Fig 1: Intratumoral heterogeneity in gluconeogenesis gene expression and relation to tumor size. (A) Differences in glycolysis and gluconeogenesis gene expression according to T stage *P < 0.05, **P < 0.01 on Mann–Whitney U‐test versus T1. n.s., not significant; (B) Expression intensity of PCK2 as compared between tumor margin (arrow) and center. Normal lung tissue is indicated with asterisks. Scale bar = 100 µm. Strongly stained macrophages (orange circles) were automatically excluded by setting an appropriate cutoff value. Right: Results of automated analysis by means of the cellprofiler software.
Fig 2: Glycolysis and gluconeogenesis markers in NSCLC and differences according to histology and tumor size. (A) Examples of strong or moderate (1st row) versus weak/negative IHC staining (2nd row) for PCK1, PCK2, LDHB, and GLUT1 in tumors and normal lung tissue (3rd row). Scale bar = 50 µm. (B) Comparison of IHC scores in adenocarcinoma (AD) and squamous cell carcinoma (SC). Group comparisons were performed with the Mann–Whitney U‐test in LUAD and LUSC patients with evaluable PCK1 (n = 258/85), PCK2 (n = 264/94), LDHB (n = 257/92), and GLUT1 staining (n = 276/77). *** P < 0.001. (C) Frequency of glycolytic (GLUT1 positive, PCK1/2 negative), gluconeogenic (GLUT1 negative, PCK1 or PCK2 positive), and mixed phenotypes (GLUT1 positive and PCK1 or PCK2 positive). Samples without immunopositivity for either PCK1, PCK2, or GLUT1 were defined as 'unspecified'. LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma. (D) Waterfall plot visualizing combined PCK1/2 IHC scores and GLUT1 scores in a total of 317 NSCLC patients.
Fig 3: Association of gluconeogenic enzyme expression in lung adenocarcinoma with overall survival. (A) Kaplan–Meier survival functions +/‐ 95% confidence interval in LUAD patients with positive or negative PCK1, PCK2, LDHB and GLUT1 staining (cutoff = 1). (B) Kaplan–Meier estimators of the publicly available TCGA LUAD data for PCK2 and GLUT1 (SLC2A1) mRNA, respectively (n = 491; data were retrieved via the UCSC Xena platform, https://xenabrowser.net/). Median values were used as the cutoff. (C) Overall survival in LUAD patients according to the metabolic phenotype. (A–C) Differences in survival were calculated using the Logrank test.
Fig 4: Gluconeogenesis and glycolysis enzyme expression in primary tumors and metastases. (A) A total number of 42 primary tumors from metastatic patients and 54 samples of NSCLC metastases were analyzed. No significant differences in IHC expression between primaries and metastases were observed in the separate analysis of PCK1, PCK2, LDHB, and GLUT1. The combined score of PCK1 and PCK2 (sum of the two scores) is significantly higher in the metastases. Group comparisons were performed with the Mann–Whitney U‐test in primary tumors and metastases with evaluable PCK1 (n = 39/41), PCK2 (n = 41/50), LDHB (n = 40/48), GLUT1 staining (n = 42/53), or PCK1/2 combined scores (n = 39/41). *P < 0.05. (B) distribution of glycolytic, gluconeogenic, and mixed phenotypes. Samples without immunopositivity for either PCK1, PCK2, or GLUT1 were defined as 'unspecified'.
Fig 5: PCK2 and GLUT1 expression are modulated by hypoxia in lung cancer cell lines. Expression of PCK2 protein in H460 (A) and A549 (B) lung cancer cell lines, cultured in high glucose (10 mm) or low glucose (1 mm) media, in normoxia or hypoxia (1% oxygen). Beta‐actin and GAPDH were used as loading controls. Integrated density was normalized to first sample and the loading control. (C and D) GLUT1 (SLC2A1) expression analyzed by quantitative PCR in H460 (C) and A549 (D) cells. Results, displayed as mean +/‐ SEM, were obtained in four (A, B), six (C), or five (D) independent experiments. Group comparisons were performed using Student’s t‐test or one‐group Student’s t‐test as applicable. *P < 0.05, **P < 0.01, ***P < 0.001.
Supplier Page from Abcam for Anti-PCK2 antibody