Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance. Homo sapiens

Current preclinical models in tumor biology are limited in their ability to recapitulate relevant (patho-) physiological processes, including autophagy. Three-dimensional (3D) growth cultures have frequently been proposed to overcome the lack of correlation between two-dimensional (2D) monolayer cell cultures and human tumors in preclinical drug testing. Besides 3D growth, it is also advantageous to simulate shear stress, compound flux and removal of metabolites, e.g. via bioreactor systems, through which culture medium is constantly pumped at a flow rate reflecting physiological conditions. Here, we show that both Staticic 3D growth and 3D growth within a bioreactor system modulate key hallmarks of cancer cells, including proliferation and cell death as well as macroautophagy, a recycling pathway often activated by highly proliferative tumors to cope with metabolic stress. The autophagy-related gene expression profiles of 2D- and 3D-grown cells are substantially different, with the 3D-grown cells exhibiting an expression profile closely resembling the (patho-) physiological Statice of a tumor. Underscoring the importance of this pathway, autophagy-controlling transcription factors, such as TFEB and FOXO3, are upregulated in tumors, and 3D-grown cells have increased expression compared with cells grown in 2D conditions. Three-dimensional cultures depleted of the autophagy mediators BECN1, ATG5 or ATG7 or the transcription factor FOXO3, are more sensitive to cytotoxic treatment. Accordingly, combining cytotoxic treatment with compounds affecting late autophagic flux, such as chloroquine, renders the 3D-grown cells more susceptible to therapy and increases intracellular doxorubicin concentration to the level of 2D-grown cells. Altogether, 3D cultures are a valuable tool to study drug response of tumor cells, as these models recapitulate (patho-) physiologically relevant pathways, such as autophagy. Overall design: Total RNA was isolated from neuroblastoma cell (BE(2)-C) cultures grown under 2D, 3D-Staticic and 3D-bioreactor conditions from three independent experiments and human neuroblastoma tissue samples chosen to resemble characteristics of BE(2)-C cells (each in triplicate)

当前肿瘤生物学领域的临床前模型在重现相关（病理生理）过程（包括自噬）方面存在显著局限。三维（3D）培养体系常被提出用于克服二维（2D）单层细胞培养与临床前药物测试中人体肿瘤相关性不足的问题。除三维培养外，模拟剪切应力、化合物通量及代谢物清除（例如通过生物反应器系统，以贴合生理条件的流速持续泵入培养基）也具备显著优势。本研究证实，静态3D（Staticic）培养及生物反应器系统内的3D培养均可调控癌细胞的关键特征，包括增殖、细胞死亡以及巨自噬——这是一类常被高增殖肿瘤激活以应对代谢应激的回收通路。二维与三维培养细胞的自噬相关基因表达谱存在实质性差异，三维培养细胞的表达谱更贴近肿瘤的（病理生理）状态。进一步凸显该通路重要性的是，自噬调控转录因子（如TFEB与FOXO3）在肿瘤中呈上调状态，且三维培养细胞的相关表达水平高于二维培养条件下的细胞。当敲除自噬介导因子BECN1、ATG5或ATG7，抑或是转录因子FOXO3后，三维培养细胞对细胞毒性治疗的敏感性显著提升。据此，将细胞毒性治疗与影响晚期自噬通量的化合物（如氯喹）联合使用，可使三维培养细胞对治疗更易感，并将细胞内阿霉素浓度提升至二维培养细胞的水平。总而言之，三维培养是研究肿瘤细胞药物反应的宝贵工具，因为这类模型能够重现（病理生理）相关通路，例如自噬。整体实验设计：总RNA提取自3次独立实验中分别于二维、静态3D（Staticic）及生物反应器3D培养条件下生长的神经母细胞瘤细胞（BE(2)-C），同时提取与BE(2)-C细胞特征相似的人神经母细胞瘤组织样本的总RNA，所有样本均设置3次重复。