Animal Models For Cancer Research Research Articles

Traditional Approaches and Innovative Strategies in Laboratory Animal Models for Cancer Research: A Comprehensive Review

Traditional Approaches and Innovative Strategies in Laboratory Animal Models for Cancer Research: A Comprehensive Review

Abstract PO1-25-09: A new generation of breast cancer modeling has been achieved through somatic precision gene editing, offering high flexibility and efficiency

Abstract Breast cancer, along with many other cancers, is primarily caused by genetic mutations, typically involving the alteration of nucleotides (A, G, C, T) within genes (with a total of 12 possible mutations). Gene sequencing advancements have facilitated the identification of numerous gene mutations in tumor samples. However, comprehending the precise mechanisms through which these gene mutations contribute to tumor initiation, development, and their impact on tumor characteristics cannot be solely determined from these findings. Therefore, it is crucial to observe the influence of mutations on tumor initiation and development from the moment they occur. Since conducting such observations in human subjects is impractical, animal models with simulated gene mutations have become essential for studying tumor initiation and development. Mice, with a genome comprising approximately 2.7 billion nucleotides, are commonly used as animal models in cancer research due to their cost-effectiveness and high similarity to humans. About 90% of their genes share similar functions, particularly those associated with cancer development. However, achieving precise alterations to specific nucleotides among the 2.7 billion nucleotides within live mouse cells (with a total of 12 possible changes) has presented significant technical challenges. In this study, we present modifications to the CRISPR/Cas9 vector system, enabling homology-directed repair-mediated precise editing of any proto-oncogene in murine somatic mammary epithelial cells, thereby generating mammary tumor models with exceptional flexibility and efficiency. The resulting tumors exhibit significant advantages over traditional mouse models. This technological breakthrough bridges the gap between the potential of CRISPR technology and the accuracy of mouse models, facilitating the study of human tumor evolution and preclinical drug testing. Citation Format: Wen Bu, Yi li. A new generation of breast cancer modeling has been achieved through somatic precision gene editing, offering high flexibility and efficiency [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-25-09.

Three-Dimensional In Vitro Cell Cultures as a Feasible and Promising Alternative to Two-Dimensional and Animal Models in Cancer Research.

Cancer represents one of the diseases with the highest mortality rate worldwide. The burden of cancer continues to increase, not only affecting the health-related quality of life of patients but also causing an elevated global financial impact. The complexity and heterogeneity of cancer pose significant challenges in research and clinical practice, contributing to increase the failure rate of clinical trials for antitumoral drugs. This is partially due to the fact that preclinical models still present important limitations in faithfully recapitulating human tumors to serve as reliable indicators of drug effectiveness. Up to now, research and development strategies employ expensive animal models (including the so-called "humanized mice") that not only raise ethical concerns, but also frequently fail to accurately predict responses to anticancer drugs because they do not faithfully replicate human physiology as well as the patient's tumor microenvironment. On the other side, traditional two-dimensional (2D) cell cultures fail to adequately reproduce the structural organization of tumor and the cellular heterogeneity found in vivo. The growing necessity to develop more accurate cancer models has increasingly emphasized the importance of three-dimensional (3D) in vitro cell cultures, such as cancer-derived spheroids and organoids, as promising alternatives to bridge the gap between 2D and animal models. In this review, we provide a brief overview focusing on 3D in vitro cell cultures as preclinical models capable of properly reproducing the tissue organization, biological composition, and complexity of in vivo tumors in a fine-tuned microenvironment. Despite their limitations, these models collectively enhance our understanding of the mechanisms underlying cancer and may offer the potential for a more reliable assessment of drug efficacy before clinical testing and, consequently, improve therapeutic outcomes for cancer patients.

