Organoid Acquired Resistance

项目摘要 该U 54申请的项目3的总体目标是获得性免疫缺陷机制的基于类器官的定义。 非小细胞肺癌（NSCLC）的靶向治疗耐药（AR）。NSCLC的治疗采用 针对EGFR、ALK、KRAS G12 C、ROS 1和PD-1/PD-L1通路的多种疗法。然而，在这方面， 尽管最初的反应，这些分子靶向治疗是没有治愈性的，几乎所有的患者， 最终死于疾病进展。获得性耐药在复发中表现突出，例如 吉非替尼或奥希替尼治疗后EGFR T790 M或C797 S突变;然而，已知的AR机制 对于接受靶向治疗的大部分患者，不能确定。进一步加剧了这一点 两难是肿瘤内在与肿瘤微环境（TME）影响的不同贡献。的 TME特别含有细胞外基质（ECM）、成纤维细胞、血管和免疫成分， 严重影响AR的发展和表现。TME依赖性与非依赖性研究 AR的机制还受到明显缺乏体外人类实验系统的阻碍， 患者活检中TME的整体重现。 我们的应用充分利用了Sourav Bandyopadhyay和卡尔文郭集团的协同优势 在类器官和蛋白质组学技术，以解决EGFR和KRAS G12 C AR在NSCLC中的机制。我们 进行先进的蛋白质组学方法和类器官培养， 耐药性（来自项目1）以及来自在人免疫系统中生长的患者来源的异种移植（PDX）肿瘤 重组小鼠（来自项目2）。我们的类器官要么是“仅上皮细胞”，要么是新型气液界面（ALI） 与肿瘤浸润成纤维细胞和免疫细胞一起保存肿瘤上皮的类器官培养物 亚群（T、B、NK、髓样）。目的1利用NSCLC类器官CRISPR筛选的结果来确定 奥希替尼致敏基因座可以克服、延迟或改变临床活检/PDX AR类器官中的获得性耐药， 强调TME机制的命中。Aim 2使用我们的质谱平台来表征 首次治疗、残留和获得性耐药类器官中的蛋白质组和磷酸化蛋白质组变化， PDX，同时追求领先的候选人TROP 2和新的目标提名的遗传，机械和 药理学研究最后，项目1已经鉴定了MIF/CD 74/CD 44和CD 47/SIRPa肿瘤成纤维细胞- 巨噬细胞途径在AR期间上调，然后在Aim 3中使用ALI类器官对其进行功能探索 从AR临床活检和PDX肿瘤。总的来说，我们提出了一个全面的，翻译的方法， 利用互补的类器官和蛋白质组学专业知识直接研究NSCLC的活检和PDX 对靶向治疗具有获得性耐药性的患者，以开发改进的治疗方法。

PROJECT SUMMARY The overall goal of Project 3 of this U54 application is the organoid-based definition of mechanisms of acquired resistance (AR) to targeted therapy in non-small cell lung cancer (NSCLC). The treatment of NSCLC employs numerous therapies directed against the EGFR, ALK, KRAS G12C, ROS1 and PD-1/PD-L1 pathways. However, despite initial responses, these molecularly-targeted therapies are not curative, and virtually all patients eventually succumb to progressive disease. Acquired resistance features prominently in relapse, such as with EGFR T790M or C797S mutations following gefitinib or osimertinib treatment; however, known AR mechanisms cannot be identified for a significant proportion of patients receiving targeted therapy. Further compounding this dilemma are the distinct contributions of tumor-intrinsic versus tumor microenvironmental (TME) influences. The TME in particular contains extracellular matrix (ECM), fibroblast, vascular and immune components that can vitally impact the development and manifestations of AR. The study of TME-dependent and -independent mechanisms of AR have been further hindered by a distinct lack of in vitro human experimental systems allowing holistic recapitulation of the TME in patient biopsies. Our application leverages the synergistic strengths of the Sourav Bandyopadhyay and Calvin Kuo groups in organoid and proteomics technology to address mechanisms of EGFR and KRAS G12C AR in NSCLC. We conduct advanced proteomic approaches and organoid culture both from primary tumor biopsies at acquired resistance (from Project 1) as well as from patient-derived xenograft (PDX) tumors grown in human immune reconstituted mice (from Project 2). Our organoids are either “epithelial-only” or novel air-liquid interface (ALI) organoid cultures that co-preserve tumor epithelium en bloc with tumor-infiltrating fibroblasts and immune subsets (T, B, NK, myeloid). Aim 1 leverages the results of a NSCLC organoid CRISPR screen to determine if osimertinib-sensitizing loci can overcome, delay or alter acquired resistance in clinical biopsy/PDX AR organoids, emphasizing hits with TME mechanisms. Aim 2 uses our mass spectrometry platforms to characterize total proteome and phospho-proteomic changes in treatment-naive, residual and acquired resistance organoids and PDX, while pursuing lead candidate TROP2 and new target nominations by genetic, mechanistic and pharmacologic studies. Lastly, Project 1 has identified MIF/CD74/CD44 and CD47/SIRPa tumor-fibroblast- macrophage pathways upregulated during AR which are then functionally explored in Aim 3 using ALI organoids from AR clinical biopsies and PDX tumors. Overall, we propose a comprehensive, translational approach that exploits complementary organoid and proteomics expertise to directly study biopsies and PDX from NSCLC patients with acquired resistance to targeted therapy, towards development of improved therapeutic approaches.