Developing a novel in vivo gene therapy intervention for solid tumours by targeting tumour-associated-macrophage plasticity.

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There is an unmet clinical need for primary liver cancer treatment, which currently relies on resection and transplantation as standards of care. Up to 80% of these tumours are unresectable and approximately 20% of those who undergo transplantation experience tumour recurrence. Combine this with the difficulty of finding donors, transplant rejection and post-transplant complications, it is evident that a non-surgical intervention is needed.Recent advances in chimeric-antigen-receptor (CAR) based immunotherapies have proved efficacious in treating numerous 'liquid' blood cancers. However, the complex nature and density of the tumour microenvironment (TME) in solid tumours produces an immunosuppressive environment that both aids tumour progression and confers protection against therapies. A multiplicity of host immune cells are recruited to the TME and macrophages constitute a large proportion of these in most solid tumours. Macrophages can be subdivided into two major phenotypic groups. The M1 population produces a pro-inflammatory cytokine response and their localisation around a cancer mass has been shown to increase the effectiveness of chemotherapies in vitro. The other major macrophage group, M2, is associated with wound healing and tissue repair by inducing collagen production and building extracellular matrices. Within the TME these M2 cells promote angiogenesis and metastasis and have been shown to increase tumour cell survival against chemotherapies. Macrophages demonstrate high plasticity between phenotypes depending on their environmental stimuli, however they appear to skew towards the M2 phenotype in the TME which contributes to its immunosuppressive characteristic. Therefore, a therapeutic strategy to treat solid tumours is to target this population of tumour-associated macrophages (TAMs) and induce a phenotypic change into the cytotoxic M1 population to promote an anti-tumour environment within the TME. A model for polarising macrophages between phenotypes can be achieved using a two-step process. First, the monocytic cell line THP-1 is treated with Phorbol 12-myristate 13-acetate (PMA) to induce a macrophage M0 state. This is followed by incubation with separate cytokine cocktails for either M1 (IFN-g and LPS) or M2 (IL-4 and IL-13). Markers for each phenotype are assessed by qPCR, flowcytometry or immunostaining. CD68 is a universal marker for macrophages, while IL-6 and CXCL10 or MRC1 and FN1 can further identify M1 and M2 phenotypes respectively. Gene therapy viral vectors will be assessed in this model and further engineered for specificity towards M2 cells. Then, CAR construct libraries will be screened to identify suitable candidates to elicit a shift in phenotype from M2 to M1 cells. Once these two systems are combined, the resulting therapy will be tested in 3D in vitro models called Tumouroids. These models comprise of different spatially segregated compartments to engineer both the tumour and surrounding stroma, however migration and invasion of cells between these compartments is possible. The matrix of these compartments is generated using dense collagen-I matrices, where the stiffness matches that of human liver tissue. This model will be developed to embody a primary liver cancer TME as closely as possible. 3D in vitro models are more relevant than ever following the FDA ruling removing the necessity for new drugs to be tested in animals and as such are a suitable method for testing this therapy in the first instance.