The Effects of Hypoxia on the Immune–Metabolic Interplay in Liver Cancer
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.1.1. Liver Tumor Cells
2.1.2. Monocytes and Macrophages
2.2. Cell Viability Assay
2.3. Analysis of the Tumor Microenvironment (TME)
2.3.1. Extracellular pH (pHe)
2.3.2. Collection of Tumor-Conditioned Medium
- Liver cancer cells (HepG2 and VX2) were incubated under hypoxia (0.1% O2) with complete medium (final concentration of glucose: 2 g/L) for 48 h, referred to as 0.1%HepG2-TCM and 0.1%VX2-TCM.
- Liver cancer cells (HepG2 and VX2) were incubated under hypoxia (0.1% O2) with high-glucose complete medium (final concentration of glucose: 4.5 g/L) for 48 h, referred to as 0.1%HepG2-TCMHG and 0.1%VX2-TCMHG.
- Liver cancer cells (HepG2 and VX2) were incubated under normoxia (21% O2) with complete RPMI 1640 medium (final concentration of glucose: 2 g/L) for 48 h, referred to as 21%HepG2-TCM and 21%VX2-TCM.
- Liver cancer cells (HepG2 and VX2) were incubated under normoxia (21% O2) with high-glucose complete medium (final concentration of glucose: 4.5 g/L) for 48 h, referred to as 21%HepG2-TCMHG and 21%VX2-TCMHG.
2.3.3. GC-MS-Based Metabolic Profiling of TCM
2.4. Effects of TCMs on the Polarization of Macrophages
2.4.1. Arginase Assay
2.4.2. Nitrite Assay
2.5. Effects of 2-Amino-Butanoic Acid (2A-BA) on the Polarization of Macrophages
2.5.1. Cell Viability Assay
2.5.2. Arginase Assay
2.6. Statistical Analysis
3. Results
3.1. Cell Viability Assay
3.2. Extracellular pH
3.3. Lactate Concentration of Liver Cancer Tumor Microenvironment (TME)
3.4. GC-MS-Based Metabolic Profiling of Liver Cancer Tumor Microenvironment (TME)
3.5. Effects of Tumor-Conditioned Media (TCMs) on Macrophage Polarization
3.5.1. Arginase Assay
3.5.2. Nitrite Assay
3.6. Effects of 2-Amino-Butanoic Acid (2A-BA) on Macrophage Polarization
3.6.1. Cell Viability Assay of 2-Amino-Butanoic Acid (2A-BA)
3.6.2. Arginase Assay of 2-Amino-Butanoic Acid (2A-BA)
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Rumgay, H.; Arnold, M.; Ferlay, J.; Lesi, O.; Cabasag, C.J.; Vignat, J.; Laversanne, M.; McGlynn, K.A.; Soerjomataram, I. Global burden of primary liver cancer in 2020 and predictions to 2040. J. Hepatol. 2022, 77, 1598–1606. [Google Scholar] [CrossRef]
- Niu, Z.S.; Wang, W.H.; Niu, X.J. Recent progress in molecular mechanisms of postoperative recurrence and metastasis of hepatocellular carcinoma. World J. Gastroenterol. 2022, 28, 6433–6477. [Google Scholar] [CrossRef]
- Llovet, J.M.; Kelley, R.K.; Villanueva, A.; Singal, A.G.; Pikarsky, E.; Roayaie, S.; Lencioni, R.; Koike, K.; Zucman-Rossi, J.; Finn, R.S. Hepatocellular carcinoma. Nat. Rev. Dis. Primers 2021, 7, 6. [Google Scholar] [CrossRef]
- Mizuno, K.; Beltran, H. Future directions for precision oncology in prostate cancer. Prostate 2022, 82 (Suppl. S1), S86–S96. [Google Scholar] [CrossRef] [PubMed]
