Journal: Cell Reports Medicine

Article Title: Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer

doi: 10.1016/j.xcrm.2023.101162

Figure Lengend Snippet: Metabolite profiling stratifies PDAC into two subtypes (A) PDAC organoid subgrouping based on widely targeted metabolite abundance. Differential metabolites were identified by the Wilcoxon rank-sum test (p < 0.05, fold change > 1.2). Samples and metabolites are displayed as columns and rows, respectively, and the color of each organoid shows the relative abundance of the metabolites. (B) Relative enrichment of the six metabolic ontology classes in class 1 and class 2 organoids, presented as the difference (class 2 versus class 1) in the ssGSEA score. Positive scores represent terms enriched in metabolites with high intensities in class 2. (C) Fractions of labeled metabolites in glycolysis from [U- 13 C 6 ] glucose in representative organoids of class 1 (n = 6) and class 2 (n = 6). (D) Fractions of labeled metabolites in the TCA cycle from [U- 13 C 6 ] glucose in representative organoids of class 1 (n = 6) and class 2 (n = 6). (E and F) Fractions of labeled metabolites in oxidative PPP (E) and nonoxidative PPP (F) from [U- 13 C 6 ] glucose in representative organoids of class 1 (n = 6) and class 2 (n = 6). (G) Heatmap of widely targeted lipidomics assay showing the abundance of differentially lipid metabolites in class 1 (n = 6) and class 2 (n = 6). (H and I) Class 1 (n = 13) and class 2 (n = 15) organoids were exposed to oligomycin, FCCP, and rotenone/antimycin A to measure the oxygen consumption rate (OCR) by XF Cell Mito Stress Test. (J and K) Dose-response curves and normalized area under the curve (AUC) distribution for TVB-3166 (fatty acid synthase inhibitor) on class 1 and class 2. (L and M) Dose-response curves and normalized AUC distribution for Gboxin (inhibitor of oxidative phosphorylation) on class 1 (n = 13) and class 2 (n = 15). (N) Heatmap of RNA sequencing (RNA-seq) showing the expression of differentially expressed genes (DEGs) in glucomet-PDAC (n = 15) and lipomet-PDAC (n = 13) (p was calculated with limma, p < 0.05 and fold change > 1.5). Representative genes associated with lipid or glucose metabolism are shown in the box on the right. (O) Profile plot and heatmap of assay for transposase-accessible chromatin with sequencing (ATAC-seq) results showing the distribution of differential peaks around the transcriptional start site (TSS) of signature genes in lipomet-PDAC (n = 13) and glucomet-PDAC (n = 15). (P) KEGG pathways enriched in the lipomet subtype (left) and glucomet subtype (right) identified by GSEA. The significance of the difference was determined by Student’s t test (∗p < 0.05; ∗∗p < 0.01) (B). Data are presented as the mean values ± SEMs, and statistical significance was computed by unpaired Student’s t test (C–F). Statistical significance was computed by unpaired Student’s t test (I, K, and M). αKG, α-ketoglutarate; G6P, glucose-6-phosphate; 6PG, 6-phosphate gluconate; F6P, fructose-6-phosphate; R5P, ribose 5-phosphate; S7P, sedoheptulose-7-phosphate; E4P, erythrosine 4-phosphate.

Article Snippet: PDAC organoids in Matrigel (CORNING, 356231) were collected.

Techniques: Labeling, Phospho-proteomics, RNA Sequencing, Expressing, Sequencing

Journal: Cell Reports Medicine

Article Title: Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer

doi: 10.1016/j.xcrm.2023.101162

Figure Lengend Snippet: Glucomet-PDAC is associated with worse prognosis and chemoresistance (A) Heatmap of tumors in the Bailey PDAC cohort (n = 55, only squamous and pancreatic progenitor samples were included) split by glucomet and lipomet signature genes. (B) Kaplan-Meier survival curves of the Bailey PDAC cohort showing differential prognosis among patients with different subtypes. (C) Heatmap of tumors in the TCGA PDAC cohort (n = 156, only ductal pancreatic cancer samples were included) split by glucomet and lipomet signature genes. (D) Kaplan-Meier survival curves of the TCGA PDAC cohort showing differential prognosis among patients with different subtypes. (E) Normalized AUC distribution for GEM, 5-FU, OXA, and IRI on glucomet-PDAC (n = 15) and lipomet-PDAC (n = 13). The Z scores of the obtained normalized AUC values are depicted in the heatmap. High values (indicating resistance) are depicted in red, and low values (indicating sensitivity) are depicted in blue. (F) Comparison of AUCs of four agents among the two metabolic subtypes. The boxplot shows the median (central line) and the 25%–75% interquartile range (box limits). (G and H) Representative radiation examination of both the surgical area and liver in lipomet-PDAC (DAC-5 and DAC-18) and glucomet-PDAC (DAC-42 and DAC-22) at the time of diagnosis and 6 months postsurgery. The arrow marks the tumor position. (I–L) 5-FU responsiveness test in the indicated ODX models (n = 6 per group). Tumor volumes measured by calipers at the indicated time points in tumor-implanted mice subjected to treatments with control or 5-FU (25 mg/kg, every 2 days). Statistical significance was computed by log rank test (B and D). Significance was computed by a one-sided paired t test (F). Data are presented as the mean values ± SEMs, and statistical significance was computed by unpaired Student’s t test (I–L). GEM, gemcitabine; 5-FU, 5-fluorouracil; IRI, irinotecan; OXA, oxaliplatin.

