Deconstructing the collective invasion pack to define Myo10 function

项目摘要 集体侵袭是在大多数实体瘤类型的患者中观察到的主要转移模式。如何 集体入侵包作为一个单一的内聚单位运作，通信和导航仍然不清楚。到 为了解决这个问题，我们发表了一个图像引导的基因组学平台，以分离集体中的任何活细胞。 入侵包装，并扩大人口的基因组和分子分析，一种技术称为 时空细胞和基因组分析（佐贺）。我们使用佐贺来剖析分子，表观遗传， 作为一个层次的凝聚性单位入侵的领导者和追随者细胞的基因组图谱。以确定如何 表观遗传重编程驱动了这种表型异质性，我们解构了集体入侵包， 使用佐贺，然后整合全基因组启动子甲基化和转录组数据， 在领导者和追随者表型内的甲基化区域。我们观察到领导者的全球表观基因组重新布线 细胞支持表观遗传基础的表型异质性内的集体入侵包。然后我们 发现Myo 10（myosinX）是一个甲基化和表达差异最大的基因， 启动子是低甲基化的，并且几种肺癌细胞系中的前导基因过表达Myo 10。Myo 10是一个典型的 丝状伪足伸长的调节因子，我们发现它驱动丝状伪足伸长，集体入侵，领导细胞驱动 纤连蛋白微图案化（纤维形成），并由Jag 1/Notch转录激活。我们将使用这个 信息，以测试具有总体假设的机制模型，即Myo 10通过启动子激活 前导细胞中的低甲基化驱动纤连蛋白的基于丝状伪足的微图案化， 我们认为，这导致肺癌细胞的侵袭优势， 转移性疾病在目标1中，我们测试了前导序列中Myo 10低甲基化允许Jag 1/Notch 1- 驱动转录激活、驱动丝状伪足伸长和纤连蛋白微图案化。在目标2中，我们测试 使用体内转移模型和第一个患者，这种集体侵袭途径如何影响转移- 衍生的前导细胞。在整个过程中，我们利用这里开发的独特资源，包括SaGA衍生的细胞 细胞系、离体成像和患者来源的肺癌前导细胞。我们推测这些数据将提供 集体侵袭机制及其对理解肺癌患者的转化价值 领导细胞生物学。

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.