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

微立方体肿瘤解剖的高内涵功能性癌症药物测试

• 批准号: 10025143
• 负责人: ALBERT FOLCH
• 金额: $ 60.2万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-06 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10025143
• 关键词: Address; Agonist; Antibodies; Antitumor Drug Screening Assays; Autologous; Behavior; Bioinformatics; Biopsy; Breast Cancer Model; Breast Cancer Patient; CTLA4 gene; Cell Death; Cells; Cellular immunotherapy; Clinic; Collaborations; Collagen; Cytotoxic agent; Development; Devices; Diffuse; Dissection; Drug Combinations; Drug Delivery Systems; Drug Exposure; Ensure; Enzyme-Linked Immunosorbent Assay; Evaluation; Excision; Extracellular Matrix; Fluorescence; Future; Gel; Glioma; Goals; Growth; Human; Hydrogels; Immune; Immune checkpoint inhibitor; Immune system; Immunohistochemistry; Immunomodulators; Immunotherapy; In Situ; Individual; Innate Immune System; Interleukin-10; Label; Lasers; Liquid substance; Mammary Neoplasms; Methodology; Microdissection; Microfluidic Microchips; Microfluidics; Modeling; Mouse Mammary Tumor Virus; Mus; Optics; Organoids; Patients; Pharmaceutical Preparations; Pharmacotherapy; Phase; Polymethyl Methacrylate; Preparation; Production; Reproducibility; Retrieval; Sampling; Shapes; Slice; Stains; T cell therapy; T-Cell Activation; T-Lymphocyte; Testing; Tissue Microarray; Tissue Viability; Tissues; Tumor Tissue; Tumor-Derived; Tumor-infiltrating immune cells; Xenograft Model; base; cancer immunotherapy; cancer therapy; cell killing; cell type; cost; cytokine; design; drug development; drug response prediction; genetic analysis; human tissue; macrophage; malignant breast neoplasm; micrography; next generation; particle; personalized cancer therapy; preservation; prevent; programmed cell death ligand 1; programmed cell death protein 1; prototype; response; screening; tumor; tumor microenvironment; wasting

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).

摘要 该项目的目标是对数百名患者的药物和免疫治疗反应进行高内容分析。 完整的，活的培养片段分离自一个活的肿瘤活检。近年来，患者源性肿瘤 “类器官”已经显示出预测个性化癌症治疗的药物反应的巨大希望。 免疫疗法，包括细胞免疫疗法，代表了下一代癌症疗法，许多 相关药物作用于肿瘤局部微环境（TME）。迫切需要功能 测试平台使用人类，完整和活的肿瘤组织，以更好地预测传统和免疫疗法 应答这些平台还应尽可能多地保留原生TME。当前高通量 具有这些特征中的一些特征的测试平台，例如基于患者来源的肿瘤类器官，需要 改变TME的生长步骤。另一方面，将肿瘤组织显微切割成“球体”， 含有完整的TME的细胞已经显示出对天然免疫细胞上的免疫调节剂的有希望的应答。我们 提出了一种微流体平台，使药物治疗，外源性T细胞治疗，和高含量 使用数百至数千个大小相似的精密切片立方形微组织（Cµ T）进行分析 从一个肿瘤样本中产生。 在这里，我们提出了两种方法的组合，以证明我们的方法的可行性：1）精度 从单个肿瘤中产生大量立方状微组织（Cµ T）的切片方法 活组织检查;以及2）在多孔平台中微流体捕获Cμ T，允许将药物应用于每个 单个CµT或Cµ T组。我们将能够从每一个患者中获得数百个患者来源的Cµ T。 肿瘤切除术Cµ T的尺寸（最初为400 µm×400 µm×400 µm）将是可重现的，并选择为 优化TME的活力和保留。随着Cµ T的培养，它们的立方体形状将松弛成更大的形状。 圆一。我们将研究Cµ T的可行性及其TME组成作为各种尺寸的函数。 培养条件，包括胶原凝胶。 我们将专注于使用同基因小鼠乳腺肿瘤模型进行乳腺癌免疫治疗。为了这个目标，我们 将递送各种浓度和组合的免疫调节药物，包括基于抗体的 药物，乳腺肿瘤CµTs在微流体设备，并检查对居民免疫系统的影响。 我们将评估细胞因子的产生，并使用高含量的免疫组织化学和生物信息学分析， 评估免疫细胞与不同细胞类型的接合以及细胞死亡。我们将把这个平台应用于 递送免疫检查点抑制剂（CTLA 4、PD-L1、PD-1）和其他免疫调节剂（例如IL-10）， 检查对免疫状态、细胞死亡和常驻T细胞行为的影响（活化和 本地化）。在R33阶段，我们计划将我们的微流体平台应用于从乳腺肿瘤中获得的Cµ T 患者（正在与V.K.博士合作）Gadi，Fred Hutch）.