Extended Data Fig. 3: Segmentation of the mass spectrometry images.
a) Scatter plot of individual pixel intensities in mass spectrometry images of tumour sections from GB1 and GB2. The x-axes shows [13C2]glutamate and the y-axes [U-13C]lactate signal intensities. Colours correspond to the k-means segmentation of pixels into three vs. four groups. b) K-means segmentation maps of all the GB1 and GB2 tumour sections assuming three metabolic states. The three metabolic states are represented in red (low activity in glycolysis and TCA cycle), yellow (high TCA cycle activity) and blue (high glycolytic activity). The sections were arranged randomly on the slides for data acquisition. They have been arranged here to match their respective parent tumour in order to assist visualisation. c) K-means segmentation maps of all the GB1 and GB2 tumour sections assuming four metabolic states shown in a. d) Representative GB sections showing the three metabolic states and corresponding signal for 13C labelled metabolites in glycolysis and the TCA cycle. e) Relative abundance of TCA cycle and glycolytic intermediates in the 3 metabolic states in GB1, GB2 and normal appearing brain (Normal brain n = 4; Region 1 n = 22; Region 2 n = 14; Region 3 n = 17) expressed as mean ± SD. f) GB1 had a higher proportion of oxidative regions in the tumor sections (Region 2, yellow) while GB2 had more glycolytic regions (Region 3, blue). The three metabolic phenotypes were present in both patients. Asterisks refer to P values obtained from one-way ANOVA test followed by Tukey’s multiple comparisons test or unpaired T-test (*P < 0.05; **P < 0.005; ***P < 0.0005; ****P < 0.00005).
