Anti-Complement Immunotherapy for Pancreatic Cancer

项目总结/摘要 胰腺导管腺癌（PDAC）是一种几乎一致致死的疾病， 百分之十尽管在癌症治疗的所有领域都取得了治疗进展，包括免疫疗法，但总生存率仍然很低。 在PDAC方面没有显著改善，这代表了开发新的治疗药物的迫切需要。 应对这种疾病的策略。PDAC中的肿瘤微环境（TME）的特征在于 免疫抑制性浸润导致T细胞排斥和致密间质结缔组织增生。补体 级联在PDAC TME中被激活，并且可以通过向免疫系统发出信号来促进这种独特的挑战性TME。 表达补体受体的细胞和成纤维细胞。补体激活以前是 证实促进免疫细胞浸润，这有助于多种肿瘤类型中的T细胞排斥 以上下文相关方式。该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，并阐明补体与PDAC之间的相互作用。 放疗（RT）和补体阻断PDAC。建立了一种新型PDAC本土小鼠模型 产生并与补体缺陷型C3敲除（KO）小鼠杂交，以允许研究， 补体在PDAC中的作用。补体熟练野生型（WT）PDAC小鼠和C3 KO小鼠中的肿瘤生长 将使用连续PET/CT成像跟踪PDAC小鼠。将在3 - 6个月内对肿瘤进行组织学评估 在用他莫昔芬诱导后测定组织学分级、正常腺泡面积和纤维化面积。流式细胞术 将用于确定补体对浸润免疫细胞的影响，而单细胞RNA测序 将提供深入了解癌症相关成纤维细胞的发展和激活。我们亦会评估 在表达YFP的KPC细胞（KPCY）中肿瘤细胞与间质来源的C3的作用， 在WT和C3 KO小鼠中的侧腹肿瘤生长研究作为第二模型以证实这些结果。评估 光子、质子和碳离子RT对补体激活和免疫耐受影响 KPCY使用每种放射方式。将通过培养经辐照的 和未处理的肿瘤细胞在血清作为补体源存在下，然后通过 流式细胞仪接下来，树突状细胞将与具有血清的照射和未照射的KPCY细胞共培养 为了确定补体在被照射的肿瘤细胞上的沉积是否减少树突细胞活化标志物， 通过流式细胞术测量。将使用中和抗体和小分子抗体进行转化研究。 在原位KPCY模型中单独和与RT一起使用分子抑制剂阻断补体信号传导。小鼠将 用中和抗体和抑制剂治疗以阻断补体信号传导，并用X-射线照射。 strahl小动物辐射研究平台或Varian ProBeam质子治疗机， 由IVIS追踪。将通过蛋白质印迹和免疫荧光以及流式细胞术评估补体激活。 流式细胞术将评估照射后浸润免疫细胞的差异。

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.