Immunosurveillance of pancreatic adenocarcinoma: Insights from genetically engineered mouse models of cancer

Cancer Letters 279 (2009) 1–7

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Cancer Letters journal homepage: www.elsevier.com/locate/canlet

Mini-review

Immunosurveillance of pancreatic adenocarcinoma: Insights from genetically engineered mouse models of cancer Carolyn E. Clark, Gregory L. Beatty, Robert H. Vonderheide * Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Department of Medicine, 551 BRBII/III, 421 Curie Blvd., Philadelphia, PA 19104, USA

a r t i c l e

i n f o

Article history: Received 8 July 2008 Accepted 30 September 2008

Keywords: Pancreatic cancer Kras Macrophage Myeloid derived suppressor cell Immunosurveillance

a b s t r a c t The resurgent theory of cancer immunosurveillance holds that the immune system plays an important role in the suppression of tumors, particularly in the elimination of early neoplastic lesions. Tumors with reduced immunogenicity or those that have acquired mechanisms to suppress immune effector functions, however, can emerge from this selection pressure and grow progressively. This is an especially important issue in pancreatic cancer, which although inflammatory in vivo is nevertheless highly aggressive and nearly always lethal. Here, we review emerging data obtained from novel genetically defined mouse models of pancreatic adenocarcinoma that suggest that the immune system may be complicit in the inception and progression of pancreatic cancer. Host immune cells with suppressive properties infiltrate the pancreas early during tumorigenesis, even at the earliest stages of neoplasia, preceding and effectively undermining any lymphocytes with potential antitumor function. Thus, in pancreatic adenocarcinoma, the failure of immunosurveillance is likely an early event during tumorigenesis, a concept that carries important implications for the design of novel immunotherapeutics in this disease. Ó 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Pancreatic ductal adenocarcinoma (PDA) is an exceptionally aggressive and lethal form of cancer, with an extremely poor 5-year survival [1]. Experimental immunotherapy has demonstrated promise as adjuvant therapy after surgery [2], but has had limited efficacy in patients with a heavy disease burden. In order to successfully manipulate the immune system for the treatment of pancreatic cancer, the endogenous immune response to the developing tumor must be understood. Until recently, studying the dynamics of the immune response from preinvasive through invasive disease has not been feasible due to the lack of physiologically relevant mouse models and the limited tissue available from human specimens. However, the recent development of genetically modified

* Corresponding author. Tel.: +1 215 573 4265; fax: +1 215 573 2652. E-mail address: [email protected] (R.H. Vonderheide). 0304-3835/$ - see front matter Ó 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2008.09.037

mouse models of PDA that recapitulate both the preinvasive and invasive features of the human disease [3,4] have afforded the opportunity to explore these questions. In this mini-review, we will first discuss current thinking in the field of tumor immunology regarding the theory of immunoediting then review data specifically relating to the role of the immune system in pancreatic tumorigenesis. 2. The theory of immunoediting The immune system is intimately involved during tumorigenesis, with certain immune populations thought to have host-protective roles and others implicated in tumor-promoting roles. Host-protective roles are proposed in the immunoediting hypothesis [5], a revised version of the classic cancer immunosurveillance hypothesis [6]. According to this theory the immune system can and does recognize and eliminate cancer cells naturally thereby exerting a selective pressure that tumor cells must evade in order to grow.

