5-ALA localises to the autophagy compartment and increases its fluorescence upon autophagy enhancement through caloric restriction and spermidine treatment in human glioblastoma

Kim Fredericks; Jurgen Kriel; Lize Engelbrecht; Petra Andreea Mercea; Georg Widhalm; Brad Harrington; Ian Vlok; Ben Loos

doi:10.1016/j.bbrep.2024.101642

5-ALA localises to the autophagy compartment and increases its fluorescence upon autophagy enhancement through caloric restriction and spermidine treatment in human glioblastoma

Biochem Biophys Rep. 2024 Jan 13:37:101642. doi: 10.1016/j.bbrep.2024.101642. eCollection 2024 Mar.

Authors

Kim Fredericks¹, Jurgen Kriel², Lize Engelbrecht², Petra Andreea Mercea³, Georg Widhalm³, Brad Harrington⁴, Ian Vlok⁴, Ben Loos¹

Affiliations

¹ Department of Physiological Sciences, Stellenbosch University, Stellenbosch, South Africa.
² Central Analytical Facility, Microscopy Unit, Stellenbosch University, South Africa.
³ Department of Neurosurgery, Medical University of Vienna, Vienna, Austria.
⁴ Department of Neurosurgery, Stellenbosch University, Cape Town, South Africa.

Abstract

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.

Keywords: 5-Aminolevulinic acid fluorescence; Autolysosomes; Autophagic activity; Autophagosomes; Autophagy; Glioblastoma; Lysosomes.