Glutaminase 2 expression is associated with regional heterogeneity of 5-aminolevulinic acid fluorescence in glioblastoma

Abstract

Fluorescence-guided surgery using 5-aminolevulinic acid (5-ALA) is now a widely-used modality for glioblastoma (GBM) treatment. However, intratumoral heterogeneity of fluorescence intensity may reflect different onco-metabolic programs. Here, we investigated the metabolic mechanism underlying the heterogeneity of 5-ALA fluorescence in GBM. Using an in-house developed fluorescence quantification system for tumor tissues, we collected 3 types of GBM tissues on the basis of their fluorescence intensity, which was characterized as strong, weak, and none. Expression profiling by RNA-sequencing revealed 77 genes with a proportional relationship and 509 genes with an inverse relationship between gene expression and fluorescence intensity. Functional analysis and in vitro experiments confirmed glutaminase 2 (GLS2) as a key gene associated with the fluorescence heterogeneity. Subsequent metabolite profiling discovered that insufficient NADPH due to GLS2 underexpression was responsible for the delayed metabolism of 5-ALA and accumulation of protoporphyrin IX (PpIX) in the high fluorescence area. The expression level of GLS2 and related NADPH production capacity is associated with the regional heterogeneity of 5-ALA fluorescence in GBM.

Spectral complexity of 5-ALA induced PpIX fluorescence in guided surgery: a clinical study towards the discrimination of healthy tissue and margin boundaries in high and low grade gliomas

“…Indeed, a strong 5-ALA induced fluorescence intensity has been observed in a patient showing only inflammatory cells without tumor cells [47]. Furthermore, PpIX accumulation is known to be sensitive to gene expression [48] or metabolic pathways [49]. Our study shows the predominance of State 620 of PpIX in these low-density tumor cells regions.…”

Optical Characterization of Neurosurgical Operating Microscopes: Quantitative Fluorescence and Assessment of PpIX Photobleaching

“…We have also noted high variability in the rate of PpIX photobleaching in experimental gliomas that depended on the initial fluorescence intensity 26 . Such a large range of reported photobleaching decay is most likely due to glioma heterogeneity 40 . Another factor that affects PpIX fluorescence decay is the variability in light power output of the operating microscopes.…”

In order for the light to shine so brightly, the darkness must be present—why do cancers fluoresce with 5-aminolaevulinic acid?

“…Two recent studies suggest that aberrant NADPH production is associated with 5-ALA-induced fluorescence in glioma cells. 66,67 NADPH is generated during the formation of α-ketoglutarate, feeding into the TCA cycle by isocitrate dehydrogenases (IDH1 and IDH2) and glutaminases (GLS and GLS2). IDH1-mutant human glioma cells (U87MG) with reduced levels of NADPH showed enhanced PpIX fluorescence after incubation with 5-ALA compared with their wild-type counterparts.…”

“…66 Conversely, overexpression of GLS2 in glioma cells (T98G, LN18, U87MG) increased the levels of NADPH but reduced 5-ALA-induced fluorescence. 67 The question therefore arises as to whether PpIX accumulates in cancer cells because the demand for NADPH outstrips supply. Both of the studies in glioma cells show rapid consumption of NADPH after 5-ALA incubation in all cell lines, with strong PpIX fluorescence associated with the lowest levels of NADPH in glioblastoma tissue.…”