Embryonic Carcinoma Cells Show Specific Dielectric Resistance Profiles during Induced Differentiation

Induction of differentiation in cancer stem cells by drug treatment represents an important approach for cancer therapy. The understanding of the mechanisms that regulate such a forced exit from malignant pluripotency is fundamental to enhance our knowledge of tumour stability. Certain nucleoside analogues, such as 2′-deoxy-5-azacytidine and 1β-arabinofuranosylcytosine, can induce the differentiation of the embryonic cancer stem cell line NTERA 2 D1 (NT2). Such induced differentiation is associated with drug-dependent DNA-damage, cellular stress and the proteolytic depletion of stem cell factors. In order to further elucidate the mode of action of these nucleoside drugs, we monitored differentiation-specific changes of the dielectric properties of growing NT2 cultures using electric cell-substrate impedance sensing (ECIS). We measured resistance values of untreated and retinoic acid treated NT2 cells in real-time and compared their impedance profiles to those of cell populations triggered to differentiate with several established substances, including nucleoside drugs. Here we show that treatment with retinoic acid and differentiation-inducing drugs can trigger specific, concentration-dependent changes in dielectric resistance of NT2 cultures, which can be observed as early as 24 hours after treatment. Further, low concentrations of nucleoside drugs induce differentiation-dependent impedance values comparable to those obtained after retinoic acid treatment, whereas higher concentrations induce proliferation defects. Finally, we show that impedance profiles of substance-induced NT2 cells and those triggered to differentiate by depletion of the stem cell factor OCT4 are very similar, suggesting that reduction of OCT4 levels has a dominant function for differentiation induced by nucleoside drugs and retinoic acid. The data presented show that NT2 cells have specific dielectric properties, which allow the early identification of differentiating cultures and real-time label-free monitoring of differentiation processes. This work might provide a basis for further analyses of drug candidates for differentiation therapy of cancers.

药物诱导癌症干细胞分化，是癌症治疗的重要策略之一。阐明调控癌细胞被迫脱离恶性多能状态的机制，对于深化我们对肿瘤稳定性的认知至关重要。部分核苷类似物，如2′-脱氧-5-氮杂胞苷与1β-阿拉伯呋喃糖基胞嘧啶，可诱导胚胎癌症干细胞系NTERA 2 D1（NT2）发生分化。这类诱导分化过程与药物依赖性DNA损伤、细胞应激以及干细胞因子的蛋白水解耗竭密切相关。为进一步阐明此类核苷类药物的作用机制，本研究采用细胞基质阻抗传感（electric cell-substrate impedance sensing, ECIS）技术，实时监测体外培养的NT2细胞在分化过程中其介电特性的特异性变化。我们实时记录了未处理组与视黄酸处理组NT2细胞的阻抗值，并将其阻抗谱与经多种经典诱导分化物质（包括核苷类药物）处理的细胞群体的阻抗谱进行了对比。本研究结果显示，视黄酸与分化诱导药物处理均可触发NT2细胞培养物的介电阻抗发生特异性、浓度依赖性变化，此类变化最早可在给药后24小时内被检测到。此外，低浓度核苷类药物诱导产生的分化相关阻抗值与视黄酸处理组相当，而高浓度核苷类药物则会引发细胞增殖缺陷。最后，本研究证实，经化学物质诱导分化的NT2细胞，其阻抗谱与通过敲低干细胞因子OCT4（Octamer-binding transcription factor 4）诱导分化的细胞高度相似，这表明降低OCT4水平在核苷类药物与视黄酸诱导的分化过程中发挥主导作用。本研究数据表明，NT2细胞具有特异性介电特性，可用于早期识别正在发生分化的细胞培养物，并实现分化过程的实时无标记监测。本研究可为癌症分化治疗领域的候选药物后续研究提供重要基础。