outline the current evidence for TMEinfluences on multiple aspects of PDA. These include pro-liferation and survival,metastasis, resistance to therapy, andescape from immune control. We have limited our analysisto those studies carried out in the most relevant GEMMs ororthotopic tumor models as well as clinical data because

© 2012 American Association for Cancer Research

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Figure 1. PDA contains an abundant stroma. A,mouse PDA tissuewas stainedwith picrosirius red and imaged under polarized light to visualize collagen fibers(�20 magnification). B, Masson trichrome staining highlights connective tissue distribution in an example of mouse PDA. Collagen fibers are staining in bluewith the cytoplasm appearing in red (�40 magnification). C, immunohistochemistry for SPARC on murine PDA (a-SPARC polyclonal antibody, proteintech catalog number: 15274-1-AP,�10magnification). D, histochemical staining of hyaluronan (biotinylated hyaluronan binding protein, Calbiochem385911,�10 magnification) reveals pan-stromal deposition in murine PDA.

TME in Pancreatic Ductal Cancer

www.aacrjournals.org Clin Cancer Res; 18(16) August 15, 2012 4267



xenograft/allograft models have a fundamentally differentcomposition and functionality of the microenvironmentincluding dramatic differences in the ECM components,immune cells, and tumor vasculature. This fact must beconsidered in the design of future research and interpreta-tion of published data.

Stromal Fibroblasts—The Pancreatic StellateCell

Pancreatic stellate cells (PaSC) were identified in 1998 asa rare stromal cell type in the healthy pancreas (17, 18).Their periacinar star-shaped morphology, characteristicmarker protein expression, and storage of fat droplets richin vitamin A resembled hepatic stellate cells and inspiredthe name. Under homeostatic conditions, PaSCs are qui-escent, and their physiologic role has yet to be delineated.Acute and chronic inflammatory conditions cause acti-vation of PaSCs, which is characterized by morphologicchanges, increased proliferation, deposition of ECM, andexpression of a-smooth muscle actin (a-SMA) as well asthe loss of the fat droplets (19). On the basis of theobservation that activated PaSCs are detected in areas withhigh collagen content, it was postulated that PaSCs may becausally involved in the pathogenesis of pancreatic fibrosis(20). Although the embryonic origin of PaSCs has not yetbeen addressed, mesenchymal cells in the bonemarrow area likely source for PaSCs in adult mice after injury (pancre-atitis and partial pancreatectomy) and in 7,12-dimethyl-benz(a)anthracene (DMBA)-initiated sarcomatoid pancre-atic tumors (21–23).

The scarcity of PaSCs and their limited life span in culturehas prompted the generation of immortalized PaSC linesfrom human, rat, and mouse pancreata (24–30; Table 1).Such immortalized PaSCs have enabled the dissection ofimportant cross-talk pathways between PaSCs and neoplas-tic PDA cells by coculturing inmonolayers or 3-dimension-al models. Indeed, it was recently reported that all-transretinoic acid induced the quiescence of PaSCs, and this ledto decreased proliferation and survival of pancreatic cancercells in 3-dimensional coculture and in a GEMM (31).Therefore, PaSCs represent a resource thatmaybeharnessedto explore the tumor-promoting aspects of tumor fibro-blasts in PDA.

Cocultures of PaSCs and PDA cells have generally shownan enhancement of pancreatic cancer cell proliferation andmigration by release of growth factors and cytokines (32).In vivo studies corroborate those findings, revealing that thecoinjection of PaSCs with tumor cells in orthotopic modelsof PDA increases tumor size and causes ahigher incidence ofmetastasis (25, 33). In a subsequent study, Xu and collea-gues investigated the role of PaSCs in the metastatic processand found that theyorchestratemetastatic dissemination bycomigratingwithneoplastic cells to potentially establish theappropriate metastatic niche or "soil" (34). Two recentpublications may provide an additional explanation for theenhanced tumorigenicity of tumor cell/PaSC cotransplants.In vitro experiments showed that PaSCs increase the stemcell phenotype of pancreatic cancer cells and suggest thatpharmacologic targeting of PaSCs could have unrecognizedadditional benefits (35, 36). The identification of majorsignaling pathways activated inPaSCs in response to contactwith cancer cells will be an interesting platform onwhich todevelop therapies targeting PaSCs. For instance, the mito-gen-activated protein kinase pathway plays a prominentrole in the response to mitogenic stimuli of which platelet-derived growth factor (PDGF) seems most potent (37, 38).Other potential targets include stimulators of the fibrino-genic program such as fibroblast growth factor, downstreameffectors generated by transforming growth factor b, con-nective tissue growth factor, and EGF. Additional insightsmay be gleaned through investigation of pathways knowntobe relevant forhepatic stellate cell activation (39). Finally,targeting specific pathways germane for PaSC–neoplasticcell cross-talk may also modulate other aspects of the TME,including the vascular and immune system.

