Organoid Acquired Resistance

项目总结 本U54申请的项目3的总体目标是基于有机物的获得性机制的定义 非小细胞肺癌(NSCLC)对靶向治疗的耐药性(AR)。非小细胞肺癌的治疗采用 针对EGFR、ALK、KRAS G12C、ROS1和PD-1/PD-L1通路的多种治疗方法。然而， 尽管最初有反应，但这些分子靶向治疗并不是治愈的，而且几乎所有的患者 最终死于进行性疾病。获得性耐药是复发的显著特征，例如 吉非替尼或奥西美替尼治疗后EGFR T790M或C797S突变；然而，已知的AR机制 对于接受靶向治疗的相当大比例的患者来说，无法确定。进一步加剧了这一点 两难境地是肿瘤内在与肿瘤微环境(TME)影响的不同贡献。这个 TME尤其含有细胞外基质(ECM)、成纤维细胞、血管和免疫成分，这些成分可以 对AR的发展和表现有至关重要的影响。TME依赖与非依赖的研究 AR的机制由于明显缺乏体外人体实验系统而进一步受阻 患者活检组织中TME的整体回顾。 我们的应用程序利用了Sourav Bandyopadhyay和Calvin Kuo集团的协同优势 在有机体和蛋白质组学技术方面，研究非小细胞肺癌中EGFR和KRAS G12C AR的机制。我们 进行先进的蛋白质组学方法和有机物培养，均来自获得性原发肿瘤活检组织 抗药性(来自项目1)以及来自患者来源的异种移植瘤(PDX)在人类免疫中生长 重组小鼠(摘自项目2)。我们的有机类物质要么只存在于表面，要么具有新颖的气液界面(ALI)。 与肿瘤浸润性成纤维细胞和免疫共保存肿瘤上皮的有机培养物 亚群(T、B、NK、髓系)。AIM 1利用NSCLC有机物CRISPR筛查的结果来确定 奥西美替尼增敏基因可以克服、延迟或改变临床活检/PDX AR有机物的获得性耐药， 强调使用TME机制进行命中。Aim 2使用我们的质谱学平台来表征Total 治疗初期、残留和获得性耐药有机物的蛋白质组和磷酸蛋白质组变化 PDX，同时追求领先候选人TROP2和新的目标提名通过遗传，机械和 药理学研究。最后，项目1已经确定了MIF/CD74/CD44和CD47/SIRPA肿瘤成纤维细胞- 巨噬细胞通路在AR期间上调，然后在AIM 3中使用ALI类有机物进行功能探索 来自AR临床活检和PDX肿瘤。总体而言，我们提出了一种全面的翻译方法， 利用互补的有机体和蛋白质组学专业知识直接研究非小细胞肺癌的活检组织和PDX 对靶向治疗产生获得性抵抗力的患者，转向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.