Cancer and wound healing - in-vitro insights

New non-animal research methods are generating some excitement among scientists working in medical research and testing. In particular, advances in cell models of human health problems offer fresh insights and therapeutic opportunities.

Three-dimensional in vitro glandular structures comprising luminal and myoepithelial cells from breast tissue.

The Dr Hadwen Trust was established in 1970 to develop and promote the use of valid techniques to replace animal use in medical research and testing1. Most researchers, institutions, funders and regulatory authorities today accept the need to replace animal procedures, and this approach is supported by legislation and related policies around the world2. For the Dr Hadwen Trust and many other players, the goal is to achieve excellence in medical research and testing without using living animals.

As well as making medical progress with a range of approaches from computer modelling and mass spectrometry to neural imaging, the Dr Hadwen Trust’s researchers have developed techniques focused on the cellular and molecular aspects of several human health problems. For example, researchers have constructed an in vitro model of the adherence of Neisseria meningitidis to human meningeal cells in culture, as an alternative to animal experiments. N. meningitidis is a human pathogen that does not naturally infect animals which makes findings from animal-based studies of limited relevance to the human disease.

Instead, understanding how N. meningitidis interacts with the natural host cells of the human meninges provided clues to new treatments and prevention strategies. The work, headed by Dr Dlawer Ala’Aldeen and Dr Karl Wooldridge at the University of Nottingham, showed that pro-inflammatory and apoptosis-regulating genes in meningeal cells are affected by meningococcal challenge. This provided the first evidence that the meninges actively resist the effects of pathogenic bacteria3. An improved in vitro model has subsequently been created, comprising a bilayer of human brain microvascular endothelial cells and meningeal cells, infected with meningococci. This more complex and realistic model of the blood-brain barrier is now being used to study meningococcal pathogenesis.

The following two of the Dr Hadwen Trust’s newly completed research projects have advanced cell culture technologies in areas of medicine that have a real impact on human morbidity and mortality - breast cancer and wound healing.

Professor Louise Jones and Dr Deborah Holliday at the Institute of Cancer, University of London, have developed physiologically relevant, three-dimensional, multicellular in vitro models of normal breast tissue and pre-invasive breast cancer, to replace animal models in this area of cancer research.

Pre-invasive ductal carcinoma in-situ (DCIS) is a growing health problem and now accounts for up to 40% of clinical cases. Yet knowledge of DCIS remains patchy, and there are no targeted therapies as yet4. Most research into DCIS has used animal systems – either xenograft models, where tumour fragments are implanted into nude mice5, or tumour models, involving the genetic modification of animals. Animal models provide the ability to analyse disease progression in a complex environment, but species differences are a concern6. A major strength of this non-animal approach is its direct relevance to human disease.

Professor Jones prefers to use human-based systems in her research. She says: “In my field, the widely held perception that animal studies represent the ‘gold standard’ may have contributed to a delay in developing robust in vitro methods to replace animal studies. There is growing interest in complex in vitro models of disease, particularly in the clinical setting. However, it’s important that the value of such models is emphasised in the academic community. It’s only by demonstrating the power of alternative models that the scientific world will be persuaded that there are viable replacements.”

The Dr Hadwen Trust grant allowed Louise Jones and her colleagues to develop cell-based alternatives to animal research, recapitulating the complex tumour micro-environment and allowing direct manipulation of the cellular components. This is the first time a complex, multi-cell in vitro model of DCIS has been developed and maintained in culture to study breast cancer progression.

To develop the model, the research team isolated, purified and co-cultured three different cell types from donated normal and cancerous breast tissue – myoepithelial cells, luminal cells (which become malignant in breast cancer) and fibroblast cells. Interactions between cells are important in tumour cell invasion. Normal myoepithelial cells play a central modulatory role in the breast, having a broad tumour-suppressor activity, while fibroblasts frequently promote tumour growth and invasion. Meaningful models must include these interacting cell populations, and the relative influence of each cell type may vary at different stages of breast cancer progression. The successful isolation and culture of DCIS-associated myoepithelial cells is a novel and significant step forward.

