C H A P T E R

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SCREENING USING ANIMAL SYSTEMS

Angelika M. Burger* Heinz-Herbert Fiebig*,
*Tumor Biology Center at the University of Freiburg and

Institute for Experimental Oncology GmbH Freiburg, Germany

Summary 1. Introduction 2. Choice of in Vivo Systems for Large-Scale Drug Development 3. Combined in Vitro/in Vivo Testing Procedure Using Human Tumor XenograftsThe Freiburg Experience

4. Use of Transgenic Animals in the Search for New Drugs 5. Screening for Angiogenesis Inhibitors References

Summary
Anticancer drug development is historically based on in vivo screening. Although transplantable murine tumors were used until the mid-1970s, human tumor xenografts and transgenic mice dominate the disease-oriented and molecular-target-directed screening approaches of today. Xenografts have proven predictive for clinical response, and virtually any specic target can be found indigenously overexpressed in individual xenografts of large in vivo model panels such as the U.S. National Cancer Institute (NCI) 60-cell-line panel or our own panel of more than 60 xenografts. The Freiburg models were developed by subcutaneous grafting of patients tumors into immunodecient mice. More than 350 tumors, covering a broad spectrum of tumor types, were transferred into serial passage. Sixty tumors were characterized in detail for sensitivity against standard agents; for expression of oncogenes, tumor suppressor genes, growth factors, and receptors; for parameters of angiogenesis, invasion, and metastasis; or for resistance associated proteins. The response to standard agents in nude mice and in patients tumors was similar, and correct prediction was found in 90% of sensitive and 97% of resistant tu-

mors. We initially employ in vitro testing using xenograft tissues in the clonogenic assay, enabling a target (and/or surrogate)response combination in large tumor numbers. Only the most sensitive and target-expressing tumors are selected for in vivo studies, thus increasing the likelihood of response and reducing the number of animal experiments. Compounds discovered as active in our screen that have entered clinical trials include agents with diverse modes of action and specic molecular targets, such as methotrexate coupled to human serum albumin (MTX-HSA), flavopiridol, 17-allylaminogeldanamycin, and rViscumin. An overview of the available screens is provided for particular therapeutic strategies, such as the targeting of tumor angiogenesis or the application of chemopreventive agents, which require specialized in vivo assays and animal systems such as transgenic mouse models.

1. Introduction
The advent of modern cancer chemotherapy in the middle of the 20th century is exemplified by the introduction of alkylating agents, particularly nitrogen mustards, and antiCopyright 2002 by Academic Press. All rights of reproduction in any form reserved.

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metabolites into clinical use (Goldenberg and Moore, 1997). Subsequently, most of the currently available anticancer drugs have been developed in transplantable rodent tumor models. Thus, cancer drug discovery traditionally has utilized in vivo screening. The earliest models were the syngeneic transplantable murine leukemias L1210 and P388 (Fig. 1, Table 1). (Boyd, 1989; Schepartz, 1976). These tumors were the backbone of the U.S. National Cancer Institute (NCI) preclinical drug development program from the 1950s to 1985 (Boyd, 1989). The rapidly growing L1210 and P388 leukemias were implanted intraperitoneally and grew as ascites as well as systematically with survival as an end point, providing a rapid and reproducible mean of identifying cytotoxic drugs (Teicher, 1997). In 1976 solid murine tumor models, such as the B16 melanoma, the C38 colon tumor, the Lewis lung cancer, and in 1981 the M5076, a reticulosarcoma of the ovary, were added (Table 1). Three human tumor xenografts representing the major solid tumor typeslung (LX-1), colon (CX-1), and breast (MX-1)were used to test active compounds. While these in vivo models identied 35 therapeutic agents up to the early 1980s, it became evident that the classes of agents found active were limited and comprised mainly alkylating agents and some other DNAinteracting drugs. Novel structures had not been discovered for more than 20 years (Fiebig et al., 1999a; Goldin et al., 1981; Ovejera, 1987). In addition, public awareness of labo-

Unknown Compounds

In Vivo Murine P388 Leukemia Primary Screen (Disease-specific)

In Vivo Tumor Panel

ratory animal welfare, particularly in Europe (Tierschutzgesetz in Germany and United Kingdom Animals Scientific Procedures Act, both 1986), prohibited the further use of survival end points and demanded alternative end points as well as minimal use of animals for maximal gain of information (Workman et al., 1997). The above events led to a renement of the screening systems in that compound-oriented preclinical drug development (Fig. 1) was replaced by tumor type-oriented drug discovery strategies (Fig. 2) (Boyd, 1989). Increasing knowledge of genes responsible for cancer causation also facilitated renement. Hence, molecular targets for the discovery of more effective and selective anticancer agents (Sausville and Feigal, 1999; Workman et al., 1997) were dened, and the emphasis is now placed on demonstrating activity in accordance with target expression and modulation. Consequently, contemporary preclinical small-molecular drug development strategies employ a sequential test cascade that includes high-throughput compound screening in biochemical (usually with recombinant target protein) and target cell assays in vitro, followed by a very limited and carefully selected number of compounds tested in surrogate in vivo models (Fig. 2). This would greatly increase the likelihood that agents tested in vivo would then have the desired antitumor properties (Sausville and Feigal, 1999; Workman et al., 1997). Prompted by the need to identify tumor types that express the target for the new-generation cancer medicines, in vivo models used in preclinical development today are both disease oriented and target characterized, and comprise either human tumor explants/xenografts or specifically bred transgenic mice (Malakoff et al., 2000). However, due to high running costs and limited availability, transgenic mice are not suitable for large-scale drug testing, so that xenografts have become the gold standard in cancer drug development. Their use is highly recommended by regulatory agencies such as the European Agency for the Evaluation of Medicinal Products (EMEA) in the Note for guidance on the pre-clinical evaluation of anticancer medicinal products (EMEA, 1998). This chapter will review the variety of contemporary animal screening systems and the selection of in vivo models for large-scale anticancer drug development.

Formulation Toxicology

2. Choice of in Vivo Systems for LargeScale Drug Development

Two model systems with established cell lines growing in nude mice, the hollow-fiber assay and human tumor xenografts, are currently being used for large-scale in vivo testing. For compounds acting potentially via an intact immune system, the transplantable murine leukemias and solid tumors still have an accepted role (Table 2). Cancers induced by chemical carcinogens comprise another category of ex-

Clinical Trials (Disease-oriented)

FIGURE 1 Compound-oriented preclinical drug development strategy used by the National Cancer Institute, 19751985. (Adopted from Boyd, 1989).

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TABLE 1 Murine Tumor Models Used for Anticancer Drug Development at NCI Tumor type Leukemias L1210 P388 Melanoma B16 Lung Lewis Colon Colon 38 Ovary M5076 Induced by Host mice Year established Histology Main use

MCA MCA Spontaneous Spontaneous DMH DMH

DBA/2 DBA/2 C57Bl/6 C57Bl/6 C57Bl/6 C57Bl/6

1948 1952 1954 1957 1953 1981

Acute lymphoblastic Acute lymphoblastic Amelanotic Carcinoma Poorly diff. adeno cancer Reticulosarcoma

19551985 19681985 19761986 19761986 19781985 19811985

DMH, 1,2-dimethylhydrazine; MCA, methylcholanthrene.