Article Snippet: PDAC organoids in Matrigel (CORNING, 356231) were collected.

Techniques: Comparison, Biomarker Discovery, Control

Journal: Cell Reports Medicine

Article Title: Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer

doi: 10.1016/j.xcrm.2023.101162

Figure Lengend Snippet: The GLUT1 and ALDOB drives glucose metabolic reprogramming in glucomet-PDAC (A) Boxplot of ALDOB and GLUT1 expression levels stratified by metabolic subgroup (p was calculated with limma). (B) Scatterplot showing the correlation of lactate intensities and GLUT1 or ALDOB gene expression (Pearson correlation analysis). Each dot represents an individual sample. Color represents the metabolic subgroup (red dot represents glucomet-PDAC, and blue dot represents lipomet-PDAC). (C) Extracellular glucose consumption and lactate secretion were evaluated in representative organoids of glucomet-PDAC (n = 5) and lipomet-PDAC (n = 5). (D and E) Fractions of labeled metabolites in glycolysis (D) and the TCA cycle (E) from [U- 13 C 6 ] glucose in control and GLUT1 knockdown organoids (n = 3 per group). (F and G) Control and GLUT1 knockdown organoids were exposed to rotenone/antimycin A and 2-DG to measure the ECAR at the basal level and compensatory level by the Seahorse XF Glycolytic Rate Assay (n = 3 per group). (H and I) Fractions of labeled metabolites in glycolysis (H) and the TCA cycle (I) from [U- 13 C 6 ] glucose in control and ALDOB -overexpressing organoids (n = 3 per group). (J and K) Control and ALDOB -overexpressing organoids were exposed to rotenone/antimycin A and 2-DG to measure the ECAR at the basal level and compensatory level by the Seahorse XF Glycolytic Rate Assay (n = 3 per group). (C–E, G–I, and K) Data are presented as mean values ± SEMs. Statistical significance was computed by unpaired Student’s t test.

Article Snippet: PDAC organoids in Matrigel (CORNING, 356231) were collected.

Techniques: Expressing, Gene Expression, Labeling, Control, Knockdown

Journal: Cell Reports Medicine

Article Title: Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer

doi: 10.1016/j.xcrm.2023.101162

Figure Lengend Snippet: The GLUT1/ALDOB/G6PD axis drives glucose metabolic reprogramming in glucomet-PDAC (A) Relative G6PD enzyme activity in representative organoids of glucomet-PDAC (n = 6) and lipomet-PDAC (n = 6). (B) Relative G6PD enzyme activity in vector- and ALDOB -overexpressing organoids (n = 3). (C) Relative abundance of oxidative PPP metabolites in control and ALDOB -overexpressing organoids (n = 3). (D and E) Relative abundance of oxidative PPP metabolites in control, GLUT1 knockdown, ALDOB overexpression, GLUT1 knockdown, and ALDOB overexpression organoids (n = 3). (F) Endogenous ALDOB and G6PD interactions in PDAC organoids detected by immunoprecipitation (IP) experiments. (G–I) TCGA patients with PDAC (n = 156) were divided into GLUT1 high / ALDOB low , GLUT1 low / ALDOB high , and others based on GLUT1 and ALDOB expression levels. Samples with expression of GLUT1 in the top 40% and expression of ALDOB in the last 40% were named GLUT1 high / ALDOB low , while samples with expression of ALDOB in the top 40% and expression of GLUT1 in the last 40% were named GLUT1 low / ALDOB high . GSEA enrichment plot for GLUT1 high / ALDOB low group versus GLUT1 low / ALDOB high group of fatty acid metabolism (G), pentose phosphate pathway (H), and glycolysis/gluconeogenesis (I) signature genes. (J) Representative images of immunohistochemical staining show high or low GLUT1 staining and high or low ALDOB staining in PDAC TMA (n = 285). Scale bar, 50 μm. Patients were divided into four groups based on ALDOB and GLUT1 expression levels: ALDOB high (++), ALDOB expression >50%; ALDOB low (+), ALDOB expression <50%; GLUT1 high (++), GLUT1 expression >50%; and GLUT1 low (+), GLUT1 expression <50%. (K) Kaplan-Meier survival curves based on the expression of GLUT1 and ALDOB in 285 patients with PDAC. (L) Summary scheme highlighting the roles of the GLUT1/ALDOB/G6PD axis in glucose reprogramming. Data are presented as the mean values ± SEMs, and statistical significance was computed by unpaired Student’s t test (A–E). Statistical significance was computed by log-rank test (K).

