Crosstalk between AML and stromal cells triggers acetate secretion through the metabolic rewiring of stromal cells
- Institute of Cancer and Genomic Sciences, University of Birmingham, United Kingdom
- Stem Cells, Ageing and Cancer Research Group, Department of Medical Biology, Faculty of Health Sciences, UiT – The Arctic University of Norway, Norway
- Department of Laboratory Medicine (hematology), Faculty of Applied Medical Sciences. Albaha University, Kingdom of Saudi Arabia, Saudi Arabia
- Centre for Computational Biology, University of Birmingham, United Kingdom
- Institute of Chemistry and Metabolomics, University of Lübeck, Germany
- Department of Experimental Hematology, University Medical Center Groningen, University of Groningen, Netherlands
- Department of Medicine, Hematology/Oncology, University Hospital Frankfurt, Goethe University Frankfurt, Germany
- Frankfurt Cancer Institute, Goethe University Frankfurt, Germany
- German Cancer Consortium (DKTK), partner site Frankfurt/Mainz, and German Cancer Research Center (DKFZ), Germany
- Centre for Clinical Haematology, University Hospitals Birmingham NHS Foundation Trust, Queen Elizabeth Hospital, Queen Elizabeth Medical Centre, United Kingdom
Figures
Figure 1 with 2 supplements
Acetate secretion by stromal cells increases in AML-stroma co-cultures of several AML cell lines and primary AML cells in direct contact.
(A) Section of 1H-NMR spectra, corresponding to the methyl group of acetate, from extracellular medium samples of SKM-1 cells cultured alone (blue), MS-5 cells cultured alone (red) and SKM-1 and …
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Figure 1—source data 1
Values and stats for panels included in Figure 1.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Other extracellular metabolite levels in co-cultures with SKM-1 and MS-5 cells.
Sections of 1H-NMR spectra from extracellular medium samples of SKM-1 cells cultured alone (blue), MS-5 cells cultured alone (red) and SKM-1 and MS-5 cells in co-culture (green) after 24 hr of …
Figure 1—figure supplement 2
Proliferation in co-culture, co-culturing AML cells with an unrelated cell line (HeLa).
(A) CFSE cell proliferation analysis in AML cell lines alone or in co-culture with MS-5 cells. The population of living cells was gated, and 1500 cells for each condition were randomly selected and …
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Figure 1—figure supplement 2—source data 1
Values obtained for cell proliferation with CFSE in AML cell lines cultured alone vs in coculture (A) and raw extracellular acetate values obtained for SKM-1 grown in cocultured with HeLa cells.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig1-figsupp2-data1-v3.xlsx
Figure 2 with 5 supplements
AML cells can import the secreted acetate in co-culture to use it in TCA cycle and lipid biogenesis.
(A) Schematic of label distribution arising from [2-13C]acetate in TCA cycle intermediates. Black circles correspond to positions expected to be labelled. (B) 13C percentages of label incorporation …
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Figure 2—source data 1
Data and stats for panels included in Figure 2.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig2-data1-v3.xlsx
Figure 2—figure supplement 1
Titration of acetate concentration in co-culture and acetate consumption by AML cells.
(A) Linear regression of acetate concentrations and detected intensities in 1H-NMR spectra (Intensity = 1.38·106±1.6·104 x acetate concentration +2.27·105±3.3·104; R2=0.9987). The intensities …
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Figure 2—figure supplement 1—source data 1
Raw values for acetate titration (A) and acetate consumption by SKM1 (B).
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig2-figsupp1-data1-v3.xlsx
Figure 2—figure supplement 2
Label incorporation from [2-13C]acetate in SKM-1 and MS-5 cells cultured alone.
(A) Example of metabolites assigned in a 1H-13C-HSQC spectrum from a polar extract from SKM-1 cells cultured with [2-13C]acetate for 2 hr. 13C percentages were calculated in 13 carbons from 11 …
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Figure 2—figure supplement 2—source data 1
Raw values for different metabolites showing label incorporation from acetate in coculture 30 min incubation.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig2-figsupp2-data1-v3.xlsx
Figure 2—figure supplement 3
Acetate label incorporation analysis in lipids in AML cells in co-culture after 8 hr of labelling.
1H 1D NMR of lipids extracted from SKM-1 (A), Kasumi-1 (B) and HL-60 (C) cell lines after being in co-culture with MS-5 cells for 24 hr and being labelled with 4 mM acetate for extra 8 hr (n=3). …
Figure 2—figure supplement 4
Acetate label incorporation analysis in lipids in AML cells in co-culture after 48 hr of labelling.