- Topalian, S.L.; Taube, J.M.; Anders, R.A.; Pardoll, D.M. Mechanism-driven biomarkers to guide immune checkpoint blockade in cancer therapy. Nat. Rev. Cancer 2016, 16, 275–287. [Google Scholar] [CrossRef] [PubMed]
- Harbeck, N.; Gnant, M. Breast cancer. Lancet 2017, 389, 1134–1150. [Google Scholar] [CrossRef] [PubMed]
- Liu, A.; Curran, M.A. Tumor hypermetabolism confers resistance to immunotherapy. Semin. Cancer Biol. 2020, 65, 155–163. [Google Scholar] [CrossRef] [PubMed]
- Lunt, S.Y.; Vander Heiden, M.G. Aerobic glycolysis: Meeting the metabolic requirements of cell proliferation. Annu. Rev. Cell Dev. Biol. 2011, 27, 441–464. [Google Scholar] [CrossRef]
- Warburg, O. On respiratory impairment in cancer cells. Science 1956, 124, 269–270. [Google Scholar] [CrossRef]
- Cao, X.; Geng, Q.; Fan, D.; Wang, Q.; Wang, X.; Zhang, M.; Zhao, L.; Jiao, Y.; Deng, T.; Liu, H.; et al. m6A methylation: A process reshaping the tumour immune microenvironment and regulating immune evasion. Mol. Cancer 2023, 22, 42. [Google Scholar] [CrossRef]
- Michl, J.; Wang, Y.; Monterisi, S.; Blaszczak, W.; Beveridge, R.; Bridges, E.M.; Koth, J.; Bodmer, W.F.; Swietach, P. CRISPR-Cas9 screen identifies oxidative phosphorylation as essential for cancer cell survival at low extracellular pH. Cell Rep. 2022, 38, 110493. [Google Scholar] [CrossRef]
- Qin, H.; Yu, H.; Sheng, J.; Zhang, D.; Shen, N.; Liu, L.; Tang, Z.; Chen, X. PI3Kgamma Inhibitor Attenuates Immunosuppressive Effect of Poly(l-Glutamic Acid)-Combretastatin A4 Conjugate in Metastatic Breast Cancer. Adv. Sci. 2019, 6, 1900327. [Google Scholar] [CrossRef] [PubMed]
- Xiang, W.; Shi, R.; Zhang, D.; Kang, X.; Zhang, L.; Yuan, J.; Zhang, X.; Miao, H. Dietary fats suppress the peritoneal seeding of colorectal cancer cells through the TLR4/Cxcl10 axis in adipose tissue macrophages. Signal Transduct. Target. Ther. 2020, 5, 239. [Google Scholar] [CrossRef]
- Yan, L.; Xu, F.; Dai, C.L. Relationship between epithelial-to-mesenchymal transition and the inflammatory microenvironment of hepatocellular carcinoma. J. Exp. Clin. Cancer Res. 2018, 37, 203. [Google Scholar] [CrossRef] [PubMed]
- Bai, R.; Li, Y.; Jian, L.; Yang, Y.; Zhao, L.; Wei, M. The hypoxia-driven crosstalk between tumor and tumor-associated macrophages: Mechanisms and clinical treatment strategies. Mol. Cancer 2022, 21, 177. [Google Scholar] [CrossRef] [PubMed]
- Wei, K.; Zhang, H.; Yang, S.; Cui, Y.; Zhang, B.; Liu, J.; Tang, L.; Tan, Y.; Liu, S.; Chen, S.; et al. Chemo-drugs in cell microparticles reset antitumor activity of macrophages by activating lysosomal P450 and nuclear hnRNPA2B1. Signal Transduct. Target. Ther. 2023, 8, 22. [Google Scholar] [CrossRef] [PubMed]
