Breast Cancer Modeling

Breast cancer is one of the most common cancers in women worldwide. Breast cancer is a heterogeneous cancer, and it has been distinguished into four subtypes: luminal A (ER positive, HER2 negative, low proliferation), luminal B (ER positive, HER2 negative, high proliferation), HER2-positive (HER2-positive, ER and PgR negative), basal-like (HER2, ER, and PgR negative, known as triple-negative breast cancer)^[1][2].

Although the exact pathogenesis is still unknown, it is reported that molecular mutations, epigenetic alterations, hormone exposure and immune microenvironment are related to the progression of breast cancer^[2].

Firstly, in molecular mutations, the most frequently mutated and amplified genes in early breast cancer cells are TP53 (41%), PIK3CA (30%), MYC (20%), PTEN (16%), CCND1 (16%), ERBB2 (13%), FGFR1 (11%) and GATA3 (10%). These genes encode cell-cycle modulators that are inhibited (p53) or activated (Cyclin D1), thereby sustaining proliferation and inhibiting apoptosis. In Luminal A subtype, tumors have a high prevalence of PIK3CA mutations (49%), whereas a high prevalence of TP53 mutations exists in basal-like subtype (84%).

Secondly, in epigenetic alterations, genes can be globally hypomethylated or focally hypomethylated. The former leads to gene activation, oncogenes upregulation and chromosomal instability, and the latter leads to gene repression and gene instability. Other epigenetic mechanisms involve histone tail modifications by DNA methylation. The modifications induce chromatin structure changes which silence gene expression and nucleosomal remodeling.

Thirdly, hormone exposure is also a risk factor that contributes to breast cancer. Specifically, oestrogen can promote carcinogenesis when binding to the ER located in the nucleus. During the menstrual cycles, the imbalance between oestrogen and progesterone enhances cell proliferation and may cause DNA damage, which may lead to mutations in pre-malignant, and then in malignant cells.

Finally, the microenvironment containing several cells and ECM can influence the development and progression of breast cancer. In the early stage of carcinogenesis, the immune microenvironment plays an anti-tumor role via the cytokine derived from activated CD8+ and CD4+ T cells. Once the tumor becomes invasive, the microenvironment including cancer-associated fibroblasts and cytokines promotes tumor, and ‘hacked’ by breast cancer cells.

MCE has not independently verified the accuracy of these methods. They are for reference only.

Animal models of breast cancer manly involve transplantation models (CDX and PDX model), induced models (by chemical, physical and biological method), and engineered models (transgenic and knockout model)^[3].

• 1. Transplantation models^[4]

Transplantation models can be classified into cell-derived xenografts (CDX) and patient-derived xenografts (PDX). CDX and PDX models can be established by subcutaneous or orthotopical injection. Subcutaneous injection model facilitates monitoring tumor growth, orthotopical injection model is suitable for studying tumor metastatic.

In CDX transplantation models, tumor cells are transplanted into nude mice. Luminal B cell line (BT474) and triple negative cancer cell lines (MDA-MB-231, MDA-MB-435) have been used to establish subcutaneous CDX model. In orthotopical CDX models, tumor cells are transplanted to a mammary fat pad of NOD/SCID mice. Several triple negative cancer cell lines (MDA-MB-231, MDA-MB-435, SUM1315) and luminal A cell lines (MCF7 and T47D) have been used to in orthotopical CDX model.

In PDX transplantation models, primary breast cancer tumor tissue are implanted into immune-deficient mice (nude, NOD/SCID, or NSG mice). PDX models are able to mimic the histology, genomic signature and heterogeneity of the patients' tumors, and predict the drug response.

PDX model (subcutaneous transplantation) can refer as below^[5]

1. NOD-SCID or NSG mice are kept under specific pathogen-free (SPF) conditions.

2. Mice are pre-treated with 0.16 µg/mL 17β-Estradiol (HY-B0141) in water at least one week before xenografting. Drinking water containing estradiol should keep fresh (change to fresh one every week).

3. Percutaneous biopsy specimen and post treatment surgical samples are kept on ice and delivered in sterile PBS. The implantation should be done within 1 h.