CanISO: a database of genomic and transcriptomic variations in domestic dog (Canis lupus familiaris)

BackgroundThe domestic dog, Canis lupus familiaris, is a companion animal for humans as well as an animal model in cancer research due to similar spontaneous occurrence of cancers as humans. Despite the social and biological importance of dogs, the catalogue of genomic variations and transcripts for dogs is relatively incomplete.ResultsWe developed CanISO, a new database to hold a large collection of transcriptome profiles and genomic variations for domestic dogs. CanISO provides 87,692 novel transcript isoforms and 60,992 known isoforms from whole transcriptome sequencing of canine tumors (N = 157) and their matched normal tissues (N = 64). CanISO also provides genomic variation information for 210,444 unique germline single nucleotide polymorphisms (SNPs) from the whole exome sequencing of 183 dogs, with a query system that searches gene- and transcript-level information as well as covered SNPs. Transcriptome profiles can be compared with corresponding human transcript isoforms at a tissue level, or between sample groups to identify tumor-specific gene expression and alternative splicing patterns.ConclusionsCanISO is expected to increase understanding of the dog genome and transcriptome, as well as its functional associations with humans, such as shared/distinct mechanisms of cancer. CanISO is publicly available at https://www.kobic.re.kr/caniso/.

Disease Animal Models for Cancer Research.

Despite nonanimal methods (NAMs) are more and more exploited and new NAMs are developed and validated, animal models are still used in cancer research. Animals are used at multiple levels, from understanding molecular traits and pathways, to mimicking clinical aspects of tumor progression, to drug testing. In vivo approaches are not trivial and involve cross-disciplinary knowledge: animal biology and physiology, genetics, pathology, and animal welfare.The aim of this chapter is not to list and address all animal models used in cancer research. Instead, the authors would like to guide experimenters in the strategies to adopt in both planning and performing in vivo experimental procedures, including the choice of cancer animal models.

The Barretos Cancer Hospital Animal Facility: Implementation and Results of a Dedicated Platform for Preclinical Oncology Models.

The Barretos Cancer Hospital Animal Facility (BCHAF) is a unique facility in Brazil exclusively dedicated to working with animal models for cancer research. In this article, we briefly present our modern facility and the main experiments performed, focusing on mutant strains of mice (PTCH-knockout and ApcMin mice), xenograft models, and patient-derived xenografts (PDXs). Our results show the progress and challenges in establishing these models and the need for having an appropriate representation of our cancer population to better understand tumor biology and to identify cancer biomarkers, which could be putatively targeted, allowing for personalized therapy.

Bioluminescent Zebrafish Transplantation Model for Drug Discovery.

In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing.

Zebrafish Xenograft Model to Study Human Cancer.

The zebrafish, Danio rerio, has been an important animal model for cancer research over the last decade. The capability of a high-throughput screen in zebrafish and a wide range of pharmacologically active compounds elicit physiological responses in zebrafish embryos comparable to those in mammalian systems, making zebrafish ideal for identifying clinically relevant drug targets and compounds that regulate tumor progression. The zebrafish model is suitable for patient-derived xenograft (pdx) and large-scale screening of lead compounds against specific malignancies. This established vertebrate model has many advantages, including fast response time, cost efficiency for drug testing, efficient manipulation of the host microenvironment by genetic tools, suitable for small molecule drug screening in high-throughput setting, easy maintenance, transparency for easy observation, high fecundity, and rapid generation time. The zebrafish model is a good alternative in vivo model to mammals for robust testing of drug candidates for cancer therapy.

Swine models for translational oncological research: an evolving landscape and regulatory considerations.

Swine biomedical models have been gaining in popularity over the last decade, particularly for applications in oncology research. Swine models for cancer research include pigs that have severe combined immunodeficiency for xenotransplantation studies, genetically modified swine models which are capable of developing tumors in vivo, as well as normal immunocompetent pigs. In recent years, there has been a low success rate for the approval of new oncological therapeutics in clinical trials. The two leading reasons for these failures are either due to toxicity and safety issues or lack of efficacy. As all therapeutics must be tested within animal models prior to clinical testing, there are opportunities to expand the ability to assess efficacy and toxicity profiles within the preclinical testing phases of new therapeutics. Most preclinical in vivo testing is performed in mice, canines, and non-human primates. However, swine models are an alternative large animal model for cancer research with similarity to human size, genetics, and physiology. Additionally, tumorigenesis pathways are similar between human and pigs in that similar driver mutations are required for transformation. Due to their larger size, the development of orthotopic tumors is easier than in smaller rodent models; additionally, porcine models can be harnessed for testing of new interventional devices and radiological/surgical approaches as well. Taken together, swine are a feasible option for preclinical therapeutic and device testing. The goals of this resource are to provide a broad overview on regulatory processes required for new therapeutics and devices for use in the clinic, cross-species differences in oncological therapeutic responses, as well as to provide an overview of swine oncology models that have been developed that could be used for preclinical testing to fulfill regulatory requirements.

