Pinaki Mondal

Katie L Bailey

Sara B Cartwright

Vimla Band

Mark A Carlson
email: macarlso@unmc.edu

Large Animal

Large Animal Models of Breast Cancer

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However, limitations associated with these models, including growth in the absence of a functioning host immune system, have made them poor predictors of clinically-relevant biologic behavior. [START_REF] Holen | In vivo models in breast cancer research: progress, challenges and future directions[END_REF][START_REF] Kawaguchi | Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research[END_REF] They have reduced intra-tumoral heterogeneity which does not optimally represent a human breast tumor. [START_REF] Holen | In vivo models in breast cancer research: progress, challenges and future directions[END_REF] . When compared to PDX and original patient tumors, cell lines have shown a loss of genetic expression profile and loss of tumor-specific genes, [START_REF] Daniel | A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro[END_REF] , an alteration in growth and invasive properties, loss of specific cell populations, and selection of cell population. [START_REF] Kawaguchi | Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research[END_REF] They are frequently derived from highly aggressive malignant tumors or pleural effusions and thus are less useful in studying the early stages of disease. [START_REF] Holen | In vivo models in breast cancer research: progress, challenges and future directions[END_REF] The lack of a functional host immune system means that CDX tumors do not undergo any appreciative immunoediting, [START_REF] Vesely | Natural innate and adaptive immunity to cancer[END_REF][START_REF] Wagner | Cancer Immunoediting by Innate Lymphoid Cells[END_REF] which is increasingly recognized as important during tumor growth and evolution. Overall, CDX models are useful for BC genetics and basic biology, but are poorly predictive of human response to test interventions.

Patient-Derived Xenograft Models

With the limitations of CDX, there has been significant growth in the use of patientderived xenograft (PDX) murine models for translational cancer research. These models involve the transplantation of fragmented primary human tumor from surgical resection or biopsy into immune deficient mice. One of the main advantages of PDX models is that the grafted tumor remains biologically stable. Biologic stability is seen in relation to tumor architecture, gene expression, mutational status, growth kinetics, invasiveness, metastatic potential, and drug responsiveness. [START_REF] Kawaguchi | Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research[END_REF][START_REF] Tentler | Patient-derived tumour xenografts as models for oncology drug development[END_REF][START_REF] Roarty | Laboratory Models for Investigating Breast Cancer Therapy Resistance and Metastasis[END_REF] . In breast cancer models, they have been shown to conserve ER, PR, and HER2 expression particularly when grafted directly into mammary ducts, [START_REF] Dobrolecki | Patient-derived xenograft (PDX) models in basic and translational breast cancer research[END_REF][START_REF] Richard | The mammary ducts create a favourable microenvironment for xenografting of luminal and molecular apocrine breast tumours[END_REF] and they have shown similar metastatic progression compared to human tumors. [START_REF] Roarty | Laboratory Models for Investigating Breast Cancer Therapy Resistance and Metastasis[END_REF][START_REF] Derose | Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes[END_REF] . These features have made PDX models useful for pre-clinical evaluation of drug therapy including new treatment targets, novel therapy combinations, treatment schedules, and even personalized drug therapy. [START_REF] Kawaguchi | Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research[END_REF] However, PDX models are not without limitations. PDX models pose a slightly higher cost than CDX models and require access to fresh patient material. [START_REF] Holen | In vivo models in breast cancer research: progress, challenges and future directions[END_REF] The prolonged length of time it takes to generate tumors does not always match clinical or research needs. [START_REF] Holen | In vivo models in breast cancer research: progress, challenges and future directions[END_REF][START_REF] Kawaguchi | Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research[END_REF][START_REF] Paez-Ribes | Development of Patient Derived Xenograft Models of Overt Spontaneous Breast Cancer Metastasis: A Cautionary Note[END_REF] Despite early studies showing success with different breast cancer subtypes with orthotopic implantation, [START_REF] Dobrolecki | Patient-derived xenograft (PDX) models in basic and translational breast cancer research[END_REF][START_REF] Richard | The mammary ducts create a favourable microenvironment for xenografting of luminal and molecular apocrine breast tumours[END_REF] ideal location of implantation remains uncertain [START_REF] Kawaguchi | Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research[END_REF][START_REF] Paez-Ribes | Development of Patient Derived Xenograft Models of Overt Spontaneous Breast Cancer Metastasis: A Cautionary Note[END_REF] and engraftment failure rates remain high in hormone receptor positive tumors favoring more aggressive subtypes. [START_REF] Kawaguchi | Current Update of Patient-Derived Xenograft Model for Translational Breast Cancer Research[END_REF][START_REF] Roarty | Laboratory Models for Investigating Breast Cancer Therapy Resistance and Metastasis[END_REF][START_REF] Moon | Prognostic and functional importance of the engraftment-associated genes in the patient-derived xenograft models of triple-negative breast cancers[END_REF] Another concern is that by the third in vivo passage murine stroma replaces the human grafted stroma, which may result in changes in paracrine regulation as well as physical properties. [START_REF] Wever | Role of tissue stroma in cancer cell invasion[END_REF][START_REF] Junttila | Influence of tumour micro-environment heterogeneity on therapeutic response[END_REF] With PDX models favoring more aggressive subtypes, they have been used extensively to study triple negative breast cancer (TNBC) and metastasis. However, limitations have been apparent, including (i) the need to utilize immunodeficient mice which do not mimic the clinical effect of the immune system (immunoediting) on the development of metastasis, 17 (ii) the prolonged time for studying metastasis, [START_REF] Roarty | Laboratory Models for Investigating Breast Cancer Therapy Resistance and Metastasis[END_REF][START_REF] Paez-Ribes | Development of Patient Derived Xenograft Models of Overt Spontaneous Breast Cancer Metastasis: A Cautionary Note[END_REF] and (iii) the rarity of spontaneous metastasis development. [START_REF] Paez-Ribes | Development of Patient Derived Xenograft Models of Overt Spontaneous Breast Cancer Metastasis: A Cautionary Note[END_REF] Metastatic tumors do occur in PDX models, primarily in lungs and lymph nodes, but less so in the brain and bone (the latter are common sites seen in human breast cancer metastasis). [START_REF] Holen | In vivo models in breast cancer research: progress, challenges and future directions[END_REF] There is also high rates of tumor regrowth at primary implant sites which reduce survival time and failure of the metastatic sites to manifest the expected more aggressive phenotype when reimplanted (in contrast with what occurs with established cell lines). [START_REF] Roarty | Laboratory Models for Investigating Breast Cancer Therapy Resistance and Metastasis[END_REF] In summary, PDX models may be more predictive for preclinical drug evaluation, but ultimately are limited by the same issue afflicting the CDX model: a lack of a functional host immune system.

