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

对于原发性肝癌治疗存在未满足的临床需求，其目前依赖于切除和移植作为护理标准。高达80%的这些肿瘤是不可切除的，大约20%接受移植的患者会出现肿瘤复发。联合收割机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证明在治疗许多"液体"血癌中有效。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，又能保护患者免受治疗。多种宿主免疫细胞被募集到TME，巨噬细胞在大多数实体瘤中占很大比例。巨噬细胞可细分为两个主要的表型组。M1群体产生促炎细胞因子反应，并且它们在癌块周围的定位已被证明可以增加体外化疗的有效性。另一个主要的巨噬细胞群M2通过诱导胶原蛋白产生和构建细胞外基质与伤口愈合和组织修复相关。在TME内，这些M2细胞促进血管生成和转移，并已显示出增加肿瘤细胞对化疗的存活。巨噬细胞表现出取决于其环境刺激的表型之间的高可塑性，然而，它们似乎倾向于TME中的M2表型，这有助于其免疫抑制特性。因此，治疗实体瘤的治疗策略是靶向肿瘤相关巨噬细胞（TAM）群体，并诱导细胞毒性M1群体发生表型变化，以促进TME内的抗肿瘤环境。可以使用两步过程来实现用于使巨噬细胞在表型之间极化的模型。首先，用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理单核细胞系THP-1以诱导巨噬细胞M0状态。随后与M1（IFN-g和LPS）或M2（IL-4和IL-13）的单独细胞因子混合物一起孵育。通过qPCR、流式细胞术或免疫染色评估每种表型的标志物。CD68是巨噬细胞的通用标志物，而IL-6和CXCL10或MRC 1和FN 1可分别进一步鉴定M1和M2表型。基因治疗病毒载体将在该模型中进行评估，并进一步工程化以对M2细胞的特异性。然后，将筛选CAR构建体文库以鉴定合适的候选物，以引起表型从M2细胞向M1细胞的转变。一旦这两种系统结合起来，所产生的治疗将在称为Tumouroids的3D体外模型中进行测试。这些模型包括不同的空间隔离区室，以工程化肿瘤和周围基质，然而这些区室之间的细胞迁移和侵入是可能的。这些隔室的基质是使用致密的胶原蛋白-I基质产生的，其中硬度与人肝组织的硬度相匹配。将开发该模型以尽可能接近地体现原发性肝癌TME。3D体外模型比以往任何时候都更相关，因为FDA的裁决取消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。

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.