Development and implementation of multiplex methods to understand the biology and heterogeneity of patient-derived cancer models

开发和实施多重方法来了解源自患者的癌症模型的生物学和异质性

• 批准号: 10004385
• 负责人: William C. Hahn
• 金额: $ 100.49万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-15 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004385
• 关键词: Address; Affect; Aftercare; Archives; Biological Assay; Biological Markers; Biology; Biopsy Specimen; CRISPR library; CRISPR screen; CRISPR/Cas technology; Cancer Biology; Cancer Model; Cancer cell line; Cell Line; Cell Proliferation; Cells; Cellular biology; Clinical Trials; Collection; Communities; Complex; Coupled; Data; Dependence; Development; Disease; Drug resistance; Evaluation; Evolution; Exhibits; Experimental Models; Future; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Genetically Engineered Mouse; Genomic Instability; Genomics; Goals; Heterogeneity; Human; Immunofluorescence Immunologic; Informatics; Investigational Therapies; Kinetics; Libraries; Malignant Neoplasms; Malignant neoplasm of pancreas; Maps; Methodology; Methods; Modeling; Molecular; Molecular Biology; Mutation; Oncogenes; Organoids; Pancreas; Pathway interactions; Patients; Phase; Phenotype; Primary Neoplasm; Property; Proteins; Protocols documentation; Reagent; Research; Resistance profile; Screening for cancer; Signal Transduction; Techniques; Testing; Therapeutic; Therapeutic Agents; Tissue Microarray; Tissues; Validation; Work; analysis pipeline; anticancer research; base; cancer initiation; cancer type; clinically relevant; cost effective; design; drug sensitivity; effective therapy; experience; functional genomics; genetic manipulation; genome-wide; improved; innovation; innovative technologies; insight; loss of function; matrigel; mouse model; new technology; new therapeutic target; next generation; novel; novel marker; novel strategies; novel therapeutics; patient screening; precision oncology; predictive marker; programs; screening; single cell sequencing; single-cell RNA sequencing; small molecule; small molecule therapeutics; technology development; therapeutic target; three dimensional cell culture; transcriptome sequencing; treatment response; tumor; tumor progression

Abstract Experimental models of cancer provide the means both to decipher the molecular basis of cancer and to develop new therapeutic agents. To date, most cancer research has employed established cancer cell lines and genetically engineered mouse models. Although these models have provided tremendous insight into many aspects of cancer initiation and progression, each of these models has important limitations, including adaptation to culture (cell lines), lack of genomic instability (mouse models), and inadequate representation of the spectrum of mutations and subtypes of human cancers. Next generation cancer models (NGCMs) such as organoid models have recently been developed. NGCMs address many deficits of prior models and promise to accelerate cancer research and experimental therapeutic efforts. Recent methodological advances now make it possible to create patient-derived cancer cell lines and organoids with increased efficiency. When coupled with genomic analysis, these new models may facilitate new insights into human cancers. However, organoids require complex culture conditions and display distinct properties that pose challenges for implementation of standard molecular and cell biology techniques. To facilitate widespread use of organoid models within the research community, we must develop innovative technologies to overcome these challenges and enable study of organoids for a range of cancer phenotypes. In this Project, we will build on our expertise in the development of genome scale and informatic methods as well as our work to derive many of the HCMI models with the goal of developing high throughput approaches to perform genetic and small molecule screens in patient-derived organoids created by the Human Cancer Models Initiative (HCMI). In addition, we will use innovative methods to interrogate cell state plasticity and heterogeneity in these models. These studies will allow the cancer research community to perform both high and low throughput analyses in patient-derived models and to provide deep insight into the stability and phenotypes represented by these models. While we will focus our technology development efforts using pancreatic cancer organoids, we anticipate that the approaches developed in this proposal will be widely applicable to many different models from a range of cancer types. In Aim 1, we will develop and implement a highly multiplexed method to screen patient-derived organoid models with both small molecules and genetic reagents. These studies will provide a powerful approach to interrogating HCMI models at high throughput. In Aim 2, we will build on our preliminary studies that indicate that patient-derived organoids exhibit heterogeneity and rapid shifts in expressed phenotypes. We will interrogate the dynamics of these state changes and assess the degrees of heterogeneity in these models using newly developed physical and sequencing methodology. In Aim 3, we will build on Project Achilles and the DepMap (www.DepMap.org) to create and implement an optimized genome scale CRISPR-Cas9 library that permits the systematic genetic interrogation of genetic dependencies in patient-derived organoids. We anticipate that these studies will create new methods that permit rigorous evaluation of HCMI models as well as the discovery of novel biomarkers and therapeutic targets in pancreatic cancer. More broadly, these studies will provide critical proof of principle that these methods can be used by others to study specific phenotypes in next generation cancer models such as organoids.

抽象的 癌症的实验模型提供了破译癌症分子基础和发展的手段 新的治疗剂。迄今为止，大多数癌症研究都采用了既定的癌细胞系，并且 基因工程的鼠标模型。尽管这些模型为许多人提供了深刻的见解 癌症开始和进展的各个方面，这些模型都有重要的局限性，包括适应 培养（细胞系），缺乏基因组不稳定性（小鼠模型）和光谱的表示不足 人类癌症的突变和亚型。下一代癌症模型（NGCM），例如类器官 最近开发了模型。 NGCMS解决了先前模型的许多缺陷，并有望加速 癌症研究和实验性治疗工作。 现在的方法学进步使得创建患者衍生的癌细胞系和 效率提高的器官。当与基因组分析相结合时，这些新模型可能会促进新的新模型 对人类癌症的见解。但是，器官需要复杂的培养条件，并显示出不同的 对实施标准分子和细胞生物学技术构成挑战的特性。到 促进研究社区内的类器官模型的广泛使用，我们必须开发创新 克服这些挑战并能够研究类器官的技术。 在这个项目中，我们将基于我们在基因组量表和信息方法的发展方面的专业知识， 以及我们为推导许多HCMI模型的工作，目的是开发高通量的方法 在由人类癌症模型创建的患者衍生的类器官中执行遗传和小分子筛选 倡议（HCMI）。此外，我们将使用创新方法来询问细胞状态的可塑性和异质性 在这些模型中。这些研究将使癌症研究界能够表现出高和低通量 在患者衍生模型中进行分析，并深入了解由代表的稳定性和表型 这些模型。虽然我们将使用胰腺癌器官重点，但我们 预计本提案中开发的方法将广泛适用于许多不同的模型 一系列癌症类型。 在AIM 1中，我们将开发和实施一种高度多路复用的方法来筛选患者衍生的类器官 具有小分子和遗传试剂的模型。这些研究将为 在高吞吐量时询问HCMI模型。在AIM 2中，我们将基于我们的初步研究，表明 患者衍生的类器官在表达表型中表现出异质性和快速变化。我们会审问 这些状态的动态变化并使用新模型评估异质性的程度 开发了物理和测序方法。在AIM 3中，我们将建立在阿喀琉斯项目和DepMap的基础上 （www.depmap.org）创建和实施优化的基因组规模CRISPR-CAS9库，该库允许 对患者衍生的类器官中遗传依赖性的系统遗传审查。 我们预计这些研究将创建新的方法，以允许对HCMI模型进行严格评估 以及在胰腺癌中发现新型生物标志物和治疗靶标。更广泛地说，这些 研究将提供原则的关键证据，证明其他人可以使用这些方法来研究特定 下一代癌症模型（例如器官）中的表型。