Unknown Compounds

Biochemical Screen (High throughput tests with recombinant reagents)

Target Cell Assays (Disease-oriented in vitro primary screen in human cell line panels, to measure activity, selectivity, mode of action)

perimental tumors. In the rat it is possible to induce autochthonous tumors in most organs (Table 3) with a tumor incidence after 420 weeks ranging from 50% to 100%. 7,12Dimethylbenzanthracene- or ethylnitrosourea-induced mammary cancers in rats were useful models for the identication of new endocrine therapies (Sugamata et al., 1999). The other autochthonous models did not gain a broader application because of the requirement for long pretreatment with a carcinogen, the long induction time, and the asynchronous occurrence of the tumors. Furthermore, testing in rats was much more expensive than in mice and higher amounts of the test compound are required. A. Hollow-Fiber Assay

Establishment of Pharmacokinetic and/or Surrogate Endpoints (in blood, tumor, and normal tissue)

In Vivo Disease Model

Formulation/Toxicology

Clinical Trials (Disease-specific)

FIGURE 2 Current test cascade of disease-oriented, targeted anticancer drug development. Adopted from UKCCCR Guidelines for the Welfare of Animals in Experimental Neoplasia (2nd ed., 1997, Appendix 4a, p. 25).

The NCI favors the hollow-ber assay in which more than 50 human tumor cell lines have been grown successfully in polyvinyl fibers of 1 mm inner diameter. Normally three bers with three different cell lines are implanted intraperitoneally and subcutaneously per mouse. Each fiber (2 cm length) contains 50200,000 cells in 20 l. The treatment is normally administered intraperitoneally using a daily (for 4 days) schedule, starting on the third or fourth day after ber implantation. The bers are harvested on day 58, and the viable cell number is determined using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assay (Hollingshead et al., 1995, 1999). The NCI has validated the hollow ber assay in comparing the activity of 84 compounds in permanent cell lines growing in the hollow-ber assay and as subcutaneous xenografts in nude mice. In the hollow-ber assay 29 compounds were rated as active whereas in the same tumors growing subcutaneously only four compounds were active. Therefore, as an in vivo screen, the hollow-ber assay

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TABLE 2 In Vivo Models for Large-Scale Anticancer Drug Testing Host NuNu mice NuNu mice Type Hollow-ber assay Established human solid tumor models Localization IP, SC SC, orthotopic Reference Hollingshead et al., 1999 Fiebig et al., 1999a Fiebig and Burger, 2001 Hoffman, 1999 Waud, 1997 Corbett et al., 1997 Berger, 1999

Mice Mice Rats

Transplantable leukemias Transplantable tumors Chemically induced cancers

IP, IV, SC SC, IV See Table 3

DMBA, 7,12-dimethylbenzanthracene.

is feasible for the rapid identication of inactive compounds and for prioritization of active compounds at reasonable cost. However, active compounds must be studied subsequently in human tumor xenografts growing subcutaneously or orthotopically before a decision for development should be taken. The latter must be carefully considered under the view that the requirements for Good Manufacturing Practice drug synthesis, Good Laboratory Practice toxicology, pharmacokinetics, safety pharmacology, and clinical trials imply a multimillion dollar investment. B. Human Tumors Growing in Nude Mice As a second well-established testing procedure for largescale in vivo testing, tumors can be derived from cancer patients and grown subcutaneously or orthotopically in nude mice. Such xenografts can be generated from almost all solid cancers. Subcutaneous implantation is much easier to perform than orthotopic implantation, allowing for testing of broad tumor panels (Fiebig et al., 1992, 1999a). Patient-derived hu-

man tumor xenografts, growing subcutaneously, have been shown to maintain typical histologic characteristics, chemosensitivity proles, expression of relevant oncogenes, growth factors/growth factor receptors, and parameters associated with angiogenesis, invasion, and metastasis. Chemosensitivity studies of novel molecular targeted therapies are normally performed in well-characterized models expressing the target against which the new compound should act. Orthotopically growing tumors have the advantage that metastases occur in much higher frequency and the invasion seems to be more pronounced compared with subcutaneously growing models (Hoffman, 1999). However, the implantation technique is more time consuming, allowing only a limited number of compounds to be tested per experiment. Usually serial size measurements are not possible for tumors growing in internal organs. Therefore, the mice have to be sacriced to measure tumor volume that allows only a one-point measurement. This is a major drawback of this otherwise very attractive, more clinically relevant disease model. Broad testing of standard agents or novel compounds has not been

TABLE 3 Autochthonous Chemically Induced Cancers in Rats Tumor type Mammary Sarcoma Hepatoma Esophagus Stomach Colon Bladder Brain, CNS Leukemias Histology Adeno cancer Fibrosarcoma Hepatocellular Squamous cell Adeno cancer Adeno cancer Carcinoma Glioblastoma Acute myelocytic Carcinogen DMBA, ethyl nitrosourea Benzo(a)pyrene Diethyl nitrosourea Phenylethyl nitrosamine Methyl nitrosourea 1,2-Dimethylhydrazine Butylbutanol nitrosamine Butyl nitrosamine DMBA Ethyl nitrosourea
DMBA, 7,12-dimethylbenzanthracene.

Incidence (%) 100 190 190 180 190 190 190 190 190 5080

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reported using orthotopic implantation, which means that the procedure is too time consuming and expensive for largescale testing. However, the orthotopic model provides the most appropriate evaluation of specic inhibitors of metastases or invasion.

3. Combined in Vitro/in Vivo Testing Procedure Using Human Tumor XenograftsThe Freiburg Experience
The testing of novel components for in vivo activity in randomly selected models has a low probability of success, apart from series of closely related analogues of an active compound. Therefore, additional criteria for selecting appropriate models are necessary, and two approaches are particularly promising. If the target or mechanism of action is known, tumors overexpressing this target should be selected. If the mechanism is not known, in vitro testing in a panel of xenografts using the clonogenic assay will allow identica-

tion of the most sensitive tumors, which are subsequently studied in vivo (Fig. 3). We have rened the clonogenic assay and have observed growth of colonies in more than 86% of xenografted tumors. This percentage is markedly higher than that for fresh tumor material derived from patients (4050%). We were able to use 24-well plates, and at least 20 colonies were required for an assay to be considered as evaluable. Details of the methodology have been described previously (Fiebig et al., 1992, 1999a). A. Combined in Vitro/in Vivo Testing Procedure We have recently developed a combined in vitro/in vivo test procedure for anticancer drug development (Fiebig et al., 1992, 1999a), and an outline of the five-step evaluation is shown in Fig. 3. In the rst stage, large-scale tests (primary screening) are performed in vitro using a modified clonogenic assay in six human tumors obtained from serial passage in nude mice. Secondary in vitro screening is performed in a total of 20 human tumor xenografts, 14 being sensitive against

Stage I

Inhibition of tumor stem cells in vitro In 3 resistant and 3 sensitive human tumor xenografts

Stage II

Inhibition of tumor stem cells in vitro In 6 resistant and 14 sensitive human tumor xenografts

Stage III

In vitro effect in human hematopoietic stem cells (CFU-GM)

In vivo effect in 23 tumors growing s.c. in nude mice

Stage IV

Tumor type-oriented testing in vitro In 60 well-characterized representative human tumors, 36 models from 1012 types Phase II Response prediction

Stage V

Tumor type-oriented testing in vivo In most sensitive and target expressing tumors from stage IV, parallel measurement of target expression under therapy and/or surrogate markers

FIGURE 3 Combined in vitro/in vivo testing procedure with human tumor xenografts.

