Three-dimensional organoid models to study breast cancer progression

通过乳房X线摄影检测到的乳腺癌中约有20%是浸润前导管癌， 原位（DCIS）。如果不及时治疗，大约20-50%的DCIS会发展为更致命的浸润性导管癌。 癌（IDC）。没有预后生物标志物可以可靠地预测从DCIS进展到IDC的风险。类似 匹配的浸润前DCIS和IDC的基因组图谱表明，进展不是由遗传驱动的， DCIS细胞中的畸变，但微环境因素，如缺氧和代谢应激普遍存在， DCIS可能会推动转型。我们需要创新模型来研究如何阻止DCIS进展的步骤 侵袭性表型和随后的原发部位转移。该提案直接针对 通过开发一种新的三维体外类器官模型， DCIS进展为IDC的标志：肿瘤大小诱导的缺氧和代谢应激，肿瘤异质性 以及在没有任何额外刺激的情况下在相同亲本细胞中自发出现迁移表型。一 所提出的类器官模型的明显优势是能够精确和可重复地研究细胞如何在体内生长。 缺氧微环境诱导肿瘤在真实的时间内迁移，并与存在于 体内，提供了独特的机会来定义DCIS到IDC进展的肿瘤内在机制。我们 初步的观察导致了一个中心假设，即肿瘤大小诱导的缺氧建立了一个“缺氧 分泌组”，它启动迁移表型;缺氧分泌组，然后与细胞内 信令网络以独立地维持细胞迁移。我们提出三个独立但相互关联的 旨在将缺氧分泌组与迁移表型的启动、维持和空间分布联系起来。 Aim 1将设计尺寸可控的DCIS类器官（150-600 µm），并控制缺氧微环境， 鉴定和检测低氧分泌体如何启动迁移表型。我们将联合收割机 利用延时成像和计算方法研究类器官迁移的类器官模型。目标2将 证明迁移细胞可以重新建立分泌组并保持迁移表型独立 缺氧。我们将利用细胞内信号转导系统重建由缺氧分泌蛋白激活的细胞内信号转导网络。 微阵列数据。我们将在分选的迁移和非迁移细胞中验证这些基因表达特征， 并使用分泌抑制研究来验证它们。目标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，以及深度学习算法，以跟踪和可视化迁移的出现， 表型从缺氧核心向外到周边或从迁移前沿。 这个3D类器官模型的成功开发和拟议工作的完成将提供 对浸润性乳腺癌进展中的两个基本问题的回答：1）是什么导致了一些DCIS 细胞迁移并发展成侵袭性肿瘤？2)迁移表型如何以及在何处 (IDC)出现？从这些研究中获得的机制理解将改善诊断， 制定治疗策略，在癌前阶段阻止侵袭，从而防止患者 过度治疗将我们的系统推广到其他肿瘤类型，肿瘤/间质细胞的发展是直接的。 共培养和药物筛选。

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.