Three-dimensional organoid models to study breast cancer progression

研究乳腺癌进展的三维类器官模型

• 批准号: 10438709
• 负责人: Shilpa Sant
• 金额: $ 41.88万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10438709
• 关键词: 3-Dimensional; Address; Agreement; Biomedical Engineering; Breast; Breast Cancer Patient; Cancer cell line; Carcinoma in Situ; Cause of Death; Cell Line; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Coupled; Data; Development; Diagnosis; Drug Screening; E-Cadherin; Engineering; Epidermal Growth Factor Receptor; Experimental Models; Fibronectins; Gene Expression Profile; Genes; Genetic; Heterogeneity; Hypoxia; Image; Image Analysis; In Vitro; Individual; Knock-in; Knowledge; Label; Lead; Left; Link; Maintenance; Malignant - descriptor; Malignant Neoplasms; Mammography; Matrix Metalloproteinases; Metabolic stress; Metastatic breast cancer; Modeling; Neoplasm Metastasis; Noninfiltrating Intraductal Carcinoma; Organoids; Outcome; Parents; Pathway interactions; Patients; Peripheral; Phenotype; Pleural effusion disorder; Prognostic Marker; Proteins; Reproducibility; Resolution; Risk; Sampling; Signal Pathway; Signal Transduction; Site; Spatial Distribution; Stimulus; Stress; Stromal Neoplasm; System; Testing; Therapeutic; Time; Vimentin; Woman; Work; automated image analysis; base; breast cancer progression; cell motility; clinically relevant; confocal imaging; deep learning; deep learning algorithm; design; effective therapy; gene regulatory network; genetic signature; genomic profiles; imaging approach; improved; in vitro Model; in vivo; infiltrating duct carcinoma; innovation; malignant breast neoplasm; migration; neoplastic cell; new therapeutic target; novel; overtreatment; paracrine; premalignant; prevent; therapy development; treatment strategy; tumor; tumor heterogeneity; tumor hypoxia; tumor progression

Approximately 20% of breast cancers detected through mammography are pre-invasive Ductal Carcinoma in situ (DCIS). If left untreated, approximately 20-50% of DCIS will progress to more deadly Invasive Ductal Carcinoma (IDC). No prognostic biomarkers can reliably predict the risk of progression from DCIS to IDC. Similar genomic profiles of matched pre-invasive DCIS and IDC suggests that the progression is not driven by genetic aberrations in DCIS cells, but microenvironmental factors, such as hypoxia and metabolic stress prevalent in DCIS, may drive the transition. We need innovative models to investigate how to halt steps of DCIS progression to invasive phenotypes and subsequent metastasis from the primary site. This proposal directly addresses this unmet need by developing a novel three-dimensional in vitro organoid model that recapitulates key hallmarks of DCIS to IDC progression: tumor-size induced hypoxia and metabolic stress, tumor heterogeneity and spontaneous emergence of migratory phenotype in the same parent cells without any additional stimulus. A tangible advantage of the proposed organoid models is the ability to precisely and reproducibly study how the hypoxic microenvironment induces tumor migration in real time and in isolation from non-tumor cells present in vivo, providing unique opportunity to define tumor-intrinsic mechanisms of DCIS to IDC progression. Our preliminary observations lead to central hypothesis that tumor size-induced hypoxia establishes a “hypoxic secretome”, which initiates the migratory phenotype; the hypoxic secretome then cooperate with intracellular signaling networks to independently maintain cell migration. We propose three independent but inter-related aims to link hypoxic secretome with the initiation, maintenance and spatial distribution of migratory phenotypes. Aim 1 will engineer size-controlled DCIS organoids (150-600 µm) with controlled hypoxic microenvironments to identify and examine how hypoxic secretome initiates migratory phenotype. We will combine experimental organoid models with time-lapse imaging and computational approaches to study organoid migration. Aim 2 will demonstrate that migratory cells can re-establish the secretome and maintain migratory phenotype independent of hypoxia. We will reconstruct an intracellular signaling network activated by the hypoxic secretome using microarray data. We will verify these gene expression signatures in sorted migratory and non-migratory cells, and validate them using secretome inhibition studies. Aim 3 will investigate, for the first time, the spatial distribution and origin of the migratory phenotype. We will use CRISPR-based gene knock-in (FP-labeling), automated image analyses, and a deep-learning algorithm to track and visualize the emergence of migratory phenotypes from the hypoxic core outward to the periphery or from the migratory front. The successful development of this 3D organoid model and completion of the proposed work will provide answers to two fundamental questions in the progression of invasive breast cancer: 1) What causes some DCIS cells to become migratory and develop into invasive tumors? 2) How and where does the migratory phenotype (IDC) emerge? The mechanistic understanding gained from these studies will improve diagnosis, lead to the development of treatment strategies to arrest invasion at the pre-malignant stage, and thus prevent patient overtreatment. It is straightforward to generalize our system to other tumor types, development of tumor/stromal co-culture, and drug screening.

