The study of cancer implicates the iterative testing of hypotheses. To mimic healthy and cancerous conditions in humans, a wide spectrum of experimental models of cancer is used including animal models, patient samples, genetically homogenous permanently growing cell lines, and genetically heterogeneous primary cells derived from tissues of human patients or animals ( Cancer Res 77:2557-2563, 2017. Review.). Cells are studied in vitro (in culture dishes) or in vivo after transplantation into animals. Additional animal models of cancer range from spontaneous to induced models in wildtype or genetically engineered organisms. Predominantly mice are used, but also lower-model organisms such as yeast, Xenopus laevis, Caenorhabditis elegans, Drosophila melanogaster and zebrafish Danio rerio. All have substantially contributed to the understanding of the molecular basis of the complex disease.
With MiTO©, a tool is offered by the German Cancer Research Center as a core of the German Cancer Consortium to the scientific community for facilitated exchange of preclinical model data and enhanced exchange of living organisms relevant in translational cancer research. Fact sheets can be created and obtained for cell lines, genetically modified organisms including reporter and recombinase strains, transplantation models, carcinogen-induced models, spontaneous tumor models, with the possibility to record metastasis and interventions. Know-how can be protected by the ownerprior to publication by setting a defined accessibility level for any given model.
Research is the best way to combat cancer. Aiming for a life without cancer, basic, epidemiological, and clinical researchers of many disciplines have made tremendous progress during the last 5 decades in improving our knowledge about the mechanisms of cancer development at the systemic, tissue, cellular, and molecular level. To the best of our knowledge, cancer is extremely heterogeneous with respect to different tumor entities and a single cancer type is comprised of a number of different subtypes. Furthermore, every individual cancer is unique in its cellular and genetic composition and even different areas of one and the same tumor are heterogeneous. Thus, cancer researchers are not confronted with a single but with more than 200 diseases, a diversity that translates into a high complexity. Despite this fact, tumor initiation, growth and dissemination of tumor cells to other sites in the body are common mechanisms of tumor development. According to Robert Weinberg and Douglas Hanahan (Cell 100, 57-70, 2000; Cell 144, 646-674, 2011), the complexity of cancer can be reduced to a number of underlying principles, the hallmarks of cancer.
- 1. Cancer cells stimulate their own growth by producing autocrine growth factors (Self-sufficiency in growth signals)!
- 2. Cancer cells resist inhibitory signals that normally stop their growth (Insensitivity to anti-growth signals)!
- 3. Cancer cells resist programmed cell death (Evading apoptosis)!
- 4. Cancer cells multiply indefinitely (Limitless replicative potential resulting in infinite generation of descendants)!
- 5. Cancer cells stimulate the growth of blood and lymphatic vessels to supply nutrients to tumors and to disseminate (Sustained [lymph]angiogenesis)!
- 6. Cancer cells invade local tissue and spread to distant organs and tissues (Tissue invasion and metastasis)!
- 7. Cancer cells use abnormal metabolic pathways to generate energy! (Warburg postulated this already in 1924, Biochemische Zeitschrift 152, 319-344, 1924)!
- 8. Cancer cells get invisible to the body’s immune system (Evading the immune system directed against the tumor)!
- 9. Cancer cell development (in many cancer types) is promoted under persistent inflammation (Chronic inflammation)!
- 10. Cancer cells can’t keep their DNA stable (Unstable DNA)!
In the course of transformation of a normal into a malignant cell fundamental traits are coming up, fueled in part by changes in the
cancer cell itself and in part by changing interactions with various cell types and matrix molecules in the environment as well as changes of systemic
factors (e.g. hormones, nutrients) in the circulation.
Today, cancer research of basic scientists and clinical researchers is switching in iterative cycles from the bench to the bedside. Networking with other fields of research in biology, systems biology, bioinformatics, medicine, chemistry, pharmacology, and physics is common.
Here, cancer researchers frequently use experimental animal models of cancer to test their hypothesis. However, a complex system like a benign or a malignant tumor is known to be high-dimensional and non-linear, and hence is difficult to model. In addition, complex systems can only be recapitulated as well as they are described and understood on functional and molecular levels. Often not a single model but a battery of models is needed to mimic such complex systems.
Models in translational oncology range from tumor cell lines in culture over in vivo equivalents (e. g. living skin equivalents, organ explants) to various model organisms.
Researchers make use of various experimental animal models for the dissection of underlying basic mechanisms of human cancer initiation,
progression and metastasis, for the improvement of prevention, detection and diagnosis as well as the translation of promising candidate
therapies from discovery to the clinic. Here, the animal models should faithfully recapitulate the human conditions and the most
appropriate for the validation of hypothesis. Upon selection of a model, many points need to be considered such as animal species,
type of cell(s) and tissue(s) affected involvement of genes, molecular pathways and networks, genetic diversity of the human disease,
as well as time course and major clinical symptoms of the disease.