Diagnostic Ability of Methods Depicting Distress of Tumor-Bearing Mice.

Simple SummaryExperiments on animals can provide important information for improving the life expectancy and life quality of patients. At the same time, the welfare of these animals is a growing public concern. Therefore, many laws and international guidelines were established with the goal of minimizing the harm inflicted on these animals. A prerequisite of improving animal welfare is to correctly measure how much distress the experiments cause to these animals. However, it is often unknown as to which methods are appropriate to assess distress. Mice bearing subcutaneous tumors are the most frequently used animal model to study the therapeutic effects of drugs. We evaluated if body weight, faecal corticosterone metabolites concentration, burrowing activity and a distress score were capable of differentiating between mice before cancer cell injection and mice bearing large tumors. We observed that only adjusted body weight change and faecal corticosterone metabolites concentration were capable of measuring distress caused by large subcutaneous tumors. Therefore, these two methods are appropriate to assess the welfare of mice with subcutaneous tumors. This knowledge provides a solid basis to optimize animal welfare in future studies. For example, both methods can define the ideal time point when an experiment should end by finding a good compromise between minimal distress for the animals and maximal knowledge gain for mankind.Subcutaneous tumor models in mice are the most commonly used experimental animal models in cancer research. To improve animal welfare and the quality of scientific studies, the distress of experimental animals needs to be minimized. For this purpose, one must assess the diagnostic ability of readout parameters to evaluate distress. In this study, we evaluated different noninvasive readout parameters such as body weight change, adjusted body weight change, faecal corticosterone metabolites concentration, burrowing activity and a distress score by utilising receiver operating characteristic curves. Eighteen immunocompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice were used for this study; half were subcutaneously injected with A-375 cells (human malignant melanoma cells) that resulted in large tumors. The remaining mice were inoculated with SCL-2 cells (cutaneous squamous cell carcinoma cells), which resulted in small tumors. The adjusted body weight and faecal corticosterone metabolites concentration had a high diagnostic ability in distinguishing between mice before cancer cell injection and mice bearing large tumors. All other readout parameters had a low diagnostic ability. These results suggest that adjusted body weight and faecal corticosterone metabolites are useful to depict the distress of mice bearing large subcutaneous tumors.

Zebrafish as a Neuroblastoma Model: Progress Made, Promise for the Future.

For nearly a decade, researchers in the field of pediatric oncology have been using zebrafish as a model for understanding the contributions of genetic alternations to the pathogenesis of neuroblastoma (NB), and exploring the molecular and cellular mechanisms that underlie neuroblastoma initiation and metastasis. In this review, we will enumerate and illustrate the key advantages of using the zebrafish model in NB research, which allows researchers to: monitor tumor development in real-time; robustly manipulate gene expression (either transiently or stably); rapidly evaluate the cooperative interactions of multiple genetic alterations to disease pathogenesis; and provide a highly efficient and low-cost methodology to screen for effective pharmaceutical interventions (both alone and in combination with one another). This review will then list some of the common challenges of using the zebrafish model and provide strategies for overcoming these difficulties. We have also included visual diagram and figures to illustrate the workflow of cancer model development in zebrafish and provide a summary comparison of commonly used animal models in cancer research, as well as key findings of cooperative contributions between MYCN and diverse singling pathways in NB pathogenesis.

