Targeting mechanosignaling in pediatric brain cancer

项目摘要 尽管采取多种治疗方式，但儿童脑癌的癌症相关死亡率仍然很高，幸存者 经常遭受严重的、终生的、与治疗相关的副作用和继发性恶性肿瘤。有一种明确的 需要更有效的治疗方法，包括最常见的儿童恶性脑肿瘤- 母细胞瘤，一种起源于小脑的肿瘤。机械敏感信号通路已经出现为 在抗癌药物发现中的强大靶点，包括治疗髓母细胞瘤。然而，当目标是 作为肿瘤细胞微环境传感器的信号通路，传统的单层培养 都是最常用的基于细胞的高通量药物发现，不能准确概括关键 环境提示，如组织僵硬或细胞外基质组成。针对关键的药物发现 机械敏感信号的介体需要在细胞培养环境中进行基于细胞的筛选分析， 更接近于体内组织。三维(3D)细胞培养已经走到了 努力创造更多类似活体的实验环境，以模拟复杂的细胞-细胞和细胞外-细胞 在组织中发现的基质相互作用。我们之前的合作工作证明了自我 组装和水凝胶MAX8β-发夹多肽作为三维细胞培养支架用于自动化高效培养 吞吐量药物发现。我们证明了MAX8在功能和可调性方面结合了生物兼容性和可调性 具有独特机械性能的刚性(例如，剪切变稀、可注射固体并可立即修复)， 通常允许使用标准高通量筛分(HTS)液体处理设备进行自动处理 在药物研发实验室里发现的。这项提议的主要目标是使用通用的和可调的 MAX8肽将开发一种3D细胞培养支架，模拟脑细胞外基质的关键特征，同时 还保留了与自动化液体处理设备一起使用的关键材料特性，所有这些都是为了更高的 以机械信号为靶点的吞吐量药物发现方法。目标一号将为一口井建立一种靶向检测- 表征了与MAX8多肽水凝胶支架相容的机械敏感信号通路- 在高通量兼容的设置中基于3D细胞培养。目标2将研究如何调整水凝胶硬度 具有脑细胞外基质成分的多肽功能化会影响分析性能和 小脑神经元和儿童脑癌细胞的表型。AIM 3将验证新开发的检测方法 通过对候选化合物进行试点药物筛选和体内疗效测试来建立平台。其结果是 这些研究将成为一个3D细胞培养平台，将提供对细胞外如何 基质组成和组织硬度调节正常神经元和儿童脑中的机械信号 癌细胞。此外，这些研究将为未来的高通量药物发现奠定基础 针对儿童脑肿瘤的支架3D培养中机械信号转导的方法。

Project Summary Despite multimodal treatment, cancer-related mortality in pediatric brain cancers remains high and survivors often suffer from serious, life-long, therapy-related side effects and secondary malignancies. There is a clear need for more effective therapies, including for the most common malignant pediatric brain cancer medullo- blastoma, a tumor that originates in the cerebellum. Mechanosensitive signaling pathways have emerged as powerful targets in cancer drug discovery, including for the treatment of medulloblastoma. Yet, when targeting signaling pathways that serve as sensors for a tumor cell's microenvironment, traditional monolayer cultures that are most commonly used in cell-based high-throughput drug discovery, do not accurately recapitulate critical environmental cues such as tissue stiffness or extracellular matrix composition. Drug discovery aimed at key mediators of mechanosensitive signaling require cell-based screening assays in a cell culture environment that more closely resembles in vivo tissue. Three-dimensional (3D) cell cultures have moved to the forefront in the effort to create more in vivo-like experimental environments that can mimic intricate cell-cell and cell-extracellular matrix interactions found in tissue. Our previous collaborative work demonstrated the suitability of the self- assembling and hydrogelating MAX8 β-hairpin peptide as a 3D cell culture scaffold for automated high- throughput drug discovery. We demonstrated that MAX8 combines biocompatibility and tunability in function and stiffness with unique mechanical properties (e.g., shear-thinning, injectable solid with immediate rehealing) that allow automatic handling with standard high-throughput screening (HTS) liquid handling equipment commonly found in a drug discovery laboratory. The primary objective of this proposal is to use the versatile and tunable MAX8 peptide to develop a 3D cell culture scaffold that mimics key features of brain extracellular matrix while also retaining material properties critical for use with automated liquid handling equipment, all for a high- throughput drug discovery approach targeting mechanosignaling. Aim 1 will establish a targeted assay for a well- characterized mechanosensitive signaling pathway that is compatible with MAX8 peptide hydrogel scaffold- based 3D cell cultures in a high throughput-compatible setup. Aim 2 will examine how tuning hydrogel stiffness and peptide functionalization with brain extracellular matrix components affects assay performance and phenotype of cerebellar neurons and pediatric brain cancer cells. Aim 3 will validate the newly developed assay platform by performing a pilot drug screen and in vivo efficacy testing of candidate compounds. The outcome of these studies will be a 3D cell culture platform that will provide fundamental understanding of how extracellular matrix composition and tissue stiffness regulate mechanosignaling in both normal neurons and pediatric brain cancer cells. Additionally, these studies will lay the foundation for a future high-throughput drug discovery approach targeting mechanosignaling in scaffold-based 3D cultures optimized for pediatric brain tumors.