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Pancreatic cancer tumor organoids exhibit subtype-specific differences in metabolic profiles - PubMed

  • ️Mon Jan 01 2024

Pancreatic cancer tumor organoids exhibit subtype-specific differences in metabolic profiles

Hassan A Ali et al. Cancer Metab. 2024.

Abstract

Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease characterized by complex metabolic rewiring that enables growth in changing nutrient availability and oxygen conditions. Transcriptome-based prognostic PDAC tumor subtypes, known as 'basal-like' and 'classical' subtypes are associated with differences in metabolic gene expression including genes involved in glycolysis. Tumor subtype-specific metabolism phenotypes may provide new targets for treatment development in PDAC, but their functional relevance has not been fully elucidated. We aimed to investigate differences in metabolic profiles and transcriptomes in tumor models derived from patients with basal-like and classical tumors.

Methods: Patient-derived organoids (PDOs) were established from tumor biopsies collected from patients with metastatic PDAC, including three PDOs from basal-like and five PDOs from classical tumors. Metabolic analyses included assessment of differences in metabolic activity using Seahorse Glycolysis and Mito Stress tests and 13C-glucose metabolites tracing analysis. In order to investigate the influence of mitochondrial pyruvate transport on metabolic differences, PDOs were treated with the mitochondrial pyruvate carrier 1 (MPC1) inhibitor UK-5099. Prognostic relevance of MPC1 was determined using a tumor tissue microarray (TMA) in resectable, and proteomics profiling in metastatic PDAC datasets. Whole genome and transcriptome sequencing, differential gene expression and gene set enrichment analyses were performed in PDOs.

Results: Metastatic PDAC PDOs showed subtype-specific differences in glycolysis and oxidative phosphorylation (OXPHOS). Basal-like tumor-derived PDOs had a lower baseline extracellular acidification rate, but higher glycolytic reserves and oxygen consumption rate (OCR) than classical tumor-derived PDOs. OCR difference was eliminated following treatment with UK-5099. In the 13C-glucose metabolites tracing experiment, a basal-like tumor PDO showed lower fractions of some M + 2 metabolites but higher sensitivity to UK-5099 mediated reduction in M + 2 metabolites than a classical tumor PDO. Protein level analyses revealed lower MPC1 protein levels in basal-like PDAC cases and association of low MPC1 levels with clinicopathologic parameters of tumor aggressiveness in PDAC. PDO differential gene expression analyses identified additional subtype-specific cellular pathways and potential disease outcome biomarkers.

Conclusions: Our findings point to distinct metabolic profiles in PDAC subtypes with basal-like tumor PDOs showing higher OXPHOS and sensitivity to MPC1 inhibition. Subtypes-specific metabolic vulnerabilities may be exploited for selective therapeutic targeting.

Keywords: Glycolysis; MPC1; Metabolic profiling; OXPHOS; Organoids; PDAC; PDAC tumor subtype.

© 2024. The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1

PDOs capture genomic heterogeneity of patient tumors and display subtype specific alterations in metabolism. (a) Oncoprint comparing somatic mutations (SNV/indel) and copy number variation (CNV) events in frequently mutated and metabolic genes between patient tumors (T) and matched PDOs (O). (b) Scatter plot depicting correlation in expression of glycolytic genes between patient tumors and derived PDOs with basal-like or classical subtypes (spearman correlation; Benjamini–Hochberg multiple test correction). (c) Extracellular acidification rate (ECAR) curves of PDOs after injections of glucose, oligomycin and 2-DG during a Glycolysis Stress Test. There are multiple phases recorded, baseline glycolysis refers to ECAR after glucose and before oligomycin injection; maximum glycolysis refers to ECAR after oligomycin injection and before 2-DG injection; and non-glycolytic acidification (NGA) refers to ECAR before injection of glucose or after injection of 2-DG. (d) Bar plots of grouped comparison between PDOs from basal-like and classical tumors showing differences in glycolysis measurements. (e) Oxygen consumption rate (OCR) curves of PDOs after injections of oligomycin, FCCP and Rotenone + Antimycin-A during a Mito Stress Test. Multiple assay phases were recorded, baseline respiration refers to OCR prior to oligomycin injection; maximum respiration refers to OCR after FCCP injection and before Rotenone + Antimycin-A injection; ATP-linked respiration is the difference between basal respiration and OCR after oligomycin injection and before FCCP injection; and non-mitochondrial respiration (NMR) refers to OCR after injection of Rotenone + Antimycin-A. (f) Bar plots of grouped comparison between PDOs from basal-like and classical tumors showing differences in OXPHOS measurements. *p < 0.05, **p < 0.01, ***p < 0.001 (Students t-test)

Fig. 2
Fig. 2

Reduced MPC1 levels are associated with disease aggressiveness and shifts in metabolic activity in PDOs. (a) Immunohistochemistry images showing low (left) and high (right) MPC1 protein levels in PDAC TMA (b) Kaplan-Meier survival analysis of patient tumors with high and low MPC1 protein levels. (c) Bar plots illustrating the association of MPC1 levels with histological grade (p < 0.0001), lymphovascular invasion (p = 0.01) and perineural invasion (p = 0.0009). (d) Box plots illustrating H-scores between basal-like and classical subtypes in TMA of resected PDAC tumors. (e) Box plots showing expression of MPC1 (left) and MPC2 (right) protein in biopsies of metastatic PDAC patient tumors (n = 45). (f) Scatter plots illustrating OCR and ECAR changes in basal-like and classical PDOs treated with a MPC1 inhibitor (UK-5099) following glucose injection (left) and oligomycin injection (right) during a Glycolysis Stress Test. Basal-like PDOs had increased baseline glycolysis levels upon inhibition of MPC1. (g) Scatter plots illustrating OCR and ECAR changes in basal-like and classical PDOs treated with a MPC1 inhibitor (UK-5099) during basal respiration (left) and post FCCP injection (right) during a Mito Stress Test. Basal-like PDOs had increased OCR during basal respiration whereas both basal-like and classical PDOs had increased OCR upon inhibition of MPC1. All ECAR and OCR data points are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3
Fig. 3

UK-5099 leads to a subtype-specific decrease in 13C labelling of glycolysis and TCA cycle metabolites. (a) Illustration of metabolites generated during glycolysis and TCA cycle with carbon backbone lengths. Black dots represent 12C and red dots 13C. (b) Bar plots showing fractions of labelled (M + 2/M + 3) carbon pool for glycolysis and TCA cycle metabolites along with select amino acids; following DMSO and UK-5099 (5𝜇M) treatment in PCBC-015 (basal-like) and PCBC-006 (classical). *p < 0.05, **p < 0.01, ***p < 0.001 (students t-test)

Fig. 4
Fig. 4

Differential gene expression analysis between PDOs from basal-like and classical tumors. (a) Volcano plot of differentially expressed genes between PDOs from basal-like and classical tumors. Expression in basal-like PDOs is shown in reference to classical PDOs. (b) Bar plots showing gene set enrichment analysis performed on up (top; red) and down (bottom; blue) regulated genes in basal-like PDOs compared to classical PDOs. Genes enriched in basal-like PDOs are belong to pathways that associate with aggressive tumors (c) Kaplan-Meier survival curves depicting association of median overall survival with gene expression quantiles in a cohort of PDAC metastatic biopsies (PanGen, n = 69)

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