Mouse models of breast cancer metastasis

Breast cancer metastatic mouse models are experimental approaches in which mice are

Breast cancer metastasis

• In 1909,
  Clarence C. Little developed the first inbred strain, the DBA
  (Dilute, brown non-Agouti) mouse.
• In 1915, N.M Haldane identified first linkage in mouse between Albino mice and pink eye dilution on chromosome seven.
• In 1921, C57BL became one of the most widely used mice in genetics and was the first strain to have its genome sequenced.
• In 1982,
  transgenic mice genetically engineered to express dominant oncogenes.^[20]
• In 1982, the stimulation of expression from the
  proto-oncogene, c-myc.^[21]

Genetic studies of common diseases in humans suffer significant limitations for practical and ethical reasons.^[22] Human cell lines can be used to model disease but it is difficult to study processes at the tissue level, within an organ or across the entire body. Mice can be a good representation of diseases in humans because:.^[23]

• There are close similarities of physiology, development and cell biology between mice and humans.
• Humans and mice both have around 30,000 protein-coding genes. The number of mouse genes without a corresponding human homologue is less than 1%.
• 90% of the human and mouse genomes are syntenic.
• 40% of both human and mouse genomes can be aligned at the nucleotide level.
• Mice have relatively short gestation periods.
• Mice take a brief time to reach sexual maturity.
• Mice have large litter sizes.
• The availability of hundreds of mutations affecting almost every tissue and aspect of development.

Mice may not be an ideal model for breast cancer. This is mainly due to the lack of precision in many of the models. When looking at metastasis, it is difficult to determine the precise location as well as its frequency. Another issue revolves around the epithelial sub types and the inability to specifically target them when targeting a mutation. An example of this would be determining the development of tumors in K14-Cre BRCA2 mice. In a standard case, the excision of BRCA2 resulted in no tumorgenesis, but if p53 was mutated and inactivated, tumorgenesis would occur. Therefore, there is not a definitive answer in terms of the origin of the tumor, due to the extra mutation in p53.^[24]

Metastatic mouse mammary carcinoma cell lines

Various mouse mammary carcinoma cell lines, like 4T1^[25] and TS/A, are metastatic in syngeneic immunocompetent mice and can be used to identify genes and pathways involved in the metastatic process.^[26]

Simple tumor transplantation models

Transplantation of tumor cells into

The specific immunodeficient mice that were used were the NOD/SCID mouse (non-obese diabetic/severe conditional immunodeficient). These mutations allow for the integration of new xenograft tissue. The mouse must first have their mammary fat pads humanized by injecting human telemorase-immortalized human mammary stromal fibroblasts(RMF/EG fibroblasts) into the mammary fat pads. Without this injection, the human mammary epithelial cells en-grafted onto the pad are unable to colonize and grow. The RMF/EG fibroblast must then be irradiated to allow the expression of key proteins and growth factors. After 4 weeks of development, the newly en-grafted human mammary epithelial cells expanded within the fat pad.[35]

Genetically engineered mice to study metastasis

Genetically engineered mice are constructed to model human

• The use of bacteria-derived tetracycline-inducible system permitting the switching on or off (Tet-On/Tet-Off system)^[36]
• Targeted mutations by
  knock in gene and knock out sequence by using Cre-Lox recombination system^[37]
• Introduction of retro viral mutations^[38]
• Introduction of chemically induced mutations

Transgenic mouse models of breast cancer

The mice undergoing the process of transgenesis are known as transgenic mice. A basic transgene has a promoter region, Protein coding sequence, Intron and a stop codon. Mouse mammary tumor virus (MMTV), is a retro virus that has been a known promoter to cause breast tumors once activated.^[39] MMTV is a heritable somatic mutagen whose target range is limited. It harbors a regulatory DNA sequence called the long terminal repeat (LTR), which promotes steroid-hormone-inducible transcription.^[40][41] Tumorgenesis that was induced by the mouse mammary tumor virus can also be done by integration of the viral genome. The sites of integration have been known to be critical genes for cellular regulation.^[42] Whey acidic protein (WAP),^[43] is another common promoter used to generate mouse mammary cancer models. For a list of other mammary gland specific promoters and mouse models see.^[44]

MMTV-PyMT

MMTV-PyMT is the model of breast cancer metastasis, in which MMTV-LTR is used to drive the expression of mammary gland specific

• PI3K/Akt signalling in metastasis can be demonstrated in MMTV-PyMT; Akt1−/− mice.^[46]
• Chemoattractive
  paracrine loop of colony-stimulating factor-1 (CSF-1) and EGF ligands between tumor-associated macrophages (TAMs) and tumor cells, and the lung metastasis can be studied by crossing MMTV-PyMT mice with Csf-1−/− mice.^[47]
• The role of an innate and adaptive immune response to assist metastasis can be studied in MMTV-PyMT; Rag1−/− mice in which CD4+ T cells are selectively lost. Interleukin-4 (IL4) lacking model of MMTV-PyMT; IL4−/− mice.^[48]
• Role of the adhesion molecule CD44 in lung metastasis.^[49]
• Conditional ablation in MMTV-PyMT breast cancer cells has been done to reveal pro-metastatic actions of the angiogenic factors, Vascular endothelial growth factor A (VEGF-A).^[50]
• The role of
  autocrine transforming growth factor beta 1(TGF-β1) signaling on motility and survival in PymT cells derived from an MMTV-PymT mouse mammary cancer.^[51]
• Others are MMTV-PyMT; uPA-/-[52] and MMTV-PyMT; MEKK1-/-.^[53]

