Synopsis

Understanding metabolic aberrations in IDH-mutated gliomas requires xenograft models growing in a relevant tissue microenvironment and resembling its human genetic counterparts. We performed in vivo ¹H MRSI of human-derived oligodendroglioma xenograft models to map lactate and total choline concentrations. Lactate levels were significantly lower and total choline higher in mutated tumor tissue compared to non-tumor brain in the same animal, or to its wild-type counterpart model. This outcome was correlated with expression levels of enzymes and transporters in both lactate- and phospholipid-related metabolic pathways. The findings point to a metabolic reprogramming of aerobic glycolysis and lipid synthesis by the IDH1 mutation.

Introduction

Isocitrate dehydrogenase (IDH) mutation has become a key element in the diagnosis of gliomas and the associated metabolic network a potential treatment target. For non-invasive detection of IDH mutations and to understand metabolic reprogramming, MR spectroscopy is an important approach. The patient-derived oligodendroglioma mouse brain xenograft model E478 carries the IDH1^R132H/WT mutation and closely resembles the genetic make-up and diffuse infiltrative phenotype of the tumor it was derived from¹. Previously, we demonstrated increased phosphocholine compound levels in this model². ¹H MR spectroscopic imaging has been used in spatial metabolic profiling of IDH mutated gliomas in humans, but not in relevant brain xenografts.

In this study we applied ¹H MRSI to obtain spatially resolved metabolite maps of the mouse brain with IDH1^R132H/WT mutated E478 gliomas, focusing on the assessment of lactate and total choline (tCho) levels with wild type counterpart model E434 as control. Outcome of these studies was correlated to transcript levels of genes encoding the relevant metabolic enzymes and transporters.

Materials and Methods

All experiments were approved by the National Animal Research Authority. Patient-derived high-grade oligodendroglioma xenografts (E434 IDH1^WT/WT and E478 IDH1^R132H/WT) were injected intracerebrally in female Balb/c-nu/nu mice¹. Anesthetized animals (n=3 for each group) were investigated on a 7T MR system (ClinScan, Bruker) with a mouse brain receive-coil. Multi-slice T2-weighted images were made in three directions parallel to the MRSI grid. 3D MRSI was performed with semi-LASER using 12x12x16 elliptical weighted phase encoding interpolated to 16x16x16. The 11x11x16mm FOV contained 0.85mm³ nominal voxels, TE=144ms, TR=1500ms, TA=27m55s. After the measurement animals were sacrificed and brains were formalin-fixed and paraffin embedded for histology assessment with H&E staining. Spectra were fitted with LCmodel using a simulated basis set. Metabolite levels were calibrated to the water signal from a separate scan and mapped to the anatomical images. For statistical analysis multiple voxels containing either tumor or normal brain were selected guided by T2-weighted images, histology and a Cramér-Rao lower bound fit value <20. Significant differences in mean values were evaluated with unpaired t-tests (p<0.05).

RNA isolated from frozen sections of E478 and E434 xenografts was subjected to next gen sequencing (ServiceXS, Leiden) and yielded 30-50 million reads per sample (single end sequencing protocol). The dataset was analyzed with the Tuxedo protocol³. Mouse reads were filtered from the data prior to data mapping against the RefSeq human genome (hg19).

Results

Metabolic maps of steady state lactate pools showed decreased lactate concentrations in IDH1^WT/R132H tumor tissue compared to IDH1^WT/WT mice (Fig1). Surrounding non-tumor tissue in IDH1^WT/R132H did not have significantly different lactate levels compared to either tumor and normal brain tissue of IDH1^WT/WT animals. Similarly, tCho concentrations were significantly increased in IDH1^WT/R132H tumor tissue compared to all other selected regions (Fig2).

RNA-seq analysis revealed decreased LDHA expression and increased LDHB expression in IDH1^WT/R132H compared to IDH1^WT/WT (Fig3). Cytidylyltransferase beta (CCTβ) and choline transporter-like protein1 (CTL1) expression were decreased and phospholipaseA2 (PLA2) and CTL2 increased. No evident increase was observed in expression of choline kinase-alpha (CHKα). Expression of other choline transporters was low in both models.

Discussion and Conclusion

We assessed the spatially resolved choline and lactate levels in tumorous and healthy brain by in vivo ¹H-MRSI. The increase of tCho in the E478 IDH1^R132/WT glioma is in line with our previous ³¹P MRS observation of increased glycerolphosphocholine (GPC) and slightly increased phosphocholine (PC)², with in vivo MRS studies of human IDH1 mutated tumors^4,5 and by ex-vivo MRS showing GPC and PC increases⁶. This seems to contrast with IDH1 mutations in U87/NHA and HOG cells in which PC is decreased^7,8, although HOG cells do show increased GPC. Our findings of altered choline pathway enzymes and transporters expression may explain the choline compound increases.

The observation of near normal lactate levels in E478 IDH1^R132/WT and increased lactate in E434 IDH1^WT/WT are in line with in vivo studies of ¹³C hyperpolarized pyruvate-to-lactate conversion in the BT142 patient-derived IDH1 mutant glioma model⁹. The decrease in the expression of LDHA in E478 IDH1^R132/WT is also in agreement with findings in BT142 glioma¹⁰.

Altogether our findings point to a consistent metabolic reprogramming of aerobic glycolysis and lipid synthesis by the IDH1 mutation in gliomas. The agreement of our results with clinical observations in patients stresses the applicability of E478 as a relevant in vivo IDH1^R132H/WT glioma model for further preclinical examinations.

Acknowledgements

References

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