Microtubular Assessment of C6 Rat Glioma Cell Spheroids Developed in Transparent Liquid Marbles or Hanging Drops

SIMPLE SUMMARY: Due to its high refractoriness to therapies, glioblastoma brain tumour is frequently used as a model to develop new therapeutic approaches. Many of these treatments may target the microtubular network of the cell, also considering that tubulin post-translational modifications (PTMs) are markers of tumour plasticity. The two-dimensional (2D) culture systems are now being replaced by three-dimensional (3D) systems capable of mimicking in vivo conditions. In this work, spheroids were developed from C6 rat glioma cells (RGCs) using two 3D systems: liquid marbles (LMs) or hanging drops (HD) and analysed in terms of the morphology and behaviour of the two main tubulin PTMs, tyrosinated α-tubulin (Tyr-T) and acetylated α-tubulin (Ac-T). RGCs spontaneously formed spheroids more rapidly in the LM than in the HD system. An increase in Tyr-T and Ac-T was observed in both the HD and LM system during IVC, with the highest values shown in LM spheroids. In conclusion, the present work shows that the LM 3D system boosts the induction and maintenance of a high plasticity state in glioma cells and could provide a novel approach to set up a biological system to evaluate new anticancer therapies and advance knowledge on glioblastoma. ABSTRACT: 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.