DataSheet1_A multiparametric analysis including single-cell and subcellular feature assessment reveals differential behavior of spheroid cultures on distinct ultra-low attachment plate types.docx

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.

球状体（Spheroid）已成为研究癌症、发育过程及药物疗效的主流三维模型。单细胞分析技术已成为评估此类模型中细胞应答复杂性的理想工具。然而，基于三维显微数据对荧光标记物（如转录因子的核质比）的亚细胞分布进行单细胞定量评估，在很大程度上仍难以实现。在球状体培养中，超低吸附培养板（ultra-low attachment plate）因其操作简便、适配自动化实验以及实验与商业可及性良好而备受关注。然而，目前尚不明确培养板类型是否以及在何种程度上会影响球状体的形成与生物学特性。本研究开发了一种新型基于人工智能（AI）的分析流程，可在整体标本、单细胞及亚细胞层面分析经光学透明化处理的大型球状体的三维共聚焦数据。为筛选适配该流程的样本，研究采用自动化明场显微镜，系统比较了四种不同人类细胞系在六种不同培养板中形成的球状体的尺寸与偏心率。结果显示，所有培养板类型均具备相近的球状体形成能力，且接种后4天内的整体生长或收缩模式基本一致。但特定细胞系与培养板类型间的球状体尺寸及偏心率存在系统性差异。基于此次预筛选，研究进一步评估了HaCaT角质形成细胞球状体与HT-29癌细胞球状体。在HaCaT细胞球状体中，深度分析揭示了球状体尺寸、细胞增殖与转录辅激活因子YAP1的核质比之间的正相关关系，以及与细胞分化之间的负相关关系。此项基于球状体模型与单细胞层面的研究结果，验证了此前关于YAP1在人类皮肤角质形成细胞增殖与分化中作用的相关理论。此外，研究结果表明，培养板类型可能会影响实验结果，因此在特定研究中，建议对不同培养板类型进行筛选以确定最优配置。