To study the EMT contributions in tumor metastasis and chemoresistance by using lineage tracing models

利用谱系追踪模型研究 EMT 在肿瘤转移和化疗耐药中的贡献

• 批准号: 10651750
• 负责人: Dingcheng Gao
• 金额: $ 47.92万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-17 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651750
• 关键词: Apoptosis; BT 474; Bar Codes; Blood; Breast Cancer Cell; Breast Cancer Model; Breast cancer metastasis; CCL7 gene; CRISPR/Cas technology; Cell Line; Cells; Chemoresistance; Circulation; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Development; Disease; E-Cadherin; Embryonic Development; Epithelium; Event; Exhibits; Fluorescence; Genes; Genetic; Homing; Human; Individual; Invaded; Knock-in; Lung; MDA MB 231; Mediating; Mesenchymal; Mesenchymal Cell Neoplasm; Metastatic Neoplasm to the Lung; Modeling; Mouse Mammary Tumor Virus; Mus; Nature; Neoplasm Metastasis; Phenotype; Primary Neoplasm; Process; Property; Recurrence; Reporter; Reporting; Resistance; Role; Site; Snails; Transgenic Mice; Vimentin; chemotherapy; epithelial to mesenchymal transition; improved; in vivo; migration; neoplastic cell; novel; polyoma middle tumor antigen; programs; stemness; transcription factor; transdifferentiation; tumor; tumor progression

Epithelial to mesenchymal transition (EMT) has been enthusiastically proposed as an essential mechanism for tumor metastasis, since the EMT-associated features such as migration, invasion, resistance to apoptosis and stemness properties, adequately meet the requirements for metastasis. Taken the challenges of tracing the EMT process in vivo, we developed a strategy of using a mesenchymal-specific Cre-mediated switch of fluorescent markers in a multiple-transgenic mouse (MMTV-PyMT:Fsp1-Cre:Rosa26mT/mG, Tri-PyMT). Surprisingly, we found that lung metastases were predominantly composed of pre-EMT RFP+ tumor cells exhibiting epithelial phenotypes under normal conditions. Importantly, the post-EMT tumor cells did exhibit resistance to chemotherapy, significantly contributed to recurrent lung metastases after chemotherapy. These findings pointed to the complexity of EMT contributions in tumor progression and revived vigorous discussions in the community. Given the transient, reversible and dynamic natures of the EMT process, and concerns about the Tri-PyMT model, we proposed new lineage tracing models to study the roles of EMT in metastasis and chemoresistance. Aim 1. To explore the contributions of EMT mechanism in tumor metastasis and chemoresistance by using Snail-CreERT2 mediated EMT lineage tracing model. The Fsp1-Cre mediated Tri-PyMT model may not be sufficient to report all EMT events. Metastatic cells could undergo EMT by activating distinct EMT transcription factors (TFs) such as Snail. Therefore, we have established a Snail-CreERT2–mediated EMT lineage tracing model. In-depth analyses will be performed to clarify the roles of EMT in metastasis and chemoresistance with this model. Aim 2. To explore the dynamic changes of EMT statuses in human breast cancer metastasis and chemoresistance. EMT reporter cell lines (MDA-MB-231:Vim/RFP and BT-474:ECAD/GFP cells) carry knockin fluorescent tags within EMT marker genes (Vimentin and E-cadherin, respectively). Unlike the Cre-mediated models, the fluorescence expression in these cells is quantitative and reversible, which allows us to analyze the dynamic changes of EMT status with and without chemotherapy in human breast cancer models. Aim 3. To assess the evolutionary lineages of metastasis-initiating cells and the involvement of EMT mechanism via genetic barcoding models. In addition to using EMT markers, we will genetically barcode the Tri-PyMT cells using the homing-CRISPR technique. This model will allow us to depict the evolutionary trajectories from primary tumor cells to individual metastases, and determine the origins of the metastasis (pre- EMT vs. post-EMT cells). Further, we will develop genetic barcoding mice (MARC:CRISPR:PyMT) for lineage tracing of spontaneous metastatic cells and assessing the contributions of the EMT program to metastasis. Impact: Resolving the controversies in the field will not only improve our mechanistic understanding of tumor metastasis but also provide novel targets/opportunities in combatting the deadly disease.

上皮到间质转化（EMT）已被热情地提出作为一种重要机制 肿瘤转移，因为 EMT 相关的特征，如迁移、侵袭、细胞凋亡抵抗和 干性特性，充分满足转移的要求。应对追踪急救人员的挑战 在体内过程中，我们开发了一种策略，使用间充质特异性 Cre 介导的荧光开关 多重转基因小鼠中的标记（MMTV-PyMT:Fsp1-Cre:Rosa26mT/mG、Tri-PyMT）。令人惊讶的是，我们 发现肺转移主要由 EMT 前 RFP+ 肿瘤细胞组成，表现出上皮细胞 正常条件下的表型。重要的是，EMT 后的肿瘤细胞确实表现出对 化疗，显着导致化疗后肺转移复发。这些发现指出 认识到 EMT 在肿瘤进展中的贡献的复杂性，并在社区中重新引发了激烈的讨论。 鉴于 EMT 过程的瞬态、可逆和动态性质，以及对 Tri-PyMT 的担忧 模型中，我们提出了新的谱系追踪模型来研究 EMT 在转移和化疗耐药中的作用。 目的1.探讨EMT机制在肿瘤转移和化疗耐药中的贡献 使用 Snail-CreERT2 介导的 EMT 谱系追踪模型。 Fsp1-Cre 介导的 Tri-PyMT 模型可能不会 足以报告所有 EMT 事件。转移细胞可以通过激活不同的 EMT 转录来经历 EMT 因子 (TF)，例如 Snail。因此，我们建立了 Snail-CreERT2 介导的 EMT 谱系追踪 模型。将进行深入分析以阐明 EMT 在转移和化疗耐药中的作用 这个模型。 目的2.探讨EMT状态在人类乳腺癌转移和转移过程中的动态变化 化学耐药性。 EMT 报告细胞系（MDA-MB-231:Vim/RFP 和 BT-474:ECAD/GFP 细胞）携带敲入 EMT 标记基因（分别为波形蛋白和 E-钙粘蛋白）内的荧光标签。与 Cre 介导的不同 模型中，这些细胞中的荧光表达是定量且可逆的，这使我们能够分析 在人类乳腺癌模型中，有或没有化疗的 EMT 状态的动态变化。 目标 3. 评估转移起始细胞的进化谱系以及 EMT 的参与 通过遗传条形码模型的机制。除了使用 EMT 标记外，我们还将对 使用归巢 CRISPR 技术的 Tri-PyMT 细胞。这个模型将使我们能够描绘进化过程 从原发肿瘤细胞到个体转移的轨迹，并确定转移的起源（预 EMT 与 EMT 后细胞）。此外，我们将开发用于谱系的遗传条形码小鼠（MARC：CRISPR：PyMT） 追踪自发转移细胞并评估 EMT 计划对转移的贡献。 影响：解决该领域的争议不仅可以提高我们对肿瘤机制的理解 转移，但也提供了对抗这种致命疾病的新目标/机会。