Animal models of pancreatic cancer for drug research

Review

10.1517/17460440802365359 © 2008 Informa UK Ltd ISSN 1746-0441 1177All rights reserved: reproduction in whole or in part not permitted

Animal models of pancreatic cancer for drug research Matthias Kapischke † & Alexandra Pries † Vivantes Hospital Spandau, Department of Surgery, Neue Bergstrasse 06, D-13585 Berlin, Germany

Background : The operative and conservative results of therapy in pancreatic ductal adenocarcinoma remain appallingly poor. This underlines the demand for further research for effective anticancer drugs. The various animal models remain the essential method for the determination of efficacy of substances during preclinical phase. Objective : Unfortunately, most of these tested substances showed a good efficacy in pancreatic carcinoma in the animal model but were not confirmed during the clinical phase. Methods : The available literature in PubMed, Medline, Ovid and secondary literature was searched regarding the available animal models for drug testing against pancreatic cancer. The models were analyzed regarding their pros and cons in anticancer drug testing. Conclusion : The different modifications of the orthotopic model (especially in mice) seem at present to be the best model for anticancer testing in pancreatic carcinoma. The value of genetically engineered animal model (GEM) and syngeneic models is on debate. A good selection of the model concerning the questions supposed to be clarified may improve the comparability of the results of animal experiments compared to clinical trials.

Keywords: animal model , anticancer drugs , pancreatic cancer

Expert Opin. Drug Discov. (2008) 3(10):1177-1188

1. Introduction

Cancer represents an increasing cause of morbidity and mortality in the Western world. Carcinoma of the exocrine pancreas remain the fourth common cause of cancer death in this region [1] . Our understanding of the molecular mechanisms of carcinogenesis, tumor growth, metastasis and tumor recurrences has improved over the past decades – but pancreatic cancer still remains a poorly understood malignancy [2] . The clinical results achieved, for example, in clinical trials for the patients suffering from pancreatic cancer remain disastrous. The only curative option available is a radical resection with extensive lymphadenectomy as well as granting a 5-year survival rate of only 6 – 20% [3] . Local recurrence accounts for > 70% of the relapses even after R0-resection underlining the demand of adjuvant therapy approaches. Even more difficult is the situation for > 60% of patients, whose disease is already inoperable at the time of diagnosis. Their life expectancy is between 3 and 6 months [4] .

Facing these dismal numbers it is obvious that the focus of anticancer drug research is clearly on the development of new agents and substances, which hopefully may lead to an improved prognosis for those patients suffering from ductal pancreatic cancer. Development costs of a new drug are about $100 million and require both patience and time [5,6] . Primary intention should be separating efficient from inefficient compounds to identify those substances promising the most probability of efficacy in the human organism [5] . An intrinsic tool to achieve this is the animal model in pancreatic cancer also.

This review provides an overview of the existing animal models for testing anticancer drugs in pancreatic cancer. The options and constraints of the

1. Introduction

2. Clinically and histological

particularities of the exocrine

pancreatic cancer

3. General requirements of

an animal model for

pancreatic cancer

4. Animal models for

pancreatic cancer

5. Humanized mice

6. Mouse pancreatic

intraepithelial neoplasia

7. Metastatic models

8. Possibilities for imaging

of tumor growth during

drug therapy

9. Histopathology and pancreatic

cancer cell lines

10. Future possibilities

11. Mechanisms for drug resistance

in xenotransplant model

12. Conclusions

13. Expert opinion

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Animal models of pancreatic cancer for drug research

1178 Expert Opin. Drug Discov. (2008) 3(10)

various models are analyzed and possible upcoming developments provided.

2. Clinically and histological particularities of the exocrine pancreatic cancer

More than 90% of the carcinoma of the exocrine pancreas is defined as ‘ductal adenocarcinomas’. Metastases are common, particularly in the lymph nodes, liver, lung, adrenal glands, kidney and stomach. The production of a desmoplastic stroma tissue is specific for pancreatic adenocarcinomas. This desmoplastic reaction includes that tumor cells produce growth factors enforcing the growth of fibroblasts as well as the forming of matrix proteins [6,7] . Tumor cells are able to modify the gene expression in fibroblasts maintaining the modifications of apoptotic characteristics as well as other effects [8] .

About two-thirds of the tumors of the exocrine pancreas are located in the head of the gland. Even small and early detected tumors show a tendency to spread in the lymph nodes, infiltrating the perineural tissue and mesenteric blood vessels. Interestingly ductal adenocarcinomas are often associated with a macroscopically visible fibrosis as an expression of the desmoplastic reaction.