Extracellular Matrix as an Obstacle to TherapyPancreatic ductal adenocarcinoma is histologically char-

acterized by the abundance of ECM, commonly alsoreferred to as desmoplasia (Fig. 1A–D). ECM componentsinclude collagen, fibronectin, proteoglycans, and hyaluro-nic acid, as well as catalytically active enzymes and protei-nases. The accumulation of ECM components distorts thenormal architecture of pancreatic tissue inducing an abnor-mal configuration of blood and lymphatic vessels (40–42).One factor potentially contributing to therapeutic resistance

Table 1. Overview of published pancreatic stellate cell lines of human, mouse, or rat origin

Name of cell line Source Method of immortalization Reference

RLT-PaSC Primary human from chronic pancreatitis SV40 large T antigen þ hTERT 2625HPaSC Human PDA SV40 large T antigen þ hTERT 25PS-1 Human pancreas (transplantation tissue) hTERT 24irPaSC Sprague–Dawley rat SV40 large T antigen 28SAM-K Male Wistar rat SV40 large T antigen 29SIPS Male Wistar rat Spontaneous immortalization 27LTC-7 and LTC-14 Male LEW.1W SV40 large T antigen 30imPaSC C57BL/6 mouse SV40 large T antigen 28

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in PDA may be the rigidity of the ECM that compressesblood vessels, leading to reduced perfusion that ultimatelyimpedes the delivery of drugs to neoplastic cells. Indeed, wepreviously reported that the concentration of an activeintracellular metabolite of gemcitabine, 20,2-difluorodeox-ycytidine triphosphate (dFdCTP), was high in stroma-poorsubcutaneous or orthotopic xenografts/syngeneics buthardly detectable in stroma-rich PDA tumors in a GEMM(11). Further analysis revealed that transplanted tumorsexhibited an increased vascular content and function ascompared with primary GEMM tumors and human PDA.Because sonic hedgehog (SHH) signaling has been shownto be restricted to the stromal compartment and to enhancethe desmoplastic reaction (43, 44), our laboratory reasonedthat pharmacologic inhibition of the Shh pathway mayhave a positive impact on gemcitabine delivery. As pre-dicted, the combination of a smoothened inhibitor (IPI-926) with gemcitabine caused depletion of tumor stromaand resulted in increased microvessel density and patency(11). This alteration of the TME was paralleled by signifi-cantly enhanced intratumoral concentrations of dFdCTP,transient disease stabilization, and a survival benefit (11).Several clinical trials have been initiated as a result of thisandare recruitingpatients to investigate themechanismandtreatment effect of pharmacologic SHH inhibitors inpatients with pancreatic cancer (http://clinicaltrials.gov;NCT01195415, NCT01064622, NCT01130142, andNCT01096732). Unfortunately, Infinity Pharmaceuticalsannounced in January 2012 that it was halting its phase IItrial of the smoothened inhibitor IPI-926 plus gemcitabine(NCT01130142). This is very surprising in light of theencouraging results of 31% partial response rate (10% forgemcitabine only) reported in a previous phase Ib trial(ASCO 2011, abstract 4114). An analysis of this trial isunder way by the investigators conducting this trial.Secreted protein acidic and rich in cysteine (SPARC)