Application of Animal Models in Cancer Research: Recent Progress and Future Prospects.

Animal models refers to the animal experimental objects and related materials that can simulate human body established in medical research. As the second-largest disease in terms of morbidity and mortality after cardiovascular disease, cancer has always been the focus of human attention all over the world, which makes it a research hotspot in the medical field. At the same time, more and more animal models have been constructed and used in cancer research. With the deepening of research, the construction methods of cancer animal models are becoming more and more diverse, including chemical induction, xenotransplantation, gene programming, and so on. In recent years, patient-derived xenotransplantation (PDX) model has become a research hotspot because it can retain the microenvironment of the primary tumor and the basic characteristics of cells. Animal models can be used not only to study the biochemical and physiological processes of the occurrence and development of cancer in objects but also for the screening of cancer drugs and the exploration of gene therapy. In this paper, several main tumor animal models and the application progress of animal models in tumor research are systematically reviewed. Finally, combined with the latest progress and development trend in this field, the future research of tumor animal model was prospected.

Transgenic Animal Models of Bladder Cancer

Traditional animal models for cancer research represent an excellent experimental tool in bridging the gap between pre-clinical and clinical studies. The advances in gene transfer technologies allow the generation of transgenic animal models through the integration of the gene of interest into the animal models through gene transfer methods. Overcoming the limitations of traditional animal models, transgenic animal models are beneficial for studying carcinogenesis with respect to the genes. Besides, the utilization of transgenic animal models is also necessary for the development of novel targeted therapy. Bladder cancer (BC) is the most common cancer type worldwide and effective therapies targeting BC are urgently needed. Thus, with the delineation of molecular signatures of BC pathogenesis, the research involving transgenic animal models of BC will facilitate the development of clinical medicine, thereby improving the clinical outcome of BC patients in the future.

Spontaneous and Induced Animal Models for Cancer Research.

Considering the complexity of the current framework in oncology, the relevance of animal models in biomedical research is critical in light of the capacity to produce valuable data with clinical translation. The laboratory mouse is the most common animal model used in cancer research due to its high adaptation to different environments, genetic variability, and physiological similarities with humans. Beginning with spontaneous mutations arising in mice colonies that allow for pursuing studies of specific pathological conditions, this area of in vivo research has significantly evolved, now capable of generating humanized mice models encompassing the human immune system in biological correlation with human tumor xenografts. Moreover, the era of genetic engineering, especially of the hijacking CRISPR/Cas9 technique, offers powerful tools in designing and developing various mouse strains. Within this article, we will cover the principal mouse models used in oncology research, beginning with behavioral science of animals vs. humans, and continuing on with genetically engineered mice, microsurgical-induced cancer models, and avatar mouse models for personalized cancer therapy. Moreover, the area of spontaneous large animal models for cancer research will be briefly presented.

The humanized mouse: Emerging translational potential.

The humanized mouse (HM) has emerged as a valuable animal model in cancer research. Engrafted with components of a human immune system and subsequently implanted with tumor tissue from cell lines or in the form of patient-derived xenografts, the HM provides a unique platform in which the tumor microenvironment (TME) can be evaluated in vivo. This model may also be beneficial in the assessment of potential cancer treatments including immune checkpoint inhibitors. However, to maximize its utility, researchers need to understand the critical factors necessary to ensure that the tumor immune interactions in the HM are representative of those within cancer patients. In most current HM models, the human T cells residing in the HM are educated in a murine thymus, allogeneic to implanted tumor tissue, and/or alloreactive to mouse tissues, making their interaction and reactivity with tumor cells suspect. There are several strategies underway to harmonize the immune-tumor environment in the HM. Once the essential components of the HM-tumor TME interface have been identified and understood, the HM model will permit not only the discovery of effective immunotherapy treatments, but it can be used to predict patient responses to great clinical benefit.

