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Blaszczak W, White B, Swietach P. Protocol for separating cancer cell subpopulations by metabolic activity using flow cytometry. STAR Protoc 2024; 5:103105. [PMID: 38824638 PMCID: PMC11176822 DOI: 10.1016/j.xpro.2024.103105] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/29/2024] [Revised: 04/20/2024] [Accepted: 05/13/2024] [Indexed: 06/04/2024] Open
Abstract
Cells, even from the same line, can maintain heterogeneity in metabolic activity. Here, we present a protocol, adapted for fluorescence-activated cell sorting (FACS), that separates resuspended cells according to their metabolic rate. We describe steps for driving lactate efflux, which produces an alkaline transient proportional to fermentative rate. This pH signature, measured using pH-sensitive dyes, identifies cells with the highest metabolic rate. We then describe a fluorimetric assay of oxygen consumption and acid production to confirm the metabolic contrast between subpopulations. For complete details on the use and execution of this protocol, please refer to Blaszczak et al.1.
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Affiliation(s)
- Wiktoria Blaszczak
- Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, OX1 3PT Oxford, UK
| | - Bobby White
- Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, OX1 3PT Oxford, UK
| | - Pawel Swietach
- Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, OX1 3PT Oxford, UK.
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Blaszczak W, White B, Monterisi S, Swietach P. Dynamic IL-6R/STAT3 signaling leads to heterogeneity of metabolic phenotype in pancreatic ductal adenocarcinoma cells. Cell Rep 2024; 43:113612. [PMID: 38141171 PMCID: PMC11149489 DOI: 10.1016/j.celrep.2023.113612] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2023] [Revised: 09/29/2023] [Accepted: 12/07/2023] [Indexed: 12/25/2023] Open
Abstract
Malignancy is enabled by pro-growth mutations and adequate energy provision. However, global metabolic activation would be self-terminating if it depleted tumor resources. Cancer cells could avoid this by rationing resources, e.g., dynamically switching between "baseline" and "activated" metabolic states. Using single-cell metabolic phenotyping of pancreatic ductal adenocarcinoma cells, we identify MIA-PaCa-2 as having broad heterogeneity of fermentative metabolism. Sorting by a readout of lactic acid permeability separates cells by fermentative and respiratory rates. Contrasting phenotypes persist for 4 days and are unrelated to cell cycling or glycolytic/respiratory gene expression; however, transcriptomics links metabolically active cells with interleukin-6 receptor (IL-6R)-STAT3 signaling. We verify this by IL-6R/STAT3 knockdowns and sorting by IL-6R status. IL-6R/STAT3 activates fermentation and transcription of its inhibitor, SOCS3, resulting in delayed negative feedback that underpins transitions between metabolic states. Among cells manifesting wide metabolic heterogeneity, dynamic IL-6R/STAT3 signaling may allow cell cohorts to take turns in progressing energy-intense processes without depleting shared resources.
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Affiliation(s)
- Wiktoria Blaszczak
- Department of Physiology, Anatomy & Genetics, University of Oxford, Sherrington Building, Parks Road, OX1 3PT Oxford, UK
| | - Bobby White
- Department of Physiology, Anatomy & Genetics, University of Oxford, Sherrington Building, Parks Road, OX1 3PT Oxford, UK
| | - Stefania Monterisi
- Department of Physiology, Anatomy & Genetics, University of Oxford, Sherrington Building, Parks Road, OX1 3PT Oxford, UK
| | - Pawel Swietach
- Department of Physiology, Anatomy & Genetics, University of Oxford, Sherrington Building, Parks Road, OX1 3PT Oxford, UK.