represents another proposed target to facilitate depletionof the tumor stroma in pancreatic cancer. SPARC is over-expressed by fibroblasts in the TME of human and murinePDA (Fig. 1C) and has been shown to inversely correlatewith survival (45, 46). A novel drug formulation consistingof paclitaxel associated with albumin [Abraxane (Celgene)or nab-paclitaxel] has been hypothesized to accumulatein and potentially deplete PDA tumor stroma via bindingof albumin to SPARC-positive fibroblasts, thus represent-ing a mechanism for targeting a specific cell type withinthe PDA TME (47). The first clinical trial of gemcitabinein combination with nab-paclitaxel showed a promisingoverall survival of 12.2 months, and the subset of pati-ents with elevated SPARC expression was correlated withincreased survival in this study. The potential role of SPARCas a predictive biomarker for positive responsive to nab-paclitaxel and gemcitabine contrasts with a separate reportthat showed poor prognosis for patients who had SPARC-enriched tumors resected and received standard adjuvanttherapy (45, 46). In this trial, patients with high SPARClevels had a mean overall survival of 17.8 months ascompared with 8.1 for low SPARC (48). A preclinical study

in a GEMM from our laboratory confirmed the remarkableefficacy of nab-paclitaxel in combination with gemcitabine,although in contrast to the data originating from patient-derived xenografts, we did not observe stromal depletion inour preclinical setting. Instead, we reported a mechanisminvolving impaired gemcitabinemetabolismdue to reactiveoxygen species–mediated degradation of cytidine deami-nase (49). Furthermore, in depth investigations are requiredto elucidate the exact role of SPARC as a novel biomarker forpatients with PDA, in particular, whether treatment withnab-paclitaxel represents the sole determinant for its prog-nostic impact.

Another possible strategy to relieve vessel compressionand aid drug delivery is to enzymatically break down theECM scaffold. Many cancers are rich in hyaluronan (HA), amegadalton glycosaminoglycan that retains water due toits high colloid osmotic pressure (Fig. 1D; ref. 50). Thisprovides elasticity to connective tissue in healthy organs,but excessive HA accumulation in solid tumors raises inter-stitial fluid pressure and compresses blood vessels. Weand others have recently shown in a GEMM of PDA thatenzymatic remodeling of the ECM is indeed a promis-ing avenue. Hyaluronan degradation by hyaluronidasePEGPH20 decreased interstitial fluid pressure in murinePDA tumors (51), as shown previously in a prostate cancerxenograft model (52). Consequently, increased vesselpatency, drug delivery, and survival were observed (51, 53).It will be interesting to see if this concept holds true inhuman pancreatic cancer when the results of an ongoingphase I/II trial of hyaluronidase (PEGPH20) plus gemci-tabine will be released (NCT01453153).

TheConundrumofHypovascularity in PancreaticCancer

As alluded to in theprevious paragraph, the vasculature inPDA is profoundly affected by the excessive desmoplasia. Asa consequence, vascular dysfunction represents a majorobstacle to pharmacodelivery. Moreover, how cancer cellsmaintain their nutrient demands to fuel the rapid growthdespite the lack of adequate perfusion is elaborated uponin the accompanying article by Le and colleagues in thisCCR Focus section (54). The discovery of the hypovascular-ity and perfusion impairment has broken with the generalassumption of an "angiogenic switch" required for tumorprogression (55, 56). Unlike pancreatic neuroendocrinetumors, which are clearly dependent on angiogenic factors,their exocrine counterparts seem to thrive without therequirement for excessive angiogenesis (11, 57). In fact,hypovascularity and perfusion impairment have longserved as diagnostic tools in the imaging of pancreaticmasses (58, 59), but mechanisms behind these histopath-ologic features have not been fully elucidated.