Abstract LB-042: Successful tumor formation following xenotransplantation of primary human ovarian cancer cells into severe combined immunodeficient (SCID) pigs

Abstract Ovarian cancer (OvCa) is the most lethal gynecologic malignancy, with 2/3 of patients having late-stage disease (stages II-IV) at diagnosis. Preclinical animal models for cancer research have primarily been restricted to rodents, but many preclinical cancer drug trials that succeed in mice fail in humans, potentially due to vast differences in physiology and size. Therefore, we sought to determine if pigs, which are more similar to humans in terms of anatomy and physiology, would be a viable preclinical animal model for OvCa. Our group has pioneered the development of severe combined immunodeficiency (SCID) pigs for biomedical research, a key technological advancement for xenotransplantation of human ovarian cancer cells to form tumors. In this study, we injected human OSPC-ARK-1 cells, a chemotherapy-resistant primary ovarian serous papillary carcinoma cell line, into the neck muscle and ear tissue of four SCID and two non-SCID pigs. Tumors developed in the ears of three SCID pigs, while two SCID pigs developed tumors in neck tissue, all of which were confirmed at pathology as true neoplasms. Non-SCID animals did not develop tumors in any location. Confirmation that OSPC-ARK1 cells can form tumors in SCID pigs substantiates further development of orthotopic models of OvCa in pigs for researching improved OvCa therapeutic and diagnostic methods. Citation Format: Adeline N. Boettcher, Matti Kiupel, Malavika Adur, Emiliano Cocco, Alessandro Santin, Sara Charley, John Risinger, Christopher Tuggle, Erik Shapiro. Successful tumor formation following xenotransplantation of primary human ovarian cancer cells into severe combined immunodeficient (SCID) pigs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-042.

Transcriptional Profiling of Saccharomyces cerevisiae Reveals the Impact of Variation of a Single Transcription Factor on Differential Gene Expression in 4NQO, Fermentable, and Nonfermentable Carbon Sources.

Cellular metabolism can change the potency of a chemical’s tumorigenicity. 4-nitroquinoline-1-oxide (4NQO) is a tumorigenic drug widely used on animal models for cancer research. Polymorphisms of the transcription factor Yrr1 confer different levels of resistance to 4NQO in Saccharomyces cerevisiae. To study how different Yrr1 alleles regulate gene expression leading to resistance, transcriptomes of three isogenic S. cerevisiae strains carrying different Yrr1 alleles were profiled via RNA sequencing (RNA-Seq) and chromatin immunoprecipitation coupled with sequencing (ChIP-Seq) in the presence and absence of 4NQO. In response to 4NQO, all alleles of Yrr1 drove the expression of SNQ2 (a multidrug transporter), which was highest in the presence of 4NQO resistance-conferring alleles, and overexpression of SNQ2 alone was sufficient to overcome 4NQO-sensitive growth. Using shape metrics to refine the ChIP-Seq peaks, Yrr1 strongly associated with three loci including SNQ2. In addition to a known Yrr1 target SNG1, Yrr1 also bound upstream of RPL35B; however, overexpression of these genes did not confer 4NQO resistance. RNA-Seq data also implicated nucleotide synthesis pathways including the de novo purine pathway, and the ribonuclease reductase pathways were downregulated in response to 4NQO. Conversion of a 4NQO-sensitive allele to a 4NQO-resistant allele by a single point mutation mimicked the 4NQO-resistant allele in phenotype, and while the 4NQO resistant allele increased the expression of the ADE genes in the de novo purine biosynthetic pathway, the mutant Yrr1 increased expression of ADE genes even in the absence of 4NQO. These same ADE genes were only increased in the wild-type alleles in the presence of 4NQO, indicating that the point mutation activated Yrr1 to upregulate a pathway normally only activated in response to stress. The various Yrr1 alleles also influenced growth on different carbon sources by altering the function of the mitochondria. Hence, the complement to 4NQO resistance was poor growth on nonfermentable carbon sources, which in turn varied depending on the allele of Yrr1 expressed in the isogenic yeast. The oxidation state of the yeast affected the 4NQO toxicity by altering the reactive oxygen species (ROS) generated by cellular metabolism. The integration of RNA-Seq and ChIP-Seq elucidated how Yrr1 regulates global gene transcription in response to 4NQO and how various Yrr1 alleles confer differential resistance to 4NQO. This study provides guidance for further investigation into how Yrr1 regulates cellular responses to 4NQO, as well as transcriptomic resources for further analysis of transcription factor variation on carbon source utilization.

