Engineering an immune response in melanoma by targeting cytokine signalling

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BackgroundTargeting immunosuppression in the tumour microenvironment allows a latent immune reaction to be unleashed resulting in tumour regression. GM-CSF stimulates the activity of antigen presenting cells and IL12 stimulates cell mediated immunity. Recently, we have observed infiltration of melanoma nodules in zebrafish by macrophages and CD4+ T-cells. Further, we have constructed a vector for inducible gene expression in melanoma nodules and found that induction of IFNy results in the outgrowth of unpigmented tumour cells. As viral vectors (e.g. T-VEC) have now been developed for directing cytokine expression in melanoma tumour nodules that show clinical activity, it would be desirable to have an easily engineered model system to evaluate cytokine efficacy and synergistic action.HypothesisThe combinatorial action of cytokines can profoundly modulate the immune response to a melanoma tumour nodule resulting in its rejectionObjectivesTo engineer constructs for expression of IL12 and GM-CSF, as well as a combination of these two factors or a combination of IL12 with IFNy or GM-CSF with IFNy and to evaluate the effect of cytokine production on tumour maintenance.To contrast the expression profiles of mononuclear phagocytes and T cells isolated from control tumours and tumours expressing cytokines and hopefully undergoing regressionTo corroborate cytokine cooperation in a mouse transplantation model of melanomaMethodsUsing a tamoxifen-responsive Cre construct, we have been able to induce gene expression in established melanoma tumour nodules in zebrafish. We have also created or acquired fluorescent reporter constructs that allow flow isolation of mononuclear phagocytes and T cells. Combining these reagents we shall induce expression of IL12 or GM-CSF alone, together, or combined with IFNy and evaluate changes in tumour volume. We shall isolate leukocytes from tumour nodules and analyse gene expression by RNA seq. Cytokine combinations will also be evaluated in a mouse melanoma transplantation model by injecting tumour nodule with recombinant protein.Potential outcome/impactThis study will establish zebrafish as a simple preclinical model for evaluating the efficacy of cytokines and cytokine combinations to induce an immune response to melanoma