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standard agents and 6 being resistant. In stage III, the effect on human hematopoietic stem cells (granulocyte-macrophage colony-forming units, (CFU-GM) from 24 healthy donors is studied. Compounds with a greater or similar effect on tumor cells in comparison with hematopoietic stem cells are subsequently studied in vivo in 23 of the most sensitive tumors transplanted subcutaneously into nude mice. The comparison of in vitro and in vivo activity enables assessment of the relevant in vitro dose, based on in vivo pharmacology. If remission or tumor stasis is observed in vivo, the compound undergoes tumor-type-oriented testing, usually in 4060 xenografts. Since our other studies have suggested that the tumors selected for stage IV have chemosensitivity profiles similar to those of clinical cancers, target tumors for clinical studies as well as tissue-specic preclinical models can be identied. B. Take Rates and Growth Behavior of Patient Tumor Explants in Nude Mice Of 1600 patient tumors, the growth behavior of 1227 different human malignancies growing subcutaneously in nude mice has been characterized in detail (Tables 4 and 5). Histologic examination of the tumors showed that 79% contained viable tumor tissue. However, only 41% of the tumors showed a rapid growth with tumor diameters of more than 8 mm after 90 days (Table 4) equal to length width of more than 60 mm2. Thus, only these rapidly growing tumors were suitable for establishing stably growing models and for further investigation. Nonetheless, it must be noted that the growth of human tumors of the latter described category is still much slower than that seen in established murine models. Four hundred (33% of the total explants) of the 499 rapidly growing human tumor explants were successfully transferred in serial passage and most of them were cryopreserved in liquid nitrogen with a recovery of 90% (Table 5). Tumors in serial passage can be divided into three categories. The rate was highest (4060%) in tumors of the esophagus, cervix and corpus uteri, colon, lung (small cell), and melanomas. Intermediate growth rates between 20% and 40% were observed for carcinoma of the lung (non-small cell); soft-tissue sarcoma; carcinoma of the ovaries, head and neck, pancreas, testicle, stomach, and bladder; and pleuramesothelioma. The lowest rates (519%) were observed in renal cell cancers and in the hormone-dependent mammary and prostate carcinomas. The growth of breast and prostate cancers was increased when intramuscular estrogen or dihydrotestosterone depots were given every 2 weeks. Among the over 400 models, 264 have been selected for further characterization, such as by determination of chemosensitivity proles. These models showed a consistent growth behavior and typical histologic and differentiation

TABLE 4 Growth of Human Tumors in Nude Mice (Freiburg Experience 19801994) Rapid growtha Tumor origin Esophagus Cervix uteri Colorectal Corpus uteri Melanomas Lung, small cell Lung, non small cell Sarcomas, soft-tissue Ovary Head and neck Pancreas Brain Hepatocellular Neuroblastoma Osteosarcoma Miscellaneous Testicle Mesothelioma Stomach Bladder Renal Mammary Prostate Total Total n 10 10 152 8 63 39 227 79 22 47 6 6 3 3 13 100 48 36 68 44 124 74 41 1227 n 7 7 88 4 39 14 118 36 7 16 2 2 1 1 5 33 14 14 17 17 37 13 7 499 % 70 70 58 50 62 36 52 46 32 34 33 33 33 33 38 33 29 39 25 39 30 18 17 41 Serial passageb n 6 6 78 4 27 16 87 29 8 16 2 2 1 1 4 26 12 9 17 10 24 13 2 400 % 60 60 51 50 43 41 38 37 36 34 33 33 33 33 31 26 25 25 25 23 19 18 5 33

characteristics. Rapidly and slowly growing tumors were included, as were models that were sensitive and resistant to standard agents. At the University of California, in a similar effort to establish human tumor models from surgical specimens, a total of 323 explants was collected from patients (Reid et al., 1978). The latter yielded in the development of only 27 (8%) transplantable models. Although the California group had similar good results with respect to retaining patient characteristics, their markedly lower success rate (8%) in obtaining stable, transplantable models versus that of our program (33%) might be due to the processing of the specimens. While they minced and then injected the tumor suspensions subcutaneously into nude mice without paying attention to the gender of the patient from which the tumors were derived or the age of the mice, we established our models from tumor fragments in very young mice (age 4 weeks) of the same gender as the donor patient. By doing that we assured that the xenografted tumor speci-

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TABLE 5 Human Tumors Available as Tumor Models in Nude Mice Median doubling time (days) 418 619 416 614 424 423 316 718 617 820 422 Total 324 Frozen in nitrogen takea/total 21/25 17/18 12/14 9/12 42/44 13/14 14/15 10/11 14/18 8/9 40/43 200/223 (90%)
a

Category Lung Lung Lung Lung Colorectal Melanomas Soft tissue Renal Stomach Mammary Miscellaneous Total

Total no. 38 20 12 7 50 24 17 16 14 8 58 264

Histology Epidermoid Adeno Large cell Small cell Adeno Melanoma Soft tissue Adeno Adeno Adeno and solid

Response totalb 7/18 3/5 1/3 6/7 2/20 1/10 3/6 0/7 6/13 6/8 13/27 48/124 (39%)

After being frozen. Partial remissions following single-drug therapy.

mens were provided with the most optimal environment such as their own extracellular matrix (ECM), appropriate hormonal conditions, high exposure to murine endogenous growth factors, and lowest probability of graft rejection. C. Selection and Characterization of Tumor Models During the last 12 years we have established more than 300 regularly growing human tumor xenografts, which are frozen in liquid nitrogen (Fiebig et al., 1992). After a detailed characterization (Table 6), 6 xenografts were selected for stage I, 20 for stage II, and 60 for stage IV in vivo testing. These 60 models were characterized in detail for oncogenes, tumor suppressor genes, growth factors and receptors, invasion- and metastasis-related proteins, as well as parameters for angiogenesis-, immortality-, and drug-resistance-associated proteins, e.g., k-ras, c-myc, p53, EGF-R, TGF-, TGF, VEGF, telomerase as well as mdr1, mdr3, GST (Table 6). Data for 29 molecular targets are available in our database, and we are able to correlate the expression of potential molecular targets with in vitro effective dose levels derived from clonogenic assays using the xenograft material. This bioinformatic approach is based on the COMPARE algorithm developed by the NCI (Paull et al., 1989); it allows the determination of any statistical interaction with the potential molecular targets (Spearman rank coefcient), thus providing a clue as to the possible mechanism of action of the drug. It is crucial for a new drug to demonstrate a differential selectivity against human tumors compared with the most

sensitive normal tissue. In this respect, human tumor xenografts are considered to be the most relevant models since, like patient-derived tumors, they (1) grow as a solid tumor, (2) develop a stroma, (3) develop vasculature, (4) develop central necrosis, (5) show some degree of differentiation, and (6) have relevant response rates (Fiebig et al., 1992, 1999a). In most cases, the tumor xenografts architecture, cell morphology, and molecular characteristics mirror those of the original cancers. This is in marked contrast to xenografts derived from cell lines, which in general show a homogeneous undifferentiated histology and as a result are very resistant to most of the standard agents (Fiebig et al., 1999a; Goldin et al., 1981). This is most likely due to the high selection pressure in vitro during long-term culture, resulting in aggressive subclones. D. Correlation of Drug Response As a rst step, clinically established anticancer drugs were tested in broad doseresponse studies. The relevant concentrations or doses reecting clinical efcacy were determined for each drug separately by comparing the in vitro/in vivo response rates of sensitive tumor types (Fiebig et al., 1987, 1992; Scholz et al., 1990; Steel, 1987). The following correlations were performed: Nude mouse models versus patient response Clonogenic assays from xenografts versus patient response Clonogenic assays from xenografts versus in vivo testing in the nude mouse after subcutaneous transplantation.