Acinar cell carcinomas are presenting in < 10% of the patients. Genetically and histological they show similar effects.

3. General requirements of an animal model for pancreatic cancer

Tumor modeling has a long history in cancer research [9] . Killion et al. outlined the characteristics of a successful preclinical animal tumor model [10] .

The model is supposed to:

reproduce the biology of the human cancer • allow the study of relevant cellular and molecular events • (e.g., growth and metastasis) reproduce the problems associated with type and location of • tumor and metastasis be reliable, reproducible, available and affordable • possess objective and quantitative end points of therapeutic • responses.

The models now presented cover the above topics to a different extent.

4. Animal models for pancreatic cancer

4.1 Chemical and environmental carcinogenesis model of pancreatic cancer About 70% of the human tumors are induced by carcinogens. Especially many environmental factors are known to enhance pancreatic carcinogenesis (chemicals owing to the human lifestyle, caffeine, ethanol etc.) [11,12] . Despite the significance of spontaneous and environmental models to biomedical research

the relatively long latency of these models makes them impractical for most preclinical tumor studies in general [13] .

4.1.1 Hamsters Hamsters are the most widely used animal models for studying exocrine pancreatic cancer induced by carcinogenesis.

The administration of N -nitroso–bis (2-oxypropyl)amine or N -nitroso–bis (2-hydroxipropyl)amine induces pancreatic ductal adenocarcinoma. With regard to histological (desmo-plastic reaction), biological (paraneoplastic syndrome) and genetical (ductal phenotype) features hamster tumors are comparable with human tumors [14,15] . Beside this the frequency of certain mutational events (p53) does not reflect the situation in humans [16,17] . The tumor latency is ∼ 8 weeks and 80 – 100% of the animals develop a tumor. Details about processing and mutation are available in [14] . Hamsters are more costly and have a longer generation time compared to mice.

For testing of anticancer drugs this model at present plays a minor role [15,18] .

4.1.2 Rats Asazerine is an alkylating carcinogen that damages DNA. This is the most common model in chemical pancreatic cancer induction in rats [19,20] . Inducement takes place by repetitive application of the carcinogen. Between 2 and 15 months after treatment with asazerine in 17 – 58% of the rats acinar cell neoplasms are found. The tumor growth is relatively slow and metastasizes infrequently. This is one of the main disadvantages of this model [21] .

A second rat model is the implantation of crystalline 7,12-dimethylbenz[a]anthracene in the head of the pancreatic gland. Local administered 7,12-dimethylbenz[a]anthracene induces the development of ductular and glandular adeno-carcinomas in 80% of the treated animals. The latency is between 5 and 8 months [22,23] .

Tumors in rats are biologically and phenotypically distinct from those in humans (acinar phenotype, slow growth, infrequent metastasis, no K-ras mutations etc.) [12,24] .

The latency and the low incidence of carcinomas as well as the infrequent metastasis are the great lack of this model. Cancer drug testing is rarely described [25,26] .

4.1.3 Mouse Mouse models are not really available for chemical cancer induction. Only single observations of carcinogenesis in mice are described [19,20] . The pancreas of the mouse is resistant to most of the available carcinogenic substances.

4.2 Genetically engineered animal models Most of these models apply mice owing to their resistance to exogenic carcinogen treatment. Genetically engineered animal models (GEM) are the most sophisticated animal models of human cancer and many now existing mimic the pathophysiological and molecular features of human pancreatic

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Expert Opin. Drug Discov. (2008) 3(10) 1179

cancer. They were developed due to the critique that genetics and histology of xenografts do not recapitulate the genetics and histology of humans tumors [27] . GEM can simply be classified as either transgenic and endogenous (knockouts) [22] . But the classification of GEM is not static. A good overview and consensus is available in [23] .

4.2.1 Transgenic animals The principle is an introduction of a DNA transgene into a mouse embryo. The transgene will integrate into genomic DNA in a fraction of the injected eggs.

The first generation of four transgenic mouse models of pancreatic neoplasia was:

EL (elastase)- • TAg ⇒ lead to acinar carcinomas [28] EL- • H-ras ⇒ lead to diffuse acinar cell atrophy and occasional acinar carcinoma [14] EL- • c-myc ⇒ lead to mostly acinar carcinomas [29] EL-TGF- • α ⇒ lead to acinar carcinomas [30,31] .

These models took advantage of cloned DNA that contained gene regulatory elements from the rat pancreatic EL gene [32] . Most of these mice models promote the finding that pancreatic carcinogenesis can be accompanied by acino–ductal metaplasia in vivo . Owing to the histological differences (acinar versus ductal) the relevance for testing of therapeutic drugs is marginal [14] .