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Michl J, Monterisi S, White B, Blaszczak W, Hulikova A, Abdullayeva G, Bridges E, Yin Z, Bodmer WF, Swietach P. Acid-adapted cancer cells alkalinize their cytoplasm by degrading the acid-loading membrane transporter anion exchanger 2, SLC4A2. Cell Rep 2023; 42:112601. [PMID: 37270778 DOI: 10.1016/j.celrep.2023.112601] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2022] [Revised: 03/16/2023] [Accepted: 05/19/2023] [Indexed: 06/06/2023] Open
Abstract
Acidic environments reduce the intracellular pH (pHi) of most cells to levels that are sub-optimal for growth and cellular functions. Yet, cancers maintain an alkaline cytoplasm despite low extracellular pH (pHe). Raised pHi is thought to be beneficial for tumor progression and invasiveness. However, the transport mechanisms underpinning this adaptation have not been studied systematically. Here, we characterize the pHe-pHi relationship in 66 colorectal cancer cell lines and identify the acid-loading anion exchanger 2 (AE2, SLC4A2) as a regulator of resting pHi. Cells adapt to chronic extracellular acidosis by degrading AE2 protein, which raises pHi and reduces acid sensitivity of growth. Acidity inhibits mTOR signaling, which stimulates lysosomal function and AE2 degradation, a process reversed by bafilomycin A1. We identify AE2 degradation as a mechanism for maintaining a conducive pHi in tumors. As an adaptive mechanism, inhibiting lysosomal degradation of AE2 is a potential therapeutic target.
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Affiliation(s)
- Johanna Michl
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK
| | - Stefania Monterisi
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK
| | - Bobby White
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK
| | - Wiktoria Blaszczak
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK
| | - Alzbeta Hulikova
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK
| | - Gulnar Abdullayeva
- MRC Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
| | - Esther Bridges
- Department of NDM Experimental Medicine, MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, JR Hospital, Headington, Oxford OX3 9DS, UK
| | - Zinan Yin
- Department of NDM Experimental Medicine, MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, JR Hospital, Headington, Oxford OX3 9DS, UK
| | - Walter F Bodmer
- MRC Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
| | - Pawel Swietach
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK.
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Autoregulation of H +/lactate efflux prevents monocarboxylate transport (MCT) inhibitors from reducing glycolytic lactic acid production. Br J Cancer 2022; 127:1365-1377. [PMID: 35840734 PMCID: PMC9519749 DOI: 10.1038/s41416-022-01910-7] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2022] [Revised: 06/24/2022] [Accepted: 06/29/2022] [Indexed: 12/02/2022] Open
Abstract
Background Pharmacological inhibition of membrane transporters is expected to reduce the flow of solutes, unless flux is restored (i.e., autoregulated) through a compensatory increase in the transmembrane driving force. Drugs acting on monocarboxylate transporters (MCTs) have been developed to disrupt glycolytic metabolism, but autoregulation would render such interventions ineffective. We evaluated whether small-molecule MCT inhibitors reduce cellular H+/lactate production. Methods Cellular assays measured the relationship between MCT activity (expressed as membrane H+/lactate permeability; PHLac) and lactic acid production (inferred from H+ and lactate excretion; JHLac) in a panel of pancreatic ductal adenocarcinoma (PDAC) cells spanning a range of glycolytic phenotype. Results MCT activity did not correlate with lactic acid production, indicating that it is not set by membrane permeability properties. MCT inhibitors did not proportionately reduce JHLac because of a compensatory increase in the transmembrane [lactate] driving force. JHLac was largely insensitive to [lactate], therefore its cytoplasmic build-up upon MCT inhibition does not hinder glycolytic production. Extracellular acidity, an MCT inhibitor, reduced JHLac but this was via cytoplasmic acidification blocking glycolytic enzymes. Conclusions We provide mathematically verified evidence that pharmacological and physiological modulators of MCTs cannot proportionately reduce lactic acid production because of the stabilising effect of autoregulation on overall flux.
