The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research[1]
Author/Writer: Stephen J. Williams, Ph.D.©
UPDATED 3/14/2020
The need for alternate models of human cancer
Many worldwide regulatory bodies are in agreement that proper choice of animal model is necessary for adequate extrapolation of toxicity and efficacy data from animal to human, considering the varied classes of therapeutics now being developed for oncology. The inability of screens, reliant on human xenografts grown in immunocompromised mice to evaluate host-immune and species-dependent effects, has made development of alternative animal-models a priority. This is evident in the fact that ninety percent of new anticancer drugs which showed anti-tumor efficacy in mouse preclinical models failed in human clinical studies. A recently developed “humanized” mouse model may assist in testing the metabolism of cancer drugs but still relies on older “immunosuppression” mouse models (http://stehlin.org/mouse-model-development/). This inadequacy of older, accepted models is clearly evident when evaluating safety and efficacy of adenoviral based gene therapies such as oncolytic conditionally-replicative adenovirus (CRAd). Although new-generation CRAds present with a relative safe profile[2, 3], adenoviral particles, especially the Ad5 based virus used for most CRAds, have the tendency to replicate in non-tumor tissue, such as liver and lung, resulting in tissue-specific toxicities[4-7]. The manifestation of these toxicities is only evident in species permissive for viral replication, such as the pig. Indeed, one of the first clinical trials with older adenovirus gene therapy, resulting in severe hepatic toxicity and fatality, may have been prevented if more appropriate preclinical screens were conducted. Thereafter, strict regulatory guidelines for adenoviral-based clinical trials have been issued, with particular emphasis on vector dosage, safety and toxicity[8]. Indeed, at Schering-Plough, a toxicology program was initiated to evaluate SCH 58500, and adenoviral gene therapy directed against p53, which involved use of non-immunogenic rats compared with testing in Yorkshire pigs made immunoreactive to the vector[9, 10]. In fact, data from the pig study revealed a faster clearance of virus as well as toxicities not seen in non-immunogenic, non-permissive hosts such as rat and mouse.
Therefore, development of a porcine model of cancer would permit both testing of both the efficacy and safety of these therapies in the same animal.
Development of large animal models of cancer
To date, large animal tumor models have been used for studying spontaneously formed tumors in dogs and cats [11](Vail, 2000, Cancer Invest), the most common being canine [12] and feline non-Hodgkin’s lymphoma [13]. The advantages of these companion models are the outbred nature of the animals, comparable size and kinetics to human tumors [14-18], and high incidence rates. Allografts of the outbred-canine transplanted venereal tumor have been used to test the ability to detect tumors using X-ray computed tomography and MRI with the ultimate goal of imaging-guided intervention. Researchers have recently utilized the spontaneously arising canine and feline soft tissue sarcomas to study effects of hyperthermia on chemotherapy pharmocokinetics, development of hypoxic cell markers, and cancer imaging techniques [15, 19-26]
Although it appears that, for a select number of tumor types, spontaneously arising tumors in large outbred animals can be useful to model the human disease, it is disappointing these spontaneous arising tumors are limited to discrete tumor types. However, due to recent advances in sequencing of several domestic animal genomes and the development of new cloning strategies, it is now very feasible to utilize other animal models more relevant to human disease, notably the miniature pig.
The Gottingen mini-pig
Large animals in medical research: Advantages of the minipig
Due to recent advances in sequencing of several domestic animal genomes [27, 28] and the development of new organism cloning technologies [29-31], it is now very feasible to utilize other species to model human disease, notably the pig. The development of porcine models of human disease has gained much interest lately. Advantages include the resemblance in anatomy, physiology, and genetic makeup with the human, as well as new methods to manipulate the pig genome [32, 33]. To date, porcine models of human metabolic syndrome [34] and diabetes [35], aortic aneurism [36], infectious disease resistance [32, 37], seizure [38], neurologic syndromes [33], and pancreatitis [39] have been developed. Recently, a genetically-engineered porcine model of cystic fibrosis was produced in collaboration with investigators at University of Iowa and Exemplar Genetics [40-42]. Additionally, Cho et al. successfully transplanted spontaneously transformed leukemic and lymphatic tumor cells in a major histocompatibility complex (MHC)-defined inbred miniature swine model [43], suggesting feasibility of an ex vivo strategy to develop a porcine tumor model. Porcine models have, also, been used to develop, test and refine surgical [44, 45] and laparoscopic techniques [46, 47], radio- and cryoablation protocols of tissues [48-52] and robotic surgery using the da Vinci Surgical SystemÒ [53, 54]. In addition, because of the size of porcine organs and their resemblance to the human (in genetics) the minipig is very useful and abundant of a source to isolate specific cell types for in vitro studies. Below is a figure showing the comparable size of human and porcine ovaries to the mouse and ability to purify porcine ovarian epithelial cells and their similarity to human and mouse ovarian epithelial cells.
Figure 1. The human and pig ovary have similar size and can yield a greater number of isolated cells than one can get from a mouse ovary.
Figure 2. Isolation and morphology of ovarian epithelial cells from three sources:
A) Devonshire/Yorkshire pig
B) normal human ovary
c) SV129/BL6 mouse
note cobblestone epithelial morphology from all three sources©
To date, there has been no allograft or xenograft model of cancer in pigs. The consensus amongst many surgeons suggests development of a minipig tumor model would be an invaluable tool for developing surgical skills.