Transcriptional analysis has previously shown a gradientof angiogenic activation fromnormal pancreas to PDA (60).Consistent with this finding, components of the prototyp-ically angiogenic VEGF pathway are highly expressed intumor cells and associated endothelia (61, 62).Despite this,





tumor samples show substantially lower microvessel den-sities (MVD) than those of the normal pancreas (Fig. 2;ref. 11, 57). Although VEGF immunostaining is positivelycorrelated with MVDs, it has limited association withpatient survival (63, 64). Despite these contradictory find-ings, antiangiogenic therapy was tested in PDA. Initialapproaches targeted matrix metalloproteinases using mar-imastat and BAY 12-9566 as well as the integrins aVb3 andaVb5 using cilengitide. These compounds did not provideany clinical benefit in trials (65–67).More recently, targetedagents such as bevacizumab, an anti-VEGFA monoclonalantibody, have been investigated in advanced pancreaticcancer in combination with gemcitabine and did notimprove survival compared with gemcitabine plus placeboin a randomized phase III trial (5.8 vs. 5.9 months; ref. 68).In addition, there was no significant benefit to overallsurvival in combining bevacizumab with erlotinib andgemcitabine compared with the combination of the latter2 compounds (7.1 vs. 6.0months; ref. 69).Bevacizumabhasalso been evaluated in other combinations including withdocetaxel and with concurrent capecitabine and radiationwithout any proven benefit, although certain studies arestill under way (70, 71). VEGF receptor inhibition usingaxitinib in combination with gemcitabine also had nobeneficial effect on overall survival (72). In addition, thekinase inhibitor sorafenib, which targets VEGFR as well asPDGFR, c-KIT, Raf1, and FLT3, was found to be inactive inadvanced pancreatic cancer (73). Likewise, discouragingresults testing sunitinib in a preclinical trial in theLSL-KrasG12D;p53R172H/þ;Ptf1a-Cre model add to mountingevidence of angiogenic independence and dominance oftumor-driven angiostasis of PDA (57). This phenotypesuggests that endogenous inhibitors in the microenviron-ment might exert an overriding angiostatic effect during the

natural history of PDA. Many of these factors are generatedfrom plasma and ECM proteins by proteases, which arefrequently upregulated in tumor and stellate cells (PaSCs;ref. 74). For example, angiostatin and endostatin are pro-duced by PDA and detected at high concentrations inpatients’ circulation (75, 76). Moreover, whereas activatedPaSCs are ostensibly proangiogenic, their coculture withtumor cells robustly increases endostatin levels, showing theangiostatic potential of such heterotypic interactions (77).

On the whole, antiangiogenic therapies have proved notto be a viable option for pancreatic cancer, and althoughtherapeutic delivery may contribute to these failures, alter-native approaches targeting the PDA vasculature remainattractive and potentially feasible. Two scenarios are possi-ble: If PDA maintains itself on frugal use of restrictedresources as a consequence of limited perfusion, it may beconceivable that impairing perfusion even more could tipthe balance toward widespread hypoxic necrosis (78). Incontrast, increasing tumor perfusion may seem counter-intuitive but could synergize with cytotoxic therapy toincrease the intratumoral drug delivery and response.

Is PDA Hypoxic?The previous paragraph laid the groundwork for this

question.Most solid tumors contain areas of below-optimaloxygen concentration (hypoxia). This occurs as a result ofinefficient tumor vascular supply and a highmetabolic needfor oxygen (79). Many studies have provided evidence thathypoxic cells are more resistant to both chemotherapy andradiotherapy and can increase their invasive and metastaticpotential, ultimately creating a more aggressive disease (80,81). The ability of cancer cells to surviveunder thesehypoxicconditions results from the ability to co-opt pathwaysnecessary for embryonic development under hypoxic con-ditions. Themainpathway involved in thehypoxic responseis the hypoxia-inducible factor (HIF) pathway (82). HIF caninduce a wide range of gene products controlling cellularmetabolism and energetics, cell survival, migration, and pH(83). The HIF transcription factors also direct the transcrip-tion of many angiogenic growth factors (84).