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The theory of immunoediting proposes three phases: (i) elimination, (ii) equilibrium and (iii) escape. The first stage, elimination, is postulated to transpire early in disease, with some rate of complete success such that transformed cells or early neoplastic lesions are eliminated before becoming clinically apparent. However, some neoplastic lesions may not be completely eliminated and may acquire additional mutations that render them resistant to immune-mediated cytolysis. In this setting, neoplastic growth may be initially controlled in an ‘‘equilibrium” phase, during which time tumor immunogenicity is sculpted by selective pressures imposed by the immune system. Nonetheless, tumor variants will eventually arise that cannot be eliminated by immune effectors so that ultimately, these clones will grow and manifest clinically in the ‘‘escape” phase. As a result, the immunoediting theory predicts that a clinically evident tumor has reduced immunogenicity that stems from selective pressures of the immune system. Indeed, this requirement for immune escape has even been proposed as the seventh ‘‘hallmark of cancer” [7] in addition to the six cell-intrinsic properties described by Hanahan and Weinberg [8]. 3. Experimental evidence in mouse models of cancer Much of the experimental evidence supporting the concept that the immune system can eliminate neoplasias derives from immunocompromised mice lacking key immunologic molecules and signaling pathways. For example, mice with deficiencies in the recombination activating gene 2 (RAG2) [9], perforin [10], or interferon-c [11] among other molecules, develop spontaneous tumors and MCA-induced sarcomas more frequently than their wildtype counterparts. The increased incidence of spontaneous or carcinogen-induced tumors in immunocompromised mice suggests a role for immunosurveillance in inhibiting tumor development. However, data from genetically induced tumor models are less convincing. For example, while mice lacking one p53 allele develop tumors more rapidly or with increased incidence in the presence of mutations in immune molecules, such as perforin [10], IFN-cR1 [12], or the death-mediator TRAIL [13], targeting other immune molecules such as TAP1 and LMP2 have demonstrated no apparent effect on tumor development [14]. Her2/neu transgenic mice exhibit no change in tumor development when deficient in TRAIL [13]. Similarly, SV40 large T antigen transgenic mice exhibit no change in tumor development when deficient in IFN-c [15], and RIP-Tag4 transgenic mice also exhibit no change in tumor development when deficient in CD8+ T cells [16]. Strikingly, K14HPV16 gene-targeted mice demonstrate a decreased incidence in carcinoma development when crossed onto a Rag1 / background [17]. These studies underscore the uncertainty in the field regarding the role of the natural immune response in inhibiting the development of genetically derived tumors. While the reasons underlying the discrepancies seen in the above studies are uncertain, it is clear that tumors grow in immunocompetent hosts. However, tumors grown in immunocompetent hosts may differ qualitatively from those grown in immunocompromised hosts. In one provocative study, tumors raised in immunocompetent hosts dis-

played a reduced immunogenicity with capacity to grow progressively when transplanted into either wild-type or Rag2 / recipients. In contrast, while 40% of tumors raised in Rag2 / hosts were rejected when transplanted into wild-type recipients, they exhibited the capacity for growth in Rag2 / recipients and in wild-type recipients depleted of CD4+ and CD8+ T cells [9]. These results imply a role for the immune system in ‘‘editing” the immunogenicity of neoplastic lesions, thereby selecting less immunogenic clones for tumor outgrowth. 4. Clinical evidence for immunosurveillance Clinically in humans, T cell infiltration into the tumor correlates with improved patient prognosis and/or prolonged disease-free survival. This correlation has been observed in patients with melanoma [18], prostate [19], breast [20], colorectal [21], ovarian [22] and other carcinomas. One recent study found that the nature of the T cell infiltrate within resected specimens of colorectal carcinoma was a prognostic indicator superior to classical histologic staging. In this study, patients with T cells expressing markers characteristic of TH1 polarization and of cytotoxic function or memory status had a low incidence of tumor recurrence [23]. Overall, these data suggest a role for tumor immunosurveillance in human malignancies. Many clinical investigations are now aimed at exploiting this anti-tumor cellular immune response using vaccines or adoptive cellular therapy. 5. The dynamics of immunosurveillance during pancreatic tumorigenesis Despite this progress in understanding cancer immunosurveillance, the dynamics of the immune reaction to a developing cancer, particularly PDA, and the relation to disease progression remains poorly understood. The kinetics of immune recognition of neoplastic lesions and when the failure of immunosurveillance occurs are not known, in contrast to immune reactions to non-tumor antigens for which such studies have been extensive [24,25]. Mouse models using implanted syngeneic or xenogeneic tumors are not well-suited to such studies and availability of human tissues is limited, particularly tissue harboring only preinvasive disease. However, the recent development of genetically engineered mouse models of spontaneous cancer now permits investigation into such questions [26]. Using this strategy, we tracked the evolution of immunosurveillance in a slowly progressing genetically defined mouse model of pancreatic cancer that recapitulates the key clinical, histopathologic, and molecular features of human PDA, beginning with its earliest precursor lesions [27]. In this model, an activating point mutation is targeted to one Kras allele, but rendered quiescent by a floxed transcriptional and translational silencing cassette in the promoter region. Breeding these animals with mice expressing Cre recombinase under the control of a pancreatic-specific promoter (Pdx-1 or p48) results in expression of the oncogenic KrasG12D allele in the pancreas [3]. This single point mutation is sufficient to initiate preinvasive le-