Humanized CDX and PDX models

A more recent development in murine cancer modeling is the humanized mouse model, [START_REF] Jin | Development of humanized mouse with patient-derived xenografts for cancer immunotherapy studies: A comprehensive review[END_REF] which has been useful for the study of the immune system's role in cancer. In brief, an immunocompromised mouse is engrafted with components of the human immune system, generating a functional human immune system somewhat similar to that observed in the originating patient, with the intention to create a more realistic tumor microenvironment. [START_REF] Morton | The humanized mouse: Emerging translational potential[END_REF] There has been increased interest in these models for pre-clinical studies with immunotherapies and breast cancer, particularly TNBC and HER2+ cancers. Human CD34+ HSC-engrafted NSG mice harboring different types of PDX are now commercially available. Some researchers have described less expensive, in-house models via intravenous injection of hematopoietic stem cells into irradiated SCID mice, which have shown successful engraftment and metastasis. [START_REF] Rosato | Evaluation of anti-PD-1-based therapy against triple-negative breast cancer patient-derived xenograft tumors engrafted in humanized mouse models[END_REF] Studies on breast cancer immunotherapy using humanized mice have successfully shown reduction of tumor growth, [START_REF] Rosato | Evaluation of anti-PD-1-based therapy against triple-negative breast cancer patient-derived xenograft tumors engrafted in humanized mouse models[END_REF][START_REF] Compte | An Fc-free EGFR-specific 4-1BB-agonistic Trimerbody Displays Broad Antitumor Activity in Humanized Murine Cancer Models without Toxicity[END_REF] but they are not without limitations, including (i) a lack of GM-CSF in humanized mice, which is important for the differentiation and maturation of the myeloid lineage, and (ii) mismatching HLA typing between hNSG and PDX. [START_REF] Rosato | Evaluation of anti-PD-1-based therapy against triple-negative breast cancer patient-derived xenograft tumors engrafted in humanized mouse models[END_REF] Some humanized murine models have shown xenograft-versus-host effect, in which mature human T cells attack their murine host, reducing the window in which this model can be used to study an implanted tumor. [START_REF] Morton | The humanized mouse: Emerging translational potential[END_REF] So, while the lack of a functional immune system in conventional CDX/PDX models has been somewhat countered by introducing components of the human immune system in humanized mice, graft vs. host issues continue to define the utility of the humanized subjects.