Considering that the hypovascular nature of PDA has asignificant impact on perfusion and drug delivery (11), itwould be reasonable to assume a hypoxic state. However,direct evidence is sparse and the majority of publicationshave used surrogatemarkers formeasuring hypoxia, such asnecrosis or expression of HIF target genes (85–87). Onlyone small study, involving 7patientswithpancreatic cancer,has directly measured the oxygen pressure during pancrea-ticoduodenectomy by inserting needle electrodes. Thisstudy revealed a dramatic reduction in oxygenation oftumor tissue versus normal pancreas (88). Interestingly,preclinical work in an orthotopic model of pancreaticcancer has shown a lack of correlation between MVD andhypoxia, perhaps suggesting that a hypovascular pancreatictumor is not directly linked to hypoxia (89). Nevertheless,this work did predict more aggressive behavior, including amore metastatic phenotype in hypoxic tumors. Clinical


T

PT

Figure 2. Murine PDA is characterized by hypovasularity. In this exampleof CD31 immunohistochemistry, the dotted line denotes the boundarybetween tumor (T) and peritumoral diseased pancreas (PT; �20magnification).

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work is under way to assess the prognostic significance ofthese results in patients with pancreatic cancer, using thehypoxia probe, pimonidazole, administered 24 hoursbefore surgery (NCT01248637).The reason hypoxia poses a challenge to the field of

anticancer therapeutics is that it provides a niche forslow-cycling, highly drug-resistant cells, which may beidentical to the proposed cancer stem cells (90, 91). Thus,standard chemotherapy agents fail because they are unsuc-cessful at targeting the cell within the hypoxic TME, whichmight be those that most need to be eliminated (92). Inaddition, hypoxic conditions are also known to stimulatethe Notch signaling pathway, and it has recently beenshown that pancreatic cancer cells can be sensitized byNotch inhibition (78). Thus, the hypovascular state of PDAcould be exploited by novel therapeutic approaches such ashypoxia-activable prodrugs (93). Further investigations arerequired into the downstreamNotch targets that are impor-tant for tumor cell survival under hypoxic conditions.Currently, a clinical trial is investigating the benefit ofcombining Notch pathway inhibition and gemcitabine(NCT01232829 and NCT01098344).

Out of Balance—Immune Cells in PDAAs is the case with other cancer types, inflammation

seems to be crucially linked to PDA development as exem-plified by chronic pancreatitis being amajor risk factor (94).However, the molecular details are still obscure and arejust beginning to be elucidated (95, 96). A comprehensiveanalysis of the immune cell composition of PanIN and PDAin the LSL-KrasG12D;Pdx1-Cre mice defined an importantbaseline for future studies (97). Use of enzymatic tumor

digestion followed by fluorescence-activated cell sortinganalysis revealed that immune cells make up roughly50% of the tumor cell mass (Fig. 3 illustrates similarfindings by immunofluorescence). From this study, it is


x, y:100 μm

Figure 3. Prominent immune cell infiltration exists in mouse PDA. Thisimmunofluorescence staining illustrates the abundance of immune cellsmarked by CD45 expression (red) between neoplastic glandularstructures (stained for EpCam in blue) and a-SMA positive stromalfibroblasts and perivascular cells that likely represent pericytes (green;�20 magnification).


A B

Normal O2 Low O

2 Low O2

dFdCTPdFdCTP�

Normal O2

CSC CSC

α-CD40

nab-paclitaxel

PSC/CAF

PaSC/CAF

ECM ECM

Hyaluronidase

nab-paclitaxelBlood vessels

Hedgehoginhibitors

Blood vessels

Immunecells

Immunecells

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Figure 4. Schematic of the TME network, cross-talk, and interdependence in PDA with a focus on therapeutic intervention points. A, activated pancreaticstellate cells lay down vast amounts of ECM, which causes a constriction/collapse of the sparse vessel network. This impedes gemcitabine delivery.Hypoxia generates niches for slow-cycling cells that are not targeted by chemotherapeutics. Also, an immunosuppressivemicroenvironment further supportstumor growth. B, Hedgehog pathway inhibition causes stromal depletion accompanied by reduced ECM. The ECM can also be enzymatically targeted, andboth interventions lead to increased vessel patency and intratumoral gemcitabine delivery. The immune system can be stimulated to turn against cancer cells,for instance by anti-CD40 antibody treatment. CAF, cancer-associated fibroblast.





apparent that immunosuppressive cell types, such as reg-ulatory T cells and myeloid-derived suppressor cells, arepredominant, with hardly any CTLs infiltrating thetumors. This paints a picture of a striking imbalance inprotumorigenic and antitumorigenic immune cells. Toadd to the complexity, a recent study revealed a newimmunosuppressive cell type in the stroma of PDA andother cancers. This cell expresses fibroblast activationprotein a (FAPa), and FAPa cell ablation resulted inimmunologic control of tumor growth in several subcu-taneous tumor models (98).