Cultured together in gels, the cells form three-dimensional structures resembling in vivo breast tissue glands. The myoepithelial cells gather round the luminal cells and establish polarity, as demonstrated with immunofluorescent staining of basal beta-4 integrin and basement membrane formation (Figure 1). 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.

Inclusion of tumour-derived fibroblasts into these models leads to marked disruption of the glandular structure, with myoepithelial cells failing to “home” to the luminal cells. Normal fibroblasts do not have this effect. The team is now focusing on the mechanisms involved in this, which include enhanced release of matrix metalloproteinases by fibroblasts7. Such molecules, mediating the disruption of epithelial interactions, may provide novel targets for breast cancer therapy.

Currently, inflammatory cells and endothelial cells are being added to the model to enhance the complexity of the micro-environment. Systems to quantify tumour cell invasion in vitro are also being developed. Without using animals, Louise Jones and her team are forging ahead with exciting discoveries about DCIS and how it progresses to breast cancer, and other researchers and funders are showing a keen interest in their results.

Impaired wound healing affects 3% of the population over the age of 60, costing the NHS over £1 billion annually and rising. Pressure sores, venous ulcers and diabetic ulcers are among the most common chronic, non-healing wounds and cause significant distress and disability. Considerable efforts have been made to generate a reliable and robust animal model, without real success. None of the animal models reproduce the dysfunctional wound healing responses that occur in older people. They are dissimilar from the human situation, being based on crushing or chemical injuries or full-thickness burns, or being dependent on inducing ischaemia in the tissue.

Moreover, in several animal models, such as the diabetic mouse with inflicted back wounds, a subcutaneous muscle contributes to the healing process; but this muscle is absent in humans. Many of the animal experiments, including the chemical induction of ulcers in rats and guinea pigs, cause considerable pain and distress.

Despite species- and disease-related differences between animals and humans8, animal models are still used widely especially for developing new therapies – an area of animal use expected to grow with the demands of an ageing human population. But at the Cardiff Institute of Tissue Engineering and Repair, Dr Phil Stephens and PhD student Matthew Caley have a different take on the topic.

With a grant from the Dr Hadwen Trust, Phil Stephens is developing an in vitro model of human wound healing. Although the whole process of wound healing in vivo is complex, the concept is to create a simple, reproducible reporter-cell assay to permit rapid, low-cost testing of drugs and materials, and thereby to replace some of the animal efficacy tests currently conducted9.

The starting point is primary fibroblasts, which play an important role in the closure of skin wounds, replacing and remodelling the lost tissue and influencing re-epithelialisation and angiogenesis. Building on the group’s earlier work studying fibroblasts from human in vivo chronic wounds in culture10, they isolated fibroblasts from venous leg ulcers and from patient-matched healthy tissues. To overcome the limited lifespan of primary cells in culture, the cells were immortalised through forced expression of the human telomerase reverse transcriptase protein (hTERT). This technology has the advantage of producing immortal cells with a stable genotype and a phenotype indistinguishable from that of normal primary cells.

The hTERT-immortalised fibroblasts continue to proliferate in culture after 400 days, a much longer lifespan than comparable unmodified fibroblasts, and they were shown to contain active telomerase. When monolayer cultures of these wound-derived and healthy fibroblast lines are subjected to a standard scratch wound, the wound-derived cells repopulate the damaged area much more slowly than the healthy fibroblasts, indicating that the cells retain their distinct phenotypes after immortalisation.

