J Pathol Transl Med.  2023 Nov;57(6):305-314. 10.4132/jptm.2023.10.09.

Senescent tumor cells in colorectal cancer are characterized by elevated enzymatic activity of complexes 1 and 2 in oxidative phosphorylation

• Shin JS
• Kim TG
• Kim YH
• Eom SY
• Park SH
• Lee DH
• Park TJ
• Park SS
• Kim JH

• KMID: 2547928
• DOI: http://doi.org/10.4132/jptm.2023.10.09

Abstract

Background
Cellular senescence is defined as an irreversible cell cycle arrest caused by various internal and external insults. While the metabolic dysfunction of senescent cells in normal tissue is relatively well-established, there is a lack of information regarding the metabolic features of senescent tumor cells.
Methods
Publicly available single-cell RNA-sequencing data from the GSE166555 and GSE178341 datasets were utilized to investigate the metabolic features of senescent tumor cells. To validate the single-cell RNA-sequencing data, we performed senescence-associated β-galactosidase (SA-β-Gal) staining to identify senescent tumor cells in fresh frozen colorectal cancer tissue. We also evaluated nicotinamide adenine dinucleotide dehydrogenase–tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH) activity using enzyme histochemical methods and compared the staining with SA-β-Gal staining. MTT assay was performed to reveal the complex 1 activity of the respiratory chain in in-vitro senescence model.
Results
Single-cell RNA-sequencing data revealed an upregulation in the activity of complexes 1 and 2 in oxidative phosphorylation, despite overall mitochondrial dysfunction in senescent tumor cells. Both SA-β-Gal and enzyme histochemical staining using fresh frozen colorectal cancer tissues indicated a high correlation between SA-β-Gal positivity and NADH-TR/SDH staining positivity. MTT assay showed that senescent colorectal cancer cells exhibit higher absorbance in 600 nm wavelength.
Conclusions
Senescent tumor cells exhibit distinct metabolic features, characterized by upregulation of complexes 1 and 2 in the oxidative phosphorylation pathway. NADH-TR and SDH staining represent efficient methods for detecting senescent tumor cells in colorectal cancer.

Keyword

Colorectal neoplasms; Metabolism; Cellular senescence; Oxidative phosphorylation; NADH

Figure

• 

  Fig. 1. Single-cell RNA-sequencing (scRNA-seq) of human colorectal cancer tissues. (A) Uniform Manifold Approximation and Projection (UMAP) of public scRNA-seq dataset GSE166555 patient #8 who markedly expressed CDKN2A is shown. A total of 274 cells were analyzed. (B) Gene set enrichment analysis (GSEA) of “Fridman Senescence Up” (left panel) and “GOBP Cellular Senescence” gene sets was performed in CDKN2A+ vs. CDKN2A– cancer cells. (C) GSEA of a “GOBP Oxidative Phosphorylation” gene set was performed in CDKN2A+ vs. CDKN2A– cancer cells. (D) GSEA of a “GOBP Mitochondrial Electron Transport NADH to Ubiquinone” gene set was performed in CDKN2A+ vs. CDKN2A– cancer cells. (E) GSEA of a “GOBP Mitochondrial Electron Transport Ubiquinol to Cytochrome c” gene set was performed in CDKN2A+ vs. CDKN2A– cancer cells. (F) GSEA of a “GOBP ATP Synthesis Coupled Electron Transport” gene set was performed in CDKN2A+ vs. CDKN2A– cancer cells. (G) Schematic image of expected electron transport efficiency in senescent and non-senescent tumor cells. respectively. The thickness of red arrows indicates the efficiency of electron flow. ‘padj’ indicates the adjusted p-value. CRC, colorectal cancer; ES, enrichment score; NES, normalized enrichment score.

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  Fig. 2. Nicotinamide adenine dinucleotide dehydrogenase–tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH) staining results in the senescence-associated β-galactosidase (SA-β-Gal) high patient. The staining results of SA-β-Gal (A), NADH-TR (B), and SDH staining (C) are shown in the SA-β-Gal high patient.

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  Fig. 3. Nicotinamide adenine dinucleotide dehydrogenase–tetrazolium reductase (NADH-TR) and succinate dehydrogenase (SDH) staining results in the senescence-associated β-galactosidase (SA-β-Gal) low patient. The staining results of SA-β-Gal (A), NADH-TR (B), and SDH staining (C) are shown in the SA-β-Gal low patient.

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  Fig. 4. Scatter plots according to the nicotinamide adenine dinucleotide dehydrogenase–tetrazolium reductase (NADH-TR) (A) and succinate dehydrogenase (SDH) (B) staining results compared with senescence-associated β-galactosidase (SA-β-Gal) staining. p-values are obtained using the hypothesis test for regression slope.

Reference

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