The second generation incorporates the most common changes identified in pancreatic adenocarcinoma:

Mutant • K-ras transgenics [31-33] Muc-1 and choleystokininreceptor transgenic • [33-35] mCK19 transgenic • [36] .

Activated K-ras represents the most attractive target for a definitive therapy. Among the many challenges in targeting Ras is to selectively inhibit the mutant and not the wild-type form [37] . Activation of oncogenic K-ras seems to represent the rate-limiting step for the development of ductal pancreatic adenocarcinoma [38] . Beside this central position K-ras seems to be the most attractive target for the definitive therapy (e.g., farnesyl transferase inhibitors). However, all clinical settings have been disappointingly ineffective either as monotherapy or in combination with gemcitabine [39,40] .

Single therapeutic approaches show that these models are suitable for testing of anticancer drugs. A relevant break through has not been achieved yet [41-43] .

4.2.2 Tumor suppressor gene knockouts The loss of suppressor gene function can be modeled by targeted deletion of a portion of a gene locus using embryogenic stem cells.

Three tumor suppressor genes are often altered in pancreatic cancer: p53 [44] , p16 [45] and DPC (smad4) [44] . None of these mice develop intestinal abnormalities although pancreatic cancer cells have a high frequency of loss in these genes.

Their specific significance is to be seen in the combination with transgenic mice [43,45] .

The advantages of GEM are:

these mice are immune competent • these tumors are able to develop in their native • compartment and complex processes (e.g., neoangiogenesis) may be • comprehended; in general it is also possible to investigate the relapse after previous therapy.

Disadvantages of GEM are – especially during preclinical phase – the relatively long time of neoplasia development as well as the limited predictability of latency and frequency of tumor formation. Through alterations in the genetic background many GEM show an increase of the tumorigenic variance. This may cause a very wide diffusion of the results especially in the outcome of drug efficiency investigations [27] .

A further disadvantage has to be seen that GEM are usually out of a singular genetic lesion. In case this lesion is a target of a new anticancer drug there is the possibility of promising results that are not confirmed in the human situation up to now, as pancreatic cancer in general is the result of various genetic alterations. GEM are also disappointing in the modeling of certain targets with intrinsic biological differences in rodents compared with humans [46] .

All approaches to generate GEM shown already as well as those to be shown in this manuscript may be combined. The implementation of GEM in both forms has significantly improved the understanding of cancer pathogenesis.

A final judgment concerning the value of GEM especially in testing of anticancer drug against pancreatic cancer is still pending. Given all conceptual advantages there are still no valid preclinical tests.

4.3 GEM and microenvironment The role of environmental influences and stromal inflammation for the formation and development of pancreatic cancer is documented elaborately [6] . GEM offer the possibility to characterize such interactions, also under the aspect that the effects of chemotherapeutic drugs can be influenced by different tumor–stroma interactions. GEM also offer the ability to investigate interactions and use them for therapeutic approaches [22] . Obviously tumor–stroma interactions as well as other microenvironment factors play a central role in tumor growth promoting and anticancer drug resistance. Tumor invasion and metastasis occur in the context that extracellular matrix and many glycoprotein-like mediators (e.g., SPARC (secreted protein acidic and rich in cystein), osteonectin) may influence cancer cells as well as epithelial and mesenchymal cells mutually [47,48] . There are at present no validated models available.

4.4 Xenogenic transplantation models Nude mice, usually athymic ones, are the most commonly used animal model in studying the metastasis of human

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pancreatic cancer [49] . Recently, severe combined immuno-deficiency (SCID), SCIDbeige and beige nude mice are made available for research of carcinogenesis and evaluating therapeutic agents [50] .

4.4.1 Ectopic or orthotopic xenograft model Today the subcutaneous xenograft model is the most applied model in testing the efficiency of new chemotherapeutic agents in cancer research. The advantages of this model are obvious:

low costs • easy handling from a technical point of view and minor • laboratory effort fast and reproducible • observation of tumor growth easily possible. •

