Collective Cell Migration in 3D Epithelial Wound Healing

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 Notch I-D114 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 Notchl-D114 signaling in regulating 3D epithelial wound healing.

集体细胞迁移在发育、伤口愈合和转移过程中起着关键作用，但对于细胞在三维微环境中迁移的机制及协同情况知之甚少。在此，我们展示了一种通过光热消融进行的三维伤口愈合检测方法，用于研究上皮组织结构中的集体细胞迁移。纳米颗粒介导的光热技术产生局部高热以选择性地消融细胞，并诱导三维组织结构的集体细胞迁移。通过结合动态单细胞基因表达分析、活细胞肌动蛋白染色以及粒子图像测速技术，我们发现伤口愈合反应包括朝向伤口的三维涡旋运动，随后形成多细胞肌动蛋白束以及具有活跃肌动蛋白突起的前沿细胞。抑制ROCK信号通路会破坏多细胞肌动蛋白束，并增强前沿前沿细胞的形成。此外，单细胞基因表达分析、药物干预以及RNA干扰表明，Notch I - D114信号负向调节多细胞肌动蛋白束和前沿细胞的形成。综上所述，我们的研究展示了一个用于研究三维集体细胞迁移的平台，并强调了ROCK和Notch1 - D114信号在调节三维上皮伤口愈合中的重要作用。