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sions (termed pancreatic intraepithelial neoplasias, PanIN) that progress in the severity of cellular and architectural abnormalities through all of the histologic stages described for the human disease [28] and acquire additional mutations that permit the development of invasive and metastatic PDA. In this model, KrasG12D mice exhibit preinvasive PanINs as early as 2 weeks of age, which spontaneously progress to invasive and metastatic PDA [3]. An intense fibroinflammatory reaction consisting of stromal and immune cells accompanies the progression from normal histology to PDA, similar to the desmoplastic reaction classically observed in human pancreatic cancer. We used this model to track the in vivo immune response to spontaneous PDA as it evolved from preinvasive disease through invasive and metastatic cancer [27]. Using flow cytometry and immunohistochemistry, we found that the progression from normal histology to PanIN to PDA in KrasG12D mice is paralleled by a progressive infiltration of CD45+ cells, with leukocytes accounting for about half of all cells in PDA tumors. There is a prominent leukocytic infiltration even around the lowest grade preinvasive lesions. However, immunosuppressive cells, including tumor-associated macrophages (TAM), myeloid-derived suppressor cells (MDSC), and regulatory T cells (Treg), dominate in both preinvasive lesions and invasive cancer. Effector T cells are scarce in preinvasive lesions and are present in higher numbers in only a subset of advanced cancers, where they show minimal evidence of activation. Intratumoral NK cells are absent. Thus, these results did not support the hypothesis that an early, productive anti-tumor immune response is ultimately suppressed by a growing burden of invasive tumor, but rather suggest that tumor immunity may be undermined from the inception of disease. We confirmed our results in a second mouse model of spontaneous PDA involving not only a constitutively active Kras allele, but also a point mutant p53 allele targeted to the pancreas. KrasG12D/p53R172H mice succumb to PDA at around 5 months of age, while KrasG12D mice can live for one year or more [4]. In spite of the shorter time-course, the observed leukocyte infiltration is similar in composition as well as in the kinetics with which each cell population appeared relative to disease progression. Namely, we observed very few CD8+ T cells and NK cells at any stage of disease, and slightly higher numbers of CD4+ T cells that included a large fraction of Foxp3+ Treg even during PanIN. Abundant myeloid cells comprised mostly of macrophages appear during PanIN with roughly equal numbers of macrophages and MDSC by end-stage PDA. Despite the similarities between the KrasG12D mice and KrasG12D/p53R172H mice, the total leukocyte infiltration was less prominent in KrasG12D/p53R172H mice. The simplest explanation for these differences is the time associated with tumor development: since the KrasG12D mice develop PanIN within the first few weeks of life, but can live over a year, this affords a large window of time for leukocytes to accumulate in the pancreas. Differences might also be attributed to genetics: the KrasG12D/p53R172H mice have fewer hurdles to developing invasive disease (as a second genetic ‘‘hit” is already provided) and therefore do not depend as much on the potential deleterious effects of