Chemically Induced Murine Models

Although genetic models have provided insight into how particular mutations can induce tumorigenesis, these models may give an oversimplified view of how cancer develops. The occurrence of point mutations, changes in gene copy number, epigenetic modifications, and other genomic events can also be affected by environmental factors. Some human cancers develop secondary to chemical carcinogenesis; chemically induced models may be able to replicate the genetic events that occur during this process. [START_REF] Mccreery | Chemical Carcinogenesis Models of Cancer: Back to the Future[END_REF] Polycyclic aromatic hydrocarbons such as 7,12-Dimethylbenz(a)-anthracene (DMBA) and methylcholanthrene (MC) compounds have been used in mice to induce breast tumors. [START_REF] Day | Dietary genistein increased DMBA-induced mammary adenocarcinoma in wild-type, but not ER alpha KO, mice[END_REF][START_REF] Fischer | The effect of dietary fat on the rapid development of mammary tumors induced by 7,12-dimethylbenz(a)anthracene in SENCAR mice[END_REF][START_REF] Huang | Effect of dietary curcumin and dibenzoylmethane on formation of 7,12dimethylbenz[a]anthracene-induced mammary tumors and lymphomas/leukemias in Sencar mice[END_REF][START_REF] Jerry | Infrequent p53 mutations in 7,12-dimethylbenz[a]anthracene-induced mammary tumors in BALB/c and p53 hemizygous mice[END_REF][START_REF] Kouri | Evidence of a genetic relationship between susceptibility to 3-methyl-cholanthrene-induced subcutaneous tumors and inducibility of aryl hydrocarbon hydroxylase[END_REF] These compounds can bind to DNA and cause errors in replication. [START_REF] Digiovanni | Biotransformation and bioactivation of 7, 12dimethylbenz[a]anthracene (7, 12-DMBA)[END_REF] DMBA has been studied in knockout, hemizygous and SENCAR (SENsitivity to CARcinogenesis) mice, producing breast tumors after 3-34 weeks. [START_REF] Day | Dietary genistein increased DMBA-induced mammary adenocarcinoma in wild-type, but not ER alpha KO, mice[END_REF][START_REF] Fischer | The effect of dietary fat on the rapid development of mammary tumors induced by 7,12-dimethylbenz(a)anthracene in SENCAR mice[END_REF][START_REF] Huang | Effect of dietary curcumin and dibenzoylmethane on formation of 7,12dimethylbenz[a]anthracene-induced mammary tumors and lymphomas/leukemias in Sencar mice[END_REF][START_REF] Jerry | Infrequent p53 mutations in 7,12-dimethylbenz[a]anthracene-induced mammary tumors in BALB/c and p53 hemizygous mice[END_REF] MC has produced tumors after 7 months. [START_REF] Kouri | Evidence of a genetic relationship between susceptibility to 3-methyl-cholanthrene-induced subcutaneous tumors and inducibility of aryl hydrocarbon hydroxylase[END_REF] In addition to modeling genetic events of chemical carcinogenesis, the advantages of chemically induced models include (i) no need for immunodeficient subjects, (ii) autochthonous tumor development, and (iii) relatively low cost and ease of use. The disadvantages of chemically-induced models is that they are not genetically defined to the same extent as GEM models are, which can have implications on the investigator's ability to generalize from their data.

