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

通过针对肿瘤相关巨噬细胞的可塑性，开发一种针对实体瘤的新型体内基因治疗干预措施。

• 批准号: 2720553
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2022
• 资助国家: 英国
• 起止时间: 2022 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2720553
• 关键词: Developing; novel; vivo; gene; therapy

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.

原发性肝癌治疗的临床需求尚未得到满足，目前依赖于切除和移植作为治疗标准。高达80%的肿瘤是不可切除的，大约20%接受移植的患者会出现肿瘤复发。结合寻找供体的困难，移植排斥和移植后并发症，显然需要非手术干预。基于嵌合抗原受体（CAR）的免疫疗法的最新进展已被证明对治疗多种“液体”血癌有效。然而，实体肿瘤中肿瘤微环境（TME）的复杂性质和密度产生了一种免疫抑制环境，既有助于肿瘤的进展，又提供了对治疗的保护。多种宿主免疫细胞被招募到TME中，巨噬细胞在大多数实体肿瘤中占很大比例。巨噬细胞可细分为两大表型组。M1细胞群产生促炎细胞因子反应，它们在肿瘤肿块周围的定位已被证明可以提高体外化疗的有效性。另一个主要的巨噬细胞群，M2，通过诱导胶原蛋白的产生和构建细胞外基质与伤口愈合和组织修复有关。在TME中，这些M2细胞促进血管生成和转移，并已被证明可以提高肿瘤细胞对化疗的存活率。巨噬细胞在表型之间表现出高度的可塑性，这取决于它们的环境刺激，然而它们在TME中似乎倾向于M2表型，这有助于其免疫抑制特性。因此，治疗实体肿瘤的一种治疗策略是靶向肿瘤相关巨噬细胞（tam）群体，诱导细胞毒性M1群体的表型变化，以促进TME内的抗肿瘤环境。巨噬细胞极化表型之间的模型可以通过两步过程来实现。首先，用Phorbol 12-肉豆蔻酸13-醋酸酯（PMA）处理单核细胞系THP-1，诱导巨噬细胞M0状态。随后用单独的细胞因子鸡尾酒孵育M1 （IFN-g和LPS）或M2 （IL-4和IL-13）。每种表型的标记物通过qPCR、流式细胞术或免疫染色进行评估。CD68是巨噬细胞的通用标记物，而IL-6和CXCL10或MRC1和FN1可以分别进一步识别M1和M2表型。基因治疗病毒载体将在该模型中进行评估，并进一步设计针对M2细胞的特异性。然后，将筛选CAR构建文库，以确定合适的候选细胞，以诱导表型从M2细胞向M1细胞的转变。一旦这两种系统结合起来，所产生的治疗将在三维体外模型Tumouroids中进行测试。这些模型由不同的空间分离的室组成，用于设计肿瘤和周围的基质，然而细胞在这些室之间的迁移和侵袭是可能的。这些隔室的基质是使用致密胶原- 1基质生成的，其硬度与人类肝组织的硬度相匹配。该模型将被发展为尽可能地体现原发性肝癌TME。在美国食品和药物管理局（FDA）决定取消新药在动物身上测试的必要性之后，体外3D模型比以往任何时候都更有意义，因此是一种适合首次测试这种疗法的方法。