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TABLE 6 Validated and Novel Molecular Targets Characterized in the 60-HumanTumor Xenograft Panel Category Oncogenes Target k-ras c-myc c-erb-B2 Di12 p53 EGF-R TGF- TGF- EGF bFGF Estrogen receptor Progesterone receptor uPA Cathepsin D MMP-2 MMP-3 MMP-9 TIMP-2 nm23 CD44v6 VEGF Vascular porosity Microvessel density Angiogenin Cytokeratin Telomerase MDR-1 MDR-3 GST

TABLE 7 Comparison of Drug Response in Human Tumors Growing Subcuteously in Nude Mice and in Cancer Patients Mouse/patient Remission/remission No remission/remission No remission/no remission Remission/no remission Total 19 2 57 2

Tumor supressor genes Growth factors and receptors

Eighty comparisons were obtained in 55 tumors. Xenografts predicted correctly for response in 90% (19/21), resistance in 97% (57/59).

Invasion related

found also in other in vitro systems, is that the correct prediction for tumor sensitivity is in the range of 6070%. In this respect the xenografts are more reliable. Other laboratories have also demonstrated the high predictivity of the xenograft model (Steel, 1987). E. Selected Examples of Anticancer Agents in Clinical Trials Which Have Been Discovered in a Target-Oriented Approach Using the Freiburg Xenografts For the past 10 years our group has received compounds from academia, the EORTC, the NCI, and the pharmaceutical industry (Burger et al., 1997, 1998, 1999a, b; 2000; Drees et al., 1997; Fiebig et al., 1994, 1995, 1999b; Hartung et al., 1999; Hendriks et al., 1993, 1999; Klenner et al., 1992; Maly et al., 1995; Stehle et al., 1997; Winterhalter et al., 1993). They belonged to various structural classes and act by different mechanisms. An overview of compounds that showed activity in our models and where we contributed to the decision to develop the compounds clinically is shown in Table 8. Compounds that have now entered clinical trials are methotrexate coupled to human serum albumin (MTX-HSA) as synthesized at the German Cancer Research Center; pH-sensitive salicylic acid derivatives from the University of Freiburg; avopiridol, spicamycin, and 17-allylaminogeldanamycin obtained from the NCI; ecteinascidin and rhizoxin from the EORTC; and recombinant mistletoe lectin (rML rViscumin) and various other compounds from the pharmaceutical industry. rML provides a good example of a preclinical development using the Freiburg combined in vitro/in vivo xenograft approach (Figs. 35). rML is the recombinant precursor of mistletoe lectins IIII produced in Escherichia coli with a molecular weight of 57 kDa, and has been shown to possess ribosome-inactivating activity (RIP) leading to a specic induction of apoptosis (Langer et al., 1999). We found that rML was cytotoxic in the nanomolar range in permanent growing tumor cell lines in vitro. In subsequent stage IV test-

Metastases related Angiogenesis

Structural proteins Immortality Drug resistance related

Details to the methodology used, the tumors included and the procedures for evaluating combinations have been published by our group (Fiebig et al., 1987, 1992, 1999a; Scholz et al., 1990; Steel, 1987). Subcutaneously growing xenografts showed the highest correlation with the response of patients: the correct prediction for tumor responsiveness was 90% (19/21 cases) and for tumor resistance 97% (57/59 cases) (Table 6). In other words, the positive predictive value (or the true positives) was 90% and the negative predictive value (or the true negatives) was 97%. The clonogenic assay predicted correctly for tumor response in 62% and for resistance in 92% (Table 7). The in vitro/in vivo correlations obtained in the clonogenic versus the nude mouse system were similar as for the clonogenic assay versus the patient. The correlations obtained in our clonogenic assays using xenografts as donor tumor were similar to those reported by many other groups when patients tumors were used (Hamburger and Salmon, 1977; Von Hoff, 1987). One drawback,

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TABLE 8 Anticancer Drugs Discovered as Active in the Freiburg Human Tumor Xenograft Panel Mode of Action From academia: MTXHSA Lip. NOAC pH-sensitive SA Flavopiridol Quinocarmycin Spicamycin 17-AAG ET743 Rhizoxin EO9 Lobaplatin D21266 BAY 38-3441 RML/rViscumin Several discrete compounds Antimetabolite, EPR effect Antimetabolite Apoptosis Cyclin-dependent kinase inhibition DNA binding Glycoprotein synthesis HSP90 modulation DNA minor grove alkylation Tubulin binder Bioreductive alkylating DNA cross-linking and adducts PKC and PLC inhibition Topoisomerase II inhibition RIP, ribosome inactivation/apoptosis Ref. Stehle et al., 1997, Hartung et al., 1999 Fiebig et al., 1995 Burger et al., 1999a Drees et al., 1997 Fiebig et al., 1994 Burger et al., 1997 Burger et al., 1998 Burger et al., 2000 Hendriks et al., 1999 Winterhalter et al., 1993 Hendriks et al., 1993 Klenner et al., 1992 Maly et al., 1995 Fiebig et al., 1999b Burger et al., 1999b

From the U.S. NCl:

From the EORTC:

From companies:

MTX-HSA, methotrexate coupled to human serum albumin; EPR, enhanced permeability and retention effect; SA, salicylic acid; PKC, phosphokinase C; PLC, phospholipase C; NOAC, N-octadecyl-cytosine arabinoside; AAG, 17-allylaminogeldanamycin.

ing in 47 human tumor xenografts in the clonogenic assay, we found that rML has a differential cytotoxicity prole (Fig. 4), with certain tumors being more sensitive or more resistant. Among the sensitive tumor types were breast, prostate, pancreas, and small cell lung cancers (Fig. 4). Thus, the sensitive in vitro small cell lung tumor model LXFS 538 was chosen for evaluation of in vivo pharmacologic activity (Fig. 5). Marked tumor growth inhibition (T/C 27%) was observed at the maximal tolerated dose of the drug (3 g/kg) given on days 1418 and 2125. Figures 4 and 5 clearly demonstrate that the in vitro activity of rML could be translated into in vivo efcacy. F . Future Impact of Human Tumor Xenografts Inasmuch as new drug development is already focused on target-oriented and tumor-cell-specic approaches based on currently available knowledge, there is still a continuing demand for a better understanding of tumor biology. Thus, basic research can be directed to genetic tumor proling, nding more promising tumor-type-specic therapeutic targets, and individualizing cancer therapy. In accomplishing this, genomics and proteomics now provide a large number of possible new gene candidates for therapeutic intervention. However, the validation of these genes in a wide collection of human tumor specimens provides a bottleneck; in particular, the lack of sufficient tumor tissue material to perform and thoroughly repeat a set of confirmatory analyses such as Northern or Western blots is often problematic. Here our xenograft collection not only provides a rich source of the hu-

man genome and proteome, but also allows repeated growth of material of consistent quality. Tissues can be collected while fresh and immediately frozen, resulting in total RNA and mRNA of extremely high quality. Moreover, the optimal tissue-processing procedure can be evaluated and novel treatments, either small molecules or gene therapy, can be tested later on in the same tissue from which a target was isolated and validated. In addition, pre- and posttreatment specimens for a pharmacogenomic approach can easily be collected. In view of these possibilities, the usefulness of human tumor xenografts and explants will steadily increase.

