Effects of the tumour microenvironment on protoporphyrin IX accumulation in glioblastoma

Aims Glioblastoma is the most common primary brain tumour and has a poor prognosis. Standard clinical intervention involves the resection of the tumour volume, chemotherapy and radiotherapy. However, achieving gross-total resection is challenging due to poorly defined boundaries as a result of tumour infiltration. Fluorescence-guided surgery (FGS) utilises an apparently selective accumulation of protoporphyrin IX (PPIX) that occurs in areas of glioblastoma after systemic administration of the metabolite 5-aminolevulinic acid (5-ALA). We have investigated the metabolic basis for the heterogeneity of the PPIX fluorescent signal, and its implications for glioma biology. Method Using glioblastoma cell lines and patient-derived primary cells, we have monitored the uptake of 5-ALA and conversion to the fluorescent molecule PPIX. Stable isotope tracing coupled with GCMS and LCMS was used to analyse intra- and extracellular metabolite levels arising from exogenous 5-ALA administration under both normoxic (21% O2) and hypoxic (1% O2) conditions. Results Uptake of exogenous 5-ALA from culture media and conversion to PPIX is observed in a time and dose-dependent manner in both normoxia and hypoxia. High levels of PPIX accumulation are associated with reduced cell proliferation despite the majority of the PPIX synthesised not being retained within the tumour cell, but exported into the medium. Under hypoxic conditions, reduced fluorescence is observed as a result of the decrease in oxygen availability likely affecting the oxygen-dependent enzymes. Stable isotope tracing experiments indicate an increase in the glutamine-derived succinate pool in response to exogenous 5-ALA, which is dependent on flux through the heme pathway. Conclusion Our data suggest that different microenvironments within the tumour alter the activity of the heme biosynthetic pathway, resulting in differential fluorescence in glioblastoma. It paves the way by which we could work to alter the glioblastoma microenvironment in order to further improve the use of FGS in guiding surgery across these devastating tumours.

Stable Isotope Tracing In Vivo Reveals A Metabolic Bridge Linking The Microbiota To Host Histone Acetylation

The gut microbiota influences host epigenetics by fermenting dietary fiber into butyrate. Although butyrate could promote histone acetylation by inhibiting histone deacetylases, it may also undergo oxidation to acetyl-CoA, a necessary cofactor for histone acetyltransferases. Here, we find that epithelial cells from germ-free mice harbor a loss of histone H4 acetylation across the genome except at promoter regions. Using stable isotope tracing in vivo with 13C-labeled fiber, we demonstrate that the microbiota supplies carbon for histone acetylation. Subsequent metabolomic profiling revealed hundreds of labeled molecules and supported a microbial contribution to host fatty acid metabolism, which declined in response to colitis and correlated with reduced expression of genes involved in fatty acid oxidation. These results illuminate the flow of carbon from the diet to the host via the microbiota, disruptions to which may affect energy homeostasis in the distal gut and contribute to the development of colitis.

Correcting for Naturally Occurring Mass Isotopologue Abundances in Stable-Isotope Tracing Experiments with PolyMID

Stable-isotope tracing is a method to measure intracellular metabolic pathway utilization by feeding a cellular system a stable-isotope-labeled tracer nutrient. The power of the method to resolve differential pathway utilization is derived from the enrichment of metabolites in heavy isotopes that are synthesized from the tracer nutrient. However, the readout is complicated by the presence of naturally occurring heavy isotopes that are not derived from the tracer nutrient. Herein we present an algorithm, and a tool that applies it (PolyMID-Correct, part of the PolyMID software package), to computationally remove the influence of naturally occurring heavy isotopes. The algorithm is applicable to stable-isotope tracing data collected on low- and high- mass resolution mass spectrometers. PolyMID-Correct is open source and available under an MIT license.