Project 1: Inhibition of Complement C5aR1 Radioprotects Normal Tissue and Radiosensitizes Tumors

摘要(项目1-牛津) 增加放射治疗的治疗窗口可以通过使用针对癌症的靶向治疗来实现- 关联的路径。补体系统是一种先天免疫途径，在癌症中的作用正在显现。 进步。还没有研究评估补体抑制对肿瘤和正常组织的影响 辐射反应。在这里，我们展示了靶向补体受体C5aR1改善了对辐射的反应 在减少辐射毒性的同时，建立结直肠癌模型。靶向C5aR1可增加IL-10的分泌， 它减弱了核因子-κB信号，促进了肿瘤细胞的凋亡。依赖IL-10的细胞凋亡是 在体内和体外都观察到了非典型的应激特异性和可能的免疫非依赖性作用 C5aR1在调节细胞凋亡中的作用。重要的是，我们发现C5aR1耗尽会导致小肠减少。 照射后小鼠的组织学损伤、隐窝细胞凋亡和存活增加。在小肠里， C5aR1耗尽会导致非免疫细胞和免疫细胞群体中IL-10表达增加。IL-10 CX3CR1+巨噬细胞中的信号对肠道损伤防御很重要，C5aR1-/-小鼠有显著的 CX3CR1+巨噬细胞水平高于WT小鼠。作为对辐射的反应，C5aR1的耗尽不会 减弱核因子-κB信号转导但增加与细胞凋亡减少相关的AKT激活 以IL-10依赖的方式。因此，我们的数据表明，从遗传学或药理学上来说，C5aR1是靶向的 可改善大肠肿瘤模型的辐射反应，同时降低辐射诱导的小肠毒性。 综上所述，这些发现表明，抑制补体可能是一种有希望的方法来增加 放射治疗窗口期。在这个项目中，我们提出了四个具体目标，以更好地理解 C5aR1在辐射防护和放射增敏中的作用。目标1将确定广泛保护与具体保护 C5aR1-/-小鼠对正常上皮(肠、唾液腺和肺上皮)的反应分级 和低分割放射治疗，与项目2、3、4和核心B合作。在目标2中，我们将调查 当C5aR1被抑制时辐射防护的机制基础。我们的初步数据显示，这是一个 IL-10依赖机制，涉及CX3CR1+巨噬细胞的募集，促进生存和 血管生成。目的3将确定C5aR1如何与放射治疗相结合促进肿瘤细胞杀伤。 基于我们初步数据的假设是，抑制C5aR1会增加IL-10的分泌，从而 减弱NF-κB信号，导致IL-10依赖的肿瘤细胞在细胞培养和动物体内的杀伤 模特们。目的4将确定临床级别的C5aR1抑制剂对肠道、唾液的辐射保护作用 腺体和肺，并使结直肠肿瘤对放射治疗敏感。我打算每月回斯坦福大学 或每两个月一次的时间表，并将与我在斯坦福的同事分享这些数据。在牛津，我们表演我们自己的 他将与斯坦福大学的核心B合作，以确保方法的可比性。

Abstract (Project 1 - Oxford) Increasing the therapeutic window of radiotherapy may be achieved by using targeted therapies against cancer- associated pathways. The complement system is an innate immunity pathway, with emerging roles in cancer progression. No study has evaluated the effect of complement inhibition on both tumor and normal tissue radiation response. Here we show that targeting complement receptor C5aR1 improves radiation response in colorectal cancer models while reducing radiation-induced toxicity. Targeting C5aR1 increases IL-10 secretion, which attenuates NF-κB signaling and enhances apoptosis in tumor cells. IL-10-dependent apoptosis is observed both in vivo and in vitro suggesting a non-canonical stress-specific and likely immune-independent role for C5aR1 in regulating apoptosis. Importantly, we find that C5aR1 depletion results in decreased small intestinal histologic damage, crypt cell apoptosis and increased survival of mice following irradiation. In the small intestine, C5aR1 depletion results in increased IL-10 expression in both non-immune and immune cell populations. IL-10 signaling in CX3CR1+ macrophages is important for intestinal injury defense and C5aR1-/- mice have significantly higher levels of CX3CR1+ macrophages than WT mice. In response to irradiation, C5aR1 depletion does not attenuate NF-κB signaling but instead increases AKT activation correlating with decreased apoptosis occurring in an IL-10-depedent manner. Our data, therefore, indicate that genetically or pharmacologically targeting C5aR1 can improve radiation response in colorectal tumor models while reducing radiation-induced small bowel toxicity. Together, these findings suggest that inhibiting complement could be a promising approach to increasing the therapeutic window of radiotherapy. In this Project, we propose four specific aims to better understand the role of C5aR1 in radioprotection and radiosensitization. Aim 1 will determine the broad versus specific protection of normal epithelium (intestines, salivary glands, and lung epithelium) by C5aR1-/- mice in response fractionated and hypofractionated radiotherapy, in collaboration with Projects 2, 3, 4 and Core B. In Aim 2, we will investigate the mechanistic basis of radioprotection when C5aR1 is inhibited. Our preliminary data indicates that this is an IL-10 dependent mechanism and involves recruitment of CX3CR1+ macrophages that promote survival and angiogenesis. Aim 3 will determine how C5aR1 promotes tumor cell killing in combination with radiotherapy. The hypothesis based on our preliminary data is that C5aR1 inhibition increases IL-10 secretion, which attenuates NF-κB signaling, resulting in IL-10 dependent tumor cell killing both in cell culture and in animal models. Aim 4 will determine the effect of clinical grade C5aR1 inhibitors on radioprotecting intestine, salivary glands and lungs, and sensitizing colorectal tumors to radiotherapy. I intend to return to Stanford on a monthly or bimonthly schedule and will share this data with my colleagues at Stanford. At Oxford, we perform our own dosimetry and will cooperate with Core B at Stanford to ensure comparability of methods.