Project 1: FLASH vs. Standard radiotherapy for treatment of PDAC and sparing normal intestine tissues

摘要(与以前提交的文件相比有变化，用 带阴影的文本 ) 项目1将测试质子放射治疗以超快剂量率提供的总体假设(&gt；60 )，称为闪光PRT，控制胰腺肿瘤生长的效果与标准剂量率(&lt；1 质子放射治疗(S-PRT)，同时避免正常肠道组织的急性和迟发性毒性。 成功完成拟议的研究将有助于从机制上更好地理解差异 在分子、遗传和组织水平上比较F-PRT和S-PRT的作用，并将定义 启动临床试验所需的参数。在已发表的初步研究中，我们证明了 与S-PRT相比，F-PRT提高了模型小鼠的总体存活率，并改善了晚期 毒性，主要是纤维化。同时，F-PRT与S-PRT在控制病虫害方面具有等效性。 同种异体移植瘤的生长。使用单细胞RNA测序(ScRNAseq)的研究揭示 上皮性干细胞/祖细胞对F-PRT反应中基因表达谱的有趣差异 与S-PRT相比，这与其对祖细胞增殖的抑制作用降低不谋而合。在……里面 目标1，我们将定义最大限度地保护正常组织的剂量学和生物物理参数 利用同基因侧翼和原位模型及基因工程技术研究F-PRT的抗肿瘤作用 Panca小鼠模型(GEMM)。然后我们将连线表演 一项使用这些优化的剂量递增研究 参数和焦点RT在F-PRT中的确定与S-PRT相比提高总体生存率 。在目标2中，我们 将勾勒出正常肠道和肝脏组织包括上皮细胞的差异反应机制， 血管、免疫和 流通中 将细胞分别转化为S-PrT和F-PrT，用scRNAseq去卷积分化 这两种模式所引发的基因表达模式。在目标3中，我们将使用GEMM和组织特异性 P53(上皮与内皮)的缺失研究上皮和内皮细胞的作用 分别在反应S-PRT和F-PRT对急性毒性和晚期毒性的联合上皮细胞减少 障碍损失。 我们还将测试Wnt/β-连环蛋白和R-响应蛋白信号在介导F- 保留正常肠上皮的PRT 。该项目将受益于概念性项目，并为其做出贡献 其他项目的进展情况。从scrna-seq获得的信息将为皮肤毒性实验提供信息。 以及项目2中的祖细胞命运，包括狗对肉瘤的试验。结果来自上皮细胞特异性和 该项目中的内皮特异性p53基因敲除小鼠将指导项目2和项目3中的实验 肉瘤和肺部。项目3将产生碳和质子辐照的肠道和潘卡样本， 将由项目1分析。最后，项目4将开发铅笔束扫描方法，我们将 在目标1下的剂量递增实验中使用。

SUMMARY (changes from previous submission denoted with shaded text ) Project 1 will test the overall hypothesis that Proton Radiotherapy delivered in ultra-fast dose rates (>60 Gy/sec), termed FLASH-PRT, controls Pancreatic tumor growth equally well as Standard dose rate (<1 Gy/sec) Proton Radiotherapy (S-PRT), while sparing normal intestinal tissue from acute and delayed toxicity. Successful completion of the proposed studies will lead to better mechanistic understanding of the differential effects of F-PRT compared to S-PRT at the molecular, genetic and organismic level and will define the parameters necessary for the initiation of clinical trials. In preliminary published studies, we demonstrated that compared to S-PRT, F-PRT increases overall survival of model mouse models and also ameliorates late stage toxicity, primarily fibrosis. At the same time, F-PRT was shown to be equipotent to S-PRT in controlling the growth of allografted syngeneic tumors. Studies using single-cell RNA- sequencing (scRNAseq), reveal intriguing differences in gene expression profiles in the response of epithelial stem/progenitor cells to F-PRT compared to S-PRT which coincide with a reduction in the inhibitory effect on progenitor cell proliferation. In Aim 1, we will define the dosimetric and biophysical parameters which deliver maximum normal tissue sparing and anti-tumor effect of F-PRT using syngeneic flank and orthotopic models and Genetically Engineered Mouse model (GEMM) of PanCa. We will then link perform a dose-escalation study using these optimized parameters and focal RT to determine in F-PRT improves overall survival compared to S-PRT . In Aim 2, we will delineate the mechanism of differential response of normal intestinal and liver tissues including epithelium, vascular, immune and circulating cells to S-PRT and F-PRT, using scRNAseq to deconvolute the differential patterns of gene expression elicited by the two modalities. In Aim 3, we will use GEMM with tissue-specific deletion of p53 (epithelium vs. endothelium) to investigate the role of epithelial and endothelial cells respectively, in the response to S-PRT and F-PRT on acute and late toxicity coupled with reduced epithelial barrier loss. We will also test the involvement of the Wnt/β-catenin and R-Spondin signaling in mediating F- PRT sparing of normal intestinal epithelium . This project will benefit from, and contribute to, conceptual advances in the other projects. Information garnered from scRNA-seq will inform experiments on skin toxicity and progenitor cell fate in Project 2, including the canine trial on sarcoma. Results from epithelial-specific and endothelial-specific p53 knockout mice in this project will guide experiments in Projects 2 and Project 3 in sarcoma and lung. Project 3 will generate Carbon and Proton-irradiated intestinal and PanCa samples which will be analyzed by Project 1. Finally, Project 4 will develop a pencil-beam scanning approach which we will employ in the dose-escalation experiments under Aim 1.