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Michl J, Wang Y, Monterisi S, Blaszczak W, Beveridge R, Bridges EM, Koth J, Bodmer WF, Swietach P. CRISPR-Cas9 screen identifies oxidative phosphorylation as essential for cancer cell survival at low extracellular pH. Cell Rep 2022; 38:110493. [PMID: 35263578 PMCID: PMC8924371 DOI: 10.1016/j.celrep.2022.110493] [Citation(s) in RCA: 28] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2021] [Revised: 01/05/2022] [Accepted: 02/14/2022] [Indexed: 02/04/2023] Open
Abstract
Unlike most cell types, many cancer cells survive at low extracellular pH (pHe), a chemical signature of tumors. Genes that facilitate survival under acid stress are therefore potential targets for cancer therapies. We performed a genome-wide CRISPR-Cas9 cell viability screen at physiological and acidic conditions to systematically identify gene knockouts associated with pH-related fitness defects in colorectal cancer cells. Knockouts of genes involved in oxidative phosphorylation (NDUFS1) and iron-sulfur cluster biogenesis (IBA57, NFU1) grew well at physiological pHe, but underwent profound cell death under acidic conditions. We identified several small-molecule inhibitors of mitochondrial metabolism that can kill cancer cells at low pHe only. Xenografts established from NDUFS1-/- cells grew considerably slower than their wild-type controls, but growth could be stimulated with systemic bicarbonate therapy that lessens the tumoral acid stress. These findings raise the possibility of therapeutically targeting mitochondrial metabolism in combination with acid stress as a cancer treatment option.
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Affiliation(s)
- Johanna Michl
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK.
| | - Yunyi Wang
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK
| | - Stefania Monterisi
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK
| | - Wiktoria Blaszczak
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK
| | - Ryan Beveridge
- Virus Screening Facility, MRC Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK
| | - Esther M Bridges
- Department of NDM Experimental Medicine, MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, JR Hospital, Headington, Oxford OX3 9DS, UK
| | - Jana Koth
- MRC Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
| | - Walter F Bodmer
- MRC Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK
| | - Pawel Swietach
- Department of Physiology, Anatomy and Genetics, Parks Road, Oxford OX1 3PT, UK.
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Klein SG, Alsolami SM, Arossa S, Ramos-Mandujano G, Parry AJ, Steckbauer A, Duarte CM, Li M. In situ monitoring reveals cellular environmental instabilities in human pluripotent stem cell culture. Commun Biol 2022; 5:119. [PMID: 35136190 PMCID: PMC8826360 DOI: 10.1038/s42003-022-03065-w] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Accepted: 01/20/2022] [Indexed: 12/26/2022] Open
Abstract
Mammalian cell cultures are a keystone resource in biomedical research, but the results of published experiments often suffer from reproducibility challenges. This has led to a focus on the influence of cell culture conditions on cellular responses and reproducibility of experimental findings. Here, we perform frequent in situ monitoring of dissolved O2 and CO2 with optical sensor spots and contemporaneous evaluation of cell proliferation and medium pH in standard batch cultures of three widely used human somatic and pluripotent stem cell lines. We collate data from the literature to demonstrate that standard cell cultures consistently exhibit environmental instability, indicating that this may be a pervasive issue affecting experimental findings. Our results show that in vitro cell cultures consistently undergo large departures of environmental parameters during standard batch culture. These findings should catalyze further efforts to increase the relevance of experimental results to the in vivo physiology and enhance reproducibility.
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Affiliation(s)
- Shannon G Klein
- Red Sea Research Center (RSRC) and Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia
| | - Samhan M Alsolami
- Biological and Environmental Science and Engineering Division (BESE), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia
| | - Silvia Arossa
- Red Sea Research Center (RSRC) and Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia
| | - Gerardo Ramos-Mandujano
- Biological and Environmental Science and Engineering Division (BESE), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia
| | - Anieka J Parry
- Red Sea Research Center (RSRC) and Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia
| | - Alexandra Steckbauer
- Red Sea Research Center (RSRC) and Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia
| | - Carlos M Duarte
- Red Sea Research Center (RSRC) and Computational Bioscience Research Center (CBRC), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia.
| | - Mo Li
- Biological and Environmental Science and Engineering Division (BESE), King Abdullah University of Science and Technology, Thuwal, 23955, Saudi Arabia.
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