Genetically engineered murine models

There are several types of GEM models, including conventional, knockout, and conditional. Conventional GEM models are typically driven by mammary-specific promoters that direct expression of specific oncogenes (transgenes) which may not be specific to mammary epithelial cells. Common mammary promoters in use include the mouse mammary tumor virus long terminal repeat (MMTV-LTR), whey acidic protein (WAP), b-lactoglobulin (BLG) and the 5' flanking region of the C3 component of the rat prostate steroid binding protein. [START_REF] Asch | Tumor viruses and endogenous retrotransposons in mammary tumorigenesis[END_REF][START_REF] Green | The C3(1)/SV40 T-antigen transgenic mouse model of mammary cancer: ductal epithelial cell targeting with multistage progression to carcinoma[END_REF][START_REF] Webster | Tissue-specific, temporally regulated expression mediated by the proximal ovine beta-lactoglobulin promoter in transgenic mice[END_REF][START_REF] Wen | Expression of whey acidic protein (WAP) genes in tissues other than the mammary gland in normal and transgenic mice expressing mWAP/hGH fusion gene[END_REF] The first GEM model from the 1980s expressed the c-Myc oncogene under the control of the MMTV promoter, which resulted in mammary adenocarcinoma. [START_REF] Stewart | Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTV/myc fusion genes[END_REF] Since then, a number of transgenic mice have been created to study the role of proto-oncogenes in the development and progression of tumors. In addition to c-Myc, some of the oncogenes that have been overexpressed using the MMTV-LTR promoter include v-HRas, Wnt, PyMT, Neu, and ErbB2. [START_REF] Stewart | Spontaneous mammary adenocarcinomas in transgenic mice that carry and express MTV/myc fusion genes[END_REF][START_REF] Guy | Activated neu induces rapid tumor progression[END_REF][START_REF] Muller | Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene[END_REF][START_REF] Sinn | Coexpression of MMTV/v-Ha-ras and MMTV/c-myc genes in transgenic mice: synergistic action of oncogenes in vivo[END_REF][START_REF] Tsukamoto | Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice[END_REF][START_REF] Guy | Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease[END_REF] Expression of these oncogenes resulted in mammary tumorigenesis and metastatic lesions. [START_REF] Muller | Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene[END_REF][START_REF] Guy | Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease[END_REF][START_REF] Christenson | MMTV-PyMT and Derived Met-1 Mouse Mammary Tumor Cells as Models for Studying the Role of the Androgen Receptor in Triple-Negative Breast Cancer Progression[END_REF] GEM models were also generated to understand loss-of-function (knockout) of tumor suppressor genes. One of the most common knockout models involves the tumor suppressor Trp53. [START_REF] Jerry | A mammary-specific model demonstrates the role of the p53 tumor suppressor gene in tumor development[END_REF][START_REF] Kuperwasser | Development of spontaneous mammary tumors in BALB/c p53 heterozygous mice. A model for Li-Fraumeni syndrome[END_REF] Mice that were homozygous for Trp53 null allele developed lymphomas and died at around 4-6 months of age. [START_REF] Kuperwasser | Development of spontaneous mammary tumors in BALB/c p53 heterozygous mice. A model for Li-Fraumeni syndrome[END_REF] In both the conventional and knockout models, the gene targets are not specific to a certain cell lineage. These models cannot recapitulate sporadic disease in which there is an accumulation of genetic events in a specific cell lineage. There also can be off-target effects in these models, and other types of cancers may arise in them. In addition, ~30% of the conventional knockout models are lethal in the perinatal period, which makes experimentation difficult.

To overcome these problems and prevent early embryonic death, conditional GEM models have been developed that allow for the activation of oncogenes and/or deletion of tumor suppressors, specifically targeted to the mammary gland (i.e., tissue-specific edits). These models utilize systems such as the Cre/loxP and Flp/frt systems for activation or deletion of genes. [START_REF] Holen | In vivo models in breast cancer research: progress, challenges and future directions[END_REF] These systems provide for controllable recombination events, such as removal of stop codon that subsequently permits transcription of a transgene. 11 Among the first two conditional GEM models created were WAP-Cre and MMTV-Cre [START_REF] Wagner | Spatial and temporal expression of the Cre gene under the control of the MMTV-LTR in different lines of transgenic mice[END_REF][START_REF] Wagner | Cre-mediated gene deletion in the mammary gland[END_REF] These models have been able to generate hereditary breast cancer models specifically by modeling the heterozygous mutations observed in the BRCA1/BRCA2 genes. [START_REF] Trusler | BRCA1 and BRCA2 associated breast cancer and the roles of current modelling systems in drug discovery[END_REF] GEM models that contained a conditional mutant BRCA1 allele and a disruption in Trp53 have accelerated mammary tumor development. [START_REF] Trusler | BRCA1 and BRCA2 associated breast cancer and the roles of current modelling systems in drug discovery[END_REF] Trp53 in combination with other genes has been extensively studied using conditional GEM models. [START_REF] Cressman | Mammary tumor formation in p53-and BRCA1-deficient mice[END_REF] Tissue-specific genetic edits in conditional GEM models can also be induced at a specific time using the inducible Cre-ERT system or the tetracycline/ doxycycline promoter. In the Cre-ERT model the hormone-binding domain of the estrogen receptor (ERT) is fused to Crerecombinase. Upon administration of tamoxifen (an estrogen analogue) Cre-recombinase is activated, leading to the recombination event marked by the loxP sites. [START_REF] Vooijs | A highly efficient ligand-regulated Cre recombinase mouse line shows that LoxP recombination is position dependent[END_REF] GEM models have become increasingly popular in BC and other cancer research, providing insight into disease initiation, progression, evaluation of drug delivery, therapeutic response and biomarkers in their natural microenvironment. [START_REF] Day | Preclinical mouse cancer models: a maze of opportunities and challenges[END_REF] GEM models typically have a normal immune system, tumor development is autochthonous, and by definition are genetically defined. The disadvantages of GEM models are the expense and complexity involved in their creation.

