Using oncolytic viral therapy to target the tumour microenvironment in chromosomally unstable cancers

在过去的十年里，免疫疗法的成功改变了癌症治疗的格局。然而，由于肿瘤介导的免疫抑制机制，大多数患者仍然没有从这种方法中受益。其中一种免疫抑制策略涉及癌症相关的成纤维细胞--这些是正常的间质细胞，被癌细胞劫持，在这些成纤维细胞内重新布线路径，以促进肿瘤生长和免疫抑制。此前，Parkes团队已经发现，癌细胞的内在特征，如染色体的不稳定性，直接影响成纤维细胞在肿瘤微环境中的行为。溶瘤人员已经发现并开发了新的溶瘤病毒疗法，不久将进入临床环境。然而，这些治疗剂在富含成纤维细胞的、免疫抑制的肿瘤微环境中的行为目前尚不清楚，也是人们主要感兴趣的，因为这些通常是对其他免疫治疗方法具有耐药性的癌症。因此，在本项目中，利用它们的主要治疗候选细胞，2D和3D共培养模型将被用来表征肿瘤细胞特性、成纤维细胞表型和溶瘤病毒治疗反应之间的关系。目的：(1)研究肿瘤细胞和成纤维细胞在溶瘤病毒治疗反应中的相互作用。为了评估癌细胞和成纤维细胞共培养测量对溶瘤病毒的反应的直接效果，将用来自患者样本的永生化癌症相关成纤维细胞系培养染色体日益不稳定的新型同基因癌细胞系(由Parkes实验室产生)。荧光标记的癌细胞和成纤维细胞将被分解，以检测RNA和趋化因子对病毒的反应。将使用流式细胞术进一步评估成纤维细胞表型，以确定肿瘤相关成纤维细胞标志物的表达。(2)表征基质组织和僵硬在溶瘤病毒治疗反应中的作用。成纤维细胞是肿瘤微环境中基质蛋白的主要生产者，它随后会影响肿瘤的机械性能，包括硬度和细胞在肿瘤中迁移的能力。基质硬度对溶瘤病毒反应的影响将使用增加硬度的人工矩阵进行评估。在这些基质中，类有机物(代表低染色体和高染色体不稳定性)将与成纤维细胞共培养。在与肿瘤微环境相关的僵硬增加的背景下，溶瘤病毒诱导细胞死亡的能力将被测量。溶瘤病毒感染细胞和复制的活性将在基质硬度的背景下进行评估。(3)研究成纤维细胞激活和基质硬度对免疫细胞激活的影响。在癌细胞-成纤维细胞共培养的条件培养液中培养的T细胞将被分析激活、增殖和识别肿瘤抗原的能力。利用AIM(2)中的模型，将使用有机物、成纤维细胞和免疫细胞(来自血液样本)的3D共培养来表征免疫细胞的激活、增殖和细胞毒活性对溶瘤病毒治疗的反应。该项目将描述一种使用新开发的细胞系和成纤维细胞共培养的新型治疗剂，以及肿瘤微环境的全面近患者3D模型。

The cancer treatment landscape has been transformed by the success of immunotherapies over the past decade. However, most patients still receive no benefit from this approach, due to tumour-mediated immunosuppressive mechanisms. One of these immunosuppressive strategies involves cancer-associated fibroblasts - these are otherwise normal stromal cells which have been hijacked by cancer cells rewiring pathways within these fibroblasts to promote tumour growth and immunosuppression. Previously, the Parkes group has identified that intrinsic characteristics of the cancer cells, e.g. chromosomal instability, directly impact on the behaviour of fibroblasts in the tumour microenvironment.Theolytics have identified and developed novel oncolytic viral therapeutics shortly entering the clinical setting. However, the behaviour of these therapeutic agents in a fibroblast-rich, immunosuppressed tumour microenvironment is not currently known and of key interest as these are typically cancers resistant to other immunotherapeutic approaches. Therefore, in this project, using their lead therapeutic candidates, 2D and 3D co-culture models will be used to characterise the relationship between tumour cell characteristics, fibroblast phenotype and response to oncolytic viral therapy.Aims:(1) Investigate the interaction between cancer cells and fibroblasts in response to oncolytic viral therapy. In order to assess the direct effect of co-culture of cancer cells and fibroblasts measuring response to oncolytic virus, novel isogenic cancer cell lines of increasing chromosomal instability (produced from the Parkes lab) will be cultured with immortalised cancer-associated fibroblast lines derived from patient samples. Fluorescently-labelled cancer cells and fibroblasts will be disaggregated for RNA and chemokine profiling in response to virus. Fibroblast phenotype will be further assessed using flow cytometry for expression of cancer associated fibroblast marker expression.(2) Characterise the role of matrix organisation and stiffness in response to oncolytic viral therapy. Fibroblasts are the principal producers of matrix proteins in the tumour microenvironment, which subsequently affects the mechanical properties of the tumour including stiffness and the ability of cells to migrate through the tumour. The effect of matrix stiffness on oncolytic virus response will be assessed using artificial matrices of increasing stiffness. In these matrices, organoids (representing low and high-chromosomal instability) will be co-cultured with fibroblasts. The ability of oncolytic viruses to induce cell death in the context of increasing stiffness, relevant to the tumour microenvironment, will be measured. The activity of oncolytic virus to infect cells and replicate will be assessed in the context of matrix stiffness.(3) Investigate the effect of fibroblast activation and matrix stiffness on immune cell activation. T cells cultured in conditioned media from cancer cell-fibroblast co-cultures will be analysed for ability to activate, proliferate and recognise tumour antigen. Using models in aim (2), 3D co-cultures of organoids, fibroblasts and immune cells (derived from blood samples) will be used to characterise immune cell activation, proliferation and cytotoxic activity in response to oncolytic viral therapy. This project will characterise a novel therapeutic agent using newly developed cell lines and fibroblast co-culture, as well as comprehensive near-patient 3D-models of the tumour microenvironment.