Engineering an immune response in melanoma by targeting cytokine signalling

背景在肿瘤微环境中靶向免疫抑制可以释放潜在的免疫反应，导致肿瘤消退。GM-CSF刺激抗原呈递细胞的活性，IL 12刺激细胞介导的免疫。最近，我们观察到巨噬细胞和CD 4 + T细胞浸润斑马鱼黑色素瘤结节。此外，我们还构建了用于黑色素瘤结节中诱导型基因表达的载体，并发现IFNy的诱导会导致无色素肿瘤细胞的生长。病毒载体（例如T-VEC）现已被开发用于指导显示临床活性的黑素瘤肿瘤结节中的细胞因子表达，因此，需要一种易于工程化的模型系统来评估细胞因子的功效和协同作用。假设细胞因子的组合作用可以深刻地调节对黑素瘤肿瘤结节的免疫应答，导致其排斥。和GM-CSF，以及这两种因子的组合或IL 12与IFN γ或GM-1的组合。为了对比从对照肿瘤和表达细胞因子并希望经历消退的肿瘤分离的单核吞噬细胞和T细胞的表达谱，为了证实细胞因子在黑色素瘤小鼠移植模型中的合作，通过构建他莫昔芬响应性Cre构建体，我们已经能够在斑马鱼中建立的黑素瘤肿瘤结节中诱导基因表达。我们还创建或获得了荧光报告构建体，其允许单核吞噬细胞和T细胞的流动分离。将这些试剂组合，我们将诱导单独、一起或与IFN γ组合的IL 12或GM-CSF的表达，并评估肿瘤体积的变化。我们将从肿瘤结节中分离白细胞，并通过RNA测序分析基因表达。细胞因子组合也将在小鼠黑色素瘤移植模型中通过注射肿瘤结节与重组蛋白进行评估。潜在的结果/影响本研究将建立斑马鱼作为一个简单的临床前模型，用于评估细胞因子和细胞因子组合诱导对黑色素瘤的免疫应答的功效。

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