Exploring O-glycoproteomics to prevent metabolic radioresistance in the tumor microenvironment

项目总结 放射治疗(RT)通常是不能手术的肿瘤患者的唯一治疗选择。然而，辐射是 也已知会损害肿瘤代谢，导致放射抵抗，这是RT失败的主要原因。新陈代谢 癌症中的重新编程(MR)被定义为肿瘤重新连接其能量以满足以下需求的能力 肿瘤的发生和发展。我们的小组首次观察到MR对己糖胺的影响 生物合成途径(HBP)，一种未被研究的导致蛋白质糖基化的葡萄糖途径，与 肺腺癌患者存活率低。准确地说，我们发现这种代谢转换主要发生在 在癌症相关成纤维细胞(CAF)中。这表明CAF将它们的葡萄糖重定向到HBP，这 增加O-糖基化，一种已知的调节辐射抗性的翻译后修饰(PTM)。 然而，人们对1)哪些蛋白质在MR后O-糖基化成HBP和2)知之甚少。 这些PTM如何影响细胞行为和调节辐射抗性。我的初步结果显示 癌症和间质细胞之间的代谢合作改变了两种细胞中的蛋白质O-糖基化。 因此，我假设肿瘤-间质在肿瘤微环境(TME)中的串扰，导致 O-糖蛋白组的改变，在辐射抗性中起着一定的作用。为了验证这一假设，我们开发了 一种新的方法，可以精确地测量MR对HBP(例如，O-糖蛋白质组)的结果 肿瘤-间质串扰的背景。我们建议将这项技术应用于肿瘤间质器官。 这里被指定为“集合体”，概括了新陈代谢的异质细胞邻域和 RT前后的O-糖蛋白组学特征。首先，要可视化高血压患者的代谢异质性 TME，我将构建一张原发肿瘤隔间(内皮细胞、恶性细胞、成纤维细胞和 免疫)利用Codex富含HBP代谢标志物和糖类结构。食典是一种切割- 边缘多路复用成像方法，允许单细胞原位量化多达50个标记(目标1)。 然后，我将使用机器学习和聚类生物计算方法对细胞邻域进行去卷积 量化和告知哪些邻域是蛋白质O-糖基化的活跃区域。在《目标2》中，我会 使用3D组合体模型概括富含HBP的细胞邻域，照射它们，然后表征 使用CODEX的代谢辐射抗性模式。最后，在目标3中，我将分析O-糖蛋白质组和空间 抗辐射集合体的信息。涉及O-糖蛋白或O-糖基化的上游驱动因素 在危急时刻，肿瘤与间质的相互作用将被抑制，以试图恢复放射敏感性。生成的数据 将产生第一个假说合成工具，探索细胞信号传递的一个未被充分研究的维度，即O- 糖蛋白组学。它们将导致发现新的分子靶点，涉及肿瘤新陈代谢和 间质相互作用的主要目标是改善患有无法手术肿瘤的癌症患者的RT反应。

PROJECT SUMMARY Radiotherapy (RT) is often the only curative option for patients with inoperable tumors. However, radiation is also known to impair tumor metabolism, leading to radioresistance, the main reason for RT failure. Metabolic reprogramming (MR) in cancer is defined as the ability of the tumor to rewire its energy to fulfill the needs for tumorigenesis and progression. Our group observed for the first time that MR toward the Hexosamine Biosynthesis Pathway (HBP), an understudied glucose pathway leading to protein glycosylation, is associated with poor survival in the lung adenocarcinoma. Precisely, we showed that this metabolic switch happens primarily in Cancer-Associated Fibroblasts (CAFs). This suggests that CAFs redirect their glucose toward HBP, which increases O-glycosylation, a Post-Translational Modification (PTM) known to modulate radioresistance. However, very little is known about 1) which proteins are O-glycosylated after MR toward HBP and 2) how these PTMs affect the cellular behavior and modulate radioresistance. My preliminary results show that metabolic cooperation between cancer and stromal cells alters protein O-glycosylation in both cell types. Therefore, I hypothesize that tumor-stroma crosstalk in the Tumor Microenvironment (TME), leading to changes in the O-glycoproteome, plays a role in radioresistance. To validate this hypothesis, we developed a novel approach that precisely measures the outcome of MR towards HBP (e.g., O-glycoproteome) in the context of tumor-stroma crosstalk. We propose to apply this technique to tumor-stroma organoids designated here as “assembloids” that recapitulate metabolically heterogeneous cell neighborhoods and characterize their O-glycoproteome before and after RT. First, to visualize HBP metabolic heterogeneity in the TME, I will construct an in-situ map of the primary tumor compartments (endothelial, malignant, fibroblast, and immune) enriched for HBP metabolic markers and glycoform structures, using CODEX. CODEX is a cutting- edge multiplexed imaging method that allows for single-cell quantification of up to 50 markers in situ (aim 1). Then, I will deconvolute cell neighborhoods using machine learning and clustering biocomputational approaches to quantify and inform which neighborhoods are active regions of protein O-glycosylation. In aim 2, I will recapitulate HBP-enriched cell neighborhoods using a 3D assembloid model, irradiate them, then characterize metabolic radioresistance patterns using CODEX. Lastly, in aim 3, I will analyze the O-glycoproteome and spatial information of radioresistant assembloids. The O-glycoproteins or upstream drivers to O-glycosylation involved in critical tumor-stroma interactions will be inhibited in an attempt to restore radiosensitivity. The resulting data will generate the first hypothesis synthesis tool exploring an understudied dimension of cell signaling, the O- glycoproteome. They will lead to the discovery of new molecular targets involved in both tumor metabolism and stromal interactions with the primary goal of improving RT response in cancer patients with inoperable tumors.