4. Cut the samples into 4 mm³ fragments, and implant the samples subcutaneously with a 14 gauge trocar.

5. Each patient-derived sample is implanted into 2-5 mice (depending on the quality and quantity of samples received).

6. Record tumor growth daily by measuring the length (L), width (W) and height (H) with a caliper. Tumor volume (TV, mm³) is calculated as TV = π/6×L×W×H.

CDX model (subcutaneous transplantation) can refer as below^[6]

1. Prepare the female athymic nude mice (5-8 weeks old).

2. Culture the human breast cancer cell lines in a humidified 5 % CO₂ incubator at 37 °C.

3. Harvest the cultured cancer cells with cold PBS and suspend the cells into cold Matrigel quickly.

4. Inoculate mice with 100 μL of breast cancer cells. The cell suspension density can refer as 5 × 10⁶ cells for MDA-MB-468 or MDA-MB-453, and 1 × 10⁶ for MDA-MB-231 cells.

5. Record tumor growth three times per week by measuring the length (L) and width (W) with a caliper. Tumor volume (TV, mm³) is calculated as TV = 0.5×L×W².

2. Induced models^[3]

The most common method is administration of DMBA (HY-W011845) or MNU (HY-34758) (chemical method), which generally induces adenomas and type B adenocarcinomas of breast tumors in mice. Besides, radiation (physical method) and lentivirus infection (biological method) are also used. DMBA or MNU is usually administered to mice or SD rats or Fischer 344 rats by intravenous, subcutaneous, or intragastric administration. Otherwise, SD and Lewis rats are most susceptible to radiation. Biological method mainly relies on lentiviruses to overexpress oncogenes or silence tumor suppressor genes in experimental animals.

DMBA induced model can refer as below^[7]

1. Prepare the 55 days old female Charles foster rats weighing (150 ± 10 g).

2. Freshly prepare single dose of 20 mg/mL of DMBA (diluted in olive oil), and is administered by oral gavage.

3. Rats are palpated weekly starting from 4th week after DMBA administration, to check for the tumor appearance.

4. The first tumor appears in the 16th week. Tumor appears in all rats by 18th week after DMBA administration.

3. Engineered models^[3]

Engineered models can be classified into transgenic models and gene knockout model.

Transgenic models usually use promoters to achieve specific expression of oncogenes in breast epithelial cells, which avoids tumor induction in other organs. The most common used promoters include mouse mammary tumor virus long terminal repeat (MMTV-LTR) and whey acidic protein (WAP) promoters. MMTV is an important virus causing mammary tumors in mice. And breast-specific oncogenes include HER2/ERBB2, PyMT, Wnt, Myc, Ras, and PIK3CA. For example, MMTV-PyMT transgenic mice show obvious tumors at 4-8 weeks, and 84%-90% of mice develop lung metastases at 14 weeks. And MMTV-Wnt-1 transgenic mice shows extensive ductal hyperplasia in early stage, about 50% of female transgenic mice develop breast adenocarcinoma by the age of six months.

Apart from transgenic models, tumor suppressor (such as p53, BRCA1/2, and pTEN) knockout can also be used to establish breast cancer model. Breast tissue-specific gene knockout can be achieved by using the Cre/loxP recombinase system and MMTV-Cre or WAP-Cre tool mice. For example, p53 is critical in controlling genomic integrity and homeostasis. Breast-specific p53 knockout mice show spontaneous tumors after 10 weeks and rapid tumor development after 15-20 weeks.

Advantages:

Transplantation model: Short cycles, low costs, small variations, high tumor formation rates.

Induced model: High tumor incidence rates, short latencies.

Engineered model: Clear target, intact animal's immune function, similar genetic alterations with patients.

Disadvantages:

Transplantation model: Expensive humanized mouse models, time consuming, require multidisciplinary expertise.

Induced model: Low efficiencies, different incidence times, different pathological characteristics.

Engineered model: Different tumor histology from patients, expensive and time consuming, gene editing occurs in almost all mammary ductal epithelial cells.

The number of tumors, latency, and histological type in animals may be affected by age, reproductive history, and the host's endocrine environment when exposed to carcinogens^[3].