Figure 3. Transcriptomic data and [U-¹³C]glucose labelling reveal that stromal cell metabolism is shifted towards higher glycolysis and ROS upon co-culture with AML cells.

(A) Top 25 GSEA hallmark gene sets ranked by expression in MS-5 cells only vs co-culture with SKM-1 cells, analysed using the collection of hallmark gene sets from Molecular Signature Database with a false discovery rate threshold at 5%. p-Values for each pathway are presented as –log(10)p-value. (B) GSEA enrichment score plots of glycolysis and ROS generated using Sleuth 0.30.0 R statistical package. (C) Fold change values of detected gene transcripts (TPMs) related to glycolysis and pyruvate metabolism. FC values are represented as log₂FC, red values indicate upregulation and blue values indicate downregulation in MS-5 cells in co-culture. (D) Label incorporation from [U-¹³C]glucose into extracellular metabolites in AML and MS-5 cells cultured alone or in co-culture after 24 hr. Bars represent the mean of n=3 independent experiments and error bars represent standard deviations. (E), Label incorporation from [U-¹³C]glucose into intracellular acetate, alanine and lactate in MS-5 cells cultured alone or in co-culture after 24 hr. Bars represent the mean of n=3 independent experiments and error bars represent standard deviations. p-Values are represented by n.s. for not significant * for p-value <0.05, ** for p-value <0.01 and *** for p-value <0.001.

Figure 3—figure supplement 1. PCA component analysis, heat map of differentially expressed genes and qPCR for MS-5 cells cultured alone and in co-culture with SKM-1 cells.

(A) PCA plot showing the clustering of individual samples of MS-5 cells cultured alone (MO) and MS-5 cells in co-culture (MC). The x and y axis values represent the variation between the sample groups. Generated using Sleuth 0.30.0 R statistical package. (B) Heat map of differentially expressed genes in MS-5 only (red) vs MS-5 co-culture (blue) calculated using the Wald statistical test, correcting for multiple testing comparison employing the Benjamini-Hochberg method using a false discovery rate threshold of 1% (q-value <0.01). Generated using Sleuth 0.30.0 R statistical package. (C) Quantitative PCR mRNA expression values for RNA sequencing validation of MS-5 cells cultured alone and in co-culture with SKM-1 cells. The gene set chosen was Hk2, Pdhx, Pdk1, and Pdk2. mRNA quantification was normalized to B2m house-keeping gene. Bars represent the mean and error bars represent the SEM of three independent experiments (n=3). An unpaired Student’s t-test was applied for each gene and p-values are represented by * for p-value <0.05, ** for p-value <0.01 and *** for p-value <0.001.