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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.

SUBMITTER: Kim S

PROVIDER: S-EPMC5610329 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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

Kim Sojin S Kim Ja Eun JE Kim Yong Hwy YH Hwang Taeyoung T Kim Sung Kwon SK Xu Wen Jun WJ Shin Jong-Yeon JY Kim Jong-Il JI Choi Hyoungseon H Kim Hee Chan HC Cho Hye Rim HR Choi Anna A Chowdhury Tamrin T Seo Youngbeom Y Dho Yun-Sik YS Kim Jin Wook JW Kim Dong Gyu DG Park Sung-Hye SH Kim Hyeonjin H Choi Seung Hong SH Park Sunghyouk S Lee Se-Hoon SH Park Chul-Kee CK

Scientific reports 20170922 1

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 inten ...[more]

PMID: 28939850

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Project description:Simple Summary 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence is used in neurosurgery for intraoperative identification of high-grade glioma tissue. In this paper, using a fluorescence microscopy analysis on human tumor specimens, we assessed the actual number of fluorescence-positive tumor cells both in low-grade and high-grade glioma, and the ability of 5-ALA to cross the blood–brain barrier (BBB). We found that in high-grade gliomas, 32.7–75.5 percent of cells display 5-ALA induced PpIX fluorescence, whereas in low-grade gliomas the tumor cells did not fluoresce following 5-ALA. Immunofluorescence for BBB components suggested that 5-ALA does not cross the un-breached BBB. These findings are of crucial importance in planning neurosurgical resection of gliomas. Abstract 5-aminolevulinic acid (5-ALA)-induced PpIX fluorescence is used by neurosurgeons to identify the tumor cells of high-grade gliomas during operation. However, the issue of whether 5-ALA-induced PpIX fluorescence consistently stains all the tumor cells is still debated. Here, we assessed the cytoplasmatic signal of 5-ALA by fluorescence microscopy in a series of human gliomas. As tumor markers, we used antibodies against collapsin response-mediated protein 5 (CRMP5), alpha thalassemia/mental retardation syndrome X-linked (ATRX), and anti-isocitrate dehydrogenase 1 (IDH1). In grade III–IV gliomas, the signal induced by 5-ALA was detected in 32.7–75.5 percent of CRMP5-expressing tumor cells. In low-grade gliomas (WHO grade II), the CRMP5-expressing tumor cells did not fluoresce following 5-ALA. Immunofluorescence with antibodies that stain various components of the blood–brain barrier (BBB) suggested that 5-ALA does not cross the un-breached BBB, in spite of its small dimension. To conclude, 5-ALA-induced PpIX fluorescence has an established role in high-grade glioma surgery, but it has limited usefulness in surgery for low-grade glioma, especially when the BBB is preserved.

Project description:BackgroundGlutaminase isoenzymes GLS and GLS2 play apparently opposing roles in cancer: GLS acts as an oncoprotein, while GLS2 (GAB isoform) has context specific tumour suppressive activity. Some microRNAs (miRNAs) have been implicated in progression of tumours, including gliomas. The aim was to investigate the effect of GLS and GAB expression on both miRNAs and oxidative status in glioblastoma cells.MethodsMicroarray profiling of miRNA was performed in GLS-silenced LN229 and GAB-transfected T98G human glioblastoma cells and their wild-type counterparts. Results were validated by real-time quantitative RT-PCR. Oxidative status and antioxidant enzymes were determined by spectrophotometric or fluorescence assays in GLS-silenced LN229 and T98G, and GAB-transfected LN229 and T98G.ResultsMiRNA-146a-5p, miRNA-140-3p, miRNA-21-5p, miRNA-1260a, and miRNA-92a-3p were downregulated, and miRNA-1246 was upregulated when GLS was knocked down. MiRNA-140-3p, miRNA-1246, miRNA-1260a, miRNA-21-5p, and miRNA-146a-5p were upregulated when GAB was overexpressed. Oxidative status (lipid peroxidation, protein carbonylation, total antioxidant capacity, and glutathione levels), as well as antioxidant enzymes (catalase, superoxide dismutase, and glutathione reductase) of silenced GLS glioblastoma cells and overexpressed GAB glioblastoma cells significantly changed versus their respective control glioblastoma cells. MiRNA-1246, miRNA-1260a, miRNA-146a-5p, and miRNA-21-5p have been characterized as strong biomarkers of glioblastoma proliferation linked to both GLS silencing and GAB overexpression. Total glutathione is a reliable biomarker of glioblastoma oxidative status steadily associated to both GLS silencing and GAB overexpression.ConclusionsGlutaminase isoenzymes are related to the expression of some miRNAs and may contribute to either tumour progression or suppression through certain miRNA-mediated pathways, proving to be a key tool to switch cancer proliferation and redox status leading to a less malignant phenotype. Accordingly, GLS and GAB expression are especially involved in glutathione-dependent antioxidant defence.

Dataset Information

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

Publications

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

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