Dissecting innate immune mechanisms of resistance to checkpoint blockade therapy in bladder cancer

项目摘要 膀胱癌是美国第五大常见癌症，每天约有47人死亡。 令人鼓舞的是，五种PD-1/PD-L1免疫检查点阻断（ICB）疗法在2011年被批准用于膀胱癌。 2016.尽管这些ICB治疗在一部分患者（15 - 25%）中取得了持久的临床反应， 大多数患者仍未从该疗法中获益。临床迫切需要扩大 ICB对更多患者的应用导致需要研究耐药的肿瘤内在机制。肿瘤- 促进炎症是癌症发病机制的标志，已知在多种情况下促进癌症生长。 包括抑制抗肿瘤免疫。我们在治疗前的肿瘤中发现了一个基因标记 与富含炎症和先天免疫基因的骨髓细胞相关，并预测不良的 两项ICB临床试验的ICB结局和生存率。我计划继续这项工作， 膀胱癌概况和研究骨髓细胞介导的对ICB治疗的抗性的机制。 目的1旨在定义未经治疗的膀胱肿瘤的景观，并提供对免疫细胞的洞察力。 ICB耐药的潜在亚群。我将构建一个膀胱癌的转录组和分子图谱， 单细胞分辨率，目前不存在的资源。我会建立病人的肿瘤血液 使用单细胞RNA测序的尿液，通过测序对转录组和表位进行细胞索引 （CITEseq），空间转录组学和O-link蛋白质组学，并使用Seurat和其他基于R 工具.我计划解析表达这种耐药基因特征的骨髓细胞，并确定它们的细胞相互作用。 在目标2中，我将深入研究骨髓细胞中导致ICB抗性的转录途径。我们 已经在肿瘤单核-巨噬细胞（单核-巨噬细胞）中鉴定了NLRP 3炎性小体激活和IL-1 β信号传导。 M Φ）作为促进肿瘤炎症和进展的候选途径。我将对这些单M Φ进行建模， 使用GM-CSF和M-CSF在缺氧条件下用IL-10将外周血单核细胞分化为M Φ 1 β和NLRP 3炎性体激活剂，我将测试对适应性免疫的影响，通过共同培养这些单- M Φ与活化的自体CD8 + T细胞。我还将使用这个模型来测试已知的调节 IL-1 β和NLRP 3炎性体活性作为膀胱癌中ICB的潜在组合治疗。 该建议将直接的离体单细胞基因组学与体外功能实验相结合，以进行彻底的研究。 询问先天免疫对膀胱癌ICB抗性的贡献。结合起来，这些目标 阐明先天免疫途径驱动的膀胱癌对PD-1/PD-L1 ICB治疗的耐药性，这可以 用于识别关键的预测性临床生物标志物，并为新的组合治疗策略提供信息。

PROJECT SUMMARY Bladder cancer is the fifth most common cancer in the United States, accounting for around 47 deaths per day. Promisingly, five PD-1/PD-L1 immune checkpoint blockade (ICB) therapies were approved for bladder cancer in 2016. Although these ICB treatments have achieved durable clinical responses in a subset of patients (15-25%), the majority of patients have still not benefitted from this therapy. This clinical urgency to extend the benefits of ICB to more patients has led to a need to investigate tumor intrinsic mechanisms underlying resistance. Tumor- promoting inflammation, a hallmark of cancer pathogenesis, is known to contribute to cancer growth in multiple ways including restraining antitumor immunity. We discovered a gene signature from pre-treatment tumor associating with myeloid cells that is enriched in inflammation and innate immune genes and predictive of poor ICB outcomes and survival in two ICB clinical trials. I plan to follow up on this work and dissect the innate immune landscape of bladder cancer and investigate mechanisms of myeloid-cell mediated resistance to ICB therapy. Aim 1 seeks to define the landscape of untreated bladder tumors and provide insight into the immune cell subsets underlying ICB resistance. I will construct a transcriptomic and molecular atlas of bladder cancer at a single-cell resolution, a resource that does not currently exist. I will build atlases of patients’ tumor, blood, and urine using single-cell RNA sequencing, Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITEseq), spatial transcriptomics, and O-link proteomics and analyze them using Seurat and other R-based tools. I plan to resolve myeloid cells expressing this resistant gene signature and define their cellular interactions. In Aim 2, I will delve into the transcriptional pathways in myeloid cells that are contributing to ICB resistance. We have identified NLRP3 inflammasome activation and IL-1β signaling in tumor monocyte-macrophages (mono- MΦs) as candidate pathways promoting tumor inflammation and progression. I will model these mono-MΦs by differentiating peripheral blood monocytes into MΦ using GM-CSF and M-CSF under hypoxic conditions with IL- 1β and NLRP3 inflammasome activators., I will test effects on adaptive immunity by co-culturing these mono- MΦs with activated autologous CD8+ T cells. I will also use this model to test drug candidates known to modulate IL-1β and NLRP3 inflammasome activity as potential combinatorial treatments with ICB in bladder cancer. This proposal combines direct ex vivo single cell genomics with in vitro functional experiments for a thorough interrogation of the innate immune contribution to ICB resistance in bladder cancer. Combined, these aims will elucidate innate immune pathway driven resistance to PD-1/PD-L1 ICB therapy in bladder cancer, which can be used to identify critical predictive clinical biomarkers and inform new combinatorial treatment strategies.