Fluctuations in cell density alter protein markers of multiple cellular compartments, confounding experimental outcomes

The life cycle of cultured proliferating cells is characterized by fluctuations in cell population density induced by periodic subculturing. This leads to corresponding changes in micro- and macroenvironment of the cells, accompanied by altered cellular metabolism, growth rate and locomotion. Studying cell density-dependent morphological, physiological and biochemical fluctuations is relevant for understanding basic cellular mechanisms and for uncovering the intrinsic variation of commonly used tissue culture experimental models. Using multiple cell lines, we found that expression levels of the autophagic markers p62 and LC3II, and lysosomal enzyme cathepsin D were altered in highly confluent cells as a consequence of nutrient depletion and cell crowding, which led to inactivation of the mTOR signaling pathway. Furthermore, both Lamp1 and active focal adhesion kinase (FAK) were reduced in high-density cells, while chemical inhibition or deletion of FAK led to alterations in lysosomal and autophagic proteins, as well as in the mTOR signaling. This was accompanied by alterations in the Hippo signaling pathway, while cell cycle checkpoint regulator p-cdc2 remained unaffected in at least one studied cell line. On the other hand, allometric scaling of cellular compartments in growing cell populations resulted in biochemically detectable changes in the plasma membrane proteins Na+K+-ATPase and cadherin, and nuclear proteins HDAC1 and Lamin B1. Finally, we demonstrate how treatment-induced changes in cell density and corresponding modulation of susceptible proteins may lead to ambiguous experimental outcomes, or erroneous interpretation of cell culture data. Together, our data emphasize the need to recognize cell density as an important experimental variable in order to improve scientific rigor of cell culture-based studies.

体外培养增殖细胞的生命周期，以周期性传代培养（subculturing）诱导的细胞群体密度波动为典型特征。该过程会引发细胞微环境与宏观环境的相应改变，同时伴随细胞代谢、生长速率及运动能力的改变。探究依赖细胞密度的形态学、生理学及生物化学波动，有助于解析细胞的基础生物学机制，同时可揭示常用组织培养（tissue culture）实验模型的内在变异。本研究依托多种细胞系开展实验，发现高度汇合态细胞中，自噬标志物（autophagic markers）p62、LC3II以及溶酶体酶（lysosomal enzyme）组织蛋白酶D（cathepsin D）的表达水平发生改变，这一变化由营养耗竭与细胞拥挤所致，并最终导致哺乳动物雷帕霉素靶蛋白（mTOR）信号通路失活。此外，高密度培养的细胞中，溶酶体相关膜蛋白1（Lamp1）与活化型黏着斑激酶（FAK）的表达水平均有所降低；通过化学抑制或基因敲除FAK，可导致溶酶体与自噬相关蛋白以及mTOR信号通路发生改变。该变化同时伴随Hippo信号通路（Hippo signaling pathway）发生改变，而在至少一种受试细胞系中，细胞周期检查点调控因子p-cdc2的表达未受影响。另一方面，增殖细胞群体中细胞区室（cellular compartments）的异速生长缩放（allometric scaling），可通过生化手段检测到质膜蛋白（plasma membrane proteins）钠钾ATP酶（Na+K+-ATPase）、钙黏蛋白（cadherin）以及核蛋白（nuclear proteins）组蛋白去乙酰化酶1（HDAC1）、核纤层蛋白B1（Lamin B1）的表达变化。最后，本研究阐明了处理诱导的细胞密度改变，以及易感蛋白的相应调控，如何会导致实验结果模棱两可，或是对细胞培养实验数据产生错误解读。综上，本研究数据强调，需将细胞密度视为一项重要的实验变量，以提升基于细胞培养的研究的科学严谨性。