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2004-05 Cancer Research - Improved cell culture methods for anti-cancer drug development

The LDF has been funding Professor Ian Cree of the Translational Oncology Research Centre, Queen Alexandra Hospital in Portsmouth, to develop improved cell culture techniques for testing the efficacy of anti-cancer drugs, using human cancer cell lines cultured in human serum.

Animal researchers grow cell lines in culture then graft these onto animals, usually mice, which are then dosed to determine the efficacy of new anti-cancer drugs. Alternative, and more advanced, approaches use tumour cells derived from patients, or cell lines which mimic the situation in the living body. Although cultured cell lines are relatively inexpensive and give reproducible results rapidly, the relevance of these results to the efficacy of anti-cancer drugs on cancer cells in human patients is not well established. There are differences between cell lines and cells taken directly from the patient. The growth rates of cell lines and tumour-derived cells differ. Cell lines consist of rapidly dividing identical cells, whereas cells taken from tumours are generally a mixed population consisting of cancer cells, the characteristics of which are determined by their environment, and non-cancer cells.

Additionally, cell lines may lose some of the characteristics which they possess in the living body, due to prolonged culture. Because of these differences between cell lines and tumour-derived cells, there are differences in their sensitivity to drugs which kill cancer cells. For example, anti-cancer agents tend to target growing cells, so cell lines are more sensitive than tumour-derived cells.

The aim of this study is to generate defined media (the cell growth medium) and cell lines suitable for widespread use in testing new agents, in place of using animals. Professor Cree and colleagues have demonstrated previously that growth rate and drug sensitivity of the cell lines can be altered by reduced serum concentrations in culture. Cell lines are generally poor representatives of the tumours from which they are derived because they are adapted to cell culture conditions. By altering the environment in which the cells are cultured, it may be possible to make cell lines behave more like tumour-derived cells. The response of the cell lines, cultured in altered conditions, to standard anti-cancer drugs, is being compared to a data set of known effects on tumour-derived cells.

Just over ten years ago, Professor Cree’s team developed an assay called the Adenosine Triphosphate Tumour Chemosensitivity Assay (ATP–TCA), which measures ATP, the most basic fuel of living cells. The amount of ATP present in a cell population reflects the number of viable cells. This technique is being used to assess the toxic effects of the drugs on cultured cells. Professor Cree and colleagues have used ATPTCA to look at both tumour-derived cells and cell lines. Over 9 years all data collected from ATP-TCA experiments, using different drugs tested on different tumour types, has been added to a database. This currently holds data for 1,400 human tumours.

Cell lines are being derived from cells obtained from small pieces of human tumour tissue representing common tumours, such as those in the breast, lung, bowel, stomach, ovaries, prostate and skin. The majority of the cell lines to be tested are normally grown in media containing foetal bovine serum. Human serum at various concentrations is being substituted in this study. Serum-free media are also being tested.

Molecular fingerprinting of cells under culture conditions will be carried out by Affymetrix chip assays to enable comparison with representative tumour-derived cells for each of the tumour types tested. The intention is to set up a panel of cell lines whose sensitivity to anti-cancer drugs is representative of common tumour types. The results will be published and the methods will be free to scientists to use as needed. Pharmaceutical companies are likely to be able to use this data to identify new chemotherapeutic agents more accurately and effectively.

Professor Cree says: “At present cell lines are such a poor model for likely human response that xenografts are used. Xenograft experiments use tumour cells (usually cell lines injected into animals so that tumours grow either within the peritoneum [the internal abdominal membrane or lining] or skin, followed in some cases by spread to other tissues. At any one time there are approximately 1,000 investigational new anti-cancer drugs being tested in human subjects for possible clinical use: the turnover is probably around 500 per annum. Each one of these is probably the lead compound from around 20 drugs tested in xenograft experiments, most of which include around 30 mice. I would therefore estimate the number of mice used in such experiments as at least 300,000 per annum. This figure is likely to be an under-estimate, as such models are used routinely in many cancer laboratories. While the majority of animals used are likely to be mice, there will be some use of rats, guinea pigs and rabbits for these experiments.

“The proposed cell line testing methods could replace much, if not all, of the testing of the efficacy of anti-cancer drugs in animals prior to human studies...”

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