4. Use of Transgenic Animals in the Search for New Drugs

A battery of human oncogenes and tumor suppressor genes has now been identified, and the prospect of finding even more important cancer-causing and preventing genes has increased with sequencing of the human genome (Futreal et al., 2001; Lander et al., 2001; Venter et al., 2001). The functional characterization of genes and proof-of-disease hypotheses can be accomplished by designing either murine gene knockout or transgenic animals (Malakoff et al., 2000; Palmiter and Brinster, 1985). In the latter case, a gene in the form of a DNA fragment (viral or cellular origin) is linked to a suitable promoter DNA sequence and injected into a mouse egg nucleus. When the recombinant DNA molecule becomes integrated into the mouse chromosome, a transgenic mouse strain has been generated, which carries the gene in all cells. The

FIGURE 4 Mean bar graph presentation of rML activity in 47 human tumor xenografts in the clonogenic assay. Bars to the left represent more sensitive, bars to the right more resistant tumor types compared to the mean IC70 over the whole panel. Sensitive tumors were mammary carcinomas, small cell lung carcinomas, pancreas and prostate cancers. From this panel, xenografts were chosen for in vivo efcacy studies, such as the LXFS 538 small cell lung cancer model. This study represents the stage IV testing outlined in Fig. 3. The names of the xenografts resemble the tumor type (rst two letters, e.g., MA, mammary), X is the xenograft, F the Freiburg panel, followed by the continuous tumor collection number.

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1800 1600 Median Relative Tumor Volume [%] 1400 rML 0.3 g/kg 1200 1000 800 600 400 200 0 0 3 7 10 14 Time [d] FIGURE 5 rML antitumor activity in the human small cell lung cancer xenograft LXFS-538. In vivo activity of rML versus Adriamycin in the LXFS 538 small cell lung cancer human tumor xenograft. Median relative tumor volumes are compared. rML was administered intraperitoneally on a daily 5 schedule weekly for 4 weeks. Adriamycin was administered intravenously on days 1 and 15. rML (3 g) was able to induce signicant tumor growth inhibition in nude mice with an optimal T/C of 27% compared with control. In the latter group, 1.5 g of rML/kg was given initially for two cycles, and was then increased to the maximal tolerated dose levels of rML (3 g/kg) starting on day 14 (arrow). This resulted in tumor shrinkage and a statistically signicant inhibitory effect ( p 0.001). 17 21 24 28 35 42 ADR 8 mg/kg Control rML 1.5/3 g/kg

expression of the transgene is determined by the tissue specicity of the associated promoter (Alberts et al., 1994). Several transgenic models of recessive or dominant oncogenes have proven to mimic human disease and disease progression, e.g., the TRAMP mouse (transgenic adenocarcinoma mouse prostate). TRAMP mice which express high levels of the transgene (Table 9) displayed progressive forms of prostatic disease that histologically resembled human prostate cancer, ranging from mild hyperplasia to multinodular malignant neoplasia (Greenberg et al., 1995). Nevertheless, transgenic mice have not been used for large-scale anticancer drug testing so far because of high running costs and the difculties of obtaining synchronous onset of tumor growth and development. Examples of transgenic mouse models used for evaluation of drug effects on cancer growth are listed in Table 9. The wap-ras mouse is the most frequently employed transgenic model in cancer drug development and has been used to demonstrate preclinical in vivo activity of the ras/farnesyl protein transferase inhibitor SCH-66336. This agent has now advanced to clinical trial (Table 9) (Liu et al., 1998). Transgenic mice are also exploited in prophylactic chemotherapy approaches to study novel agents that, when given chronically, can prevent or reduce the growth of tumors. In TRAMP mice, daily administration of 20 mg/kg of

the nonsteroidal anti-inammatory drug R-urbiprofen significantly lowered the incidence of metastasis and reduced the occurrence of primary prostate tumors (Wechter et al., 2000). Diuoromethylornithine, given to K14-HPV16 transgenic animals, diminished the precursor lesions of ear and chest skin malignancies (Table 9) (Arbeit et al., 1999). Since the transgenic technology emerged only in the early 1980s, there is still a need to systematically characterize and validate such tumor models with respect to their predictivity of clinical response and thus to determine their ultimate usefulness in the search for new anticancer drugs. Transgenic mice will undoubtedly enlarge our in vivo testing systems for cancer drug development because they can be designed to facilitate answering questions regarding risk assessment and carcinogenesis, xenobiotic metabolism, ligand-mediated toxicity, immunotoxicity, and drug resistance.

5. Screening for Angiogenesis Inhibitors

The hallmark of angiogenesis is the formation of new blood vessels from endothelial cells, which can be regulated by inducers and inhibitors of endothelial cell proliferation and migration (Hanahan and Folkman, 1996; Risau, 1997).

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TABLE 9 Transgenic Models for Cancer Drug Development Model Bcr/abl TRAMP MMTV-ras MMTV-myc Wap-ras P53 RIP1-Tag2 K14-HPV16 BM-MDR Transgene bcr/abl p190 cDNA
a

Tumor type Acute leukemia Prostate cancer Mammary tumors Mammary tumors Lung carcinoma, lymphoma, sarcoma Pancreatic islet cancer Epidermal cancers None, bone marrow cells with multidrug transporter

Use Kinase inhibitors Chemoprevention Modeling disease progression Farnesyltransferase inhibitors Tubulin-binding agents Predisposition/mutagenesis studies Angiogenesis inhibitors Chemopreventative agents Agents that reverse MDR/ chemosensitization

Ref. Huettner et al., 2000 Greenberg et al., 1995 Wechter et al., 2000 Sinn et al., 1987 Liu et al., 1998 Porter et al., 1995 Lavigueur et al., 1989 Nichol et al., 1995 Bergers et al., 1999 Arbeit et al., 1999 Mickisch et al., 1991a Mickisch et al., 1991b

PB SV40 lage Tag

c-myc or v-Ha-ras MMTV driven Ha-ras, cwap driven

mtp53 (Val-135)
d

RIP1-SV40 Tag

HPV 16, eK14-driven MDR-1

a b c

Prostate-specic rat probasin promoter driving SV40 large T-antigen-coding region. Mouse mammary tumor virus long terminal repeat promotor. Whey acidic protein promotor. d Rat insulin promoter-SV40 tumor antigen. c Wt HPV early region cloned behind human keratin-14 promoter.

