We have accumulated a panel of more than 100 colorectal carcinoma derived cell lines, representing more than 80 different independent tumours. This panel is an invaluable resource for analysing the genetic and epigenetic changes in colorectal cancer that define its somatic evolution, and the functional consequences of these changes. Tumours, for example, which are replication error deficient (RER) essentially all have mutations in TGFßIIR, disrupting the key TGFß signalling pathway, while most others have SMAD4 mutations or other SMAD4 related changes involved in this same pathway, or LOH around that region of chromosome 18. We have also obtained extensive results on the epigenetic control of the homeobox gene CDX1 in colorectal cancers and the importance of this for columnar epithelial cell differentiation. Another interest is in the use of the cell line panel to investigate the relationships between drug responses and the genetic, epigenetic and mRNA expression profiles of the cell lines, and so the corresponding tumours.
We are also developing the use of a monoclonal antibody against a distinctive determinant on CEA for eventual use in immunotherapy against colorectal cancer.In vitro studies have shown that the antibody mediates cellular based killing of tumour cells. Similar investigations are under way with monoclonal antibodies to growth factor receptors.
Our major current research interest is in the identification and functional analysis of cancer stem cells(CSCs) in the lines. Most CSC assays have so far depended on the use of a variety of different cell surface markers, including CD133, CD44 and CD24, to enrich for CSCs from primary tumor tissue using fluorescence-activated cell sorting (FACS) technology, and then testing propagation in immunodeficient mice. Such assays are cumbersome and expensive, and do not readily enable either the characterisation of the CSCs or their functional study. A reliable in vitro assay for using CSCs derived from cell lines would have many advantages, for example, enabling relatively high throughput testing of drugs and antibody effects, as well as gene knock down and re-expression studies on specific CSC functions, rather than on all the cells in a cancer. An in vitro assay would also enable the unequivocal identification of the specific genes that control the stem cell state, which has so far proved relatively elusive.
We have devised a novel assay to enrich for cancer stem cells based on a two-pronged approach, namely colony morphology and cancer stem cell surface markers CD44 and CD24 (Yeung et al. PNAS 2010). We have demonstrated unequivocally that cancer stem cells can be isolated from colorectal cancer cell lines. These cancer stem cells are able to self-renew, differentiate into all the lineages found within the large colon, and initiate tumours in immunodeficient NOD/SCID mice. We have also demonstrated that colorectal cancer cell lines have contrasting abilities to differentiate in 3-D growth, which correlates inversely with the proportion of CSCs found within them.Using a combination of the 3-D differentiation assay and our microarray expression, we have been able to identify new markers that enrich for CSCs. We are also generating stably transfected cell lines with plasmids expressing the promoters of stem cell and differentiation markers, which will allow separation of stem-like and differentiated cells based on GFP/RFP fluorescence.
We are interested in the effects of hypoxia on maintaining the stem-like phenotype of CSCs and have found that hypoxia enriches for CSCs, increases their clonogenicity, and prevents their differentiation when grown in Matrigel. Hypoxia upregulates genes that are associated with the stem-like phenotype, including Notch and BMI1, and suppresses the differentiation markers CDX-1 and mucin production. Our results show that Hif1a plays an important role in mediating the stem-like phenotype conferred by hypoxia.
Another aspect of our work is to investigate in vitro models for studying the interaction between CSCs and myofibroblasts, which are considered to be essential for maintaining the normal colorectal stem cell niche. We are now, in addition , working on applying the results from the cell lines to fresh human tumour tissue
Our in vitro assays with enriched cancer stem cells from the cell lines, should enable relatively large scale drug screening to identify drugs that are more specific for CSCs.
We are using an automated fluorescence microscope for the detection of rare circulating cancer cells in the blood. Initial studies have shown that we can detect less than one epithelial cell per million white cells in the blood. Our hope is that, with suitable markers, we will be able to ascertain to what extent such circulating cells are cancer stem cells and so refine the use of this technology for application to the clinical assessment of tumours, their prognosis and treatment.