1H 1D NMR of lipids extracted from Kasumi-1 (A) and HL-60 (B) cell lines after being in co-culture with MS-5 cells for 24 hr and being labelled with 4 mM acetate for extra 48 hr (n=3). Lower panel …
Figure 2—figure supplement 5
Acetate label incorporation in TCA metabolites after 24 hr of ACSS2i treatment.
13C percentages on polar metabolites in SKM-1 cells. Cells were co-cultured for 24 hr with 20 µM ACSS2i before the addition of extra 4 mM sodium [2-13C]acetate and incubated for 24 hr (n=1). Bars …
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Figure 2—figure supplement 5—source data 1
Raw values of acetate labelling in SKM-1 cells +/-ACSS2 i.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig2-figsupp5-data1-v3.xlsx
Figure 3 with 1 supplement
Transcriptomic data and [U-13C]glucose labelling reveal that stromal cell metabolism is shifted towards higher glycolysis and ROS upon co-culture with AML cells.
(A) Top 25 GSEA hallmark gene sets ranked by expression in MS-5 cells only vs co-culture with SKM-1 cells, analysed using the collection of hallmark gene sets from Molecular Signature Database with …
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Figure 3—source data 1
Data and stats for panels included in Figure 3.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig3-data1-v3.xlsx
Figure 3—figure supplement 1
PCA component analysis, heat map of differentially expressed genes and qPCR for MS-5 cells cultured alone and in co-culture with SKM-1 cells.
(A) PCA plot showing the clustering of individual samples of MS-5 cells cultured alone (MO) and MS-5 cells in co-culture (MC). The x and y axis values represent the variation between the sample …
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Figure 3—figure supplement 1—source data 1
mRNA expression in MS-5 cells cocultured with SKM-1 cells relative to MS-5 alone.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig3-figsupp1-data1-v3.xlsx
Figure 4 with 1 supplement
Acetate secretion is linked to ROS transfer from AML to stromal cells.
(A) Extracellular acetate levels in SKM-1 (black) and MS-5 cells cultured alone (dark grey) and in co-culture (light grey) for 24 hr in a control medium, medium with 50 µM H2O2 or medium with 5 mM …
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Figure 4—source data 1
Values and tats for panels included in Figure 4.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Acetate secretion in thiamine free medium and after modulating ROS levels.
(A), Extracellular acetate levels in MS-5 cells cultured in control medium (black) or in thiamine-depleted medium (grey) after 24 hr. (B), Extracellular acetate levels in Kasumi-1 (navy blue) and …
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Figure 4—figure supplement 1—source data 1
Acetate values and stats for MS5 cells in thiamine-free medium vs control (A), for Kasumi and HL-60 +/-NAC or H2O2 alone vs coculture (B), and for MS-5 cells with different concentrations of catalase (C).
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig4-figsupp1-data1-v3.xlsx
Figure 5 with 1 supplement
Acetate secretion is linked to ROS transfer from AML to stromal cells via gap junctions.
(A) Fold change (FC) values of detected gene transcripts (TPMs) related to gap junctions. FC values are represented as log2FC, red values indicate upregulation and blue values indicate …
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Figure 5—source data 1
Values and stats for all panels included in Figure 5.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig5-data1-v3.xlsx
Figure 5—figure supplement 1
Calcein-AM and CD33 staining in SKM-1.
(A) Flow cytometry diagrams showing CD33 and calcein-AM green fluorescence in SKM-1 cells cultured alone or SKM-1 cells in co-culture with/without Calcein-AM-stained MS-5 cells after 3 hr. …
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Figure 5—figure supplement 1—source data 1
Percentage and stats for Calcein-AM +CD33 cells.
- https://cdn.elifesciences.org/articles/75908/elife-75908-fig5-figsupp1-data1-v3.xlsx
Figure 6
Schematic summary of our findings.
