Fredericks Kim, Kriel Jurgen, Engelbrecht Lize, Mercea Petra Andreea, Widhalm Georg, Harrington Brad, Vlok Ian, Loos Ben
Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South Africa.
Central Analytical Facility, Microscopy Unit, Stellenbosch University, South Africa.
Biochem Biophys Rep. 2024 Jan 13;37:101642. doi: 10.1016/j.bbrep.2024.101642. eCollection 2024 Mar.
Glioblastoma Multiforme (GBM) is the most invasive and prevalent Central Nervous System (CNS) malignancy. It is characterised by diffuse infiltrative growth and metabolic dysregulation that impairs the extent of surgical resection (EoR), contributing to its poor prognosis. 5-Aminolevulinic acid (5-ALA) fluorescence-guided surgical resection (FGR) takes advantage of the preferential generation of 5-ALA-derived fluorescence signal in glioma cells, thereby improving visualisation and enhancing the EoR. However, despite 5-ALA FGR is a widely used technique in the surgical management of malignant gliomas, the infiltrative tumour margins usually show only vague or no visible fluorescence and thus a significant amount of residual tumour tissue may hence remain in the resection cavity, subsequently driving tumour recurrence. To investigate the molecular mechanisms that govern the preferential accumulation of 5-ALA in glioma cells, we investigated the precise subcellular localisation of 5-ALA signal using Correlative Light and Electron Microscopy (CLEM) and colocalisation analyses in U118MG glioma cells. Our results revealed strong 5-ALA signal localisation in the autophagy compartment - specifically autolysosomes and lysosomes. Flow cytometry was employed to investigate whether autophagy enhancement through spermidine treatment (SPD) or nutrient deprivation/caloric restriction (CR) would enhance 5-ALA fluorescence signal generation. Indeed, SPD, CR and a combination of SPD/CR treatment significantly increased 5-ALA signal intensity, with a most robust increase in signal intensity observed in the combination treatment of SPD/CR. When using 3-D glioma spheroids to assess the effect of 5-ALA on cellular ultrastructure, we demonstrate that 5-ALA exposure leads to cytoplasmic disruption, vacuolarisation and large-scale mitophagy induction. These findings not only suggest a critical role for the autophagy compartment in 5-ALA engagement and signal generation but also point towards a novel and practically feasible approach to enhance 5-ALA fluorescence signal intensity. The findings may highlight that indeed autophagy control may serve as a promising avenue to promote an improved resection and GBM prognosis.
多形性胶质母细胞瘤（GBM）是最具侵袭性且最为常见的中枢神经系统（CNS）恶性肿瘤。其特征在于弥漫性浸润性生长和代谢失调，这会影响手术切除范围（EoR），导致预后不良。5-氨基乙酰丙酸（5-ALA）荧光引导下的手术切除（FGR）利用了胶质瘤细胞中优先产生的5-ALA衍生荧光信号，从而改善可视化效果并提高手术切除范围。然而，尽管5-ALA FGR是恶性胶质瘤手术治疗中广泛使用的技术，但浸润性肿瘤边缘通常仅显示模糊或无可见荧光，因此切除腔内可能会残留大量肿瘤组织，进而导致肿瘤复发。为了研究控制5-ALA在胶质瘤细胞中优先积累的分子机制，我们使用相关光电子显微镜（CLEM）和共定位分析，在U118MG胶质瘤细胞中研究了5-ALA信号的精确亚细胞定位。我们的结果显示，5-ALA信号在自噬区室——特别是自噬溶酶体和溶酶体中强烈定位。采用流式细胞术研究通过亚精胺处理（SPD）或营养剥夺/热量限制（CR）增强自噬是否会增强5-ALA荧光信号的产生。事实上，SPD、CR以及SPD/CR联合处理均显著增加了5-ALA信号强度，其中SPD/CR联合处理观察到的信号强度增加最为显著。当使用三维胶质瘤球体评估5-ALA对细胞超微结构的影响时，我们证明5-ALA暴露会导致细胞质破坏、空泡化和大规模线粒体自噬诱导。这些发现不仅表明自噬区室在5-ALA结合和信号产生中起关键作用，还指出了一种新颖且切实可行的方法来增强5-ALA荧光信号强度。这些发现可能突出表明，自噬控制确实可能是促进改善切除效果和GBM预后的一个有前景的途径。