Engineered Swine Models of Cancer.

Over the past decade, the technology to engineer genetically modified swine has seen many advancements, and because their physiology is remarkably similar to that of humans, swine models of cancer may be extremely valuable for preclinical safety studies as well as toxicity testing of pharmaceuticals prior to the start of human clinical trials. Hence, the benefits of using swine as a large animal model in cancer research and the potential applications and future opportunities of utilizing pigs in cancer modeling are immense. In this review, we discuss how pigs have been and can be used as a biomedical models for cancer research, with an emphasis on current technologies. We have focused on applications of precision genetics that can provide models that mimic human cancer predisposition syndromes. In particular, we describe the advantages of targeted gene-editing using custom endonucleases, specifically TALENs and CRISPRs, and transposon systems, to make novel pig models of cancer with broad preclinical applications.

Zebrafish: a new companion for translational research in oncology.

In an era of high-throughput "omic" technologies, the unprecedented amount of data that can be generated presents a significant opportunity but simultaneously an even greater challenge for oncologists trying to provide personalized treatment. Classically, preclinical testing of new targets and identification of active compounds against those targets have entailed the extensive use of established human cell lines, as well as genetically modified mouse tumor models. Patient-derived xenografts in zebrafish may in the near future provide a platform for selecting an appropriate personalized therapy and together with zebrafish transgenic tumor models represent an alternative vehicle for drug development. The zebrafish is readily genetically modified. The transparency of zebrafish embryos and the recent development of pigment-deficient zebrafish afford researchers the valuable capacity to observe directly cancer formation and progression in a live vertebrate host. The zebrafish is amenable to transplantation assays that test the serial passage of fluorescently labeled tumor cells as well as their capacity to disseminate and/or metastasize. Progress achieved to date in genetic engineering and xenotransplantation will establish the zebrafish as one of the most versatile animal models for cancer research. A model organism that can be used in transgenesis, transplantation assays, single-cell functional assays, and in vivo imaging studies make zebrafish a natural companion for mice in translational oncology research.

PTEN deletion potentiates invasion of colorectal cancer spheroidal cells through 3D Matrigel.

PTEN (phosphatase and tensin homolog), a tumour suppressor negatively regulating the PI3K signalling pathway, is the second most frequently mutated gene in human cancers. Decreased PTEN expression is correlated with colorectal cancer metastases and poor patient survival. Three dimensional (3D) multicellular spheroid models have been postulated to bridge the gap between 2D cell models and animal models for cancer research and drug discovery. However, little is known about the impact of PTEN deletion on the invasion of colon cancer spheroidal cells through a 3D extracellular matrix, and current techniques are limited in their ability to study in vitro 3D cell models in real-time. Here, we investigated the migration and invasion behaviours of the colon cancer cell line HCT116 and its PTEN-/- isogenic cell line using three different in vitro assays, wound healing, transwell invasion, and label-free single cell 3D(2) invasion assays enabled by a resonant waveguide grating (RWG) biosensor. Light microscopic and RWG imaging showed that PTEN deletion influences the spheroid formation of HCT116 cells at high seeding density, and accelerates the spontaneous transfer from the spheroid to substrate surfaces. In vitro migration and invasion assays showed that PTEN knockout increases the 2D migration speed of HCT116 cells, and the invasion rate of individual cells through Matrigel or cells in the spheroid through 3D Matrigel; moreover, the PI3K inhibitor treatment drastically reduces the invasiveness of both cell lines. This study suggests that PTEN knockout potentiates the invasiveness of colorectal cancer spheroidal cells through a 3D extracellular matrix, and the label-free single cell assay is a powerful tool for investigating cancer cell invasion, in particular using 3D cell models.