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.

对原发性肝癌治疗的临床需求未满足，目前依赖于切除和移植作为护理标准。这些肿瘤中有多达80％是无法切除的，大约有20％的人接受移植经历了肿瘤复发。将其与寻找捐助者，移植排斥和移植后并发症的困难相结合，很明显需要进行非手术干预措施。基于嵌合的抗原受体受体（CAR）免疫疗法的重新进展已证明有效地治疗了许多“液态”的血液癌。然而，实体瘤中肿瘤微环境（TME）的复杂性质和密度产生了一种免疫抑制环境，既有助于肿瘤进展并赋予防止疗法的保护。在大多数实体瘤中，将多种宿主免疫细胞募集到TME，巨噬细胞占其中很大一部分。巨噬细胞可以细分为两个主要的表型组。 M1群体产生促炎性细胞因子反应，它们在癌症周围的定位已被证明可以提高化学疗法在体外的有效性。另一个主要的巨噬细胞组M2通过诱导胶原蛋白产生和建造细胞外基质，与伤口愈合和组织修复有关。在TME中，这些M2细胞会促进血管生成和转移，并已被证明会增加针对化学疗法的肿瘤细胞存活。巨噬细胞表现出表型之间的高可塑性，具体取决于其环境刺激，但是它们似乎偏向TME中的M2表型，这有助于其免疫抑制特征。因此，治疗实体瘤的治疗策略是针对与肿瘤相关的巨噬细胞（TAM）的群体，并诱导表型变化到细胞毒性M1种群中，以促进TME内的抗肿瘤环境。可以使用两步过程来实现表型之间极化巨噬细胞的模型。首先，单核细胞系THP-1用佛波尔12-羟基含量13-乙酸盐（PMA）处理，以诱导巨噬细胞M0态。接下来是与单独的M1（IFN-G和LPS）或M2（IL-4和IL-13）的单独的细胞因子鸡尾酒一起孵育。每种表型的标记通过QPCR，流经术或免疫染色来评估。 CD68是巨噬细胞的通用标记，而IL-6和CXCL10或MRC1和FN1分别可以进一步识别M1和M2表型。基因疗法将在该模型中评估病毒载体，并为M2细胞的特异性进行进一步设计。然后，将筛选CAR构造文库以识别合适的候选物，以引起表型从M2转移到M1细胞的转变。一旦将这两个系统组合在一起，将在3D体外模型中测试所得的疗法。这些模型包括不同的空间隔离区，以设计肿瘤和周围的基质，但是这些隔室之间细胞的迁移和侵袭是可能的。这些隔室的基质是使用密集的胶原蛋白矩阵生成的，其中刚度与人肝组织的刚度相匹配。该模型将开发以尽可能接近原发性肝癌TME。在FDA裁定之后，3D体外模型比以往任何时候都更有意义，从而消除了在动物中测试新药的必要性，因此是首先测试这种疗法的合适方法。