AML cells present high levels of ROS that activate the formation of gap junctions. ROS is transferred from AML cells to stromal cells through these gap junctions and it is utilised by stromal cells …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus, females) | C57BL/6 J | The Jackson Laboratory | RRID:IMSR_JAX:000664 | |
| Strain, strain background (Mus musculus, males) | MLL-AF9 | The Jackson Laboratory | RRID:IMSR_JAX:009079 | Wild type littermates used as controls |
| Cell line (Homo sapiens) | SKM-1 | DMSZ | DMSZ ACC 547; RRID: CVCL_0098 | |
| Cell line (Homo sapiens) | Kasumi-1 | DMSZ and a gift from the laboratory of C.Bonifer (UoB) | DMSZ ACC 220; RRID: CVCL_0589 | |
| Cell line (Homo sapiens) | HL-60 | ATCC | ATCC CCL-240 RRID: CVCL_0002 | |
| Cell line (Mus musculus) | MS-5 | Gift from the laboratory of JJ Schuringa (Groninger University) | DMSZ ACC 441 RRID:CVCL_2128 | |
| Cell line (Homo sapiens) | HeLa | ATCC | ATCC CCL-2 RRID: CVCL_0030 | |
| Antibody | CD33 (mouse monoclonal, clone P67.6) | eBioscience | 48-0337-42 | 1:100 |
| Antibody | anti-CD34 APC (mouse monoclonal) | BD Biosciences | 560940 | 10 ul per 10 million cells |
| Antibody | anti-CD38 FITC (mouse monoclonal) | BD Biosciences | 555459 | 10 ul per 10 million cells |
| Chemical compound, drug | tert-Butyl hydroxyperoxide (TBHP) | Sigma/Merck | 478139 | |
| Chemical compound, drug | carbenoxolone (CBX) | Sigma-Aldrich | C4790 | For in vitro experiments |
| Chemical compound, drug | carbenoxolone (CBX) | Thermofisher | J63714.03 | For in vivo experiments |
| Chemical compound, drug | Catalase | Sigma-Aldrich | C100 | |
| Chemical compound, drug | 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) | Sigma-Aldrich | D6883 | |
| Chemical compound, drug | H2O2 | Merck | 386790 | |
| Chemical compound, drug | N-acetylcysteine (NAC) | Merck | 106425 | |
| Chemical compound, drug | Calcein-AM green | Invitrogen | C1430 | |
| Chemical compound, drug | CellTrace carboxyfluorescein succinimidyl ester (CFSE) | Invitrogen | C34570 | |
| Chemical compound, drug | ACSS2i | Selleck | S8588 | |
| Chemical compound, drug | Cytarabine (AraC) | Sigma-Aldrich | C1768 | |
| Chemical compound, drug | [U-13C]Glucose | CortecNet | CC860P1 | |
| Chemical compound, drug | [2-13C]acetate | Sigma-Aldrich | 279315–1 G | |
| Other | CD34 magnetic microbeads | Miltenyi Biotec | 130-046-702 | Please see Materials and Methods section under Primary Patient samples |
| Other | CD117 magnetic microbeads | Miltenyi Biotec | 130-091-332 | Please see Materials and Methods section under Primary Patient samples |
| Sequence-based reagent | Hk2 q PCR primers | Sigma-Aldrich | NM_013820 | KiCqStart primers KSPQ12012 |
| Sequence-based reagent | Pdhx q PCR primers | Sigma-Aldrich | NM_175094 | KiCqStart primers KSPQ12012 |
| Sequence-based reagent | Pdk1 q PCR primers | Sigma-Aldrich | NM_172665 | KiCqStart primers KSPQ12012 |
| Sequence-based reagent | Pdk2 q PCR primers | Sigma-Aldrich | NM_133667 | KiCqStart primers KSPQ12012 |
| Sequence-based reagent | B2m qPCR primers | Thermo Fisher Scientific | NM_009735.3 | |
| Software.algorithm | Flowjo | BD-Bioscience | ||
| Software.algorithm | MetaboLab software within the MATLAB environment (MathWorks). | Ludwig and Günther, 2011 . https://doi.org/10.1186/1471-2105-12-366 | https://www.ludwiglab.org/software-development | |
| Software.algorithm | Chenomx 7.0 software (Chenomx Inc) | Chenomx Inc https://www.chenomx.com/ | ||
| Software.algorithm | FastQC 0.11.7 software | http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ | ||
| Software.algorithm | Kallisto 0.43.0 software | Bray et al., 2016 | https://pachterlab.github.io/kallisto/ | |
| Software.algorithm | R statistical package Sleuth 0.30.0 | Pimentel et al., 2017 | https://github.com/pachterlab/sleuth | |
| Software.algorithm | the R statistical package fgsea 1.10.0 | http://bioconductor.org/packages/release/bioc/html/fgsea.html | ||
| Software.algorithm | R statistical package BioMart 2.40.3 | https://bioconductor.org/packages/biomaRt | ||
| Software.algorithm | GraphPad version 8 | https://www.graphpad.com/ |
Additional files
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Supplementary file 1
Primary AML samples’ additional information.
Table shows information regarding the type of AML, karyotype and additional mutation and risk of the different AML patient samples used during this study.
- https://cdn.elifesciences.org/articles/75908/elife-75908-supp1-v3.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/75908/elife-75908-mdarchecklist1-v3.docx