Small Non-Murine Models of BC

Rat BC Models

Rats have been considered a suitable animal model to study breast cancer due to their similarity with human mammary cancer in terms of histology, immunocytochemical markers and biological behavior of tumors. [START_REF]Significance of rat mammary tumors for human risk assessment[END_REF] The histologic characteristics of normal mammary luminal epithelium and myoepithelium is similar between rats and human. [START_REF] Jn | A comparison of the histopathology of premalignant and malignant mammary gland lesions induced in sexually immature rats with those occurring in the human[END_REF] Long term studies have shown that some rats can develop breast tumors spontaneously. [START_REF]Significance of rat mammary tumors for human risk assessment[END_REF] Use of a chemical carcinogen in rats can result in a shorter latency period to tumor development. [START_REF] Jn | A comparison of the histopathology of premalignant and malignant mammary gland lesions induced in sexually immature rats with those occurring in the human[END_REF][START_REF]Animal models of breast cancer: their diversity and role in biomedical research[END_REF][START_REF] Kl | Development and characterization of a novel rat model of estrogen-induced mammary cancer[END_REF][START_REF]Intraductal administration of N-methyl-N-nitrosourea as a novel rodent mammary tumor model[END_REF][START_REF]Suppression of 7,12-dimethylbenz(a)anthracene-induced mammary carcinogenesis in rats by resveratrol: role of nuclear factor-kappaB, cyclooxygenase 2, and matrix metalloprotease 9[END_REF] Recently, 17b-estradiol (E2) was used to induce breast tumors in August Copenhagen Irish-rats by modulating estrogen mediated mechanisms in breast cancer development. [START_REF] Kl | Development and characterization of a novel rat model of estrogen-induced mammary cancer[END_REF] In xenograft-Matrigel® implantation experiments, younger rats have experienced greater tumor growth compared to older rats. 60, [START_REF]Breast cancer models to study the expression of estrogen receptors with small animal PET imaging[END_REF] A bone metastasis immunodeficient rat model has been developed in which human breast cancer cells (MDA-MB-231) were intra-arterially injected into a hindlimb artery. [START_REF] Fp | Characterization of a rat model with site-specific bone metastasis induced by MDA-MB-231 breast cancer cells and its application to the effects of an antibody against bone sialoprotein[END_REF] Genetically engineered rat models of breast cancer have been developed in which HER2 and TGF-a were overexpressed through the mouse mammary tumor virus (MMTV) promoter. [START_REF] Davies | Development of hyperplasias, preneoplasias, and mammary tumors in MMTV-c-erbB-2 and MMTV-TGFalpha transgenic rats[END_REF] This model stochastically produced a variety of benign, hyperplastic and malignant lesions, including ductal carcinoma in situ and carcinoma within a year.

In a rat model with three copies of human HRAS proto-oncogene, induction of carcinogenesis with nitrosomethylurea resulted in large mammary tumors within 8 weeks. [START_REF] Asamoto | Transgenic rats carrying human c-Ha-ras proto-oncogenes are highly susceptible to Nmethyl-N-nitrosourea mammary carcinogenesis[END_REF] Mammary carcinogenesis in rats was induced through injection of high-titer, Neu-containing, replication-defective retrovirus which produced hormonally-responsive in situ carcinomas within 15-days post infusion, and regressed spontaneously after 20-days post infusion. [START_REF] Woditschka | A short-term rat mammary carcinogenesis model for the prevention of hormonally responsive and nonresponsive in situ carcinomas[END_REF] Injection of human adenovirus type 9 (Ad9) also is known to induce estrogen-dependent mammary tumors in rats within 7-12 months. [START_REF] Javier | Mammary tumors induced by human adenovirus type 9: a role for the viral early region 4 gene[END_REF] Overall, however, use of rats in BC research has lagged far behind the broad use of mice.

