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

摘要（与之前提交的变更， 阴影文本 ) 项目1将测试质子放射治疗以超快剂量率（>60 戈伊/秒），称为FLASH-PRT，控制胰腺肿瘤的生长同样好的标准剂量率（<1 戈伊/sec）质子放射治疗（S-PRT），同时保护正常肠组织免受急性和延迟毒性。 成功完成拟议的研究将有助于更好地了解差异的机理 F-PRT与S-PRT相比在分子、遗传和器官水平上的作用，并将定义 启动临床试验所需的参数。在初步发表的研究中，我们证明， 与S-PRT相比，F-PRT增加了模型小鼠的总体存活率，并且还改善了晚期 毒性主要是纤维化与此同时，F-PRT与S-PRT在控制 同种异体移植的同基因肿瘤的生长。使用单细胞RNA测序（scRNAseq）的研究显示， 上皮干/祖细胞对F-PRT反应中基因表达谱的有趣差异 与S-PRT相比，这与对祖细胞增殖的抑制作用的降低一致。在 目标1，我们将确定剂量和生物物理参数，提供最大的正常组织保护 使用同基因侧翼和原位模型和基因工程的F-PRT的抗肿瘤作用 PanCa的小鼠模型（GEMM）。然后我们将链接执行 使用这些优化的剂量递增研究 与S-PRT相比，F-PRT中确定的参数和局灶性RT可改善总生存期 .在目标2中， 将描述正常肠和肝组织（包括上皮）的不同反应的机制， 血管、免疫和 循环 细胞到S-PRT和F-PRT，使用scRNAseq去卷积差异表达。 这两种方式引起的基因表达模式。在目标3中，我们将使用GEMM与组织特异性 缺失p53（上皮与内皮），以研究上皮细胞和内皮细胞的作用 分别在对S-PRT和F-PRT的急性和晚期毒性反应中， 势垒损耗 我们还将检测Wnt/β-catenin和R-Spondin信号通路在介导F- 正常肠上皮的PRT保留 .该项目将受益于，并有助于， 其他项目的进展。从scRNA-seq获得的信息将为皮肤毒性实验提供信息 和祖细胞的命运在项目2中，包括对肉瘤的犬试验。上皮特异性和 本项目中内皮特异性p53基因敲除小鼠将指导项目2和项目3的实验， 肉瘤和肺。项目3将产生碳和质子辐照的肠道和PanCa样本， 将由项目1进行分析。最后，项目4将开发一种双光束扫描方法， 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.