- Guerra, A.D.; Yeung, O.W.H.; Qi, X.; Kao, W.J.; Man, K. The Anti-Tumor Effects of M1 Macrophage-Loaded Poly (ethylene glycol) and Gelatin-Based Hydrogels on Hepatocellular Carcinoma. Theranostics 2017, 7, 3732–3744. [Google Scholar] [CrossRef] [PubMed]
- Zhou, F.; Mei, J.; Han, X.; Li, H.; Yang, S.; Wang, M.; Chu, L.; Qiao, H.; Tang, T. Kinsenoside attenuates osteoarthritis by repolarizing macrophages through inactivating NF-κB/MAPK signaling and protecting chondrocytes. Acta Pharm. Sin. B 2019, 9, 973–985. [Google Scholar] [CrossRef] [PubMed]
- Han, Y.; Wang, X.; Xia, K.; Su, T. A novel defined hypoxia-related gene signature to predict the prognosis of oral squamous cell carcinoma. Ann. Transl. Med. 2021, 9, 1565. [Google Scholar] [CrossRef] [PubMed]
- Tsilimigras, D.I.; Ntanasis-Stathopoulos, I.; Moris, D.; Pawlik, T.M. Liver Tumor Microenvironment. Adv. Exp. Med. Biol. 2020, 1296, 227–241. [Google Scholar] [CrossRef]
- Clinical Guidelines Committee of Chinese College of Interventionalists. Chinese clinical practice guidelines for transarterial chemoembolization of hepatocellular carcinoma (2023 edition). Zhonghua Yi Xue Za Zhi 2023, 103, 2674–2694. [Google Scholar] [CrossRef]
- Zhong, C.; Niu, Y.; Liu, W.; Yuan, Y.; Li, K.; Shi, Y.; Qiu, Z.; Li, K.; Lin, Z.; Huang, Z.; et al. S100A9 Derived from Chemoembolization-Induced Hypoxia Governs Mitochondrial Function in Hepatocellular Carcinoma Progression. Adv. Sci. 2022, 9, e2202206. [Google Scholar] [CrossRef]
- Lewandowski, R.J.; Geschwind, J.F.; Liapi, E.; Salem, R. Transcatheter intraarterial therapies: Rationale and overview. Radiology 2011, 259, 641–657. [Google Scholar] [CrossRef] [PubMed]
- Savic, L.J.; Schobert, I.T.; Hamm, C.A.; Adam, L.C.; Hyder, F.; Coman, D. A high-throughput imaging platform to characterize extracellular pH in organotypic three-dimensional in vitro models of liver cancer. NMR Biomed. 2021, 34, e4465. [Google Scholar] [CrossRef]
- Ludwig, A.; Poller, W.C.; Westphal, K.; Minkwitz, S.; Lättig-Tünnemann, G.; Metzkow, S.; Stangl, K.; Baumann, G.; Taupitz, M.; Wagner, S.; et al. Rapid binding of electrostatically stabilized iron oxide nanoparticles to THP-1 monocytic cells via interaction with glycosaminoglycans. Basic Res. Cardiol. 2013, 108, 328. [Google Scholar] [CrossRef] [PubMed]
- Pietzke, M.; Zasada, C.; Mudrich, S.; Kempa, S. Decoding the dynamics of cellular metabolism and the action of 3-bromopyruvate and 2-deoxyglucose using pulsed stable isotope-resolved metabolomics. Cancer Metab. 2014, 2, 9. [Google Scholar] [CrossRef]
- Opialla, T.; Kempa, S.; Pietzke, M. Towards a More Reliable Identification of Isomeric Metabolites Using Pattern Guided Retention Validation. Metabolites 2020, 10, 457. [Google Scholar] [CrossRef]
- Suthen, S.; Lim, C.J.; Nguyen, P.H.D.; Dutertre, C.A.; Lai, H.L.H.; Wasser, M.; Chua, C.; Lim, T.K.H.; Leow, W.Q.; Loh, T.J.; et al. Hypoxia-driven immunosuppression by Treg and type-2 conventional dendritic cells in HCC. Hepatology 2022, 76, 1329–1344. [Google Scholar] [CrossRef]
