Deconstructing the collective invasion pack to define Myo10 function

项目摘要 集体侵袭是在大多数实体肿瘤类型患者中观察到的主要转移方式。怎么了？ 集体入侵包作为一个单一的凝聚力单元进行操作、通信和导航仍不清楚。至 针对这一问题，我们在一个图像引导的基因组学平台上发表了分离集体中任何活细胞的方法(S) 入侵包装，并扩大种群进行基因组和分子分析，这项技术被称为 时空细胞和基因组分析(SAGA)我们用SAGA分析了分子、表观遗传学和 作为一个等级凝聚单位入侵的领导者和追随者细胞的基因组图谱。要确定如何 表观遗传重新编程驱动了这种表型异质性，我们解构了集体入侵包 使用SAGA，然后整合全基因组启动子甲基化和转录组数据来定义差异 领导者和追随者表型中的甲基化区域。我们观察到Leader中的全球表观基因组重新布线 支持集体侵袭包内表型异质性的表观遗传学基础的细胞。然后我们 发现Myo10(MyosinX)是一个甲基化和表达最高的基因，其中领头细胞 启动子是低甲基化的，在几个肺癌细胞系中的领导者过表达Myo10。Myo10是一个典型的 丝状孢子伸长的调节器，我们发现它驱动丝状孢子伸长，集体入侵，先导细胞驱动 纤维连接蛋白微图案化(纤维形成)，并被Jag1/Notch转录激活。我们将使用这个 用Myo10通过启动子激活的主要假设测试机制模型的信息 前导细胞中的低甲基化驱动纤维连接蛋白的丝状足微图案化，以创建由前导细胞驱动的前导细胞 集体入侵之路。我们认为，这导致了肺癌细胞的侵袭优势，从而导致 转移性疾病。在目标1中，我们测试了Leaders中Myo10低甲基化允许Jag1/Notch1- 驱动转录激活，驱动丝状足伸长，以及纤维连接蛋白微图案化。在目标2中，我们测试 利用体内转移模型和第一位患者，这种集体侵袭途径如何影响转移- 派生的引线细胞。自始至终，我们利用这里开发的独特资源，包括佐贺衍生的细胞 细胞系、体外成像和患者来源的肺癌先导细胞。我们推测这些数据将提供 对集体侵袭的机械洞察和对了解肺癌患者的翻译价值 领头羊细胞生物学。

Project Summary Collective invasion is a major mode of metastasis observed in patients across most solid tumor types. How the collective invasion pack operates, communicates, and navigates as a single cohesive unit remains unclear. To address this, we published on an image-guided genomics platform to isolate any living cell(s) within a collective invasion pack, and expand the population for genomic and molecular analysis, a technique termed Spatiotemporal Cellular & Genomic Analysis (SaGA). We used SaGA to dissect the molecular, epigenetic, and genomic profiles of leader and follower cells invading as a hierarchical cohesive unit. To determine how epigenetic reprogramming drives this phenotypic heterogeneity, we deconstructed the collective invasion pack using SaGA, then integrated genome-wide promoter methylation and transcriptome data to define differentially methylated regions within the leader and follower phenotypes. We observe global epigenomic re-wiring in leader cells supporting an epigenetic basis for the phenotypic heterogeneity within the collective invasion pack. We then identified Myo10 (myosinX) as a top differentially methylated and expressed gene, where the leader cell promoter is hypomethylated, and leaders in several lung cancer lines overexpress Myo10. Myo10 is a canonical modulator of filopodia elongation and we show it drives filopodia elongation, collective invasion, leader cell-driven fibronectin micropatterning (fibrillogenesis), and is transcriptionally activated by Jag1/Notch. We will use this information to test a mechanistic model with the overarching hypothesis that Myo10 activation via promoter hypomethylation in leader cells drives filopodia-based micropatterning of fibronectin to create a leader cell-driven collective invasion path. We propose that this leads to an invasive advantage for lung cancer cells resulting in metastatic disease. In Aim 1 we test the model that Myo10 hypomethylation in leaders allows for Jag1/Notch1- driven transcriptional activation, driving filopodia elongation, and fibronectin micropatterning. In Aim 2 we test how this collective invasion pathway impacts metastasis using in vivo metastasis models and the first patient- derived leader cells. Throughout, we leverage unique resources developed here including SaGA-derived cell lines, ex vivo imaging, and patient-derived lung cancer leader cells. We speculate that these data will provide mechanistic insight into collective invasion and translational value towards understanding lung cancer patient leader cell biology.