Factors that govern pathologic tumor angiogenesis are produced/induced by tumor cells directly or are derived from the ECM components. Tumor angiogenesis is a complex process involving extensive interactions between tumor cells, host endothelial cells, soluble factors such as vascular endothelial growth factor (VEGF), acidic and basic broblast growth factor, and the surrounding stromal elements (Hanahan and Folkman, 1996; Jones and Harris, 1998; Risau, 1997). Therefore, the development of antiangiogenic and antivascular therapies raises a specic challenge of modeling the interplay between host and tumor cells and their response to therapy. Here, in vivo assays are needed at an early stage of drug development to provide a meaningful experimental disease model. Animal models used in the development of antiangiogenic/vascular therapies can be divided into three categories (Table 10): those that involve tumors growing in mice, those

that use normal vascular tissues as surrogates for tumor blood vessel formation (chick chorioallantoic membrane model is most widely used), and ex vivo assays such as aortic ring tube formation (Table 10) (Giavazzi et al., 1999; Hoffmann et al., 1999). While proof-of-principle mechanistic studies of potential antiangiogenic agents demonstrating inhibition of neovascularization are most important, effects in tumor-based models showing tumor shrinkage or inhibition are also needed prior to drugs entering clinical trials. For this purpose, both rodent tumors and human tumor xenografts have been used (Giavazzi et al., 1999). Rodent models have the advantage of a syngeneic environment in that the tumor, blood supply, and stromal elements are all from the same genetic background. However, they are limited by their restriction to certain rapidly growing tumor types (Table 1) and by the fact that the target, if tumor based, is nonhuman. In contrast, if human tumor

TABLE 10 Animal Models of Angiogenesis Mouse in vivo tumor models Subcutaneous tumors syngeneic human xenografts Lung metastasis B16F10, LL Orthotopic tumors Transgenic mice (e.g., RIP1) Cranial tumor window Other in vivo models Corneal pocket assay rabbit, rat, mouse Hamster cheek pouch Dorsal skin fold window chamber Hollow-ber assay Alginate beads Matrigel plugs CAM assay Ex vivo models Aortic ring assay bovine porcine rat

LL, Lewis lung; CAM, chick chorioallantoic membrane; RIP1 model, pancreatic islet cancer.

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xenograft models are used, artifacts due to host (murine vascularization)/graft (human tumor cells) interactions may arise. Positive aspects of the use of patient-derived xenografts include the large variety of available tumor types (Tables 4 and 5); the close resemblance of tumor architecture, microvessel density, and vascular permeability of clinical cancers; and the expression of angiogenic factors such as VEGF (Berger et al., 1995; Schler et al., 1999). A large number of antiangiogenic and antivascular agents are currently under preclinical and clinical development (Jones and Harris, 1998). They include agents targeting endothelial cell signaling (VEGF and its receptor), endothelial cell motility and structure (endothelial cell tubulin binders), tumor vessel physiology (liposomes and polymers exploiting the enhanced permeability and retention by tumor blood vessels), and proteases degrading the ECM (Jones and Harris, 1998; Schler et al., 1999). In most cases, several of the in vivo model systems described above have been employed. However, none of the potential antiangiogenic agents have proven clinical activity so far. Thus, the preclinical angiogenesis models remain unvalidated, and results obtained must be interpreted with great care. In the absence of a mechanistic hypothesis for a vascular effect and/or pharmacodynamic end-point assay to demonstrate that the proposed mechanism is operative, currently available models should not be considered as screens for antiangiogenic drugs. For example, studies with anti-VEGF antibodies should be performed only in tumors expressing high VEGF levels, using VEGF reduction or VEGF binding as surrogate end point. Finally, when negative clinical data are obtained, preclinical models should be revisited to address why they have failed to predict the response/outcome. For instance, the rst generation of matrix metalloproteinase inhibitors (MMPIs) did not live up to expectations and delivered discouraging results in phase III trials. Although effects on the isolated target were clearly demonstrated and a number of commonly used preclinical angiogenesis and metastasis models had produced certain activity, most MMPIs failed. Hence, the reasons need to be identied in order to improve clinical trial design and enable the optimal translation of preclinical results into the clinic.

References
Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. D. (1994). The actions of oncogenes can be assayed singly and in combinations in transgenic mice. In Molecular Biology of the Cell. 3rd ed., chp. 24, Garland Publishing, Taylor & Francis Group, New York. Arbeit, J. M., Riley, R. R., Huey, B., Porter, C., Kelloff, G., Lubet, R., Ward, J. M., and Pinkel, D. (1999). Diuoromethylornithine chemoprevention of epidermal carcinogenesis in K14-HPV16 transgenic mice. Cancer Res. 59, 36103620. Berger, M. R. (1999). Autochthonous tumor models in rats: is there still a relevance for anticancer drug development. In Relevance of Tumor Models in Anticancer Drug Development (H. H. Fiebig and A. M. Burger, eds.), Vol. 54, pp. 1527. Karger, Basel. Berger, D. P., Herbstritt, L., Dengler, W. A., Marme, D., Mertelsmann, R., and Fiebig, H. H. (1995). Vascular endothelial growth factor (VEGF) mRNA expression in human tumor models of different histologies. Ann. Oncol. 6, 817825. Bergers, G., Javaherian, K., Lo, K. M., Folkman, J., and Hanahan, D. (1999). Effects of angiogenesis inhibitors on multistage carcinogenesis in mice. Science 284, 808812. Boyd, M. R. (1989). Status of the NCI preclinical antitumor drug discovery screen. In Principles and Practice of Oncology (S. Hellman, DeVita, V. T., and S. A. Rosenberg, eds.), Vol. 3, pp. 112. Lippincott, Philadelphia. Burger, A. M., Kaur, G., Hollingshead, M., Fischer, R. T., Nagashima, K., Malspeis, L., Duncan, K. L., and Sausville, E. A. (1997). Antiproliferative activity in vitro and in vivo of the spicamycin analog KRN5500 with altered glycoprotein expression in vitro. Clin. Cancer Res. 3, 455463. Burger, A. M., Fiebig, H. H., Newman, D. J., Camalier, R. F., and Sausville, E. A. (1998). Antitumor activity of 17-allylaminogeldanamycin (NSC 330507) in melanoma xenografts is associated with decline in Hsp90 protein expression. Ann. Oncol. 9(Suppl. 2), 132. Burger, A. M., Steidle, C., Fiebig, H. H., Frick, E., Schlmerich, J., and Kreutz, W. (1999a). Activity of pH-sensitive salicylic acid derivatives against human tumors in vivo. Clin. Cancer Res. 5, 205. Burger, A. M., Mengs, U., Schler, J. B., Zinke, H., Lentzen, H., and Fiebig, H. H. (1999b). Recombinant mistletoe lectin (rML) is a potent inhibitor of tumor cell growth in vitro and in vivo. Proc. Am. Assoc. Cancer Res. 40, 399. Burger, A. M., Sausville, E. A., Camalier, R. F., Newman, D. J., and Fiebig, H. H. (2000). Response of human melanomas to 17-AAG is associated with modulation of the molecular chaperone function of Hsp90. Proc. Am. Assoc. Cancer Res. 41, 447. Corbett, T., Valeriote, F., LoRusso, P., Polin, L., Panchapor, C., Pugh, S., White, K., Knight, J., Demchik, L., Jones, J., and Lisow, L. (1997). In vivo methods for screening and preclinical testing. In Anticancer Drug Development Guide (B. A. Teicher, ed.), pp. 75100. Humana Press, Totowa, NJ. Drees, M., Dengler, W., Roth, T., Labonte, H., Mayo, J., Malspeis, L., Grever, M., Sausville, E., and Fiebig, H. H. (1997). Flavopiridol (L868275): Selective antitumor activity in vitro and in vivo for prostate carcinoma cells. Clin. Cancer Res. 3, 273279. EMEA, The European Agency for the Evaluation of Medicinal Products Human Medicines Evaluation. (1998). CPMP/SWP/997/96 Committee For Proprietary Medicinal Products (CPMP) Note for guidance on the pre-clinical evaluation of anticancer medicinal products. (http:// www.eudra.org/emea.html), London. Fiebig, H. H., and Burger, A. M. (2001). Human tumor xenografts and explants. In Animal Models in Cancer Research (B. A. Teicher, ed.), Humana Press, Totowa, NJ, in press. Fiebig, H. H., Schmid, J. R., Bieser, W., Henss, H., and Lhr, G. W. (1987). Colony assay with human tumor xenografts, murine tumors and human bone marrow. Potential for anticancer drug development. Eur. J. Cancer Clin. Oncol. 23, 937948.