- Salman, S.; Meyers, D.J.; Wicks, E.E.; Lee, S.N.; Datan, E.; Thomas, A.M.; Anders, N.M.; Hwang, Y.; Lyu, Y.; Yang, Y.; et al. HIF inhibitor 32-134D eradicates murine hepatocellular carcinoma in combination with anti-PD1 therapy. J. Clin. Investig. 2022, 132, e156774. [Google Scholar] [CrossRef]
- Yang, M.; McKay, D.; Pollard, J.W.; Lewis, C.E. Diverse Functions of Macrophages in Different Tumor Microenvironments. Cancer Res. 2018, 78, 5492–5503. [Google Scholar] [CrossRef]
- Viola, A.; Munari, F.; Sánchez-Rodríguez, R.; Scolaro, T.; Castegna, A. The Metabolic Signature of Macrophage Responses. Front. Immunol. 2019, 10, 1462. [Google Scholar] [CrossRef] [PubMed]
- Huang, S.C.; Smith, A.M.; Everts, B.; Colonna, M.; Pearce, E.L.; Schilling, J.D.; Pearce, E.J. Metabolic Reprogramming Mediated by the mTORC2-IRF4 Signaling Axis Is Essential for Macrophage Alternative Activation. Immunity 2016, 45, 817–830. [Google Scholar] [CrossRef]
- Tan, Z.; Xie, N.; Cui, H.; Moellering, D.R.; Abraham, E.; Thannickal, V.J.; Liu, G. Pyruvate dehydrogenase kinase 1 participates in macrophage polarization via regulating glucose metabolism. J. Immunol. 2015, 194, 6082–6089. [Google Scholar] [CrossRef]
- Wang, F.; Zhang, S.; Vuckovic, I.; Jeon, R.; Lerman, A.; Folmes, C.D.; Dzeja, P.P.; Herrmann, J. Glycolytic Stimulation Is Not a Requirement for M2 Macrophage Differentiation. Cell Metab. 2018, 28, 463–475.e464. [Google Scholar] [CrossRef] [PubMed]
- Colegio, O.R.; Chu, N.Q.; Szabo, A.L.; Chu, T.; Rhebergen, A.M.; Jairam, V.; Cyrus, N.; Brokowski, C.E.; Eisenbarth, S.C.; Phillips, G.M.; et al. Functional polarization of tumour-associated macrophages by tumour-derived lactic acid. Nature 2014, 513, 559–563. [Google Scholar] [CrossRef]
- Damaghi, M.; Gillies, R. Phenotypic changes of acid-adapted cancer cells push them toward aggressiveness in their evolution in the tumor microenvironment. Cell Cycle 2017, 16, 1739–1743. [Google Scholar] [CrossRef]
- Fischer, K.; Hoffmann, P.; Voelkl, S.; Meidenbauer, N.; Ammer, J.; Edinger, M.; Gottfried, E.; Schwarz, S.; Rothe, G.; Hoves, S.; et al. Inhibitory effect of tumor cell-derived lactic acid on human T cells. Blood 2007, 109, 3812–3819. [Google Scholar] [CrossRef]
- Goetze, K.; Walenta, S.; Ksiazkiewicz, M.; Kunz-Schughart, L.A.; Mueller-Klieser, W. Lactate enhances motility of tumor cells and inhibits monocyte migration and cytokine release. Int. J. Oncol. 2011, 39, 453–463. [Google Scholar] [CrossRef] [PubMed]
- Husain, A.; Chiu, Y.T.; Sze, K.M.; Ho, D.W.; Tsui, Y.M.; Suarez, E.M.S.; Zhang, V.X.; Chan, L.K.; Lee, E.; Lee, J.M.; et al. Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma. J. Hepatol. 2022, 77, 383–396. [Google Scholar] [CrossRef]