A wide range of cultured pancreatic tumor cell lines are known and characterized [51] . These cells are derived from resected primary human tumors, from tumor biopsies or from ascites fluid of pancreatic cancer patients. Cells may be injected or surgically transplanted into the subcutis (or the peritoneal cavity). Tumors are growing quickly and – especially in case of subcutaneous injection – the tumor burden is to be monitored effectively. A comparison of pancreatic cancer cells transplanted subcutaneously showed no metastasis potential in these tumors whereas orthotopically transplanted tumors developed metastases in the abdominal cavity [49,50] . It has to be seen as a disadvantage that these cells might not represent cells of origin of pancreatic cancer duct cells. There is safe evidence that heterotopic implantation of pancreatic tumor cells alters the regulation of cell cycle and apoptosis [52] . Both mechanisms are important for the action of anticancer drugs. Beside this we find a site-dependent different cytokine production in pancreatic xenograft models. This may also be a cause for drug resistance in cancer therapy [53] . The absence of the original milieu and therefore the changed conditions for the tumor–stroma interaction are also of adverse effect for the sensitivity against chemotherapeutic drugs [54,55] . This has been known even before Paget postulated his ‘seed and soil’ hypothesis [56] .

The situation in the human body is more reflected by orthotopic injection or transplantation of tumor. Pancreatic cancer is more aggressive, shows a higher potential to invade the surrounding tissue including perineural and lymphonodal invasion and produces metastases. Tumor–stroma interactions are more evident in the original milieu of the tumor. The major drawbacks are the requirement of higher technical skills for implantation and the ability to replicate the course of this human disease. The monitoring for chemotherapeutic response is also more complex [57] .

4.4.2 Cultured tumor cells or fresh tumor tissue The injection of a tumor cell suspension orthotopically is an improvement compared to a subcutaneous injection. The orthotopic injection allows relevant metastasis. The application

of this procedure is limited as it is only possible with established cell lines. Beside this, tumor born from tumor line cell suspension often showed a low rate of metastasis compared to surgically implanted harvested human tumor fragments [58-60] . The question if the mouse pancreas stroma after injection of a human tumor cell suspension shows different reaction compared to the human stroma during development of pancreatic cancer is open to debate.

Microsurgical implantation of small tumor pieces is another model. This technique produces a tumor, which is close to the human situation [61] . Histological observations of surgically implanted tumors demonstrate a richer vascular network and a higher metastatic potential. The improvement of surgical techniques leads to a high success rate with excellent reproducible results [59,60,62] as well as inoculated cells and tumor pieces have shown their efficacy in pancreatic cancer therapy [8,63-65] .

There is a multitude of characterized pancreatic tumor cell lines available for implantation. Some show their own metastatic potential after orthotopic inoculation, others do not [51] . It would go far beyond the scope of this review to survey all these cell lines and judge them concerning their applicability for anticancer drug testing. It remains important that cell lines may change genotypically and phenotypically after several passages in vitro causing a loss of resemblance of the developing tumors to the human tumors [46,66] .

A modification of both models is the resection of the tumor with consecutive start of the therapy. The improvement of surgical technique and the postoperative care have reduced postoperative lethality of the animals dramatically. This procedure allows imitating the clinical situation with neoadjuvant and adjuvant therapy before and after tumor resections. These models have shown their possibilities in preclinical evaluation of anticancer drugs [64,65,67] .

4.4.3 Xenogenic or syngeneic For nearly 25 years transplantable mouse tumors were applied for the investigation of anticancer drugs [46] . The reasons for the deficiency of the syngeneic mouse models are the limited variety of available tumor types and the rapid growth [68] . Biological agents to be studied in such models are required for testing in a species-directed way [69] . Beside this there are compound actions that seem to have intrinsically different features in mouse tumors compared with the human cognates [46] .

Independent of this there are significant advantages. The intact tumor–host interaction allows investigating therapies that require immune response or target-specific components of blood vessels or the extracellular matrix. One of the most used model in syngeneic pancreatic cancer is the C57Bl/6 mice in combination with Panc02 and Panc03 cell lines [20] . These models are especially interesting for the increasing number of experimental therapy approaches in pancreatic cancer focusing on the activation of body’s own immune system [70] .

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The availability of immune deficient mice enabled the application of xenotransplantation models in the mid-1980s [66] .

Judging both models (syngeneic and xenotransplant) according to their advantages or disadvantages is difficult. It is more efficient to know about the details of the xenogene transplantation model, as this transplantation model permits the application of fresh human tumor explantates as well as the use of established human tumor cell lines. It is important to know for the testing of anticancer drugs that in the syngeneic model blood supply and neovascularization are coming from the host; when explants are used only a part of the stroma comes from the host. Furthermore, there are the above mentioned specificities of ectopic and orthotopic xenotransplants to be considered [46,66] .

5. Humanized mice

The terminus ‘humanized’ mice includes various modulations of the animal: Injection of human stem cells in mice or transplantation of fragments of human organs into the mice to investigate the role of interaction of human stroma with human tumors during progression [71] .

