Cell Culture Methods and Xenografts for Modeling Breast Cancer Metastasis in Early Drug Discovery
Approximately 75% of breast cancers are diagnosed as hormone positive and exhibit acceptable responses to endocrine-targeted therapies, whereas 7-15% are ER-/PR- Her2+ or ER-/PR-/HER2- – which are somewhat refractory to conventional endocrine treatment.
Gene variations, different cancer subtypes, and diverse histopathologies and clinical outcomes among patients all present a challenge to currently available breast cancer treatment options. Due to the heterogeneity of this disease, finding optimal experimental models remains a major hurdle during drug development.
Breast cancer metastasis is the main cause of cancer death. Currently available experimental models for breast cancer metastasis research are breast cancer cell lines, tumor transplantation, and genetically modified mouse models.
Although genetically engineered mice that target oncogenes (e.g., Erb2, and Wnt-1), tumor suppressor genes (e.g., P53), or mutations of tumor susceptibility genes are especially valuable for elucidating the tumorigenesis in a relevant physiological environment, cell lines and xenografts can be employed in a more timely and cost-effective manner and are suitable for investigating potential therapeutics in the pre-clinical drug discovery stage.
Breast cancer cell lines
Being easily propagated and relatively tractable to genetic manipulation, breast cancer cell line models have been widely used to investigate the impact of the candidate chemical on cell proliferation, apoptosis, and cell migration with quantifiable results during cancer progression. A number of cell line panels – possessing many of the genetic and genomic alterations found in primary breast cancers and representing diverse breast cancer subtypes – have been developed and used as pre-clinical models.
A set of functional tools can then be applied to the selected cell lines. For example, the cell shape can be indicative of a metastatic pattern. In particular, the epithelial-mesenchymal transition (EMT) is a critical mechanism for acquiring migratory and invasive properties during metastasis. Researchers can determine the efficacy of the target therapeutic in breast cancer metastasis by evaluating expression levels of EMT markers, such as E-cadherine or the mesenchymal marker (e.g., vimentin).
Three-dimensional multicellular spheroids
Tumorigenesis is constantly modulated by interactions between tumor cells and the surrounding stroma, the extracellular matrix (ECM), and infiltrating inflammatory cells. Limitations of the monolayer cell culture are that it lacks the structural architecture of and differs markedly from the breast microenvironment. This drawback is overcome by the development of physiologically relevant three-dimensional (3D) culture systems in which cells grow in a reconstituted basement membrane containing extracellular matrix proteins.
Due to enhanced interactions between cells and the surrounding ECM, cancer cells cultured in a 3D environment generally recapitulate the in vivo phenotype and provide more comprehensive and biologically relevant information. In this culture system, invasion assays with the extracellular matrix resembling stroma cells can be employed, and metastasis can be examined by evaluating parameters including migratory capability, cell migratory patterns, and matrix degradation.
To investigate the growth and metastasis of human breast cancer cell lines in vivo, these cells can be injected subcutaneously, intravenously, or orthotopically into the fat pad of immunocompromised mice. The site of injection together with the chosen cancer cell line determines primary and secondary metastatic growth. For example, MDA-MB-231 cells – an estrogen-independent breast cancer cell line – have been shown to colonize the bone, liver, lung, adrenal glands, ovary, and brain after intravenous injection.
In contrast to xenograft models, transplantation of cancer cells from one mouse to another mouse with identical genetic backgrounds (syngeneic transplantation) allows for investigation of malignant tumor progression in an intact immune system. For instance, sublines of 4T1 cells – which exhibit various degrees of metastatic dissemination – have been employed to generate distinct gene expression patterns for each stage of tumor progression for the testing of new drugs designed to interfere with tumor malignancy.
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