Hamster BC Models

Similar to rats, nitrosomethylurea can induce mammary carcinoma in Syrian hamsters (Mesocricetus auratus), producing high-grade poorly-differentiated mammary adenocarcinomas. [START_REF] Coburn | Establishment of a mammary carcinoma cell line from Syrian hamsters treated with N-methyl-N-nitrosourea[END_REF] Subcutaneous allogenic implantation of cell lines established from these primary tumors generated secondary tumors. Hamster models can be useful in studies on oncolytic adenoviruses, a self-replicating cancer cell-killing virus. Oncolytic adenoviruses can replicate in immunocompetent hamsters, making the hamster model of cancer a suitable nonimmunocompromised model to study therapeutic potential of these viruses. [START_REF] Li | The Efficacy of Oncolytic Adenovirus Is Mediated by T-cell Responses against Virus and Tumor in Syrian Hamster Model[END_REF]

Tree shrew BC Models

The tree shrew (Tupaia belangeri chinensis) can develop spontaneous mammary tumors, which are similar to human papillary tumors in terms of morphology and pathology. [START_REF] Hj | Characterization of spontaneous breast tumor in tree shrews (Tupaia belangeri chinenesis)[END_REF][START_REF] Elliot | Breast cancer in a tree shrew (Tupaia glis)[END_REF] Chemical induction with DMBA plus medroxyprogesterone acetate (MPA) also produced breast cancer in tree shrews. [START_REF] Xia | PTEN/PIK3CA genes are frequently mutated in spontaneous and medroxyprogesterone acetate-accelerated 7,12-dimethylbenz(a)anthracene-induced mammary tumours of tree shrews[END_REF] Injection of a lentiviral vector with PyMT (polyomavirus middle T antigen, an oncogene that activates c-Src), into the mammary duct of tree shrews resulted in tumor development in all subjects by 7 weeks post-injection. [START_REF] Ge | Generation and characterization of a breast carcinoma model by PyMT overexpression in mammary epithelial cells of tree shrew, an animal close to primates in evolution[END_REF]

Feline BC Models

Feline mammary carcinomas (FMCs) are the third most common type of cancer in cats. [START_REF] Thomas | Cytogenomics of Feline Cancers: Advances and Opportunities[END_REF] In utero implantation of allogeneic mammary cancer cell lines into fetal cats (Felis catus) produced tumor at the injection site, followed by widespread metastasis after 6-10 weeks. 74, [START_REF] Minke | Allotransplantation of K248 feline mammary carcinoma cell line in cats. A model for monoclonal antibody guided detection and therapy of human breast cancer[END_REF] A nude mouse model of FMC demonstrated metastatic potential (bone, kidney, brain, lung and liver; i.e., common sites of metastasis in human breast cancer) after injection to the primary site. [START_REF] Hassan | Feline Mammary Cancer[END_REF] Cancer stem cell-like populations in FMCs can form mammospheres (organoids) and are tumorigenic, radioresistant, and chemoresistant. [START_REF] Barbieri | Isolation of stem-like cells from spontaneous feline mammary carcinomas: phenotypic characterization and tumorigenic potential[END_REF][START_REF] Pang | Feline mammary carcinoma stem cells are tumorigenic, radioresistant, chemoresistant and defective in activation of the ATM/p53 DNA damage pathway[END_REF] Mammospheres can be used a model to study feline breast cancer. Morphological, histological, and molecular to similarities between FMCs and human breast cancer have been described and discussed. [START_REF] Wiese | Feline mammary basal-like adenocarcinomas: a potential model for human triple-negative breast cancer (TNBC) with basal-like subtype[END_REF][START_REF] De Maria | Spontaneous feline mammary carcinoma is a model of HER2 overexpressing poor prognosis human breast cancer[END_REF][START_REF] Burrai | Spontaneous feline mammary intraepithelial lesions as a model for human estrogen receptor-and progesterone receptor-negative breast lesions[END_REF]

Large Animal Models of BC

Justification for & Advantages of a Large Animal Model of BC

Breast cancer, and TNBC in particular, can be modeled with many of the above murine and other small animal models However, none of the above models can overcome the limitation which drives the current proposal: inadequate subject size. Development of some diagnostic and interventional technologies require a human-sized model to understand how clinically relevant tumor size and tissue thickness affect the performance of the experimental technology, such as with three-dimensional scanning or with a tissue-ablation device. In addition, pharmacokinetic parameters (including absorption, distribution, metabolism, and excretion) can be vastly different between a 20 g mouse and a 70 kg patient. [START_REF] Lin | Species similarities and differences in pharmacokinetics[END_REF] Testing the effect of an infused chemical entity (e.g., a novel anti-tumor agent) in a murine tumor model may result in an inaccurate conclusion secondary to these pharmacokinetic differences. As an example, a mouse vs. large subject scenario is utilized below.