A recent advancement in porcine tumor modeling was made by collaboration between researchers from the laboratories of Dr. Stefan Bossmann and Deryl Troyer at Kansas State and Iowa State, respectively[1]. The joint collaboration resulted in the development of the first severe combined immunodeficient pig line (SCID pig) which was shown to be able to accept human tumor xenografts. The line of immunodeficient pig was discovered when Yorkshire pigs were bred for increased feed efficiency and a line of pigs exhibited SCID-like symptoms including:
- Decreased levels of circulating lymphocytes
- Atrophied thymus and lymph nodes
The SCID phenotype in mice have been ascribed to defects in a DNA-dependent protein kinase gene which prevents variable-diversity-joining [V(D)J] gene region recombination[55]. There have been multiple genetic defects found in humans resulting in SCID, including defects in adenylate kinase2, Janus kinase 3, the IL2 receptor, and the IL-7 receptor[56]. The SCID phenotype in this pig line has a simple autosomal recessive inheritance pattern which, as described below in an interview with the authors, allows for the propagation of this porcine line.
An important feature of SCID models is the ability of these animals to act as a recipient of human tumorigenic cell lines. In fact, growth of cell lines in SCID mice is a common test for tumorigenicity. Therefore, to test if these pigs could act as recipients for human cancer cell lines, the authors inoculated the SCID Yorkshire pigs with 4 million A3755M human melanoma cells or PANC1 human pancreatic carcinoma cells subcutaneously in the left and right ears respectively of three pigs. Some features of the results include:
- All injection sites showed evidence (either histologic or palpable) of tumor growth
- Tumors showed characteristic histologic features of malignant neoplasm including
- Bizarre and atypical mitotic figures
- Anisocytosis (different cell sizes and shapes; feature of malignancy)
- Anisokaryosis (different size and shape of nucleus)
- tumors stained with anti-human mitochondrial antibody (a marker of epithelial cancer cells) showed strong cytoplasmic staining of neoplastic cells
- interestingly no necrotic regions in the tumor
Figure 3. Visual evidence of human tumor cells growing in SCID pig ear (day 20). B) Same picture as A) but circle outlines growth. From reference 1. Basel et al., used with permission from Mary Liebert.
It is interesting to note that these tumors only grew roughly 10 x 5.5 mm, which is genrally large enough to do preclinical studies but may be too expensive to be of use for xenograft studies. However it would be very feasible to conduct allograft studies in these SCID pigs.
Dr. Jack Dekkers, C.F. Curtiss Distinguished Professor and Section Leader of Animal Breeding and Genetics at Iowa State University, was kind to answer a few questions about the SCID pig model.
Question: You had mentioned this line was identified after breeding Yorkshire pigs for increased feed efficiency. Have you identified or hypothesize which altered pathway or molecular defect which results in a SCID phenotype? Is this SCID phenotype a result of a metabolic syndrome these pigs could have?
Dr. Dekkers: We indeed identified the SCID phenotype in a line of pigs that we had selected for increased feed efficiency. However, I don’t think this phenotype has anything to do with the selection we practiced; it was either already present in the founders of the line or it was a random mutation that occurred in the line, independent of the selection for feed efficiency. We have narrowed the mutation that causes the SCID in our pigs down to a chromosomal region and have a very strong candidate gene in that region that we are currently pursuing.
Question: In your opinion, is it possible to produce a highly inbred immunocompromised strain of pig such as a Gottingen minipig?
Dr. Dekkers: We are working on breeding the SCID mutation into mini pigs. But in the meantime, we have used bone marrow transfer to create a male that is homozygous SCID (it’s an autosomal recessive) and reproducing. This allows us to produce litters that are 50% SCID and 50% normal (carriers) by mating him to carrier females.
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UPDATED 3/14/2020
A recent Research Article and Research Article Summary in Science discusses, by the primary author of her study that describes the utility of the pig as an excellent surrogate model of the human brain and human brain function. The study, by Dr. Evelina Sjostdedt et al., was an integrative analysis of porcine, mouse, and human transcriptomic, genomic, and proteomic data from discrete anatomical regions of the brain. The global analysis suggested that there is similar regional organization and expression patterns among the three mammalian species. The authors found interspecies variability with respect for many neurotransmitter receptors.
However, for some regions of the brain, such as the cerebellum and hypothalamus, the human global expression profile is closer to that of the pig than of the mouse, suggesting that the pig might be considered a preferred animal model to study many brain processes.
In addition, interestingly, the authors found that many signature genes canonically thought to only be expressed in certain brain cells (astrocytes, microglia, oligodendrocytes) are expressed in higher levels in peripheral organs as well as immune cells.
Please go to the full article in Science,
An atlas of the protein-coding genes in the human, pig, and mouse brain,
Science 06 Mar 2020:
Vol. 367, Issue 6482, eaay5947
DOI: 10.1126/science.aay5947
to access the data used in this study, which includes high resolution images and metadata have also been made publicly available in the open-access Human Protein Atlas (HPA) Brain Atlas. at www.proteinatlas.org.
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