- Mookerjee, S.A.; Gerencser, A.A.; Nicholls, D.G.; Brand, M.D. Quantifying intracellular rates of glycolytic and oxidative ATP production and consumption using extracellular flux measurements. J. Biol. Chem. 2017, 292, 7189–7207. [Google Scholar] [CrossRef]
- Chiarla, C.; Giovannini, I.; Siegel, J.H. Characterization of alpha-amino-n-butyric acid correlations in sepsis. Transl. Res. J. Lab. Clin. Med. 2011, 158, 328–333. [Google Scholar] [CrossRef]
- Irino, Y.; Toh, R.; Nagao, M.; Mori, T.; Honjo, T.; Shinohara, M.; Tsuda, S.; Nakajima, H.; Satomi-Kobayashi, S.; Shinke, T.; et al. 2-Aminobutyric acid modulates glutathione homeostasis in the myocardium. Sci. Rep. 2016, 6, 36749. [Google Scholar] [CrossRef] [PubMed]
- Toklu, S.; Kemerdere, R.; Kacira, T.; Gurses, M.S.; Benli Aksungar, F.; Tanriverdi, T. Tissue and plasma free amino acid detection by LC-MS/MS method in high grade glioma patients. J. Neuro-Oncol. 2023, 163, 293–300. [Google Scholar] [CrossRef] [PubMed]
- Li, F.; Xia, Y.; Yuan, S.; Xie, X.; Li, L.; Luo, Y.; Du, Q.; Yuan, Y.; He, R. α-Aminobutyric Acid Constrains Macrophage-Associated Inflammatory Diseases through Metabolic Reprogramming and Epigenetic Modification. Int. J. Mol. Sci. 2023, 24, 10444. [Google Scholar] [CrossRef] [PubMed]
- Borges, N.; Doering, T.M.; Murphy, G.; Macdonald, M.; Dunstan, R.H. Amino acid distribution in blood following high-intensity interval exercise: A preliminary study. Amino Acids 2024, 56, 4. [Google Scholar] [CrossRef] [PubMed]
- Jia, B.; Wang, W.; Lin, S.; Shi, L.; Li, Y.; Gu, Y.; Gao, F.; Qin, Y. The free amino acid profiles and metabolic biomarkers of predicting the chemotherapeutic response in advanced sarcoma patients. Clin. Transl. Oncol. Off. Publ. Fed. Span. Oncol. Soc. Natl. Cancer Inst. Mex. 2020, 22, 2213–2221. [Google Scholar] [CrossRef] [PubMed]
- Bratt, J.M.; Zeki, A.A.; Last, J.A.; Kenyon, N.J. Competitive metabolism of L-arginine: Arginase as a therapeutic target in asthma. J. Biomed. Res. 2011, 25, 299–308. [Google Scholar] [CrossRef]
- Noe, J.T.; Rendon, B.E.; Geller, A.E.; Conroy, L.R.; Morrissey, S.M.; Young, L.E.A.; Bruntz, R.C.; Kim, E.J.; Wise-Mitchell, A.; Barbosa de Souza Rizzo, M.; et al. Lactate supports a metabolic-epigenetic link in macrophage polarization. Sci. Adv. 2021, 7, eabi8602. [Google Scholar] [CrossRef]
- Menjivar, R.E.; Nwosu, Z.C.; Du, W.; Donahue, K.L.; Hong, H.S.; Espinoza, C.; Brown, K.; Velez-Delgado, A.; Yan, W.; Lima, F.; et al. Arginase 1 is a key driver of immune suppression in pancreatic cancer. eLife 2023, 12, e80721. [Google Scholar] [CrossRef]
- Kelly, B.; O’Neill, L.A. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res. 2015, 25, 771–784. [Google Scholar] [CrossRef]
- Iniesta, V.; Gómez-Nieto, L.C.; Corraliza, I. The inhibition of arginase by N(omega)-hydroxy-l-arginine controls the growth of Leishmania inside macrophages. J. Exp. Med. 2001, 193, 777–784. [Google Scholar] [CrossRef]