Another model approach is the insertion of human genes into the mouse genome.

The advantages and possibilities are:

humanization of the mouse genome with relevant human • genes, especially relevant for drug-metabolization and enzyme regulation [72] reduction of the differences in relevant genes for tumor • progression [73] infl uencing, modulating and modifi cation of the immune • system of the mice reduction of the specifi c differences in drug metabolism in • animals compared to the human situation [74,75] .

Although these models enhanced the understanding of tumor progression and metastasis significantly, an application in drug development has been impeded due to limitations in practicability.

6. Mouse pancreatic intraepithelial neoplasia

In humans clinical morphologic and molecular studies have suggested that microscopic epithelial proliferations within smaller than 5 mm pancreatic ducts progress to invasive ductal adenocarcinoma. Based on the degree of atypia pancreatic intraepithelial neoplasia (PanIN) can be graded in PanIN-1 – PanIN-3 [76] . Although the generation of genetically engineered mice show lesions that mimic human PanIN and therefore have revolutionized our understanding of the precursors to invasive pancreatic ductal carcinoma, the situation in mouse models is probably more difficult. Two points of critique have to be considered: The setting in which the lesions occur in genetically engineered mouse

model is critical to their classification and direct extrapolation from lesions in mice to humans would be dangerous [23] . Therefore, the modifier ‘mouse’ should be added to the PanIN terminology – mPanIN. These mPanIN are defined as lesions, fulfilling the following criteria:

ductal epithelial proliferation confi ned to the native • pancreatic ducts the involved ducts measure < 1 mm • occurs in an appropriate setting • the lesion does not show signifi cant acinar differentiation • evidence suggests that the lesion is neoplastic. •

Evidence exists that mPanINs may progress to invasive carcinoma because they appear earlier than the invasive component in some models.

Taken together the mPanIN model is quite interesting but a more detailed consideration would be too complex for this manuscript. An excellent overview with consensus recommendations may be found at Hruban et al. [23] .

The present impact of mPanIN model for drug discovery is marginal and their future position has to be awaited.

7. Metastatic models

In an advanced state of pancreatic cancer patients show beside lymph node metastasis dissemination in more distantly located organs such as liver and later lung. Pathogenesis of metastasis is complex and consists of several segmental steps [55] . The central role plays the invasion of tumor cells into the extracellular matrix and then lymphatic and blood vessels appear to be a necessary step for the development of dissemination. A real problem has to be considered in that most of the human pancreatic cancer cell lines metastasize infrequently in mice [77] .

Beside this there are different possibilities to build-up a metastatic model:

Selection of cell lines with known metastatic potential a) Only a small number of established pancreatic tumor

cell lines show a metastatic potential in mice [78] . Owing to this it is essential to improve the incidence of metastases in animal models. Highly metastatic cells may be selected through sequential passages of human pancreatic cell lines through the liver of nude mice. The metastatic potential increases with every passage and reaches – depending on the cell line – 100% after few passages [77] . Another approach for harvesting highly metastatic cell lines is the use of a limited dilution assay. In this case clones of single cells were harvested and tested regarding their metastatic potential [79] . Spontaneous and experimental metastasis b)

The first way is the ectopic or orthotopic inoculation of tumor cells or small tumor tissue pieces into the animal. This procedure induces a local tumor and potentially the development of metastasis. The orthotopic implantation

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is more successful than ectopic implantation [80] and the use of tumor tissue seems to be more successful than tumor cell lines [78] .

Another possibility is the implementation of tumor cells intravenously, intra-arterially or intra-cardial shortly direct into the vasculature, which finally forms experimental metastasis [50] , but this version is a more artificially model. Frequent points of criticism are:

the bolus injection does not refl ect the physiological release • of tumor cells furthermore, the lack of immune response of the animals • inhibits the development of a reaction by the immune system to eliminate the circulating tumor cells this model refl ects only conditionally the metastatic potential • of a tumor.

Independent of the above points the model of experimental metastazation has the advantage of reducing the existing variable in an experiment. It may be another advantage if one requires only the later steps of metastazation in a model [50] .

For the hepatic metastazation in the pancreatic cancer model various options are established: portal vein, superior mesenteric vein and tail vein application [81] . Beside this intraperitoneal, intra-arterial, intra-splenic, intra-hepatic and intra-pancreatic injections are described. The incidence of metastasis depends highly from the cell line used. High volume intra-splenic and intra-portal injections produce nearly 100% of liver metastasis and are the best model to test novel anticancer therapies and agents [50] .

