Anti-Complement Immunotherapy for Pancreatic Cancer

项目摘要/摘要 胰腺导管腺癌(PDAC)是一种几乎一致致命的疾病，其总存活率在 10%尽管在包括免疫治疗在内的所有癌症治疗领域都取得了治疗进展，但总存活率 在PDAC方面没有显著改善，这表明开发新的治疗方法是迫切需要的 治疗这种疾病的策略。PDAC中肿瘤微环境(TME)的特点是 免疫抑制浸润物导致T细胞排斥和致密的间质间质增生。补语 级联在PDAC TME中被激活，并可能通过向免疫发出信号来促进这一具有独特挑战性的TME 表达补体受体的细胞和成纤维细胞。补体激活之前 证实促进免疫细胞的渗透，这有助于多种肿瘤类型的T细胞排斥 以一种上下文相关的方式。这个项目的长期目标是了解微环境 补体促进PDAC肿瘤发生的机制以及两者之间的相互作用 放射治疗(RT)和补体阻断。建立了一种新的自体小鼠PDAC模型。 产生并与补体缺乏C3基因敲除(KO)小鼠杂交，以允许进行定义 补体在PDAC中的作用。补体熟练野生型(WT)PDAC小鼠和C3KO小鼠的肿瘤生长 PDAC小鼠将使用连续的PET/CT成像进行跟踪。肿瘤将在3-6个月内进行组织学评估 用三苯氧胺诱导后，确定组织学分级、正常腺泡面积和纤维化区。流式细胞术 将用于确定补体对渗透的免疫细胞的影响，同时进行单细胞RNA测序 将提供对癌症相关成纤维细胞的发展和激活的洞察。我们亦会评估 肿瘤细胞与基质来源的C3在YFP表达的KPC细胞(KPCY)中的作用 WT和C3KO小鼠的侧翼肿瘤生长研究作为第二个模型证实了这些结果。要评估 光子、质子和碳离子RT对补体激活和免疫耐受的影响 KPCY使用每种辐射方式。补体沉积分析将通过培养辐照后进行 和未处理的肿瘤细胞，在血清作为补体来源存在的情况下，在通过 流式细胞术。下一步，树突状细胞将与照射和未照射的KPCY细胞与血清共培养。 确定受照射的肿瘤细胞上的补体沉积是否会减少树突状细胞激活标志物 用流式细胞仪检测。翻译研究将使用中和抗体和小分子 在原位KPCY模型中，分子抑制剂单独和RT一起阻断补体信号。老鼠会 用中和抗体和抑制剂阻断补体信号，并用X-射线照射 施特劳尔小动物辐射研究平台或瓦里安质子治疗机和生长会 被IVIS追踪到。补体的激活将通过免疫印迹和免疫荧光来评估，以及Flow 流式细胞术将评估照射后免疫细胞渗入的差异。

PROJECT SUMMARY / ABSTRACT Pancreatic Ductal Adenocarcinoma (PDAC) is an almost uniformly lethal disease, with an overall survival under 10%. Despite therapeutic advances in all arenas of cancer treatment, including immunotherapy, overall survival has not significantly improved in PDAC, representing a critical need for the development of novel therapeutic strategies for this disease. The tumor microenvironment (TME) in PDAC is characterized by an immunosuppressive infiltrate causing T-cell exclusion, and dense stromal desmoplasia. The complement cascade is activated in the PDAC TME and may promote this uniquely challenging TME by signaling to immune cells and fibroblasts expressing complement receptors. Complement activation has previously been demonstrated to promote infiltration of immune cells which contribute to T-cell exclusion in multiple tumor types in a context-dependent fashion. The long-term objectives of this project are to understand microenvironmental mechanisms by which complement promotes oncogenesis in PDAC, and to elucidate the interactions between radiotherapy (RT) and complement blockade in PDAC. A novel autochthonous mouse model of PDAC was generated and crossed with complement deficient C3 knockout (KO) mice to allow for studies which define the role of complement in PDAC. Tumor growth in complement-proficient wild type (WT) PDAC mice and C3 KO PDAC mice will be tracked using serial, PET/CT imaging. Tumors will be assessed histologically from 3-6 months after induction with tamoxifen to determine histologic grade, normal acinar area, and fibrotic area. Flow cytometry will be used to determine the impact of complement on infiltrating immune cells while single-cell RNA-sequencing will provide insight into the development and activation of cancer-associated fibroblasts. We will also assess the role of tumor cell vs. stromally derived C3 with shRNA depletion of C3 in YFP-expressing KPC cells (KPCY) in flank tumor growth studies in WT and C3 KO mice as a second model to confirm these results. To assess the impact of photon, proton, and carbon ion RT on complement activation and immune tolerance we will irradiate KPCY using each radiation modality. Complement deposition assays will be performed by culturing irradiated and non-treated tumor cells in the presence of serum as a complement source, before measuring activation by flow cytometry. Next, dendritic cells will be co-cultured with irradiated and non-irradiated KPCY cells with serum to determine if complement deposition on irradiated tumor cells reduces dendritic cell activation markers measured by flow cytometry. Translational studies will be performed using neutralizing antibodies and small molecule inhibitors to blockade complement signaling alone and with RT in an orthotopic KPCY model. Mice will be treated with neutralizing antibodies and inhibitors to blockade complement signaling and irradiated with an X- strahl Small Animal Radiation Research Platform or a Varian ProBeam proton therapy machine and growth will be tracked by IVIS. Complement activation will be assessed by western blot and immunofluorescence, and flow cytometry will assess differences in infiltrating immune cells post-irradiation.