MMTV-

HER2/neu

The MMTV-LTR can also be used to promote receptor tyrosine-protein kinase ErbB2 to transform the mouse mammary epithelium. ErbB2 is an oncogene amplified and overexpressed in around 20% of human breast cancers. The mice harbouring this oncogene develop multifocal adenocarcinomas with lung metastases at about 15 weeks after pregnancy.^[54][55] To create a more accurate representation of HER2 gene mutations, researchers have fused the mouse gene containing neu and a rat gene containing neu. This addresses the issue in terms of modeling the amplification of HER2 in mice development. In the non-fused mouse, the mammary gland would revert to a near virgin, but with this addition the mammary gland maintained the developed function.^[56]

Bi-transgenic models

Mouse models having two transgenes are called bi transgenic. To check the cooperation of two oncogenes, Tim Stewert and group made the first bi-transgenic mouse models in 1987, MMTV-

Tri-transgenic mouse models constitute of more than two genes. Multiple combinations and genetic modifications are made in such a way that either one or all the genes are put into a continuously expressed status, or in a controlled fashion to activate them at different time points. For example, TOM( TetO-myc); TOR; MTB mice, where both the myc (M) and ras (R) genes are under the control of tetracycline operators. They can also both be activated or deactivated by adding doxycycline. Other combinations in this respect are TOM; Kras; MTB, where myc can be induced and uninduced at various time points while Kras is in continuous expressed state, and myc; TOR; MTB model is vice versa.[60]

Applications of genetically modified mice to study metastasis

Metastatic cascade can be studied by keeping the gene activation under control or by adding a reporter gene e.g. Beta actin GFP (Green fluorescent protein) or RFP (Red fluorescent protein).

Identification of genes that regulate metastasis

By knocking in/knocking out specific genes by homologous recombination, the extent of metastasis can be measured and new target genes identification can be achieved e.g. a gene that consistently regulates metastatic behavior of cancer cells is TGF-β1. Acute ablation of TGF-β signaling in MMTV-PyMT mammary tumor cells leads to a five-fold increase in lung metastasis.^[61] Certain enhancer regions can also be analyzed and can be determined to be a crucial part of cell proliferation e.g. an enhancing region that is associated with a cancer critical gene p53 which was determined via CRISPR-Cas9.^[62]

Lineage tracing in metastasis models

The quantitative lineage-tracing strategies have proven to be successful in resolving cell fates in normal epithelial tissues either using tissue –specific or

Another tool to study breast cancer metastasis is to look for circulating tumor cells in transgenic mice e.g. MMTV-PyMT mice can respond to various therapies in shedding tumor cells in the blood leading to lung metastasis.[65] Not only in blood but cells can be detected in bone marrow e.g. cytokeratin-positive cells in the bone marrow of MMTV-pyMT and MMTV-Neu transgenic mice were identified but not in the wild type controls.^[66]

Limitations

In the absence of specific markers for mammary cells, models with genetic marking of tumor cells gives the best experimental advantage, however the low volume of peripheral blood that can be obtained from live animals limits the application of this technique.

In vivo imaging of metastatic mouse models

Transgenic mouse models can be imaged by various non-invasive techniques.

Bioluminescence imaging

Bioluminescence imaging relies on the detection of light produced by the enzymatic oxidation of an exogenous substrate. The substrate luciferin, is oxidized to oxyluciferin in the presence of luciferase and emits light, which can be detected using an IVIS system such as a Xenogen machine. Dissociated mammary cells from MMTV-PyMT: IRES: Luc; MTB (Internal ribosome entry site: Luciferin) animals (which were not exposed to doxycycline) can be injected into the lateral tail veins of immunodeficient mice on a doxycycline-free diet. No bioluminescence signal will be observed in the lungs of recipient mice until they are given doxycycline food. Bioluminescence can then be detected in the chest within 2 weeks of the start of doxycycline exposure.^[31] Luciferase is injected just before taking the images.

Fluorescent imaging

Intravital microscopy with multi photon excitation is a technique to visualize genetically engineered cells directly in vivo. Multi step metastatic cascades can be visualized by labelling with unique fluorescent colour under fluorescence microscope.^[67][68]

Radioisotopic imaging

Magnetic resonance imaging requires the use of nano-particles(liposomes) and an MRI contrast agent called gadolinium. The particles were then placed in vesicles via a polycarbonate membrane filter. The nano-particles are injected into the metastases evolved mice, and left there for twenty-four hours. These mice are then scanned, and in the imaging software there are accumulations of these particles in certain areas where cells have metastasized.[22]

See also

• Ensembl
  genome database of model organisms
• Fate mapping
• Firefly luciferin
• Gene targeting
• Gene trapping
• Genetic recombination
• History of model organisms
• Homologous recombination
• Recombinase-mediated cassette exchange
• Site-specific recombinase technology

References