Research Interests

Role of Macrophages in Tumour Progression and Anti-Cancer Gene Therapy

We have used our detailed knowledge of macrophage migration and activity in tumours to design novel forms of gene therapy for cancer. These use macrophages to carry hypoxically-activated, therapeutic genes into hypoxic areas of human tumours (which are known to be relatively drug resistant and inaccessible to most conventional therapies), where the gene is then activated to be expressed. Two layers of tumour targeting are provided by this method, the homing of macrophages to hypoxic tumour sites and the expression of the therapeutic gene only in the level of hypoxia found in tumours.

Initially, we have used a replication-deficient adenovirus to transiently transfect fully differentiated macrophages with such hypoxically-regulated therapeutic genes. However, we have now extended these studies to use retroviral gene transfer methods to stably transduce macrophage precursors with these DNA constructs. The transduced stem cells are then simply differentiated into monocytes or macrophages in culture. The latter approach provides macrophages with the potential for sustained, high-level expression of the therapeutic gene within tumours. We are also using macrophages to synthesise therapeutic adenovirus in hypoxic areas of prostate tumours in vitro and in vivo.

Finally, we are an oxygen-responsive bacterial transcription factor, FNR, to regulate the expression of therapeutic genes in a modified, non-pathogenic, but invasive strain of S. typhimurium (which has been shown to replicate preferentially in hypoxic/necrotic sites in murine tumours following systemic injection). So, we are attempting to use this novel bacterial system to target genes specifically to hypoxic tumours areas.

Hypoxia is not only found in solid tumours, but is also a hallmark feature of ischaemic heart disease, central nervous tissue during cerebral malaria, arthritic joints, and retinal complications in diabetic retinopathy. Therefore, it may prove possible to also target therapeutic genes to these tissues using the above delivery vehicles. Thus, these projects could ultimately lead to a range of novel therapeutic options to control various ischaemic conditions.

Development of Anti-Vascular and Angiogenic Agents

Angiogenesis is the development of new blood vessels from an existing vascular network. This is essential for the growth of tumours beyond 2mm in diameter as it ensures the adequate supply of oxygen and nutrients for tumour cell expansion and spread. We are currently developing drugs that block angiogenesis by either a direct effect (by suppressing endothelial cell responses to tumour-derived growth factors) and indirectly (by inhibiting the pro-angiogenic activities of other cell types in tumours).

Recently, we have used various forms of angiogenesis assay to demonstrate the potent anti-angiogenic effects of a 50kDa proteolytic fragment of the central region of human fibrinogen (FgnE). This blocks two steps in the angiogenic pathway, the migration and differentiation of endothelial cells in vitro in response to VEGF, bFGF and EGF. Moreover, it inhibits the effects of VEGF with greater potency than the prominent angiogenesis inhibitor, endostatin. It also markedly inhibits the growth of tumours in both xenograft and syngeneic murine tumour models by selectively disrupting the tumour vasculature (causing the formation of widespread tumour necrosis).

Our most recent studies have identified the bioactive site on this protein and we have synthesised this part of the proteins (a 24 amino acid peptide from the alpha chain of human fibrinogen - we have called `Alphastatin´). This peptide mimics the anti-angiogenic effects of the 50kDa parent molecule in vitro and its potent anti-vascular effects in vivo. We are currently working to identify the receptor(s) on endothelial cells mediating the anti-angiogenic effects of Alphastatin.

Our anti-angiogenic drug discovery work has recently been extended to include the use of the zebrafish model of angiogenesis. This powerful in vivo method involves the use of mutant fli1-EGFP zebrafish embryos in which the development of the vasculature is readily quantified using a fluorescent microscope coupled to a computer for image analysis. As such, it can be used as a rapid, inexpensive high-throughput screen for new drug candidates.

In a second line of studies in this area, we have focussed on the role of stromal cells in the regulation of angiogenesis. For many years it was believed that tumour cells produce most of the cytokines and enzymes needed to stimulate angiogenesis in tumours. However, we and others have now provided unequivocal evidence for the contribution tumour-associated macrophages (TAMs) and neutrophils (TANs), in the stimulation of this process in human and murine tumours. Indeed, high numbers of these cells in tumours have been shown to correlate with increased levels of tumour angiogenesis and reduced patient survival in such diseases as breast cancer.

The pro-angiogenic activities of these TAMs are activated by the low levels of oxygen (hypoxia) present in many areas of human tumours. Moreover, our studies indicate that TAMs lend themselves to this influence by accumulating specifically in poorly perfused areas of tumours. Here, the hypoxia present activates their expression of genes for such pro-angiogenic cytokines as vascular endothelial growth factor. In this way, TAMs appear to co-operate with tumour cells to ensure the ongoing neovascularisation of tumours.

We have used flow cytometry and cDNA microarray technology to identify various pro-angiogenic molecules activated by hypoxia in/on TAMs. Our data indicate that hypoxia upregulates the expression of uPAR, GLUT-1 and the receptor for Neuromedin B on human macrophages. Such findings will be used to target inhibitory/ cytotoxic proteins or genes specifically to hypoxic TAMs in an attempt to ablate their pro-angiogenic activities in tumours. We have also shown recently that TAM upregulate the transcription factors, HIF-1, HIF-2, Ets-1, ATF-4 and ID-2 in response to hypoxia. So, we are currently using siRNA technology to identify the array of downstream genes regulated by these factors in hypoxic macrophages.

Finally, we are investigating the way that TAMs and TANs are attracted into tumours. We have extended this work to include the use of chemokine protein arrays, human tumour arrays, and human tumour spheroids to identify the chemokines (and other factors) regulating accumulation of these cells in hypoxic tumour sites. We plan to then design agents to specifically block these mechanisms, and thus reduce the number of these pro-angiogenic cells in hypoxic areas of tumours.

Mathematical Modelling of Tumour Inflammation and Angiogenesis

We have established an EPSRC-funded tumour mathematical modelling group in collaboration with Dr Helen Byrne in Nottingham.

These projects are simulating the complex 3-D cellular interactions that regulate tumour angiogenesis in vivo, and provide important insights into the spatio-temporal patterning of hypoxia in tumours. We are using the predictive power of these models to maximise the efficacy of our gene therapy protocols.