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chronic inflammation. The rate-limiting step for pancreatic lesions in KrasG12D/p53R172H mice to become invasive appears to be loss of heterozygosity of the wild-type p53 allele, whereas the lesions of KrasG12D mice need multiple genetic alterations. Indeed, if the complement of mutations is different between the two models, presumably the signaling pathways and other genetic programs activated in the neoplastic lesions are also different. This could affect cytokine and chemokine secretion, and other variables that would influence leukocyte trafficking, growth, and survival. A number of other genetically defined mouse models of PDA have been developed in which the dynamics of the immune infiltration could be investigated [26,29]. Some models do not explicitly rely on oncogenic ras alleles, which may be an important variable to test. Beyond models of PDA, similar analyses have also been performed in models of other solid tumors such as skin and cervical carcinoma. Interestingly, the dynamics of the immune infiltrate in K14-HPV16 skin carcinoma are quite similar to those that we observed in PDA: more than 50% of cells in the invasive tumors in this model are CD45+ leukocytes and CD11b+ or Gr-1+ myeloid cells appear early and dominate, with a scarcity of CD8+ effector T cells [30]. 6. Tumor-associated macrophages Multiple deleterious roles have been attributed to each of the three major leukocyte subtypes (i.e., TAM, MDSC and Treg) present in preinvasive lesions of KrasG12D and KrasG12D/p53R172H mice. The most prominent leukocytic subset we observed was CD11b+ macrophages, which clustered around neoplastic ducts in preinvasive lesions, including those of lowest grade, and persisted throughout PDA. Their early accumulation was likely a direct consequence of chemokines secreted downstream of hyperactive Kras signaling [39,40]. TAM can inhibit anti-tumor T cell responses by production of indoleamine dioxygenase metabolites [31,32] and reactive oxygen species (ROS), and indirectly by attracting Treg to the tumor site [33]. TAM also assist in matrix remodeling, which facilitates tumor invasion. They can be found along the basement membrane in neoplastic lesions, associated with areas of membrane breakdown, suggesting that they aid in tumor cell invasion of the basement membrane [34]. High expression of matrix metalloproteinases in TAM supports this role. Invasive tumor cells and macrophages co-migrate in rodent models of mammary carcinogenesis in response to EGF and CSF-1 [35]. Furthermore, TAM may also produce pro-angiogenic factors such as VEGF, which stimulate formation of new tumor vasculature. They also facilitate intravasation of tumor cells into these blood vessels, an important step in metastasis [36]. Indeed, mice genetically devoid of macrophages due to deletion of CSF-1 exhibit reduced rates of mammary tumor progression and virtually no incidence of metastasis [37]. Macrophages have such a crucial role in promoting tumor angiogenesis and tumor cell invasion and metastasis that they represent key targets in the rational design of new therapies for cancer [38].