Acknowledgments
We are grateful to our colleagues Cornelia Steidle and Elke Simon for their important contributions to this project. We thank Hildegard Willmann for software development and assistance with data evaluation as well as Ines Fernandez for her help with the manuscript. The work was supported by grants from the German Ministry for Research and Technology (HHF); the U.S. National Cancer Institute, Biological Testing Branch (HHF); and the Deutsche Froschungsgemeinschaft DFG (AMB).

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Fiebig, H. H., Berger, D. P., Dengler, W. A., Wallbrecher, E., and Winterhalter, B. R. (1992). Combined in vitro/in vivo test procedure with human tumor xenografts. In Immunodecient Mice in Oncology (H. H. Fiebig and D. P. Berger, eds.) Vol. 42, pp. 321351, Karger, Basel. Fiebig, H. H., Berger, D. P., Dengler, W. A., Drees, M., Mayo, J., Malspeis, L., and Grever, M. (1994). Cyanocyclin A and the quinocarmycin analog NSC 607 097 demonstrate selectivity against melanoma xenografts invitro and in-vivo. Proc. Am. Assoc. Cancer Res. 35, 2794. Fiebig, H. H., Dengler, W. A., Drees, M., Schwendener, R. A., and Schott, H. (1995). In vivo activity of N4-octadecyl-Ara-C in human solid tumors and leukemias. Proc. Am. Assoc. Cancer Res. 36, A2434. Fiebig, H. H., Dengler, W. A., and Roth, T. (1999a). Human tumor xenografts: predictivity, characterization and discovery of new anticancer agents. In Relevance of Tumor Models for Anticancer Drug Development (H. H. Fiebig and A. M. Burger, eds.), Vol. 54, pp. 2950. Basel, Karger. Fiebig, H. H., Steidle, C., Burger, A. M., and Lerchen, H. G. (1999b). Anticancer activity of novel camptothecin glycoconjugates in human tumor xenograft models. Clin. Cancer Res. 5, 667. Futreal, A. P., Kasprzyk, A., Birney, E., Mullikin, J. C., Wooster, R., and Stratton, M. (2001). Cancer and genomics. Nature 409, 850852. Giavazzi, R., Valoti, G., Ferri, C., and Nicoletti, M. J. (1999). Preclinical models to evacuate angiogenesis inhibitors. In Relevance of Tumor Models for Anticancer Drug Development (H. H. Fiebig and A. M. Burger, eds.), Vol. 54, pp. 157168, Karger, Basel. Goldenberg, G. J., and Moore, M. J. (1997). Nitrogen mustards. In Cancer Therapeutics: Experimental and Clinical Agents (B. A. Teicher, ed.), pp. 322, Humana Press, Totowa, NJ. Goldin, A., Venditti, J. M., MacDonald, J. S., Muggia, F. M., Henney, J. E., and DeVita, V. T. (1981). Current results of the screening program at the division of cancer treatment, National Cancer Institute. Eur. J. Cancer 17, 129142. Greenberg, N. M., DeMayo, F., Finegold, M. J., Medina, D., Tilley, W. D., Aspinall, J. O., Cunha, G. R., Donjacour, A. A., Matusik, R. J., and Rosen, J. M. (1995). Prostate cancer in a transgenic mouse. Proc. Natl. Acad. Sci. USA 92, 34393443. Hamburger, A. W., and Salmon, S. E. (1977). Primary bioassay of human tumor stem cells. Science 197, 461463. Hanahan, D., and Folkman, J. (1996). Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353364. Hartung, G., Stehle, G., Sinn, H., Wunder, A., Schrenk, H. H., Heeger, S., Krnzle, M., Edler, L., Frei, E., Fiebig, H. H., Heene, D. L., and Queisser, W. (1999). Phase I trial of methotrexate-albumin in a weekly intravenous bolus regimen in cancer patients. Clin. Cancer Res. 5, 753759. Hendriks, H. R., Pizao, P. E., Berger, D. P., Kooistra, K. L., Bibby, M. C., Boven, E., Dreef-van der Meulen, H. C., Henrar, R. E. C., Fiebig, H. H., Double, J. A., Hornstra, H. W., Pinedo, H. M., Workmann, P., and Schwartsmann, G. (1993). E09, a novel bioreductive alkylating indoloquinone with preferential solid tumor activity and lack of bone marrow toxicity in preclinical models. Eur. J. Cancer 29, 897906. Hendriks, H. R., Fiebig, H. H., Giavazzi, R., Langdon, S. P., Jimeno, J. M., and Faircloth, G. T. (1999). High antitumor activity of ET743 against human tumour xenografts from melanoma, non-small-cell lung and ovarian cancer. Ann. Oncol. 10, 12331240. Hoffman, R. M. (1999). Visualization of metastasis in orthotopic mouse models with green uorescent protein. In Relevance of Tumor Models in Anticancer Drug Development (H. H. Fiebig and A. M. Burger, eds.), Vol. 54, pp. 8187. Karger, Basel. Hoffmann, J., Schirner, M., Menrad, A., and Schneider, M. R. (1999). Animal models for determination of anti-angiogenic drug effects. In Relevance of Tumor Models for Anticancer Drug Development (H. H. Fiebig and A. M. Burger, eds.), Vol. 54, pp. 169180. Karger, Basel. Hollingshead, M. G., Alley, M. C., Camalier, R. F., Abbott, B. J., Mayo, J. G., Malspeis, L., and Grever, M. R. (1995). In vivo cultivation of tumor cells in hollow bers. Life Sci. 57, 131141.