A key aim of the research is to identify genes that are differentially expressed in wound-derived and healthy fibroblasts, and then to use fluorescent proteins to generate reporter-cell assays. Gene expression profiles of quiescent and stimulated immortalised cells reveal four groups of genes whose activity is modified in wound-derived fibroblasts, including genes involved in extracellular matrix, immune system and inflammatory pathways. The microarray data has been confirmed using quantitative real time polymerase chain reaction technology and significant wound-healing marker genes have been selected.

Now, Phil Stephens and his team have conducted the first tests using immortalised cells transfected with a fluorescent protein, linked to a chronic wound-marker gene. They were able to quantify changes in fluorescence, and hence gene expression, over time. Phil is optimistic that the immortalised human cell lines created can form the basis of a reproducible in vitro assay that will replace some animal efficacy tests classically used to study potential wound-healing therapies. He plans to develop and expand the range of assays, to include more of the significant reporter genes identified for delayed wound healing.

The work has been presented in a number of fora so far, including recent national and international conferences, and it’s already attracting industry interest. That’s hardly surprising because, as Phil says, “The advantages of this new model of wound healing are that it will be highly reproducible, efficient, easy to use and inexpensive, with strong potential to replace painful tests on animals”. An attractive proposition indeed, and one which might not have come to fruition without funding from the Dr Hadwen Trust. It can only be good for medicine, people and animals that the initiative to replace animal experiments is gathering pace worldwide.

References

1. Dr Hadwen Trust’s Science Room website for researchers is at www.scienceroom.org 2. For example, European Directive 86/609/EEC; and Policy no. 12 under the US Animal Welfare Act 1966, as amended. 3. Robinson K, Taraktsoglou M, Rowe KSJ, Wooldridge KG and Ala'Aldeen DA (2004). Secreted proteins from Neisseria meningitidis mediate differential human gene expression and immune activation. Cell Microbiol. 6:927-938. 4. Jones JL (2006). Overdiagnosis and overtreatment of breast cancer: progression of ductal carcinoma in situ: the pathological perspective. Breast Cancer Res. 8:204. 5. Wilson CL, Sims AH, Howell A, Miller CJ and Clarke RB (2006). Effects of oestrogen on gene expression in epithelium and stroma of normal human breast tissue. Endocr. Relat. Cancer 13:617-628. 6. Utama FE, LeBaron MJ, Neilson LM, Sultan AS, Parlow AF, Wagner KU and Rui H (2006). Human prolactin receptors are insensitive to mouse prolactin: implications for xenotransplant modeling of human breast cancer in mice. J. Endocrinol. 188:589-601. 7. Holliday DL, Hughes S, Shaw JA, Walker RA and Jones JL (2007). Intrinsic genetic characteristics determine tumor-modifying capacity of fibroblasts: matrix metalloproteinase-3 5A/5A genotype enhances breast cancer cell invasion. Breast Cancer Res. 9:R67. 8. Madeddu P, Emanueli C, Spillmann F, Meloni M, Bouby N, Richer C, Alhenc-Gelas F, Van Weel V, Eefting D, Quax PH, Hu Y, Xu Q, Hemdahl AL, van Golde J, Huijberts M, de Lussanet Q, Struijker Boudier H, Couffinhal T, Duplaa C, Chimenti S, Staszewsky L, Latini R, Baumans V and Levy BI (2006). Murine models of myocardial and limb ischemia: diagnostic end-points and relevance to clinical problems. Vascul. Pharmacol. 45:281-301. 9. Poonawala T, Levay-Young BK, Hebbel RP and Gupta K (2005). Opioids heal ischemic wounds in the rat. Wound Repair Regen. 13:165-174. 10. Cook H, Davies KJ, Harding KG and Thomas DW (2000). Defective extracellular matrix reorganization by chronic wound fibroblasts is associated with alterations in TIMP-1, TIMP-2, and MMP-2 activity. J. Invest. Dermatol. 115:225-233.

By Dr Gill Langley. Gill is Science Director of the Dr Hadwen Trust for Humane Research, and has led the Trust’s research and scientific outreach programmes since 1981.