MICA: Investigating the role of the adenosinergic pathway in anti-tumour immune dysfunction in cutaneous squamous cell carcinoma (cSCC)

皮肤鳞状细胞癌(CSCC)是世界上最常见的癌症之一，在英国每年有44000例确诊病例。大多数鳞状细胞癌都是通过手术治疗的，但当癌症进入晚期或扩散到其他器官(称为转移)时，治疗选择有限。癌症被免疫细胞包围，免疫细胞在杀死癌症方面起着至关重要的作用，但这些细胞经常被癌症“瘫痪”，使肿瘤得以发展。癌症免疫疗法鼓励免疫细胞攻击癌症，并正在彻底改变晚期/转移性癌症的治疗方法。在CSCC中，针对PD-1途径的免疫疗法(Cymplimab)已被证明可提高生存率，但仅在约15%的患者中有完全反应。因此，我们迫切需要寻找新的免疫治疗靶点，以帮助增强CSCC的抗癌免疫反应。腺苷是一种在CSCC中产生的化学物质，可导致癌症中的免疫抑制。我预测，使用旨在阻断腺苷途径的治疗方法将停止腺苷对免疫细胞的抑制，并增强CSCC的免疫反应，从而为这种癌症带来潜在的新疗法。此外，我预测通过联合腺苷阻断和抗PD-1治疗，有可能增强抗PD1治疗的效果。目的1：阻断CSCC中腺苷途径的治疗如何影响抗癌免疫细胞反应？使用一种新的‘组织切片培养’(TSC)系统，我将用三种不同的腺苷途径阻滞剂(CD39抑制剂、抗CD73抗体和A2AR抑制剂)培养非常薄的新鲜取出的活的CSCC，它们可以阻断途径中不同位置的腺苷的产生或作用。这些不同治疗方法对CSCC免疫细胞的影响将通过观察蛋白质变化、肿瘤杀伤和基因表达水平来评估，后者使用一种名为单细胞转录组学的最先进方法。这是一项显示样本中每个单个细胞中哪些基因活跃的技术，因此它告诉我们单个细胞和免疫细胞组是如何反应的。TSC系统的优势是研究免疫细胞在肿瘤环境中的行为，同时允许我专注于免疫细胞的个别亚群如何对不同的腺苷途径阻滞剂做出反应，以确定它们在CSCC中的单独作用。目的2：联合使用腺苷途径阻滞剂是否比单独使用腺苷途径阻断腺苷途径更能增强CSCC的抗癌免疫反应？我将用所有腺苷阻滞剂(CD39抑制剂、抗CD73抗体和A2AR抑制剂)一起处理CSCC切片(使用TSC系统)，并与单独使用这些处理(如上所述)进行比较。我将通过观察关键基因的基因表达，免疫细胞是否产生某些已知可增强抗癌效果的蛋白质，以及腺苷途径阻滞剂的组合是否会杀死更多的癌细胞，来看看这是如何改变免疫细胞活性的。目标3：腺苷阻断与抗PD-1联合使用是否比单独使用抗PD-1改善抗癌免疫反应？我将结合腺苷途径阻滞剂与抗PD-1免疫疗法，看看这种组合是否比单独使用任何一种方法对免疫细胞产生更大的促进作用。我将以与目标2相同的方式评估免疫反应和癌细胞杀伤。未来的好处：这些结果将增强我们对腺苷途径在CSCC中的作用以及将该途径的阻断与现有的抗PD-1免疫治疗相结合是否有用的理解。这将为这些药物未来的临床试验指明方向。此外，该项目开发的技术可能对未来评估其他类型癌症的不同免疫疗法组合的研究有用。

Cutaneous squamous cell carcinoma (cSCC) is one of the commonest cancers worldwide, with >44,000 diagnosed in the UK annually. Most cSCCs are treated with surgery, but when the cancer is advanced or has spread to other organs (called metastasis) treatment options are limited. Cancers are surrounded by immune cells which play a crucial role in killing the cancer, but these cells are frequently "paralysed" by the cancer, allowing the tumour to progress. Cancer immunotherapies encourage the immune cells to attack cancers, and are revolutionising the treatment of advanced/metastatic cancer. In cSCC, an immunotherapy targeting the PD-1 pathway (Cemiplimab) has been shown to improve survival, but only gives a complete response in around 15% of patients. Therefore, we urgently need to identify new immunotherapy targets to help enhance anti-cancer immune responses in cSCC. Adenosine is a chemical produced in cSCC that can cause immune suppression in cancer. I predict that using treatments designed to block the adenosine pathway will stop adenosine inhibiting the immune cells and boost the immune response in cSCC, leading to a potential new treatment for this cancer. Additionally, I predict that by combining adenosine blockade with anti-PD-1 treatment, it may be possible to enhance the effect of anti-PD1 treatment. Objective 1: How do treatments blocking the adenosine pathway in cSCC affect anti-cancer immune cell responses? Using a novel 'tissue slice culture' (TSC) system, I will culture very thin slices of freshly removed, live cSCC with three different adenosine pathway blockers (CD39 inhibitor, anti-CD73 antibody, and an A2AR inhibitor), that block the production or action of adenosine at different positions in the pathway. The effects of these different treatments on cSCC immune cells will be assessed by looking at protein changes, tumour killing and gene-expression levels, the latter using a state-of-the-art approach called single cell transcriptomics. This is a technique that shows which genes are active in each individual cell within the sample, so it tells us how individual cells and groups of immune cells respond. The TSC system has the advantage of investigating the immune cells' behaviour within the tumour environment, while allowing me to focus on how individual subsets of immune cells respond to the different adenosine pathway blockers to determine their individual effects in cSCC.Objective 2: Does combining adenosine pathway blockers boost the anti-cancer immune response in cSCC more than blocking the adenosine pathway using individual agents? I will treat cSCC slices (using the TSC system) with all the adenosine blockers together (CD39 inhibitor, anti-CD73 antibody, and an A2AR inhibitor) and compare it with using the treatments separately (as above). I will see how this changes the immune cell activity by looking at the gene expression of key genes, whether the immune cells make certain proteins that are known to increase their anti-cancer effects, and whether the combination of adenosine pathway blockers kills more cancer cells.Objective 3: Does combining adenosine blockade with anti-PD-1 improve the anti-cancer immune response over that seen with anti-PD-1 alone?I will combine adenosine pathway blockers with an anti-PD-1 immunotherapy to see if this combination gives a bigger boost to the immune cells than using either approach alone. I will assess the immune responses and cancer cell killing in the same way as outlined in Objective 2.Future Benefits: The results will enhance our understanding of how the adenosine pathway acts in cSCC and whether combining blockade of this pathway with existing anti-PD-1 immunotherapy may be useful. This will inform the direction of future clinical trials with these agents. In addition, the techniques developed in this project may be useful for future research assessing different immunotherapy combinations for other types of cancers.