Metabolic concentration and flux maps from DGE-DMI in mouse GBM.

Example of a CT2A tumor (C1). A T2-weighted reference image (top-left) displaying the tumor region (dashed lines) and representative peritumor and tumor voxels (back dots), and respective spectral quantifications (right-side): bottom, raw spectrum (black) with overlaid estimation (purple); center, individual components for each metabolite peak (black - semi-heavy water, DHO (black); deuterated glucose, Glc (red); and glucose-derived glutamate-glutamine and lactate, Glx (green) and Lac (blue)); top, residual. B Time-course de novo concentration maps for each metabolite (mM) following Glc i.v. injection (red arrow). C Average concentration maps for each metabolite after Glc injection. D Time-course concentration plots for each metabolite (dots) and respective kinetic fitting (straight lines), displayed for the peritumor and tumor voxels shown in A (same color codes) and applied to all the voxels to generate glucose flux maps: maximum consumption rate (Vmax); and respective individual rates for lactate synthesis (Vlac) and elimination (klac), and glutamate-glutamine synthesis (Vglx) and elimination (kglx).

Histopathologic and immunohistochemical assessment in two mouse models of GBM.

A H&E-stained sections with high magnification to highlight annotations of tumor, infiltrative zones in the tumor margin (blue), and secondary lesion (red), in CT2A and GL261 tumors (subjects C4 and G4, respectively). B Iba-1 immunostained sections showing microglia/macrophage (Mgl/Mp) infiltration in CT2A and GL261 tumors: left panels, tumor core (black arrowhead) and tumor margin (white arrow) relative to the adjacent brain parenchyma; middle and right panels, depicting more infiltration by microglial/macrophage in CT2A tumors, also with clearer well-demarcated margin where IBA-1-positive cells are more densely concentrated compared to the more diffuse and irregular infiltration seen in the GL261 model; GL261 show poorly demarcated tumor border where tumor cells infiltrate the brain parenchyma (yellow diamonds); center panels, Iba-1 ROI quantification in tumor and peritumoral margin (P-Margin, yellow lines), and with red mask overlay of Iba-1 positive cells; right panel, quantification of mean Iba-1 positive area in Tumor and P-Margin regions of CT2A and GL261 cohorts – C2 sample excluded due to peritumoral hemorrhage/vascular ectasia, which distorted the peritumoral area and impaired proper assessment of peritumoral infiltration. C Ki67 immuno-stained sections with overlaid detection of positive (red) and negative (blue) cells; and high magnification to highlight annotations of tumor and peritumor border (P-Margin, yellow lines), in CT2A and GL261 tumors (subjects C1 and G3, respectively); and GBM cohort differences in tumor/P-Margin ratios of cell density and cell proliferation (dots representative of average values for each subject).. CT2A vs GL261 (ki67, n= 5 vs 5; Iba-1, n= 4 vs 5): * p<0.05; ** p<0.01; *** p<0.001; unpaired t-test. Error bars: standard deviation.

Mouse GBM models with different histopathologic phenotypes underlied by regional differences in lactate metabolism.

A Metabolic maps of de novo lactate accumulation (mM) and respective consumption/elimination rates (mM/min), in tumor and peritumor border regions (P-Margin, delineated by dashed lines) of CT2A and GL261 tumors (subjects C1 and G3, respectively). B GBM cohort differences in de novo lactate accumulation (Lac) and consumption/elimination rates (klac). C Strong linear correlations (indicated by the Person correlation coefficient, R) of: top-left, Tumor lactate consumption/elimination rates with P-Margin infiltration of microglia/macrophages in pooled cohorts; top-right, Tumor-to-P-Margin ratios of lactate accumulation and cell density in pooled cohorts; bottom, lactate consumption/elimination rates with (left-side) time post-tumor inoculation in each cohort, and (right-side) tumor vascular permeability in pooled cohorts. CT2A (n=5) vs GL261 (n=5): * p<0.05; ** p<0.01; unpaired t-test. Tumor (n=5, each cohort) vs P-Margin (n=5, each cohort): # p<0.05; ## p<0.01; paired t-test. Error bars: standard deviation. Bar plot dots representative of average pixel values for each subject.

Peritumoral metabolic changes consistent with recycling of the glutamate-glutamine pool mirror GBM infiltration and migration leading to secondary brain lesions.

A Metabolic maps (Glx) of peritumoral regions without and with secondary brain lesions (C4 and G4 tumors, respectively). B Histogram distributions of peritumoral Glx accumulation in pooled GL261 and CT2A cohorts displaying secondary brain lesions (n=4) vs without (n=6). C Bar plot comparison of mean values, showing significant decreases in peritumoral glutamate-glutamine accumulation (Glx) and increases in its consumption/elimination (kglx) in pooled GL261 and CT2A cohorts displaying secondary brain lesions (n=4; vs n=6 without): * p<0.05; unpaired t-test. Error bars: standard deviation. Bar plot dots representative of average pixel values for each subject.