BACKGROUND Tumor microtubes (TMs) are ultralong membrane protrusions of tumor cells in astrocytic gliomas, including glioblastomas. TMs are used as routes for brain invasion and for cells to interconnect over long distances resulting in a functional network that allows multicellular communication. This network mediates resistance against the cytotoxicity of radiation and chemotherapy. One explanation for TM network protection is a better homeostasis of calcium ions that would otherwise increase to toxic intracellular levels in response to these therapies. Our working hypothesis is that interfering with the integrity of the glioblastoma cell network is key to a potential breakthrough in glioma therapy. Many cellular structures are polarized and composed of charged elements and are thus potential subjects to electrical forces; this might also influence the complex intercellular calcium waves (ICWs) that are characteristic for glioma networks. We were therefore interested in the effect of TTF on glioma network maintenance. MATERIAL AND METHODS To examine the effect of TTF on glioma TMs we have established a 2D in vitro glioma model using glioblastoma stem cells (GBSCs) grown in high-glucose medium and a 3D model using glioma tumor organoids. Both models reliably reproduce functionality and complexity of morphological features we observe in our mouse model. We analyzed the disruption of tumor network complexity and disruption of functionality by measuring intercellular calcium waves. Tumor cell death and proliferation was investigated in the 2D in vitro glioma model using the inovitroTM-System. RESULTS A peculiar “cricked-TM” phenotype that rarely (0.2% ±0.14) occurred under standard or control conditions was observed in TTF-treated cells (16.22% ±5.12). Cell number was reduced by 75% in two lines of GBSCs after 5 days of TTF exposure; predominantly TM-rich GBSCs (> 4 TMs) were affected. This reduction in tumor cell number corresponded with an increase in cell death (0.3% ±0.09 in untreated cells; 1.4% ±0.45 at day 5 of TTF exposure). The frequency of intercellular calcium transients, a measurement for calcium wave frequency in the glioma networks, was instantly reduced after TTF exposure to 58% ±20.42 of control levels in the primary GBSC 2D culture, and to 57.78% ±12.34 in tumor organoids derived from 3 glioblastoma patients. CONCLUSION This data suggests a potential effect of TTF application on tumor cell networks, at least in vitro. Interestingly, particularly those glioblastoma cells that have so far been proven to be resistant to radio- and chemotherapy appeared to be affected. We will confirm the observed effects of TTFs on tumor cell calcium signaling in our in vivo chronic cranial window mouse model. We anticipate that the results of our project will provide important insights into the underlying mechanism of TTF therapy.

中文翻译：

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P13.12 肿瘤治疗场对胶质瘤肿瘤微管的影响

背景技术肿瘤微管(TM)是星形胶质细胞瘤(包括胶质母细胞瘤)中肿瘤细胞的超长膜突起。TM 被用作大脑入侵和细胞长距离互连的路线，从而形成允许多细胞通信的功能网络。该网络介导对放射和化学疗法的细胞毒性的抗性。对 TM 网络保护的一种解释是钙离子的更好稳态，否则钙离子会响应这些疗法而增加至毒性细胞内水平。我们的工作假设是，干扰胶质母细胞瘤细胞网络的完整性是胶质瘤治疗潜在突破的关键。许多细胞结构是极化的，由带电元素组成，因此可能会受到电力的影响；这也可能影响神经胶质瘤网络特有的复杂细胞间钙波 (ICW)。因此，我们对 TTF 对神经胶质瘤网络维护的影响感兴趣。材料和方法 为了检查 TTF 对胶质瘤 TM 的影响，我们建立了使用在高葡萄糖培养基中生长的胶质母细胞瘤干细胞 (GBSC) 的 2D 体外胶质瘤模型和使用胶质瘤肿瘤类器官的 3D 模型。两种模型都可靠地再现了我们在小鼠模型中观察到的形态特征的功能和复杂性。我们通过测量细胞间钙波分析了肿瘤网络复杂性的破坏和功能的破坏。使用 inovitroTM 系统在 2D 体外神经胶质瘤模型中研究肿瘤细胞死亡和增殖。结果 一种罕见的特殊“cricked-TM”表型（0.2% ±0. 14) 在标准或对照条件下在 TTF 处理的细胞中观察到 (16.22% ±5.12)。在 TTF 暴露 5 天后，两行 GBSC 的细胞数量减少了 75%；主要是富含 TM 的 GBSC (＞4 个 TM) 受到影响。肿瘤细胞数量的减少与细胞死亡的增加相对应（未处理细胞中为 0.3% ±0.09；TTF 暴露第 5 天为 1.4% ±0.45）。细胞间钙瞬变的频率，即神经胶质瘤网络中钙波频率的测量值，在 TTF 暴露后立即降低到原代 GBSC 2D 培养中对照水平的 58% ±20.42，在源自肿瘤器官的肿瘤类器官中降低到 57.78% ±12.34 3 名胶质母细胞瘤患者。结论 该数据表明 TTF 应用对肿瘤细胞网络的潜在影响，至少在体外是这样。有趣的是，特别是那些迄今为止已被证明对放射和化学疗法具有抗性的胶质母细胞瘤细胞似乎受到了影响。我们将在我们的体内慢性颅窗小鼠模型中证实观察到的 TTF 对肿瘤细胞钙信号传导的影响。我们预计，我们项目的结果将为 TTF 治疗的潜在机制提供重要的见解。