Mus musculus is still the predominant species in cancer research. Here genetically engineered mice or animals treated with chemical/physical carcinogens and/or tumor promoters serve basic and translational research. Moreover, immunocompromised mouse strains are used for investigations of cancer traits in human tumors. Tumor outgrows either after xenotransplantation of permanent cell lines or patient-derived primary tumor cells, while syngeneic transplantations into immunocompetent hosts are a regular need for studies of tumor-immune cell interactions. Lower-model organisms such as yeast, Xenopus laevis, the nematode worm Caenorhabditis elegans, the fruitfly Drosophila melanogaster and zebrafish are also substantially contributing to the understanding of the molecular basis of the disease. Moreover, the latter serve as platforms to identify new therapeutics. Of note, for each human gene the corresponding orthologues(s) have been or are being defined in various model organisms by various databases (OrthoDisease). Addressing about 2500 human disease genes derived from Online Mendelian Inheritance in Man, the rate of orthologues genes is found to be about 80 % in Mus musculus, 70 % in Zebrafish, y % in Xenopus, 50 % in D. melanogaster, and 20 % in C. elegans.
In fact, our current view on cell cycle progression and maintenance of cell polarity is based on many studies in yeast. Studies with X. laevis on the other hand provided major insights into normal cellular homeostasis, while studies on C. elegans were fundamental to our understanding of programmed cell death. Key functions of human cancer-related proteins were understood very often by analyzing the corresponding orthologues in D. melanogaster. Zebrafish Danio rerio have shown up as models for specific cancer types such as melanoma skin cancer and leukemias.
Most popular animal cancer models
Mouse cancer models
The mouse (Mus musculus) is one of the most important models to understand the function of the mammalian genome. More than 95 % of the mouse genes have a homologues sequence in the genome (Mouse Genome Database). Moreover there are fundamental similarities between human and mice in anatomy and physiology. Many differences between the two species such as lifespan, the length of telomeres or metabolism of xenobiotics as well as many others are also obvious, demanding for refinements and adjustments to generate more realistic mouse models of human cancer. Genetic studies on mice are available since more than 100 years. Most importantly, meanwhile a comprehensive “toolkit” is available to manipulate the mouse genome, in part in high-throughput-procedures ( EUCOMM, or NorCOMM, or KOMP, or TIGM , or IMPC) to produce thousands of medically relevant mouse mutants (see also EOMODIC . These may be archived by cryoconservation and distributed upon request. See also EMMA)
Inbred mice as spontaneous tumor models
In certain experimental studies, as much homogeneity as possible is a prerequisite. Therefore, mice are inbred (brother-sister matings in a systematic manner over at least 20 generations) with the aim to receive genetically identical individuals. In the last 100 years more than 100 strains of inbred mice lines were established, some of which develop distinct types of cancer with high prevalence spontaneously.
Genetically engineered mouse models (GEMM)
For transgenesis various techniques have been developed and of note, these approaches are done most successfully in the mouse. Besides classical random integration of transgenes after pronuclear DNA injection and non-homologous DNA end joining or “random integration of transposons (Moriarity B.S., Largaespada D.A., (2015) Current Opinion in Genetics and Development, 30 , pp. 66-72.), gene targeting in embryonic stem (ES) cells via homologous DNA recombination plays a fundamental role to engineer mutations in specific genes. If genetic modification of ES cells is done in a consecutive manner or by additional genetic modification of ES cells derived from a mouse strain that has already a complex mutational profile, time-consuming and expensive crossbreeding of mice can be reduced to generate complex genotypes (Dow and Lowe, Cell 148: 1099-1109, 2012). Conditional activation or inactivation of genes in distinct cell types at any desired time point is also common. Furthermore, regulation of expression of genes of interest or shRNA is possible by transient application of small molecules such as doxycycline and tamoxifen. With the same techniques, distinct cell types or distinct lineages can be tagged or traced, respectively, by fluorescent reporters. Using fluorescent or bioluminescent reporters, distinct signaling pathways can also be followed up in vivo. More advanced tools for genome engineering are the Zinc finger- and TALE nucleases as well as the recently introduced Crisp/Cas9-mediated transgenesis (Xue W et al, Nature 514, 380–384 (16 October 2014)). Finally, genetic manipulation of ES cells partially may be replaced by somatic gene transfer by means of a broad range of adenoviral or lentiviral vectors.
The transplantation of cancer cell lines, or primary patient-derived tumor cell lines or tumor tissue into the mouse or rat has been and is still a basic tool to gain knowledge about the pathomechanisms of cancer including various stages of the multi-step-process of metastasis.
