High-content functional cancer drug testing on micro-cuboidal tumor dissections

摘要 这个项目的目标是对数百名患者进行药物和免疫治疗反应的高含量分析。 从一次活体肿瘤活检中分离出来的完整的活培养片段。近年来，患者来源的肿瘤 “有机化合物”在预测个性化癌症治疗的药物反应方面显示出巨大的希望。 免疫疗法，包括细胞免疫疗法，代表了下一代癌症疗法，许多 相关药物的作用于局部肿瘤微环境(TME)。迫切需要功能强大的 使用人体、完整和活的肿瘤组织更好地预测传统和免疫治疗的测试平台 回应。这样的平台还应该尽可能多地保留原生TME。目前的高吞吐量 具有其中一些特征的测试平台，例如基于患者衍生的肿瘤有机化合物，需要 改变TME的增长步骤。另一方面，对肿瘤组织的微观解剖变成了可以 含有完整的TME对天然免疫细胞上的免疫调节剂表现出良好的反应。我们 提出一个微流控平台，使药物治疗、外源性T细胞治疗和高含量 使用成百上千个大小相似、切片精确的立方体微组织(C？)进行分析 从单一的肿瘤样本中产生。 在这里，我们提出了两种方法的组合来证明我们方法的可行性：1)精度 将从单个肿瘤中产生大量立方微组织(C？)的切片方法 活组织检查；以及2)在多孔平台中对C？T进行微流控捕获，允许在每个孔平台上应用药物 单个C？T或C？组。我们将能够从每个设备获得数百个患者派生的C？ 肿瘤切除。C？的大小(最初为400微米×400微米×400微米)将是可复制的，并可选择 优化TME的生存能力和保留率。随着C？的培养，它们的立方体形状将松弛成更多 四舍五入的一。我们将研究CµT及其TME组成随大小的函数在不同的 培养条件，包括胶原蛋白凝胶。 我们将重点研究使用同基因小鼠乳腺肿瘤模型进行乳腺癌的免疫治疗。为此，我们 将提供不同浓度和组合的免疫调节药物，包括基于抗体的药物 在微流控设备中，对乳腺肿瘤进行药物治疗，并检测其对常驻免疫系统的影响。 我们将评估细胞因子的产生，并使用高含量免疫组织化学和生物信息学分析来 评估免疫细胞与不同细胞类型的接触以及细胞死亡。我们将把该平台应用于 提供免疫检查点抑制剂(CTLA4、PD-L1、PD-1)和其他免疫调节剂(如IL-10)和 检查对免疫状态、细胞死亡和常驻T细胞行为(激活和 本地化)。在R33阶段，我们计划将我们的微流控平台应用于从乳腺肿瘤获得的C？ 患者(正在与V.K.加迪博士、弗雷德·哈奇合作)。

ABSTRACT The goal of this project is to perform high-content analysis of drug and immunotherapy responses on hundreds of intact, live cultured fragments isolated from a single live tumor biopsy. In recent years, patient-derived tumor “organoids” have shown great promise to predict drug responses for personalized cancer treatment. Immunotherapy, including cellular immunotherapy, represents the next generation of cancer therapy, and many of the relevant drugs act on the local tumor microenvironment (TME). There is a pressing need for functional testing platforms that use human, intact and live tumor tissue to better predict traditional and immunotherapy responses. Such platforms should also retain as much of the native TME as possible. Present high-throughput testing platforms that have some of these features, e.g. based on patient-derived tumor organoids, require a growth step that alters the TME. On the other hand, the micro-dissection of tumor tissue into “spheroids” that contain the TME intact has shown promising responses to immunomodulators on native immune cells. We propose a microfluidic platform that enables drug treatment, exogenous T cell therapy, and high-content analysis using hundreds to thousands of similarly sized, precision-sliced cuboidal micro-tissues (CµTs) produced from a single tumor sample. Here we propose a combination of two methodologies to demonstrate the feasibility of our approach: 1) precision slicing methodology that will produce large numbers of cuboidal micro-tissues (CµTs) from a single tumor biopsy; and 2) microfluidic trapping of the CµTs in a multi-well platform, allowing for drug application to each individual CµT or groups of CµTs. We will be able to obtain several hundred patient-derived CµTs from each tumor resection. The size of the CµTs (initially 400 µm×400 µm×400 µm) will be reproducible and chosen to optimize viability and retention of the TME. As the CµTs are cultured, their cuboidal shape will relax into a more rounded one. We will study the viability of the CµTs and their TME composition as a function of size in various culture conditions, including collagen gels. We will focus on breast cancer immunotherapy using a syngeneic mouse breast tumor model. For this Aim, we will deliver various concentrations and combinations of immunomodulatory drugs, including antibody-based drugs, to breast tumor CµTs in the microfluidic device, and examine the effects on the resident immune system. We will assess cytokine production and use high-content immunohistochemistry and bioinformatics analysis to assess immune cell engagement with different cell types as well as cell death. We will apply the platform to deliver immune checkpoint inhibitors (CTLA4, PD-L1, PD-1) and other immunomodulators (such as IL-10) and examine the effect on the immune state, cell death, and the behavior of resident T cells (activation and localization). In the R33 phase we plan on applying our microfluidic platform to CµTs obtained from breast tumor patients (ongoing collaboration with Dr. V.K. Gadi, Fred Hutch).