Article Snippet: PDAC organoids in Matrigel (CORNING, 356231) were collected.

Techniques: Activity Assay, Plasmid Preparation, Control, Knockdown, Over Expression, Immunoprecipitation, Expressing, Immunohistochemical staining, Staining

Journal: Cell Reports Medicine

Article Title: Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer

doi: 10.1016/j.xcrm.2023.101162

Figure Lengend Snippet: The GLUT1/ALDOB/G6PD axis contributes to drug resistance (A) Heatmap showing the relative abundance of nucleoside and nucleoside derivatives in glucomet-PDAC (n = 15) and lipomet-PDAC (n = 13). (B) Heatmap showing Pearson correlation coefficients between the RNA expression levels of ALDOB / GLUT1 and nucleoside derivatives. (C and D) DAC-18 (lipomet) and DAC-42 (glucomet) organoids were treated with 5-FU alone or in combination with pyrimidine nucleosides (uridine, cytidine, and thymidine, 240 μM) for 5 days, and cell viability was determined by CellTiter-Glo assays. (E and F) DAC-18 (lipomet) and DAC-42 (glucomet) organoids were treated with PTX alone or in combination with pyrimidine nucleosides (240 μM) for 5 days, and cell viability was determined by CellTiter-Glo assays. (G) Effect of GLUT1 knockdown on 5-FU responsiveness of DAC-34 (glucomet) as determined by CellTiter-Glo assays 120 h after treatment with 5-FU. (H) Effect of ALDOB overexpression on the 5-FU responsiveness of DAC-42 (glucomet) as determined by CellTiter-Glo assays 120 h after treatment with 5-FU. (I) Effect of G6PD knockdown on the 5-FU responsiveness of DAC-42 (glucomet) as determined by CellTiter-Glo assays 120 h after treatment with 5-FU. (J) Effect of ALDOB overexpression on 5-FU responsiveness in the DAC-42 (glucomet) ODX model (n = 6 per group). (K) Effect of pyrimidine nucleotide (240 μM) on 5-FU sensitivity in control and GLUT1 knockdown organoids by CellTiter-Glo assays at 120 h posttreatment. (L) Effect of pyrimidine nucleotide (240 μM) on 5-FU sensitivity in control and ALDOB overexpression organoids by CellTiter-Glo assays at 120 h posttreatment. (M) Effect of R5P (1 mM) on 5-FU sensitivity in control and GLUT1 knockdown organoids by CellTiter-Glo assays at 120 h posttreatment. (N) Effect of R5P (1 mM) on 5-FU sensitivity in control and ALDOB -overexpressing organoids by CellTiter-Glo assays at 120 h posttreatment. (O) Effect of R5P (1 mM) on 5-FU sensitivity in control and G6PD knockdown organoids by CellTiter-Glo assays at 120 h posttreatment. (P and Q) Effect of purine nucleotides (guanosine and adenosine, 200 μM) on 5-FU and GEM sensitivity in control and GLUT1 knockdown organoids by CellTiter-Glo assays at 120 h posttreatment. All dose-responsive curves were performed with 3 technical replicates. Data are presented as the mean values ± SEMs, and statistical significance was computed by unpaired Student’s t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001) (C–Q).

Article Snippet: PDAC organoids in Matrigel (CORNING, 356231) were collected.

Techniques: RNA Expression, Knockdown, Over Expression, Control

Journal: Cell Reports Medicine

Article Title: Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer

doi: 10.1016/j.xcrm.2023.101162

Figure Lengend Snippet:

Article Snippet: PDAC organoids in Matrigel (CORNING, 356231) were collected.

Techniques: Sequencing, Recombinant, SYBR Green Assay, RNA Expression, shRNA, Software

Journal: Cell reports. Medicine

Article Title: Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer.

doi: 10.1016/j.xcrm.2023.101162

Figure Lengend Snippet: Figure 4. The GLUT1/ALDOB/G6PD axis drives glucose metabolic reprogramming in glucomet-PDAC (A) Relative G6PD enzyme activity in representative organoids of glucomet-PDAC (n = 6) and lipomet-PDAC (n = 6). (B) Relative G6PD enzyme activity in vector- and ALDOB-overexpressing organoids (n = 3). (C) Relative abundance of oxidative PPP metabolites in control and ALDOB-overexpressing organoids (n = 3).

Article Snippet: Metabolon-based energy metabolism detection PDAC organoids inMatrigel were collected.

Techniques: Activity Assay, Plasmid Preparation, Control