Hollingshead, M., Plowman, J., Alley, M., Mayo, J., and Sausville, E. (1999). The hollow ber assay. In Relevance of Tumor Models in Anticancer Drug Development (H. H. Fiebig and A. M. Burger, eds.), Vol. 54, pp. 109120. Karger, Basel. Huettner, C. S., Zhang, P., van Etten, R. A., and Tenen, D. G. (2000). Reversibility of acute B-cell leukemia induced by bcr-abl1. Nat. Genet. 24, 5760. Jones, A., and Harris, A. L. (1998). New developments in angiogenesis: a major mechanism for tumor growth and target for therapy. Cancer J. Sci. Am. 4, 209213. Klenner, T., Voegeli, R., Fiebig, H., and Hilgard, P. (1992). Antitumor effect of the platinum complex D-19466 (Inn: Lobaplatin) against the transplantable osteosarcoma of the rat and other experiments. J. Cancer Res. Clin. Oncol. 118(Suppl.), p. 149. Lander, E. S., Linton, L. M., Birren, B., et al. (2001). Initial sequencing and analysis of the human genome. Nature 409, 860921. Langer, M., Mockel, B., Eck, J., Zinke, H., and Lentzen, H. (1999). Sitespecic mutagenesis of mistletoe lectin: the role of RIP activity in apoptosis. Biochem. Biophys. Res. Commun. 264, 944948. Lavigueur, A., Maltby, V., Mock, D., Rossant, J., Pawson, T., and Bernstein, A. (1989). High incidence of lung, bone and lymphoid tumors in transgenic mice overexpressing mutant alleles of the p53 oncogene. Mol. Cell Biol. 9, 39823991. Liu, M., Bryant, M. S., Chen, J., et al. (1998). Antitumor activity of SCH 6636, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wap-ras transgenic mice. Cancer Res. 58, 49474956. Malakoff, D., Vogel, G., and Marshall, E. (2000). The rise of the mouse, biomedicines model mammal. Science 288, 248257. Maly, K., Uberall, F., Schubert, C., Kindler, E., Stekar, J., Brachwitz, H., and Grunicke, H. H. (1995). Interference of new alkylphospholipid analogues with mitogenic signal transduction. Anticancer Drug Des. 10, 411425. Mickisch, G. H., Merlino, G. T., Aiken, P. M., Gottesmann, M. M., and Pastan, I. (1991a). New potent verapamil derivatives that reverse multidrug resistance in human renal carcinoma cells and in transgenic mice expressing the human MDR 1 gene. J. Urol. 146, 447453. Mickisch, G. F., Licht, T., Merlino, G. T., Gottesman, M. M., and Pastan, I. (1991b). Chemotherapy and chemosensitization of transgenic mice which express the human multidrug resistance gene in bone marrow: efcacy, potency, and toxicity. Cancer Res. 51, 54175424. Nichol, C. J., Harrison, M. L., Laposa, R. R., Gimelshtein, I. L., and Wells, P. G. (1995). A teratologic suppressor role for p53 in benzo[a]pyrenetreated transgenic p53-decient mice. Nat. Genet. 10, 181187. Ovejera, A. A. (1987). The use of human tumor xenografts in large scale drug screening. In Rodent Tumor Models in Experimental Cancer Therapy (R. F. Kallman, ed.), pp. 218220. Pergam Press, New York. Palmiter, R. D., and Brinster, R. L. (1985). Transgenic mice. Cell 41, 343345. Paull, K. D., Shoemaker, R. H., Hodes, L., Monks, A., Scudiero, D. A., Rubinstein, L., Plowman, J., and Boyd, M. R. (1989). Display and analysis of patterns of differential activity of drugs against human tumour cell lines: development of mean graph and COMPARE algorithm. J. Natl. Cancer Inst. 81, 10881092. Porter, G. M., Armstrong, L., and Nielsen, L. L. (1995). Strategy for developing transgenic assays for screening antineoplastic drugs that affect tubulin polymerization. Lab. Anim. Sci. 45, 145150. Reid, L. M., Holland, J., Jones, C., Wolf, B., Niwayama, G., Williams, R., Kaplan, N. O., and Sato, G. (1978). Some of the variables affecting the success of transplantation of human tumors into the athymic nude mouse. In Proceedings of the Symposium on the Use of Athymic (nude) Mice in Cancer Research (D. P. Houchens and A. A. Ovejera, eds.), pp. 107122. Gustav Fischer Verlag, New York. Risau, W. (1997). Mechanisms of angiogenesis. Nature 386, 671674.

SCREENING USING ANIMAL SYSTEMS

299

Sausville, E. A., and Feigal, E. (1999). Evolving approaches to cancer drug discovery and development at the National Cancer Institute. Ann. Oncol. 10, 12871292. Schepartz, S. A. (1976). Early history and development of the nitrosoureas. Cancer Treat. Rep. 60, 647649. Scholz, C. C., Berger, D. P., Winterhalter, B. R., Henss, H., and Fiebig, H. H. (1990). Correlation of drug response in patients and in the clonogenic assay using solid human tumor xenografts. Eur. J. Cancer 26, 901905. Schler, J. B., Fiebig, H. H., and Burger, A. M. (1999). Development of human tumor models for evaluation of compounds which target tumor vasculature. In Relevance of Tumor Models for Anticancer Drug Development (H. H. Fiebig and A. M. Burger, eds.), Vol. 54, pp. 181190. Karger, Basel. Sinn, E., Muller, W., Pattengale, P., Tepler, I., Wallace, R., and Leder, P. (1987). Coexpression of MMTV/v-H-ras and MMTV/c-myc genes in transgenic mice: synergistic action of oncogenes in vivo. Cell 49, 465475. Steel, G. (1987). How well do xenografts maintain the therapeutic response characteristics of the source tumor in the donor patient? In Rodent Tumor Models in Experimental Cancer Therapy (R. F. Kallman, ed.), pp. 205208. Pergam Press, New York. Stehle, G., Sinn, H., Wunder, A., Schrenk, H. H., Stewart, J. C. M., Hartung, G., Maier-Borst, W., and Heene, D. L. (1997). Plasma protein (albumin) catabolism by the tumor itself: implications for tumor metabolism and the genesis of cachexia. Crit. Rev. Oncol. Hematol. 26, 77100. Sugamata, N., Koibuchi, Y., Iino, Y., and Morishita, Y. (1999). A novel aromatase inhibitor, vozorole, shows antitumor activity and a decrease of tis-

sue insulin-like growth factor-I level in 7,12-dimethylbenz(a)anthracene induced mammary tumors. Int. J. Oncol. 14, 259263. Teicher, B. A. (ed.) (1997). Cancer Drug Discovery and Development. Cancer Therapeutics. Experimental and Clinical Agents. Humana Press, Totowa, NJ. Venter, G., Adams, M. D., Myers, E. W., et al. (2001). The sequence of the human genome. Science 291, 13041351. Von Hoff, D. D. (1987). In vitro predictive testing: the sulfonamide era. Int. J. Cell Cloning 5, 179190. Waud, R. W. (1997). Murine L1210 and P388 leukemias. In Anticancer Drug Development Guide (B. A. Teicher, ed.), pp. 5974. Humana Press, Totowa, NJ. Wechter, W. J., Leipold, D. D., Murray, E. D. Jr, Quiggle, D., McCracken, J. D., Barrios, R. S., and Greenberg, N. M. (2000). E-7869 (R-urbiprofen) inhibits progression of prostate cancer in the TRAMP mouse. Cancer Res. 60, 22032208. Winterhalter, B. R., Berger, D. P., Dengler, W. A., Hendriks, H. R., Mertelsmann, R., and Fiebig, H. H. (1993). High antitumors activity of rhizoxin in a combined in-vitro and in-vivo test procedure with human tumor xenografts. Proc. Am. Assoc. Cancer Res. 34, 376. Workman, P., Twentyman, P., Balkwill, F., Balmain, A., Chaplin, D., Double, J., Embleton, J., Newell, D., Raymond, R., Stables, J., Stephens, T., and Wallace, J. (1997). United Kingdom Co-ordinating Committee on Cancer Research (UKCCCR) guidelines for the welfare of animals in experimental neoplasy. 2nd ed., pp. 132, UKCCCR, Lincolns Inn Fields, London.