LETSSGo: Lymphoma-on-chip Engineered Technology for Single-Organoid Sequencing and Genomics

项目摘要 弥漫性大B细胞B细胞和T细胞非霍奇金淋巴瘤 细胞淋巴瘤（DLBCL）是最常见的淋巴瘤，占所有B细胞非霍奇金淋巴瘤的约30 淋巴瘤DLBCL分为不同的分子亚型，包括生发中心B细胞样（GCB） DLBCL、活化B细胞样（ABC）DLBCL和原发性纵隔B细胞淋巴瘤。ABC-DLBCL是最 化疗耐药DLBCL亚型的5年总生存率低至30%，而GCB为59和64%。 DLBCL和原发性纵隔B细胞淋巴瘤。因此，需要新的治疗方法， 改善DLBCL患者的临床结局。淋巴瘤耐药的原因尚不清楚 理解并可归因于但不限于克隆异质性，微环境信号传导， 是遗传上复杂的淋巴增生性疾病 经典和/或非经典途径如B细胞受体（BCR）的大量激活。 这种复杂性 强调需要更好地了解补充途径的作用， 微环境信号，在淋巴瘤中。然而，C 目前淋巴瘤的临床前研究依赖于 在组织培养板中用淋巴瘤细胞系的悬浮培养物测试化合物，而不考虑 考虑淋巴微环境，这些癌症的产生和居住。 本次IMAT的总体目标 R33提案是先进的开发和验证， 用于淋巴瘤细胞系的类器官平台技术 和患者来源的异种移植物，并回答与DLBCL抗性和异质性相关的未解决的问题。 这项研究将开发和验证一个实验性的治疗平台，命名为LETSSGo （淋巴瘤芯片工程技术用于单类器官测序和基因组学）。LETSSGO是一个 在微尺度淋巴样细胞中培养淋巴瘤细胞系和患者来源异种移植物的技术 微环境，并通过DNA条形码与单个类器官基因组分析整合。以下 将追求3个目标：目标1：设计和验证淋巴瘤特异性微型类器官平台， 目的2：整合药物处理的微囊化细胞， 具有用于单类器官基因组学的DNA条形码的类器官，以确定作为肿瘤功能的肿瘤抗性 目的3：工程化淋巴瘤患者来源的DLBCL和DLBCL的异种移植微类器官 确定与小鼠相比的生长速率和基因表达和DNA甲基化的异质性 个pdx 如果成功的话，我们的技术将改变科学家们对生命起源和发展的理解。 淋巴瘤，使肿瘤微环境和克隆异质性的作用机制的理解， 提供预后价值，并增加开发中药物临床前研究的“预测能力”，以及 重要的是，这将允许更快和更合理地筛选和翻译治疗方案。

Project Summary B- and T-cell Non Hodgkin lymphomas Diffuse Large B cell lymphoma (DLBCL) is the most common lymphoma representing ~30% of all B cell Non Hodgkin lymphomas. DLBCL is classified into distinct molecular subtypes, including germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal B cell lymphoma. ABC-DLBCL is the most chemo-resistant DLBCL subtype with a 5-year overall survival as low as 30% versus 59 and 64%, for GCB- DLBCL and primary mediastinal B cell lymphoma, respectively. Therefore, new treatments are needed to improve clinical outcome of the patients with DLBCL. The reasons for resistance in lymphomas are not well understood and could be attributed to, but not limited to, clonal heterogeneity, microenvironmental signaling, and are genetically complex lymphoproliferative diseases. massive activation of canonical and/or non-canonical pathways such as B cell receptor (BCR). Such complexity of drug response underscores the need for better understanding the role of complementary pathways, such as microenvironment signaling, in lymphomas. However, c urrent pre-clinical research in lymphoma has relied on testing compounds with suspension cultures of lymphoma cell lines in tissue culture plates, without taking into account the lymphoid microenvironment, where these cancers arise and reside. The overall goal of this IMAT R33 proposal is advanced development and validation of organoids platform technology for lymphoma cell lines and patient-derived xenografts, and answer unsolved questions related to DLBCL resistance and heterogeneity. This propose research will develop and validate an experimental therapeutics platform, named LETSSGo (Lymphoma-on-chip Engineered Technology for Single-Organoid Sequencing and Genomics). LETSSGO is an technology to culture lymphoma cell lines and patient derived xenografts in a microscale lymphoid-like microenvironment and integrate with single organoids genomic analysis by means of DNA barcoding. Following 3 aims will be pursued: Aim 1: Engineer and validate a lymphoma-specific mico-organoids platform for single and multi-cell encapsulation of ABC-DLBCL and GCB-DLBCL cell lines; Aim 2: Integrate drug treated micro- organoids with DNA barcoding for single-organoid genomics to determine tumor resistance as a function of tumor size and cell number; Aim 3: Engineer lymphoma patient derived xenograft micro-organoids of DLBCL and determine growth rate and heterogeneity in gene expression and DNA methylation as compared to mouse PDXs If successful, our technology will change the way scientists understood initiation and progression of lymphomas, enable mechanistic understanding of the role of tumor microenvironment and clonal heterogeneity, provide prognostic values and increase “predictive power” of pre-clinical studies for drugs in development, and importantly, will allow a faster and more rational screening and translation of therapeutic regimens.