Implantation of cells is done predominantly in an ectopic manner, meaning that cells are inoculated into a tissue that is not representing the organ of origin but a convenient anatomical location or a highly vascularized site, i. e. the subcutaneous space or the subrenal capsule. Cell transplantation into the organ of origin, the orthotopic transplantation, is rarely done, although it resembles much better the human situation. However, depending on the context, this assay needs advanced technical skills.
Syngeneic transplantation refers to the transfer of cells of a distinct species into an animal recipient of the same genetic background, predominantly mouse cells lines established from tumors grown out spontaneously in wild-type mice or genetically engineered mouse mutants, or alternatively, from carcinogen-induced tumors or transplantation tumors. Serial transplantations of mouse cell lines into mouse acceptors, i. e. serial passages of tumor cells, is an approach to select cell line derivatives with increased potential of tumorigenicity and metastasis to distant sites. In syngeneic transplantations, transplanted cells are not rejected by the host organism, since both experimental lines are immune-compatible. The presence of an intact immune system allows investigating the contribution of host immune cells within the circulation but also in the tumor microenvironment to the development of tumors and metastasis. Of note, host organisms are mostly inbred mouse strains and therefore are of less genetic heterogeneity than human patients.
Hetero(Xeno)transplantation models are given when human cells or tissues, and this is mostly done, are injected into a more or less immune-compromised acceptor animal with a different genetic background, mostly mouse. Immunodeficient hosts are a prerequisite in these settings to prevent immune rejection of the foreign cells. A major advantage of this approach is the fact, that human tumor cells and tissues are used to model the human situation. For heterotransplantation, researchers have access to a broad range of immunocompromised mouse hosts (Nude (nu/nu), SCID (severe combined immunodeficiency), SCID-Beige, NOD (non-obese diabetic)-SCID, NSG (NOD-SCID gamma), or NOG i. e. NOD/Shi-scid/IL-2Rγnull) that are characterized by different gene mutation profiles and as a consequence different levels of immune-deficiency. Of course, these models do not allow assessing the functional contribution of immune cells to tumor formation. Moreover, xenotransplantation models are not appropriate if species-specific factors such as distinct growth factors, cytokines or chemokines and their cognate receptors are subject of research. However, such limitations can partially be bypassed by humanizing the mouse acceptor by introduction of functionally active human genes into the respective mouse loci, cells, tissues, and/or organs.
Patient-derived xenografts (PDX) are used especially in the context of personalized cancer treatment and the characterization of cancer-initiating cell populations by serial transplantation and limited dilution experiments. The low rate of PDX engraftment is however still a limitation to use them in routine.
Cancer metastasis transplantation models. Syngeneic or heterogeneic cells can be inoculated via different routes into the recipient organisms. In so called experimental metastasis assays, the tumor cells are injected into the systemic circulation for example via the tail vein, the left heart ventricle, the intra-portal vein, or the carotic artery, thus mimicking only the late phases of metastasis, i. e. the survival in the circulation, organ colonization, and local outgrowth. In these colonization approaches “metastasis” shows up rapidly. Depending on the tumor cell-specific tropism and the site of injection, the “metastatic” nodules grow out predominantly in the lung (tail vein), the liver (intra-portal), the brain (intra-carotid) or different distant sites including the liver, ovaries, bone and brain (intra-cardiac).
In contrast, in spontaneous metastasis assays, tumor cells are predominantly inoculated at ectopic or orthotopic sites. It is worth to mention that the overwhelming number of ectopic transplantation models do not mimic the human counterpart with respect to tumor vascularization, tumor microenvironment, metastatic spread to the relevant distant sites, or responsiveness towards chemotherapeutics and other drugs. Consequently, orthotopic transplantation models are used, especially when the human situation of tumor-stroma-interaction or cancer metastasis needs to be modeled. In an attempt to improve cancer metastasis models, primary tumors may be resected to allow an outgrowth of metastasis (post surgery metastasis).
Cancer initiation, promotion, and progression can be faithfully recapitulated in carcinogen-induced cancers in rodents with the carcinogen being a
chemical (e. g. polycyclic aromatic hydrocarbons, tobacco-specific carcinogens, arsenic, cadmium) or physical agent (e. g. UV-irradiation, ionizing
radiation, asbestos fibers). Depending on the type and the dose of the applied carcinogen and the frequency of treatment, a single tumor type or a
spectrum of different tumors is induced. In addition to full-carcinogenic approaches, cancer-initiating protocols using subcarcinogenic doses of
the carcinogen can be combined with tumor promotion or co-carcinogenesis protocols, especially for studies of cancer in skin, liver, colon, urinary
bladder ec. Co-carcinogens or tumor promoters are agents that do not cause cancer on their own but only in synergisms with the activity of a
As a result of carcinogen treatment, mutations are induced along with other changes leading to hallmarks of cancer. Of note, incidence and multiplicity of carcinogen-induced cancer is mostly strain specific, at least in the mouse.