通过乳房X线摄影检测到的大约20％的乳腺癌是侵入性导管癌 原位（DCIS）。如果未经治疗，大约20-50％的DCI将发展为更致命的侵入性导管 癌（IDC）。没有预后的生物标志物可以可靠地预测从DCI到IDC的发展风险。相似的 匹配的侵入前DCI和IDC的基因组轮廓表明，进展不是由遗传驱动的 DCIS细胞中的畸变，但微环境因素（例如缺氧和代谢应激）普遍存在 DCIS，可能会驱动过渡。我们需要创新的模型来研究如何停止DCIS进展的步骤 从主要部位进行侵入性表型和随后的转移。该建议直接解决 通过开发一种新型的三维体外器官模型来概括钥匙，这种未满足的需求 DCI到IDC进展的标志：肿瘤大小的诱导缺氧和代谢应激，肿瘤异质性 以及同一母体细胞中迁移表型的发起效果，而没有任何额外的刺激。一个 所提出的类器官模型的切实优势是能够精确，可重复研究如何如何研究 低氧微环境可实时诱导肿瘤迁移，并从存在于中的非肿瘤细胞中分离 Vivo，提供了独特的机会来定义DCIS的肿瘤内部机制为IDC进展。我们的 初步观察结果导致中心假设，即肿瘤大小诱导的缺氧会形成“缺氧 分泌组”，启动迁移表型；低氧分泌组随后与细胞内合作 信号网络独立维护细胞迁移。我们提出了三个独立但相互关联的 旨在将低氧分泌组与迁移表型的维持和空间分布联系起来。 AIM 1将使用受控的低氧微环境来设计尺寸控制的DCIS器官（150-600 µm） 识别并检查低氧分泌组如何启动迁移表型。我们将结合实验 带有延时成像的器官模型和研究器官迁移的计算方法。 AIM 2意志 证明迁徙细胞可以重新建立分泌组并保持迁移表型独立 缺氧。我们将重建使用低氧分泌组激活的细胞内信号网络 微阵列数据。我们将在分类的迁移和非迁移细胞中验证这些基因表达特征， 并使用分泌组抑制研究对其进行验证。 AIM 3将首次调查空间 迁移表型的分布和起源。我们将使用基于CRISPR的基因敲入（FP标记）， 自动图像分析和一种深入学习算法，以跟踪和可视化迁移的出现 表型从低氧核心向外到外周或从迁徙前沿。 这个3D器官模型的成功开发和拟议工作的完成将提供 侵入性乳腺癌进展中两个基本问题的答案：1）导致某些DCIS的原因 细胞迁移并发展为侵入性肿瘤？ 2）迁移表型的方式和何处 （IDC）出现？从这些研究中获得的机械理解将改善诊断，导致 制定治疗策略以阻止恶性阶段的入侵，从而防止患者 过度治疗。将我们的系统概括为其他肿瘤类型，肿瘤/基质的发展是很简单的 共培养和药物筛查。