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7. Myeloid derived suppressor cells MDSC, another immunosuppressive myeloid cell type, were also prominent in diseased pancreata of the models that we studied. Furthermore, these cells exhibited striking accumulation in the spleens of PDA-bearing mice. MDSC, characterized by the co-expression of the surface markers Gr-1 and CD11b, have been shown to suppress specific T cell responses [41–43]. Myeloid cells coexpressing Gr-1 and CD11b comprise 20–30% of cells in normal bone marrow and can also be found to a lesser extent in normal spleen, where they comprise <4% of nucleated cells. However, the CD11b+Gr-1+ cells in normal spleen are qualitatively different from MDSC from tumor-bearing mice: when adoptively transferred into congenic recipients, Gr-1+CD11b+ splenocytes from tumor-free mice differentiate into mature macrophages and dendritic cells within 5 days, whereas many of the cells from tumor-bearing mice remain immature [44]. Therefore, in addition to their dramatically increased numbers in tumor-bearing hosts, MDSC have an impaired ability to differentiate normally. The accumulation of large numbers of MDSC in tumorbearing hosts has been linked to high levels of circulating GM-CSF in some cases [45] and VEGF in others [46]. VEGF is known to inhibit the differentiation of dendritic cells [47]. The differentiation arrest of MDSC cells has also been attributed to tumor-derived factors that activate JAK2 and STAT3 signaling pathways [48]. Chemotactic factors produced by the tumor such as MCP-1 have also been shown to attract MDSC to migrate to the tumor [49]. MDSC from tumor-bearing mice have been reported to impair T cell function in a number of ways: they can inhibit antigen-specific responses (including proliferation, cytokine production, and cytotoxic function) mediated by T cells [43]. They can cause down-regulation of the T cell receptor f chain, a key component of TCR signaling [50], or derange T cell signal transduction by other mechanisms. They have also been shown to induce the development of Treg [51]. In addition, MDSC have the capacity to elicit apoptosis in T cells, although this latter function has been attributed only to tumor-associated, and not splenic, MDSC. The mechanisms by which MDSC perform the above functions are beginning to be elucidated. L-arginine metabolism by two different enzymes appears to be crucial: NOS converts L-arginine to NO and citrulline, whereas arginase converts L-arginine to urea and L-ornithine. Enhanced arginine catabolism can result in local depletion of this amino acid, leading to inhibition of T cell proliferation [52]. Production of NO also directly impairs T cell proliferation by disrupting IL-2 receptor signaling [53]. Furthermore, arginine metabolism by both enzymes is linked to production of ROS, mainly in the form of super-oxide and its byproducts hydrogen peroxide (H2O2) and peroxynitrites. Oxidative stress caused by these ROS can impede TCRf chain expression and thereby interfere with signaling and proliferation [50]. Furthermore, peroxynitrite can cause nitration of tyrosines in TCR-CD8 complexes and impair the ability of CD8 T cells to bind peptide-MHC complexes

and to respond to a specific peptide [54]. In some cases, ROS can even induce apoptosis in T cells. Blockade of arginase and NOS activities together, as well as H2O2 or peroxynitrite scavengers, can revert the suppressive function of MDSC [48,55]. Intriguingly, splenic MDSC must first be exposed to IFN-c produced by activated T cells in order to exert their suppressive program on these T cells. In response to T cell-derived IFN-c, MDSC produce more IFN-c and IL-13, which upregulate arginase and NOS within the MDSC via an autocrine circuit [56]. Since tumor-associated MDSC constitutively produce these enzymes, their suppressive program can be activated immediately. In vitro, MDSC activity have been effectively targeted with a variety of inhibitors, including dominant negative STAT3 [57], the iNOS inhibitor L-NMMA [58], the arginase inhibitor Nor-NOHA [58], and ROS scavengers [48]. However, the utility of these agents is severely limited in vivo. The following strategies for in vivo reduction or elimination of MDSC have been reported: depletion of Gr-1+ cells with monoclonal antibody clone RB6-8C5 [59], administration of all-trans-retinoic acid (ATRA) to effect myeloid cell maturation [44], administration of 1a25-dihydroxyvitamin D3 to reduce myelopoiesis [60], administration of gemcitabine to deplete MDSC [61], and administration of phosphodiesterase-5 inhibitors to downregulate arginase and NOS [62]. These strategies have all produced measurable clinical improvements in the various animal models tested, either alone or in combination with an anti-tumor vaccine. In our studies, we found that MDSC also track with the tumor microenvironment: they were found in all metastatic lesions examined from KrasG12D/p53R172H mice including metastases to the diaphragm, liver, lung, and peritoneal wall. Importantly, these metastatic lesions do not exhibit the fibrotic, desmoplastic reaction characteristic of the primary lesions, demonstrating that MDSC are not merely coincident with inflammation. The specificity of MDSC recruitment was further underscored by the finding that these cells also accumulate dramatically in the spleen, but not in other tissues that are unaffected by disease such as the salivary gland. Within the tumor microenvironment, we found that the prevalence of MDSC inversely correlated with that of CD8+ T cells in the leukocytic infiltrate of pancreatic lesions in KrasG12D mice, raising the possibility that MDSC may negatively affect T cell trafficking or T cell survival within the neoplasm. 8. Regulatory T cells A third immunosuppressive population, Treg, has been widely observed in advanced cancer [63,64], but in our studies was also shown to participate in the early infiltration of preinvasive lesions. Although Treg exist in healthy individuals to maintain peripheral tolerance and prevent runaway immune responses [63,65], increased numbers of Treg have been observed both in the circulation of cancer patients [66] and within the tumor microenvironment itself. The growing list of cancers in which elevated numbers

