球体已经成为研究癌症的主要三维模型,发展过程,和药物功效。单细胞分析技术已成为衡量这些模型中细胞反应复杂性的理想工具。然而,基于荧光标记物亚细胞分布的三维显微数据进行单细胞定量评估,如转录因子的核/细胞质比,在很大程度上仍然难以捉摸。对于球体生成,超低连接板由于其简单而值得注意,与自动化的兼容性,以及实验和商业可达性。然而,目前尚不清楚板块类型是否以及在多大程度上影响球体的形成和生物学。这项研究开发了一种新颖的基于AI的管道,用于分析整体上光学清除的大球体的3D共聚焦数据,单细胞,和亚细胞水平。为了确定管道的相关样品,采用自动明场显微镜,使用四种不同的人类细胞系,系统地比较了在六种不同平板类型中形成的球体的大小和偏心率。这表明所有平板类型都表现出相似的球状体形成能力,并且在播种后4天内的总体生长或收缩模式是可比的。然而,大小和偏心率在特定细胞系和平板类型之间系统变化。根据这个预选,进一步评估HaCaT角质形成细胞和HT-29癌细胞的球状体。在HaCaT球体中,深入分析揭示了球体大小之间的相关性,细胞增殖,和转录共激活因子的核/细胞质比,YAP1,以及与细胞分化的逆相关。这些发现,用球体模型和单细胞水平产生,证实了YAP1在人皮肤角质形成细胞的细胞增殖和分化中的作用的早期概念。Further,结果表明,板类型可能会影响实验活动的结果,建议在特定调查期间扫描不同的板类型以获得最佳配置。
Spheroids have become principal three-dimensional models to study cancer, developmental processes, and drug efficacy. Single-cell analysis techniques have emerged as ideal tools to gauge the complexity of cellular responses in these models. However, the single-cell quantitative assessment based on 3D-microscopic data of the subcellular distribution of fluorescence markers, such as the nuclear/cytoplasm ratio of transcription factors, has largely remained elusive. For spheroid generation, ultra-low attachment plates are noteworthy due to their simplicity, compatibility with automation, and experimental and commercial accessibility. However, it is unknown whether and to what degree the plate type impacts spheroid formation and biology. This study developed a novel AI-based pipeline for the analysis of 3D-confocal data of optically cleared large spheroids at the wholemount, single-cell, and sub-cellular levels. To identify relevant samples for the pipeline, automated brightfield microscopy was employed to systematically compare the size and eccentricity of spheroids formed in six different plate types using four distinct human cell lines. This showed that all plate types exhibited similar spheroid-forming capabilities and the gross patterns of growth or shrinkage during 4 days after seeding were comparable. Yet, size and eccentricity varied systematically among specific cell lines and plate types. Based on this prescreen, spheroids of HaCaT keratinocytes and HT-29 cancer cells were further assessed. In HaCaT spheroids, the in-depth analysis revealed a correlation between spheroid size, cell proliferation, and the nuclear/cytoplasm ratio of the transcriptional coactivator, YAP1, as well as an inverse correlation with respect to cell differentiation. These findings, yielded with a spheroid model and at a single-cell level, corroborate earlier concepts of the role of YAP1 in cell proliferation and differentiation of keratinocytes in human skin. Further, the results show that the plate type may influence the outcome of experimental campaigns and that it is advisable to scan different plate types for the optimal configuration during a specific investigation.