Three-dimensional organoid models to study breast cancer progression

在通过乳房X光检查发现的乳腺癌中，大约20%是浸润性导管癌前期癌。 SITE(DCIS)。如果不治疗，大约20%-50%的DCIS将进展为更致命的侵袭性导管 癌症(IDC)。没有预测预后的生物标记物可以可靠地预测从DCIS到IDC的进展风险。类似 匹配的侵袭前DCIS和IDC的基因组图谱表明，进展不是由基因驱动的 DCIS细胞中存在异常，但微环境因素，如低氧和代谢应激，在 DCIS可能会推动这一转变。我们需要创新的模型来研究如何阻止DCIS进展的步伐 与侵袭性表型和随后的原发部位转移有关。这项提案直接针对 通过开发一种新的三维体外有机模型来满足这一尚未满足的需求 DCIS对IDC进展的特征：肿瘤大小引起的缺氧和代谢应激，肿瘤的异质性 在没有任何额外刺激的情况下，在同一亲本细胞中自发出现迁移表型。一个 所提出的有机物模型的实际优势是能够精确和可重复性地研究 低氧微环境实时诱导肿瘤迁移，并与非肿瘤细胞隔离存在 体内，提供了独特的机会，以确定肿瘤的内在机制的DCIS的IDC进展。我们的 初步观察导致了一个中心假设，即肿瘤大小引起的低氧建立了一种 分泌体“，启动迁移表型；然后低氧分泌体与细胞内协同 独立维持细胞迁移的信令网络。我们提出了三个独立但又相互关联的 旨在将低氧分泌组与迁徙表型的启动、维持和空间分布联系起来。 AIM 1将设计尺寸受控的DCIS有机化合物(150-600微米)和受控的低氧微环境，以 鉴定和研究低氧分泌体如何启动迁移表型。我们将结合实验 用时间推移成像和计算方法研究有机物迁移的有机物模型。目标2将 证明迁移细胞可以重建分泌体并保持迁移表型独立 缺氧的症状。我们将重建一个由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， 并使用分泌体抑制研究来验证它们。目标3将首次调查空间 迁徙表型的分布和起源。我们将使用基于CRISPR的基因敲入(FP标记)， 自动图像分析和深度学习算法，可跟踪和可视化迁徙的出现 表型从低氧核心向外向外或从迁徙前沿。 这一3D有机模型的成功开发和拟议工作的完成将提供 浸润性乳腺癌进展过程中两个基本问题的答案：1)是什么导致了某些DCIS 细胞会发生迁移并发展为侵袭性肿瘤？2)迁移表型是如何发生以及在哪里发生的？ (IDC)涌现？从这些研究中获得的机械性理解将改善诊断，导致 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。很容易将我们的系统推广到其他肿瘤类型、肿瘤/间质的发展 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.