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

项目摘要 B细胞和T细胞非霍奇金淋巴瘤弥漫性大B细胞 细胞性淋巴瘤(DLBCL)是最常见的淋巴瘤，约占所有B细胞非霍奇金淋巴瘤的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是最多的 耐化疗的DLBCL亚型5年总生存率低至30%，而GCB- DLBCL和原发性纵隔B细胞淋巴瘤。因此，需要新的治疗方法来 改善DLBCL患者的临床转归。淋巴瘤耐药的原因不是很好 理解并可归因于但不限于克隆异质性、微环境信号和 是一种基因复杂的淋巴增生性疾病。 大量激活规范和/或非规范途径，如B细胞受体(BCR)。 如此复杂 药物反应的研究强调需要更好地理解互补途径的作用，例如 淋巴瘤中的微环境信号。但是，c 目前淋巴瘤的临床前研究依赖于 在组织培养板上测试淋巴瘤细胞系悬浮培养的化合物，而不考虑 考虑到淋巴微环境，这些癌症发生和居住的地方。 本次IMAT的总体目标是 R33提案是高级开发和验证 用于淋巴瘤细胞系的有机物平台技术 和患者来源的异种移植，并回答与DLBCL耐药性和异质性相关的未解决问题。 本研究将开发并验证一个名为LETSSGo的实验治疗平台 (用于单一有机物测序和基因组学的芯片上淋巴瘤工程技术)。LETSSGO是一种 微淋巴样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条形码与单一有机物基因组分析相结合。跟随 将追求3个目标：目标1：设计和验证一种针对淋巴瘤的微型有机体平台 以及ABC-DLBCL和GCB-DLBCL细胞系的多细胞包埋；目的2：整合药物治疗的微 带有DNA条形码的有机化合物用于单一有机化合物基因组学以确定肿瘤耐药与肿瘤的关系 大小和细胞数量；目标3：工程淋巴瘤患者来源的DLBCL和DLBCL的异种移植微有机物 与小鼠比较，确定生长速度和基因表达和DNA甲基化的异质性 PDX 如果成功的话，我们的技术将会改变科学家们理解人类进化的方式。 淋巴瘤，使人们能够从机制上理解肿瘤微环境和克隆异质性的作用， 为正在开发的药物提供预后价值并增加临床前研究的“预测力”； 重要的是，这将允许更快、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.