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of Treg have been observed include lung [33], ovarian [67], breast [68], colorectal [69] as well as pancreatic carcinomas [68], and their presence within tumors is associated with decreased survival [67]. Tumor-associated Treg can inhibit T cell production of IFN-c and IL-2 in response to tumor-associated antigens, as well as their cytotoxic function. Thus, they represent a hurdle both to naturally occurring anti-tumor immunity and to successful immunotherapy. Indeed, many immunotherapy protocols currently in clinical trials now include strategies to deplete Treg in an effort to enhance the efficacy of vaccination or adoptive T cell therapy [70]. The mechanisms responsible for the accumulation of tumor-associated Treg in cancer lesions are not fully understood. On one hand, these cells may represent the contraction phase of an earlier, albeit ultimately ineffective, immune response. Alternatively, Treg may simply be recruited by a tumor microenvironment favorable for their existence, regardless of the presence or absence of activated T cells. We have shown that Treg infiltration is an early event that occurs during preinvasive disease, even prior to the appearance of CD8+ T cells. Overall, these data suggest a broader and more extensive role for Treg in cancer immunosurveillance than previously appreciated.

Induction

Inflammation

9. Summary An evaluation of the dynamics of the host immune response to pancreatic adenocarcinoma in mice genetically engineered to develop the disease reveals that suppressive leukocytes, rather than effector lymphocytes, infiltrate the pancreas early during tumorigenesis and may even promote disease progression. The early presence of these cell types suggests that even if T cell responses are initiated later in disease, they will immediately be quenched before full immunologic elaboration owing to multiple pre-existing components of host immunosuppression. The current theory of cancer immunosurveillance predicts that early neoplastic lesions should elicit robust immune responses in the ‘‘elimination phase”, but if elimination is not complete, clones with reduced immunogenicity or those that can effectively suppress the immune response will grow out and become a clinically evident tumor [5]. Data from KrasG12D animals, in which cancer evolves in the setting of a competent immune system, suggest a different possibility; namely, that the ‘‘elimination phase” may be nearly nonexistent and that tumor immune evasion is facilitated by an early inflammatory immune reaction that suppresses the development of an adaptive immune response (Fig. 1).

Immunosuppression

Immunosuppressive cytokines facilitate immune evasion

Immune privilege Immune reaction inhibits development of adaptive immunity

Locally y secreted secreted tumor factors recruit inflammatory y cells

Normal

PanIN

MDSC

CD8 T cell

Tumor-associated macrophage

CD4 T cell

Treg

NK cell

PDA

Immunosuppressive cytokines Tumor secreted soluble factors

Fig. 1. Proposed dynamic model of the immune reaction associated with developing pancreatic ductal adenocarcinoma. Induction – Through alterations in oncogenes and tumor suppressor genes, epithelial-derived neoplastic lesions can arise within the pancreas that progress through a series of pre-invasive stages, termed pancreatic intraepithelial neoplasias (PanIN), eventually culminating in invasive and metastatic pancreatic ductal adenocarcinoma (PDA). Inflammation – PanIN lesions secrete soluble factors that facilitate a local inflammatory reaction composed of stromal and immune cells. Immunosuppression – Infiltrating immune cells, including Treg, MDSC, and TAM, suppress the development of an adaptive immune response through both the secretion of immunosuppressive cytokines as well as direct cell-cell contact. Immune Privilege – With progression to PDA, neoplastic lesions maintain the capacity for immune evasion through both tumor-mediated and immune-mediated mechanisms of suppression thereby establishing a site of immune privilege.