Successful immunotherapy depends on the cancer cellsexpressing proteins that can be recognized as altered bythe immune system. These fall in 2 categories: Tumor-associated antigens are nonmutated self proteins that areaberrantly regulated (overexpressed or expressed in othertissues or oncofetal antigens), whereas tumor-specific anti-gens are generated as a consequence of the mutationalevents in neoplastic cells and are de novo antigens. The goal

is to induce high-affinity cytotoxic T cells (CTL or CD8T cells) without causing autoimmunity. Antigens targetedin immunotherapy clinical trials in PDA have includedMUC1, mesothelin, KRAS, carcinoembryonic antigen, sur-vivin, and telomerase, as well as whole tumor cells engineer-ed to express granulocyte macrophage colony-stimulatingfactor [GM-CSF; reviewed in Dodson and colleagues (99)].

In the first phase I clinical trial using irradiated allogeneicGM-CSF–expressing tumor cell vaccines the treatmentwas well tolerated and found to be safe for use in humans(100). This result warranted a larger phase II trial to inves-tigate the disease-free and overall survival after surgicalresection followed by chemoradiation and vaccination,which reported a median survival of 24.8 months (101).Another approach is to pulse dendritic cells with tumorantigens ex vivo and reinfuse them into patients. Muc1-pulsed dendritic cells were evaluated in a phase I/II trialin patients with resected pancreatic and biliary tumors. Thevaccine transiently increased the percentages of functional

Table 2. Summary of past and current clinical trials targeting components of the TME

Trial statusOutcome

Intervention Current phase Trial identifier Status Median survival Reference

Erlotinib þ gemcitabine III NCT00026338 Completed 6.24 vs. 5.91 mo 6Oxaliplatin þ irinotecan þleucovorin þ 5-FU

II/III NCT00112658 Completed 11.1 vs. 6.8 mo 7

GDC-0449 þ gemcitabine II NCT01064622 ActiveIPI-926 þ gemcitabine I/II NCT01130142 Stopped N/Anab-Paclitaxel þ gemcitabine I/II NCT00398086 Completed 12.2 mo 48PEGPH20 þ gemcitabine I/II NCT01453153 ActiveMarimastat þ gemcitabine III N/A Completed 5.44 vs. 5.39 mo 65Cilengitide þ gemcitabine II N/A Completed 6.7 vs. 7.7 mo 66BAY 12-9566 III N/A Completed 3.7 vs. 6.6 mo 67Bevacizumab þ gemcitabine III NCT00088894 Completed 5.8 vs. 5.9 mo 68Bevacizumab þ erlotinib þgemcitabine

III N/A Completed 7.1 vs. 6.0 mo 69

Bevacizumab þ docetaxel II N/A Completed 4.1 vs. 5.4 mo 70Radiotherapy þ capecitabine þbevacizumab, bevacizumab þgemcitabine

II NCT00114179 Completed 11.9 71

Axitinib þ gemcitabine III NCT00471146 Completed 8.5 vs. 8.3 mo 72Sorafenib þ gemcitabine II N/A Completed 4.0 mo 73RO4929097 II NCT01232829 ActiveMK0752 þ gemcitabine I/II NCT01098344 ActiveIrradiated allogeneicGM-CSF–secreting tumor vaccine

II NCT00084383 Completed 24.8 mo 101

MUC1 peptide-loadeddendritic cell vaccine

I/II N/A Completed 26 mo 102

Ipilimumab II NCT00112580 Completed N/A 103CP-870,893 þ gemcitabine I NCT00711191 Completed 7.4 mo 104Mechanistic studiesGDC-0449 þ gemcitabine 0 NCT01195415 ActiveGDC-0449 II NCT01096732 ActivePreoperative pimonidazole N/A NCT01248637 Active