In between there are transgenic mice as metastatic model in pancreatic cancer available. There are Rip1Tag2 transgenic model, the Rip-VEGF-C/RipTag and the Kras/ink4aKO model; these models are known to form tumors with a 100% certainty. With 40 – 90% the incidence of metastasis and a latency of up to 12 weeks this model is quite long and inconsistent [82] . At present this model does not permit an estimation concerning its meaning for anticancer drug testing.

8. Possibilities for imaging of tumor growth during drug therapy

Measurements of the tumor size have favored the subcutaneous transplantation of pancreatic tumors but tumor size measurement in vivo is still an uncertain parameter for the estimation of response. The most efficient estimation of tumor size is possible after the animal has been scarified. However, tumor shrinkage after therapy response can take weeks and months whereas on the cellular and molecular levels a response is possible earlier. Skinfold chamber [83] , intravital microscopy [84] and the exteriorization of organs were described before. Their disadvantages are the limited period for observation, their primary use in ectopic models and, by exteriorization of organs, the serious morbidity [85,86] .

Despite the size of the animals (mouse, rat, hamster) used in the model ultrasound examination has been established as a very efficient method over the past few years. Statements concerning the response of therapy may be made with high-resolution probes [87] . The application of microbubbles and ultrasound offers new possibilities of displaying the modifications of tumor vascularization under therapy [88] .

Micro positron electromagnetic imaging and MRI have improved the possibilities for indirect imaging of tumor growth [89] .

Luciferase based bioluminescence assays are an improved alternative to investigate tumor growth and metastasis in vivo . The sensitivity of this method and the complex equipment are rated differentially [90] . A significantly higher resolution facilitates the direct external imaging of orthotopic tumors in the fluorescent pancreatic cancer model; here detection down to a single cell and the state of angiogenesis is possible [86] . This model has shown its potential in preclinical evaluation of chemotherapeutics [91] . At present all these models are described insufficiently for the pancreatic cancer model.

Another experimental approach is the in vivo measurement of circulating tumor derived factors. The results are conflicting but the technical feasibility and the relevance in xenograft model were shown [92,93] . The relevance for the human system and the future applicability still have to be proven [94] .

9. Histopathology and pancreatic cancer cell lines

Various pancreatic tumor cell lines were characterized earlier [51] . The evaluation of these cell lines concerning their impact for the testing of anticancer drugs would be sufficient for an independent review. The same holds true for the histopathological differences of the various tumors in the different models. A very good consensus in this context is offered [23] .

10. Future possibilities

10.1 Improving the value of the prescreening In 1990 the tumor cell line screening strategy has been shifted by the NCI (National Cancer Institute) from being compound-orientated to disease-orientated. The details of the screening procedure and the information have been described previously [95,96] . This cell line screening in vitro is simple, fast and cheap and provides valuable indicative data of mechanistic activity and target interaction. It remains the peril of false-positive and false-negative results; this is the reason for the implementation of a hollow fiber assay (HFA) in 1995 [97] . This assay is supposed to be applied as secondary xenograft screening for ‘pre-screened’ compounds [96] . A HFA has been developed to test the in vivo activity of potential anticancer compounds. The HFA assesses the pharmacologic capacity of compounds to reach two physiologic

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compartments within the mouse. The current NCI HFA does not specifically define any precise mechanisms of drug action and interaction [96,98] . Overall, the results are still inconsistent. Nevertheless, it seems that robust activity in HFA has an increased likelihood of activity in clinical trials. For the application in pancreatic cancer cell lines the system requires further development [95] .

10.2 Other animal models The disadvantages of the animal models discussed, the high cost of animal experiments and the failure of many substances in clinical trials led to investigation of alternatives. One of these alternatives is the Zebrafish model. This model has some advantages:

Zebrafi sh and humans show a high degree of similarity in • molecular mechanisms of cellular physiology high reproduction rate of the Zebrafi sh • rapid and inexpensive • in vivo analysis marginal requirements concerning laboratory • the transparent Zebrafi sh may be observed by light microscopy • most transcription factors important to pancreatic cell • differentiation are expressed in mice as well as in Zebrafi sh during organogenesis embryos of the Zebrafi sh are • permeable to drugs.

This model may be used for chemical carcinogenesis, autologus transplantation and xenotransplantation as well. Studies with higher requirements towards homologenity to humans are not possible with this animal model; this has to be considered as a disadvantage [99] .