With regard to drug distribution, tumor burden in a 20 gram mouse with a 1-cm tumor is ~400-fold greater (mass:mass) than in a 70 kg subject with a 2-cm tumor (Fig. 1). If the tumor acts as a sink for a candidate anti-tumor drug, then the tumor's ability to decrease the drug's plasma concentration would be much greater in the mouse. A consequence of this tumor sink effect [START_REF] Chu | Plasma, tumor and tissue pharmacokinetics of Docetaxel delivered via nanoparticles of different sizes and shapes in mice bearing SKOV-3 human ovarian carcinoma xenograft[END_REF] would be a gross underestimation of the drug's toxicity from murine data. Alternatively, if the drug penetrates poorly into dense tumor stroma, [START_REF] Tanaka | Stromal barriers to nanomedicine penetration in the pancreatic tumor microenvironment[END_REF] then testing in a ~1 cm murine tumor may overestimate the drug's anti-tumor efficacy, as opposed to testing in a large (≥4 cm) tumor.

These drug distribution issues in a murine model could be minimized with a large animal model (e.g., a 70 kg pig) with clinically-relevant tumor size. So, a primary justification for a large animal BC model would be its ability to replicate human tumor dimension and human PK parameters.

Canine BC Models

Dog. Similar to cats, canine mammary tumors (CMTs) occur spontaneously and are the second most common cancer in dogs. [START_REF] Pinho | Canine tumors: a spontaneous animal model of human carcinogenesis[END_REF] Development of CMTs is hormone dependent and showed dysregulated expression of BRCA1, BRCA2 and TP53, [START_REF] Uva | Comparative expression pathway analysis of human and canine mammary tumors[END_REF][START_REF] Lee | Mutation and overexpression of p53 as a prognostic factor in canine mammary tumors[END_REF][START_REF] Chang | Evaluation of hormone receptor expression for use in predicting survival of female dogs with malignant mammary gland tumors[END_REF] analogous to human BC.

Transcriptional analysis of CMTs demonstrated pathways that are active in human cancer, including those involved with cell cycle regulation, apoptotic signaling, immune functions, endoplasmic reticulum stress, angiogenesis and cell migration. [START_REF] Graim | Modeling molecular development of breast cancer in canine mammary tumors[END_REF] Approximately 25% of the genetic alterations in metastatic CMTs were associated with human mammary cancer. [START_REF] Klopfleisch | Metastatic canine mammary carcinomas can be identified by a gene expression profile that partly overlaps with human breast cancer profiles[END_REF] In addition, canine spontaneous mammary DCIS and invasive cancer shows similar histologic and molecular characteristics with DCIS and invasive cancer in humans. [START_REF] Si | Ductal Carcinoma In Situ Progression in Dog Model of Breast Cancer[END_REF] Spontaneous CMTs also can metastasize to lymph nodes and lung. [START_REF] Misdorp | Canine mammary cancer. II. Therapy and causes of death[END_REF] Her2 overexpression in CMTs is controversial. [START_REF] Burrai | Investigation of HER2 expression in canine mammary tumors by antibody-based, transcriptomic and mass spectrometry analysis: is the dog a suitable animal model for human breast cancer?[END_REF] Benign tumors and mesenchymal tumors are more prevalent in CMTs; the latter are rare with human breast cancer. [START_REF] Salas | Epidemiological Study of Mammary Tumors in Female Dogs Diagnosed during the Period 2002-2012: A Growing Animal Health Problem[END_REF][START_REF] Goldschmidt | Classification and grading of canine mammary tumors[END_REF]

Non-Human Primate BC Models

Mammary gland tissue from common marmoset and rhesus macaques have been used in mammosphere culture, and can be used as a ex vivo model to study breast cancer. [START_REF] Wu | Characterization of mammary epithelial stem/progenitor cells and their changes with aging in common marmosets[END_REF][START_REF] Mariya | Mammosphere Culture of Mammary Cells from Cynomolgus Macaques (Macaca fascicularis)[END_REF] However, due to the low incidence of spontaneous tumors, long incubation periods, and high costs, nonhuman primates have not been commonly used in breast cancer research.

