Three-dimensional organoid models to study breast cancer progression

摘要 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。 在2018年7月至2022年2月ESI功勋获得期内，我们已经表明，抑制肿瘤分泌因子 有效地阻止有机物的迁移，而抑制缺氧只在一定的时间窗口内有效，而且 受肿瘤到肿瘤差异的影响，支持了我们的观点，即缺氧启动了迁移表型，但 并不能维持这种状态。我们还分析了转移性乳腺癌胸腔积液的分泌物。 CCL2/MCP1、CXCL10/IP10、IL-6、IL-8、调节性IL-10、IL-7和IL-15水平显著升高。 中和这些关键细胞因子的策略可能会在胸膜环境中产生抗肿瘤反应。 低氧诱导迁移和分泌体诱导迁移的微阵列分析提示Rho的作用 GTPase和PI3K/AKT信号通路在维持迁移中的作用。我们的结果表明，低氧有机物 模型早在第一天就表现出部分EMT特征，并以这些非迁移性器官的形式保留下来 向迁徙表型的转变。 在为期两年的延展期内，我们将继续1)优化我们的DCIS模型，包括Dductal 来自DCIS微环境的结构和其他成分；2)测试与肿瘤内在联系的新机制 低氧，部分/混合EMT和集体迁移；3)抑制信号机制，阻止 迁徙表型。 拟议工作的圆满完成将为 浸润性乳腺癌的进展：1)是什么原因导致某些DCIS细胞迁移并发展为 侵袭性肿瘤？2)迁移表型(IDC)是如何以及在哪里出现的？机械论 从这些研究中获得的理解将改善诊断，导致治疗策略的发展 在癌前阶段阻止侵袭，从而防止患者过度治疗。

Abstract 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. During July 2018-Feb 2022 ESI MERIT Award period, we have shown that inhibition of tumor-secreted factors effectively halts organoid migration, while inhibition of hypoxia is effective only within a time window and is compromised by tumor-to-tumor variation, supporting our notion that hypoxia initiates migratory phenotypes but does not sustain it. We have also analyzed secretome from metastatic breast cancer pleural effusion showing significantly higher levels of CCL2/MCP1, CXCL10/IP10, IL-6, IL-8, regulatory IL-10, and IL-7 and IL-15. Strategies to neutralize these key cytokines may generate anti-tumor responses in the pleural environment. Microarray analysis of hypoxia-induced migration and secretome-induced migration suggested role of Rho GTPase and PI3K/AKT signaling pathways in maintaining migration. Our results show that hypoxic organoid models exhibit partial EMT signatures as early as day 1, which is maintained as these non-migratory organoids transition to migratory phenotypes. During the two-year extension period, we will continue 1) to optimize our DCIS models incorporating ductal structure and other components from DCIS microenvironments; 2) to test new mechanisms linking tumor-intrinsic hypoxia, partial/hybrid EMT and collective migration; 3) to inhibit signaling mechanisms to halt emergence of migratory phenotypes. The successful 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.