- Das, P.; Lahiri, A.; Lahiri, A.; Chakravortty, D. Modulation of the arginase pathway in the context of microbial pathogenesis: A metabolic enzyme moonlighting as an immune modulator. PLoS Pathog. 2010, 6, e1000899. [Google Scholar] [CrossRef] [PubMed]
- Griffiths, H.R.; Gao, D.; Pararasa, C. Redox regulation in metabolic programming and inflammation. Redox Biol. 2017, 12, 50–57. [Google Scholar] [CrossRef]
- Reinfeld, B.I.; Madden, M.Z.; Wolf, M.M.; Chytil, A.; Bader, J.E.; Patterson, A.R.; Sugiura, A.; Cohen, A.S.; Ali, A.; Do, B.T.; et al. Cell-programmed nutrient partitioning in the tumour microenvironment. Nature 2021, 593, 282–288. [Google Scholar] [CrossRef] [PubMed]
- Nath, N.; Kashfi, K. Tumor associated macrophages and ‘NO’. Biochem. Pharmacol. 2020, 176, 113899. [Google Scholar] [CrossRef] [PubMed]
- Savic, L.J.; Schobert, I.T.; Peters, D.; Walsh, J.J.; Laage-Gaupp, F.M.; Hamm, C.A.; Tritz, N.; Doemel, L.A.; Lin, M.; Sinusas, A.; et al. Molecular Imaging of Extracellular Tumor pH to Reveal Effects of Locoregional Therapy on Liver Cancer Microenvironment. Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 2020, 26, 428–438. [Google Scholar] [CrossRef]
- Xia, P.; Zhang, H.; Lu, H.; Xu, K.; Jiang, X.; Jiang, Y.; Gongye, X.; Chen, Z.; Liu, J.; Chen, X.; et al. METTL5 stabilizes c-Myc by facilitating USP5 translation to reprogram glucose metabolism and promote hepatocellular carcinoma progression. Cancer Commun. 2023, 43, 338–364. [Google Scholar] [CrossRef] [PubMed]
- Zhang, T.; Sun, L.; Hao, Y.; Suo, C.; Shen, S.; Wei, H.; Ma, W.; Zhang, P.; Wang, T.; Gu, X.; et al. ENO1 suppresses cancer cell ferroptosis by degrading the mRNA of iron regulatory protein 1. Nat. Cancer 2022, 3, 75–89. [Google Scholar] [CrossRef]
- Galon, J.; Bruni, D. Approaches to treat immune hot, altered and cold tumours with combination immunotherapies. Nat. Rev. Drug Discov. 2019, 18, 197–218. [Google Scholar] [CrossRef]
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He, Y.; Xu, H.; Liu, Y.; Kempa, S.; Vechiatto, C.; Schmidt, R.; Yilmaz, E.Y.; Heidemann, L.; Schnorr, J.; Metzkow, S.; et al. The Effects of Hypoxia on the Immune–Metabolic Interplay in Liver Cancer. Biomolecules 2024, 14, 1024. https://doi.org/10.3390/biom14081024
He Y, Xu H, Liu Y, Kempa S, Vechiatto C, Schmidt R, Yilmaz EY, Heidemann L, Schnorr J, Metzkow S, et al. The Effects of Hypoxia on the Immune–Metabolic Interplay in Liver Cancer. Biomolecules. 2024; 14(8):1024. https://doi.org/10.3390/biom14081024
Chicago/Turabian StyleHe, Yubei, Han Xu, Yu Liu, Stefan Kempa, Carolina Vechiatto, Robin Schmidt, Emine Yaren Yilmaz, Luisa Heidemann, Jörg Schnorr, Susanne Metzkow, and et al. 2024. "The Effects of Hypoxia on the Immune–Metabolic Interplay in Liver Cancer" Biomolecules 14, no. 8: 1024. https://doi.org/10.3390/biom14081024