Harvard-MIT Division of Health Sciences and Technology , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States.
Department of Surgery , The Pennsylvania State University , Hershey , Pennsylvania 17033 , United States.
ACS Nano. 2019 Feb 26;13(2):1204-1212. doi: 10.1021/acsnano.8b06305. Epub 2019 Feb 13.
Collective cell migration plays a pivotal role in development, wound healing, and metastasis, but little is known about the mechanisms and coordination of cell migration in 3D microenvironments. Here, we demonstrate a 3D wound healing assay by photothermal ablation for investigating collective cell migration in epithelial tissue structures. The nanoparticle-mediated photothermal technique creates local hyperthermia for selective cell ablation and induces collective cell migration of 3D tissue structures. By incorporating dynamic single cell gene expression analysis, live cell actin staining, and particle image velocimetry, we show that the wound healing response consists of 3D vortex motion moving toward the wound followed by the formation of multicellular actin bundles and leader cells with active actin-based protrusions. Inhibition of ROCK signaling disrupts the multicellular actin bundle and enhances the formation of leader cells at the leading edge. Furthermore, single cell gene expression analysis, pharmacological perturbation, and RNA interference reveal that Notch1-Dll4 signaling negatively regulates the formation of multicellular actin bundles and leader cells. Taken together, our study demonstrates a platform for investigating 3D collective cell migration and underscores the essential roles of ROCK and Notch1-Dll4 signaling in regulating 3D epithelial wound healing.
细胞集体迁移在发育、伤口愈合和转移中起着关键作用，但对于三维微环境中细胞迁移的机制和协调知之甚少。在这里，我们通过光热消融展示了一种用于研究上皮组织结构中细胞集体迁移的三维伤口愈合测定法。纳米颗粒介导的光热技术可产生局部过热，从而选择性地消融细胞，并诱导三维组织结构中的细胞集体迁移。通过整合动态单细胞基因表达分析、活细胞肌动蛋白染色和粒子图像测速，我们表明伤口愈合反应包括向伤口移动的三维涡旋运动，随后形成多细胞肌动蛋白束和具有活跃肌动蛋白突起的先导细胞。抑制 ROCK 信号会破坏多细胞肌动蛋白束，并增强在前沿形成的先导细胞。此外，单细胞基因表达分析、药理学干扰和 RNA 干扰表明 Notch1-Dll4 信号负调节多细胞肌动蛋白束和先导细胞的形成。总之，我们的研究展示了一个用于研究三维细胞集体迁移的平台，并强调了 ROCK 和 Notch1-Dll4 信号在调节三维上皮伤口愈合中的重要作用。