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CD4 and CD8 T cells as well as regulatory T cells. Four of12 (33%) patients in this study were alive 5 years aftersurgery, with a median survival of 26 months (range, 13–69 months; ref. 102). Both of these studies compare favor-ably in terms of the median survival for resected pancreaticcancer, which is normally between 11 and 20 months.A third approach is the use of blocking/neutralizing anti-bodies such as ipilimumab, which targets CTLA-4, a surfaceprotein expressed by activated T cells that confers inhibitorysignals. Unfortunately, ipilimumab as a single agent wasfound to be ineffective in a phase II trial in locally advancedand metastatic pancreatic cancer. However, one of thepatients on this study experienced significant delayedregression of the primary tumor and 20 hepatic metastases,which may merit further investigation (103).GEMMs are an ideal system to evaluate immune thera-

peutic approaches in PDA; however, few reports exist to dateon this topic. This may be because the tumor antigens forPDA are unknown, making the tracing of immuneresponses very difficult. Nonetheless, a recent immunother-apy study used the "KPC" GEMM (LSL-KrasG12D;LSL-p53R172H/þ;Pdx1-Cre) to evaluatewhether activation of anti-gen-presenting cells via stimulationofCD40would result inincreased tumor antigen presentation and priming of effec-tor T cells (104). Treatment with agonistic anti-CD40achieved tumor stabilization and even regression in KPCmice but was surprisingly T-cell independent. Instead,tumor control was exerted by the activated macrophagestargeting the fibrotic stroma. Furthermore, an early-phaseclinical trial with anti-CD40 antibody showed promisingresults in patients (104).

Conclusions and Future OutlookThe influences of the stroma in pancreatic cancer are as

manifold as its components (Fig. 4). This curse may beturned into a blessing as this complexity also providesnumerous avenues for therapeutic exploration (clinical

trials mentioned in this review are summarized in Table2). Accumulating evidence suggests that the extensive des-moplastic reaction may be, at least, partly responsible forthe innate chemoresistance in pancreatic tumors by creatingbarriers that fence off tumor cells from circulating activetherapeutic compounds. Breaching this stromal barrierrepresents a promising strategy to improve the delivery andefficacy of cytotoxic drugs in the future. Therapeutic benefitmay be gained by strategies aimed at depleting the desmo-plastic stroma, exploiting the poor vasculature, or activatingthe immune system to target tumor cells. We anticipate thatfuture therapies will have to be tailored to target several ofthe described components of the microenvironment toachieve long-lasting therapeutic response.

Disclosure of Potential Conflicts of InterestD.A. Tuveson is a Senior Group Leader at the Cancer Research UK

Cambridge Research Institute. No potential conflicts of interests were dis-closed by the other authors.

Authors' ContributionsConception and design: C. Feig, D.A. Tuveson, A. NeesseAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): C. Feig, N. CookWriting, review, and/or revision of the manuscript: C. Feig, D.A. Tuve-son, A. Gopinathan, A. Neesse, D.S. Chan, N. CookAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): A. Gopinathan, D.S. ChanStudy supervision: D.A. Tuveson

Grant SupportThis study was supported by the University of Cambridge and Cancer

ResearchUK, The Li Ka Shing Foundation andHutchisonWhampoaLimited,the NIHR Cambridge Biomedical Research Centre, and the European Com-munity Grant EPC-TM-Net 256974. C. Feig was supported by the EMBOlong-term fellowship and by aMarie Curie Intra European Fellowship withinthe 7th European Community Framework Programme. A. Neesse wassupported by Deutsche Krebshilfe Mildred Scheel Postdoctoral Fellowship.D.S. Chan is funded by the Frank Edward Elmore Fund.

Received March 5, 2012; revised June 27, 2012; accepted June 27, 2012;published online August 15, 2012.

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2012;18:4266-4276. Clin Cancer Res Christine Feig, Aarthi Gopinathan, Albrecht Neesse, et al. The Pancreas Cancer Microenvironment

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