11. Mechanisms for drug resistance in xenotransplant model

The mechanisms for resistance against anticancer drugs in vivo may be classified into three groups: i) pharmacodynamic; ii) cellular; and iii) molecular [57] . Whereas a tumor model may be intrinsically resistant to an experimental agent, the combined effect of the tumor–host interaction and anatomical implant site also affect the overall drug resistance [57] . The mechanisms of drug resistance are at present poorly understood. It could be shown that the tumor–stroma interactions play a central role, especially in pancreatic cancer with its intensive desmoplastic reaction [6] . Improperly conducted studies or application of a poor model may lead to the conclusion that the tested substance shows a lack of efficacy. The mechanisms of drug resistance especially in pancreatic cancer are a very complex field and would go beyond the scope of this review.

12. Conclusions

‘Houston we have a problem’ is what one intends to say, given the results of the past investigations of chemotherapeutics tested in clinical studies against pancreatic cancer: bevacizumab

(monoclonal antibody against VEGF) showed decreased metastatic burden and increased survival in xenograft model [100] – failure in clinical testing [101] , refoxib (inhibitor of COX-2) inhibited growth in xenograft [102] – but showed no benefit in clinical trial [103] . This may be continued with cetuximab, trastuzimab and tipifarnib to extend the list of results of the performed trials against pancreatic cancer [37] . Several large-scale human tumor xenograft programs have been recently reviewed for their performance. The results are comparable: the predictive power of a xenograft study for subsequent clinical activity fluctuates between 5 and 33% [46,104,105] . The reason why preclinical results of anticancer drugs cannot be led over in good results in human species remains unclear. It seems to be a vicious cycle; on the first view most of the animal models seem to be not efficient enough to test chemotherapeutic drugs. But the problem is: we have no better system for these preclinical investigations.

Schuh has intensively analyzed the reasons for the extensive failure of therapeutic agents in clinical studies, which showed good results in preclinical testing. Consequently, design and interpretation of preclinical studies for tumor modeling should be performed carefully. The recommendations refer to study design, lack of control of the used cell lines, the site of transplantation/injection of the tumor, well analyzed host–tumor interactions and the paraneoplastic syndrome [13] . Beside this end point definition and evaluation criteria for tumor models were analyzed.

Given all these factors to be considered during the selection process for the animal model, there are other factors for the failure of preclinically effective substances in the human system. Immune deficient mice show in most cases an excellent nature of tumor response. Most of the studies did not test the therapeutic agents on advanced metastatic disease in mice. In contrast most of the Phases I – III trial patients were treated in an advanced stadium of their tumor disease with high tumor burden and metastases in many cases; whereas the therapy in mice was started in an early state of disease or right after removal of the tumor. This is drastically in contrast to the situation in humans. Most patients suffering from an advanced metastatic tumor disease have been treated previously with other chemotherapeutics or were resistant to earlier administered drugs. This is not the best initial situation to show the success of a new anticancer drug. Another reason may be that new drugs are tested as monotherapeutic application. One has to keep in mind that it is difficult to identify combination partners for gemcitabine, the only effective drug in pancreatic carcinoma. Gemcitabine is the only agent with at least a marginal but detectable effect in the human system. The insight has prevailed that tumor biology is too complex to achieve sufficient tumor growth inhibition as well as cure just by defining the therapeutic mechanisms of single substances. This has been confirmed more or less by the disastrous failure of

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matrix–metalloprotease inhibitors for pancreatic cancer in human system [106] .

Beside this most investigators have the tendency to apply concentrations of chemotherapeutic agents representing the maximum tolerated dose for mice. This doses are often significantly higher than it is for humans [107] . The fact is that drug doses in animal models are exorbitantly higher than in the human situation.

A central problem remains the tumor–stroma interaction and its influence on the anticancer drug resistance. This problem is poorly reflected in the presented models. For the future it is necessary to consider these interactions for testing of new agents. In addition to this a better understanding of these interactions offers new therapeutic approaches for the struggle against pancreatic cancer. Very attractive therapeutic agents could be antibodies against surface receptors, signaling proteins and agents influencing SPARC [6] . These problems have to be solved alongside other reasons for anticancer drug resistance in humans.

The limitations of the xenograft model are versatile but obvious. Especially for the testing of anticancer drugs it is of essential relevance in pharmacological and pharmacodynamic aspects that humans are not mice. In many cases factors such as protein binding, drug metabolism and toxicity in humans and mice are quite different [46] . It has been proved that syngeneic mice can equalize these deficits. There is no overall reply which model – xenogenic, syngeneic or GEM – is better or worse [27,46] because criteria such as ‘advantage’ and ‘disadvantage’ are difficult to summarize in a table. Each new drug has to be adjusted concerning its mechanisms and the form of the model. Moreover the future meaning of GEM concerning the testing of anticancer drugs is still judged contradictorily, and so a final evaluation is at present not possible [27,46] . It may be possible that a final evaluation will never be possible, given the momentum in the development all models.