Porcine BC Models

Swine have been used for decades as research subjects in diverse areas including transplantation, physiology, trauma, toxicology, [START_REF] Lunney | Advances in swine biomedical model genomics[END_REF][START_REF] Swindle | Swine in the Laboratory: Surgery, Anesthesia, Imaging, and Experimental Techniques[END_REF] and recently cancer. [100][101][102][103][104] The porcine genome has been sequenced, 105 and annotation is ongoing. 106 Gene editing of pigs and creation of transgenic swine is now fairly common; 102,103,107-110 some transgenics (such as the Oncopig 101 ) are commercially available. With respect to breeding, the porcine gestation period (114 d) is relatively short (goat/sheep/cow = 150/152/283 d). In regards to pharmacokinetics, the pig has the most similarity to humans among studied mammals with respect to the cytochrome P450 enzymes. 111,112 Similar pharmacokinetic behavior between humans and pigs has been reported for a number of compounds, 113 and pig is recognized as a model for enabling determinations of in vivo kinetics and drug metabolism in general. 113,114 Investigators have transformed porcine mammary epithelial cells in vitro with SV40 large T antigen insertion 115 or BRCA1 knockdown, 116 with evidence of tumorigenicity in immunodeficient mice. 115 In addition, a BRCA1 haploinsufficient Yucatan minipig was generated with somatic cell nuclear transfer, 117 but postnatal survival of cloned piglets was ≤18 days for reasons that were unclear (BRCA1+/-mice are phenotypically normal 118 ). As a proof of principle, our group has transformed porcine mammary epithelial cells with KRAS G12D and p53 R167H , and produced tumors in xenografted nude mice. Our follow-up work in this area will focus on using porcine mammary epithelial cells transformed with PIK3CA mutants with BRCA1 and TP53 disruption 119-121 to be utilized in an orthotopic porcine model of TNBC. To be clear, however, no porcine model of BC currently exists. The KRAS/p53 Oncopig 101 is not a BC model, but rather a "generic" porcine tumor model, potentially allowing transformation of all cell types. Of note, KRAS mutation is relatively uncommon (1-2%) in BC. [119][120][121] Given the recent progress in porcine modeling of pancreatic cancer by multiple groups, 101,104,122-128 it is conceivable that the best candidate for a large animal BC model will be the pig.

Disadvantages of Using Large Animal Cancer Models

In order to illustrate the potential disadvantages of using a large animal model of breast cancer, a comparison of porcine with murine models is described below. This does reveal a number of disadvantages with the porcine models, including:

1. Costs. Transgenic pigs are more expensive. Transgenic mice for BC research generally are 200-300 USD per subject (Jackson Laboratory), while transgenic pigs (e.g., the Oncopig) can be in the range of 1-2K USD. Per diem cost for pigs are generally ~10x the murine per diem at most institutions. Drug costs are higher in pigs because they require 100-1,000x the amount of drug used by mice. Labor costs are higher with pigs because of physical handling required each time a procedure is done.

2.

Husbandry. More than 100-fold mice than pigs can be housed in the same space, which can limit experimental planning at most institutions. A Sinclair mini-pig can still reach up to 50 kg at 1.5 year, so the research facility has to accommodate animals of this size. The murine gestation period (20 d) is <20% of porcine gestation period, so crossbreeding is much quicker with mice. A relatively simple maneuver of placing a 20 g mouse under general anesthesia becomes more complicated when dealing with a 30-50 kg pig.

Tools & Reagents.

The availability of antibodies, reagents, and other species-specific research tools is much greater in mice compared to pigs, though the availability for pigs has improved in the past decade.

4. Subject Age. In general, investigators only have access to young (<1 year) pigs; however, modeling epithelial tumors with young pigs may not be optimal.

5. Research Community. The number of investigators who utilize swine in cancer research is still relatively small, so other investigators may be reluctant to consider porcine experimentation.

6. Social Issues. Public reaction to swine use in research could be more negative compared to the reaction against rodent use. This possibility may demand more investigator effort devoted to public education.

Conclusion And Future Directions

The current landscape of animal modeling for breast cancer is dominated by murine models, which have developed into powerful and multi-faceted tools for the BC researcher. It would be difficult to improve on the utility that murine BC models have provided. However, there remain certain areas of research, such as device development and drug testing, which could benefit from the availability of a large animal model of BC. These BC models are still in their infancy, essentially at the point murine models were in the 1980's. While large animal BC models likely will never be able to match the proven utility and ease-of-use of murine models, the availability of validated large animal BC models could provide additional tools to the BC researcher that would address specific BC questions or BC-relevant technology development, such as those requiring a human sized subject for generation of relevant data.

Targeted drug delivery

Large sink effect Small sink effect actual size

Fig. 1 .

 1 Fig.1. Tumor Sink. In this example, murine tumor:body mass ratio is 400x the porcine ratio, even though murine tumor mass is only ~12% that of the porcine tumor. If a targeted drug preferentially concentrates in the tumor (i.e., tumor sink), then a given drug dose in the mouse may have a relatively high concentration in the tumor with respect to the plasma. The effect of tumor sink on plasma concentration at the same dose in the pig would be negligible because of the large body size. Thus, the same dose in the pig would produce a higher plasma concentration, possibly producing greater systemic toxicity than in the mouse.