Langella Arianna, Gadau Sergio Domenico, Serra Elisa, Bebbere Daniela, Ledda Sergio
Department of Veterinary Medicine, University of Sassari, Via Vienna 2, 07100 Sassari, Italy.
Biology (Basel). 2022 Mar 23;11(4):492. doi: 10.3390/biology11040492.
Glioblastoma is a brain tumour frequently used as an experimental model to exploit innovative therapeutic approaches due to its high lethality and refractoriness to therapies. Part of these innovative anticancer therapies address cytoskeletal microtubules (MTs) since specific tubulin post-translational modifications (PTMs) are considered markers of tumour plasticity. In vitro studies, which traditionally employ two-dimensional (2D) culture systems, are now being replaced by three-dimensional (3D) systems that more closely mimic in vivo physiological conditions and allow a better understanding of the signalling between cells. In this work, we compared 2 liquid base 3D methods for the generation of spheroids from C6 rat glioma cells (RGCs) using 30 µL of liquid marble (LM) or the hanging drops (HDs), which contained 2 different cell numbers (5000 or 15,000). After 24 or 48 h of in vitro culture (IVC), the morphology of the spheroids was observed and the behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T), was evaluated by fluorescence and Western blot (WB). RGCs spontaneously formed spherical agglomerates more rapidly in the LM than in the HD system. Cell density influenced the size of the spheroids, which reached a larger size (> of 300 µm Ø), with 15,000 cells compared to 5000 cells (150 µm Ø). Moreover, an increase in Tyr-T and Ac-T was observed in both the HD and LM system from 24 to 48 h, with the highest values shown in the 48 h/LM spheroids of 5000 cells (p < 0.05). In conclusion, by comparing the morphology and microtubular architecture of spheroids from C6 rat glioma cells developed by LM or HD methodology, our findings demonstrate that the use of a fumed silica microbioreactor boosts the induction and maintenance of a high plasticity state in glioma cells. RGCs cultured in LM express levels of tubulin PTMs that can be used to evaluate the efficacy of new anticancer therapies.
胶质母细胞瘤是一种脑肿瘤，由于其高致死率和对治疗的难治性，常被用作开发创新治疗方法的实验模型。这些创新的抗癌疗法部分针对细胞骨架微管（MTs），因为特定的微管蛋白翻译后修饰（PTMs）被认为是肿瘤可塑性的标志物。传统上采用二维（2D）培养系统的体外研究，现在正被三维（3D）系统所取代，三维系统更接近体内生理条件，能更好地理解细胞间信号传导。在这项工作中，我们比较了两种基于液体的3D方法，即使用30微升液体大理石（LM）或悬滴（HDs）从C6大鼠胶质瘤细胞（RGCs）生成球体，其中HDs含有两种不同的细胞数量（5000或15000）。体外培养（IVC）24或48小时后，观察球体形态，并通过荧光和蛋白质免疫印迹（WB）评估两种主要微管蛋白PTMs，即酪氨酸化α-微管蛋白（Tyr-T）和乙酰化α-微管蛋白（Ac-T）的行为。RGCs在LM中比在HD系统中更快地自发形成球形聚集体。细胞密度影响球体大小，15000个细胞形成的球体尺寸（>300微米Ø）比5000个细胞形成的球体（150微米Ø）更大。此外，在HD和LM系统中，从24小时到48小时，Tyr-T和Ac-T均有增加，5000个细胞的48小时/LM球体中显示的值最高（p<0.05）。总之，通过比较由LM或HD方法培养的C6大鼠胶质瘤细胞球体的形态和微管结构，我们的研究结果表明，使用气相二氧化硅微生物反应器可促进胶质瘤细胞中高可塑性状态的诱导和维持。在LM中培养的RGCs表达的微管蛋白PTMs水平可用于评估新抗癌疗法的疗效。
