Breast cancer in vitro
2004 – 2007 Dr Hadwen Trust Research Fellowship
Prof JL Jones and Dr D Holliday, Queen Mary’s School of Medicine and Dentistry, London University
Development of three-dimensional multicellular models of normal and pre-invasive models of breast cancer to replace animal models
Dr Deborah Holliday was supported by a postdoctoral Dr Hadwen Trust research fellowship for the duration of this project. The work was supervised by Prof Louise Jones, who is Professor of Breast Pathology at the Institute of Cancer, Queen Mary, London.
Ductal carcinoma in situ (DCIS) is a growing health problem and now accounts for up to 40% of breast cancers. However, knowledge of DCIS remains patchy, and there are no targeted therapies as yet [1]. Greater understanding of the mechanisms involved in the progression of in situ to invasive disease is needed to help more accurately predict prognosis and identify new therapeutic targets.
Much research into DCIS and progression of breast cancer has involved animal studies: usually nude mice implanted with human tumour fragments or genetically modified mice, although species differences remain a concern [2].
This Dr Hadwen Trust-funded project developed human in vitro models of DCIS which can be used to investigate factors involved in the progression of DCIS to invasive disease, and provide a realistic alternative to animal models for testing novel therapeutic agents.
Dr Holliday developed and optimised methods for isolating and maintaining primary human breast cells using animal-free reagents. She used these methods to isolate and maintain three different cell types from donated normal and cancerous breast tissue – myoepithelial cells, luminal cells (which become malignant in breast cancer) and fibroblast cells.
Co-cultures of these different cell populations were used to create three-dimensional in vitro models of normal breast and DCIS. Cultured together in gels, myoepithelial cells home around the luminal cells, establish polarity, and form structures that resemble in vivo breast tissue glands. The model system has been validated in terms of receptor and marker profiles, and in terms of responses to anti-oestrogens and growth factor inhibitors.
Normal myoepithelial cells exert a growth suppressor effect on tumour cells, an effect that is lacking in DCIS-associated myoepithelial cells. Myoepithelial cells appear to play a role in disease progression, so a novel and significant step has the isolation and short-term culture of DCIS-associated myoepithelial cells. A number of consistent differences between normal and tumour-derived myoepithelial cells have been identified, including altered integrin receptor expression and changes in extracellular matrix protein production.
Because of the difficulty of relying on primary DCIS myoepithelial cells, our researchers created an immortalised normal myoepithelial cell line manipulated to exhibit a DCIS-associated phenotype. This cell line provides an invaluable tool for modelling DCIS and dissecting out the progression of DCIS.
Interestingly, the addition of tumour-derived fibroblast cells to the in vitro model markedly disrupts the formation of glandular-like structures, with myoepithelial cells failing to home in on luminal cells. Normal fibroblasts do not have this effect, and work is continuing on uncovering the mechanisms involved in this effect [3].
Currently, the in vitro model is being further enhanced by the addition of inflammatory and endothelial cells model that will broaden their relevance and therapeutic importance.
This project has successfully created novel in vitro models of DCIS that are already shedding new light on the factors involved in the progression of DCIS, and may provide novel targets for breast cancer therapy, without using animals.
Summary
- Ductal carcinoma in situ (DCIS) now accounts for up to 40% of breast cancers, yet our understanding remains patchy.
- In vitro models for understanding the mechanisms involved in the progression of in situ to invasive disease could help identify new therapeutic targets and replace animals.
- Methods for isolating and maintaining primary breast cells have been developed and optimised.
- DCIS-associated myoepithelial cells have been isolated and maintained in short-term culture for the first time.
- Several novel differences between normal and tumour-derived myoepithelial cells have been identified.
- A manipulated and immortalised myoepithelial cell line has been generated and will be an invaluable tool for the study of DCIS progression.
- Three-dimensional models of normal breast and DCIS have been generated, characterised and validated.
- A role for tumour-associated fibroblasts in disrupting normal epithelial interactions has been demonstrated.
- Human in vitro models of normal breast and pre-invasive DCIS have been generated that can be used to investigate microenvironmental influences on breast cancer progression, and provide a viable alternative to replace animal studies.
References
1. Jones JL (2006). Overdiagnosis and overtreatment of breast cancer; progression of ductal carcinoma in situ; the pathological perspective. Breast Cancer Res 8:204.
2. Utama FE, LeBaron MJ, Neilson LM et al (2006). Human prolactin receptors are insensitive to mouse prolactin; implications for xenotransplant modeling of human breast cancer in mice. J Endocrinl 188:589-601.
3. Holliday DL, Hughes S, Shaw JA et al (2007). Intrinsic genetic characteristics determine tumour-modifying capacity of fibroblasts; matrix metalloproteinase-3 5A/5A genotype enhances breast cancer cell invasion. Breast Cancer Res 9:R67.