The xenogenic transplantation model of mice in combination with GEM-mice seems to be the most interesting system for the future [13] . It combines the human tumor with human-like genetic alterations in the host.

Independent of the applied model it has to be clarified before starting the experiments if this model is the most appropriate model to answer the investigators’ questions concerning the specific tumor localization, type of cells and the model at all. The disastrous clinical situation for patients suffering from pancreatic cancer forces every investigator to continue the efforts to find new effective anticancer drugs. The animal models have to be improved but remain the ‘workhorses’ for these investigations for the next couple of years. So we may complete the above citation: ‘Houston we have a problem – but failure is not an option!’

13. Expert opinion

The animal model plays a major role in testing of anticancer drugs in pancreatic carcinoma. For a conceivable time frame

the application of results generated from animal models will remain the bridge to clinical studies. It is deplorable that especially in testing of substances against pancreatic carcinoma the efficacy in animal models is much higher than in the clinical trials conducted thereafter, which is truly tragic for the patients suffering from pancreatic cancer. The psychological factors and the costs originated there from – on the patients’ side – have to be considered as well as the tremendous costs evolving from the development of a drug – on the industrial/clinical side. Taken together, this is a most unsatisfying situation that has to be solved sooner or later to obtain valid statements to the remaining open questions. Carcinogenesis models unfortunately do have the disadvantage of a relatively long development period of the tumor and – in comparison – marginal tumor incidence. Moreover there are relevant genetic differences in the developing tumor compared with human carcinomas. This is another situation that requires adjustment to achieve benefits for the patients. At present the transplantation model in mice represents a faster and safer alternative. The implantation of tumors evolving from cell lines as well as tissue fragments of human tumors are well investigated; it may be applied as ectop, orthotop, metastazing or resection model. Although this model seems to reflect the human tumor situation pretty well, it seems that major mechanisms of human drug resistance are still not fully identified. Therefore, their presentation in the animal model is not appropriate, as this may provide false positive results in the animal experiment. A most significant topic of the anticancer drug resistance may be explained with the protective impact of the tumor–stroma interaction. This tumor–stroma interaction requires a better analyzing of its features to obtain an application in the animal model; with GEM this is possible from a technical point of view. With GEM the researcher has the possibility to modify the single animal models. At present the experimental possibilities of ‘humanized mice’ remain unclear. Furthermore, it is still unclear how the integration of human genes into the mouse genome provides a better description of the human situation. This holds true especially for the metabolization of drugs, the immune modulation as well as the tumor–stroma interaction.

Criteria such as ‘advantage’ or ‘disadvantage’ do not measure up with the validation of the existing models Each drug supposed to be tested has to be introduced to the adequate model to obtain a valid statement concerning the efficacy of the drug.

Another topic to be considered is that the selection of the model system has to be closely correlated with the potential of the investigated drug and the questions to be answered. In pancreatic cancer orthotopic models do have clear advantages concerning the applicability and efficacy as well as considering the fact that there is a multitude of less straining possibilities to monitor the efficacy of a drug. The potential of minor models, for example, Zebrafish has to be extracted from recently achieved results.

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Given the technical and logistic complexity it would be appreciated if this specific model could be validated to preselect drugs. With a functioning Zebrafish model costs and evaluation time could be reduced significantly. Despite all deficits, especially the mouse model will still be state of the art for a certain time frame. Taken together the current situation is still not satisfying for both researchers and patients. To improve this situation the scientific community is supposed to put major efforts in the development of efficient and applicable animal

models for cancer drug research in general; results generated from other entities may be applied with just minor modifications for research in pancreatic cancer. This requires not only scientific efforts; financial support is required too.

Declaration of interest

The authors state no conflict of interest and have received no payment in preparation of this manuscript.

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Affi liation Matthias Kapischke † 1, 2 MD & Alexandra Pries 3 † Author for correspondence †1 Vivantes Hospital Spandau, Department of Surgery, Neue Bergstrasse 06, D-13585 Berlin, GermanyTel: +49 (0)30 130 132155 ; Fax: +49 (0)30130 132154 ; E-mail: [email protected] 2 University Hospital of Schleswig Holstein, Department of Surgery, Campus Luebeck, Ratzeburger Allee 160, D-23538 Luebeck, Germany 3 Chiltern International, Norsk-Data-Strasse 1, D-61352 Bad Homburg, Germany

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Animal models of pancreatic cancer for drug research