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From a translational research perspective, these observations carry important implications for the design of novel immunotherapeutic strategies for pancreatic cancer. The most important concept is that mechanisms of immunosuppression derived not only from the tumor but also from the tumor-associated leukocytic response develop early in oncogenesis and may frustrate even the most vigorous efforts to trigger an anti-tumor immune response therapeutically in end stage patients with pancreatic cancer. Consequently, the development of potent yet safe inhibitors of such immunosuppressive factors, including TAM, MDSC, and Treg, is crucial for the advancement of immunotherapies for cancer. Moreover, our data also imply that the most appropriate clinical setting to test novel immunotherapies is not in patients with advanced PDA, in whom the burden of immunosuppression may make such efforts at best poorly effective and at worse futile. Indeed, if it is true in humans as it is in genetically engineered mice that immunosuppressive factors are operative even at the inception of neoplasia, then it is possible that in order to efficiently harness the power of the immune system to combat cancer, it will be necessary to administer active immunotherapy prophylactically rather than therapeutically. The mouse models of PDA described here would be an ideal experimental platform to test this hypothesis. Acknowledgements We thank our collaborators Drs. David Tuveson, Sunil Hingorani, and Ken Olive who developed the main mouse models of pancreatic cancer described herein. References [1] A. Jemal, R. Siegel, E. Ward, T. Murray, J. Xu, C. Smigal, et al, Cancer statistics, 2006, CA Cancer J. Clin. 56 (2006) 106–130. [2] D. Laheru, E.M. Jaffee, Immunotherapy for pancreatic cancer science driving clinical progress, Nat. Rev. Cancer 5 (2005) 459–467. [3] S.R. Hingorani, E.F. Petricoin, A. Maitra, V. Rajapakse, C. King, M.A. Jacobetz, et al, Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse, Cancer Cell 4 (2003) 437–450. [4] S.R. Hingorani, L. Wang, A.S. Multani, C. Combs, T.B. Deramaudt, R.H. Hruban, et al, Trp53R172H and KrasG12D cooperate to promote chromosomal instability and widely metastatic pancreatic ductal adenocarcinoma in mice, Cancer Cell 7 (2005) 469–483. [5] G.P. Dunn, L.J. Old, R.D. Schreiber, The three Es of cancer immunoediting, Annu. Rev. Immunol. 22 (2004) 329–360. [6] F. Burnet, Cancer – a biological approach, Brit. Med. J. 1 (1957) 841– 847. [7] L. Zitvogel, A. Tesniere, G. Kroemer, Cancer despite immunosurveillance: immunoselection and immunosubversion, Nat. Rev. Immunol. 6 (2006) 715–727. [8] D. Hanahan, R.A. Weinberg, The hallmarks of cancer, Cell 100 (2000) 57–70. [9] V. Shankaran, H. Ikeda, A.T. Bruce, J.M. White, P.E. Swanson, L.J. Old, et al, IFNgamma and lymphocytes prevent primary tumour development and shape tumour immunogenicity, Nature 410 (2001) 1107–1111. [10] M.J. Smyth, K.Y. Thia, S.E. Street, D. MacGregor, D.I. Godfrey, J.A. Trapani, Perforin-mediated cytotoxicity is critical for surveillance of spontaneous lymphoma, J. Exp. Med. 192 (2000) 755–760. [11] A.S. Dighe, E. Richards, L.J. Old, R.D. Schreiber, Enhanced in vivo growth and resistance to rejection of tumor cells expressing dominant negative IFN gamma receptors, Immunity 1 (1994) 447– 456. [12] D.H. Kaplan, V. Shankaran, A.S. Dighe, E. Stockert, M. Aguet, L.J. Old, et al, Demonstration of an interferon gamma-dependent tumor surveillance system in immunocompetent mice, Proc. Natl. Acad. Sci. USA 95 (1998) 7556–7561.

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