Such approaches are often used to access chemopreventive measures and to identify risk factors and can be applied to animals with different genetic backgrounds. However, response of mice and men towards distinct carcinogens is different in many cases and extraploration from the experimental carcinogenesis study to the human situation needs caution. Mutational profiling of experimental tumors serves to compare the model system with human tumor mutation signatures.
Hamster cancer models
The Xenopus model organism database Xenbase.
Eva-Stina Edholm and Jacques Robert. Recent Research Progress and Potential Uses of the Amphibian. Xenopus as a Biomedical and Immunological Model System. ( Resources 2013, 2, 167-183 (PDF))
Nutt LK. The Xenopus oocyte: A model for studying the metabolic regulation of cancer cell death. ( Seminars in Cell Developmental Biology 2012; 23: 412-418.)
The Zebrafish Model organism database (ZFIN).
The European Zebrafish Resource Center ( EZRC).
Zebrafish International Resource Center (ZIRC).
Sanger Zebrafish Mutation Project (ZMP) Knockout for Disease Models
White R, Rose K, Zon L. Zebrafish cancer: the state of the art and the path forward. (Nature Reviews Cancer 13: 624-636, 2013 (PDF)).
Gilberto dos Santos, Andrew J. Schroeder, Joshua L. Goodman, Victor B. Strelets, Madeline A. Crosby1, Jim Thurmond2, et al. the FlyBase Consortium. FlyBase: introduction of the Drosophila melanogaster Release 6 reference genome assembly and large-scale migration of genome annotations .
A Database of Drosophila Genes & Genomes ( FlyBase).
Berkeley Drosophila Genome Project (BDGP), Release 5 genome assembly.
Cayetano Gonzalez. Drosophila melanogaster: a model and a tool to investigate malignancy and identify new therapeutics. ( Nature Reviews Cancer 13, 172-183, 2013 (PDF)).
A. Goffeau*, B. G. Barrell, H. Bussey, R. W. Davis, B. Dujon, H. Feldmann, et al. Live with 6000 Genes. (Science 1996: Vol. 274, 546-567).
Database for Dictyostelid genomics dictyBase.
Database for Tribolium genetics, genomics and developmental biology BeetleBase.
Sharma SV, Haber DA, Settleman J. Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents (Nature Reviews Cancer 10: 241-253, 2010 (PDF)).
Shoemaker, R.H. THE NCI60 human tumour cell line anticancer drug screen (Nature Reviews Cancer 6: 813-823, 2006).
Garnett, M.J. et al. Systematic identification of genomic markers of drug sensitivity in cancer cells (Nature 483: 570-575, 2012).
Barretina, J. et al. The Cabcer Cell Line Encyclopedia enables predictive modeling of anticancer drug sensitivity (Nature 483: 603-607, 2012).
3Rs: Replacement, refinement and reduction of animals in research
In the use of experimental animals for scientific purposes, achieving experimental results is as important as animal welfare (European Directive on animals used for experimental and other scientific purposes, Directive 2010/63/EU ).
To accomplish this aim, the MiTO platform has been established to facilitate the world-wide exchange of information about models in translational oncology. Moreover, the exchange of live models/animals should be facilitated since MiTO offers contact data and direct communication tools. MiTO allows specific simple and advanced searches with and without setting filters for genetically engineered organisms (recombinase and reporter tool strains etc). to ensure that the GEO of interest is available.
Especially with respect to GEO, exchange of existing mice lines (as live animals or cryo-conserved embryos/sperms) is highly relevant to avoid unnecessary repetition of the production of a desired mutant. This is particularly evident for recombinase and reporter strains.
Consequently, a reduction in animal numbers used to generate a GEO is to be expected and a number of procedures/manipulations on mice with the potential to induce distress (superovulation, collection of blastocysts, general anaesthesia, pre-emptive and top-ups analgesia, surgery/vasectomy/embryo transfer, peri/post-operative care, tissue sampling for genotyping, euthanasia) are avoided.
In case a distinct GEO has already been imported from a foreign national or international animal research facility, a repeated transport of live mice could be avoided, along with an eventually necessary embryo transfer procedures to meet the hygiene status of the acceptor facility; of course in compliance with material/animal transfer agreements and terms and conditions of use. Thus, distress due to such procedures is prevented.
Giving MiTO users also the opportunity to implement procedures as needed to create transplantation models, protocols for chemical carcinogenesis experiments or inhibitor/diet studies will allow enhanced comparison of protocols and help to find appropriate information for desired experimental animal studies. Hence, the numbers of mice needed for the establishment of models and/or procedures is reduced.