Dissecting cell-type-specific metabolism in pancreatic ductal adenocarcinoma
- Koch Institute for Integrative Cancer Research and the Department of Biology at Massachusetts Institute of Technology, United States
- Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, United States
- Department of Biology and Biological Engineering, Chalmers University of Technology, Sweden
- Cold Spring Harbor Laboratory, Cold Spring Harbor, United States
- Lustgarten Foundation Pancreatic Cancer Research Laboratory, Cold Spring Harbor, United States
- Cancer Research United Kingdom Cambridge Institute, University of Cambridge, United Kingdom
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, United States
- Department of Molecular Biology, Princeton University, United States
- Department of Medicine, University of Cambridge, United Kingdom
- Cambridge Institute for Therapeutic Immunology and Infectious Disease, University of Cambridge, United Kingdom
- Department of Pathology, Massachusetts General Hospital, United States
- Department of Medical Oncology, Dana-Farber Cancer Institute, United States
Figures
Figure 1 with 3 supplements
Glucose metabolism in PDAC tumors.
(A) Plasma glucose levels over time in autochthonous LSL-KrasG12D/+; Trp53fl/fl; Pdx1-Cre (KP-/-C) or autochthonous LSL-KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) pancreatic tumor-bearing mice infused …
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Figure 1—source data 1
Isotope labeling of tumors in U-13C- glucose-infused mice with autochthonous PDAC tumors presented in Figure 1.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Metabolite abundance and plasma labeling in U-13C- glucose-infused autochthonous PDAC tumors.
(A) Relative abundance of tumor metabolites in autochthonous LSL-KrasG12D/+; Trp53fl/fl; Pdx1-Cre (KP-/-C) (dark blue) or autochthonous LSL-KrasG12D/+; Trp53R172H/+; Pdx1-Cre (KPC) (light blue) …
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Figure 1—figure supplement 1—source data 1
Tumor metabolite abundance and plasma isotope labeling in U-13C- glucose-infused mice with autochthonous PDAC tumors presented in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig1-figsupp1-data1-v2.xlsx
Figure 1—figure supplement 2
Glucose metabolism in autochthonous PDAC tumors infused with U-13C- glucose at 30 mg/kg/min.
(A) Enrichment of fully labeled glucose (M+6) in plasma from the indicated mice following a 4 hr U-13C-glucose infusion at a rate of 30 mg/kg/min. Non-tumor bearing C57Bl6/J (WT) mice were used to …
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Figure 1—figure supplement 2—source data 1
Isotope labeling of tumors in U-13C- glucose-infused mice with autochthonous PDAC tumors presented in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig1-figsupp2-data1-v2.xlsx
Figure 1—figure supplement 3
Glucose metabolism in autochthonous and orthotopic PDAC tumors infused with U-13C-glucose at 30 mg/kg/min.
(A–K) The fractional labeling of lactate (A), alanine (B), serine (C), citrate (D), succinate (E), α-ketoglutarate (αKG) (F), fumarate (G), malate (H), aspartate (M+3 p=0.0458) (I), glutamate (J), …
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Figure 1—figure supplement 3—source data 1
Plasma isotope labeling in 13C- glucose-infused mice with autochthonous PDAC tumors and metabolite isotope labeling in orthotopic PDAC tumors presented in Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig1-figsupp3-data1-v2.xlsx
Figure 2 with 1 supplement
Assessment of pyruvate carboxylase activity in PDAC cancer cells and fibroblasts.
(A) Sections from tumors arising in LSL-KrasG12D/+; Trp53fl/fl; Pdx1-Cre (KP-/-C) mice were stained with an antibody against pyruvate carboxylase. Scale bar represents 100 μm. (B) Representative …
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Figure 2—source data 1
Metabolite isotope labeling by U-13C- glucose in unsorted organoid-PSC co-cultures.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Assessment of pyruvate carboxylase activity in PDAC cancer cells and fibroblasts.
(A) Distribution of pyruvate carboxylase staining intensity scores from a tissue microarray containing sections from 90 human pancreatic tumors. (B) Representative TMA cores containing human …
Figure 3 with 1 supplement
Turnover of polar metabolites but not protein is fast relative to the time needed to sort cells.
(A–D) Metabolite levels (total ion counts, TIC) of (A) aspartate, (B) alanine, (C) malate, and (D) citrate were measured by GC-MS in AL1376 PDAC cells extracted 0–240 min after incubation in PBS on …
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Figure 3—source data 1
Metabolite abundance, metabolite isotope labeling, and protein hydrolysate isotope labeling by U-13C- glucose in AL1376 PDAC cells.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Turnover of polar metabolites but not protein is fast relative to the time needed to sort cells.
(A–D) Metabolite levels (total ion counts) of (A) α-ketoglutarate (αKG), (B) fumarate, and (C) succinate were measured by GC-MS in cells extracted after 0–240 min in PBS on ice. (D–F) Fractional …
Figure 4 with 1 supplement
PDAC cancer cells have higher pyruvate carboxylation activity than fibroblasts in organoid co-cultures.
(A) A representative flow cytometry plot showing GFP and tdTomato expression in PSCs and cancer cells respectively from digested organoid-PSC co-cultures. Cells were gated on the single cell, live …
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Figure 4—source data 1
Isotope labeling of protein hydrolysates by U-13C- glucose in organoid-PSC co-cultures after sorting.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
PDAC cancer cells have higher pyruvate carboxylation activity than fibroblasts in organoid co-cultures.
(A–B) Fractional labeling of alanine (A) or serine (B) from protein hydrolysates from sorted organoid cancer cells (black) and PSCs (blue) after tracing with U-13C-glucose in murine organoids from LS…
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Figure 4—figure supplement 1—source data 1
Isotope labeling of protein hydrolysates by U-13C- glutamine in organoid-PSC co-cultures after sorting.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 1 supplement
PDAC cancer cells have higher pyruvate carboxylation activity in vivo.
(A–B) Tumors from LSL-KrasG12D/+; Trp53fl/fl; Pdx1-Cre; LSL-tdTomato (KP-/-CT) mice were stained with antibodies against RFP (red) and (A) α-SMA (green), a fibroblast marker, or (B) CK19 (green), a …
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Figure 5—source data 1
Isotope labeling of protein hydrolysates in mice with autochthonous PDAC tumors after 24 hr of U-13C- glucose infusion and sorting.
- https://cdn.elifesciences.org/articles/56782/elife-56782-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
PDAC cancer cells have higher pyruvate carboxylation activity in vivo.
(A) Expression of E-Cadherin was measured by qPCR in sorted cells from KP-/-CT PDAC tumors. The difference in expression between cancer cells and whole tumor was not significant (p=0.0765), but the …
Figure 6 with 2 supplements
Pyruvate carboxylase in cancer cells is required for PDAC tumor growth in vivo.
(A) Proliferation rate of AL1376 murine PDAC cells without (sgControl) or with (sgPC) deletion in standard 2D culture. Mean +/- SD is shown. (B) Fluorescent images of murine PDAC cancer cell …
Figure 6—figure supplement 1
Pyruvate carboxylase in cancer cells is required for PDAC tumor growth in vivo.
(A) Western blot for PC expression levels in AL1376 murine sgPC PDAC cell lines compared to sgControl cell lines made using CRISPRi using β-actin as a control. (B) Fractional labeling of aspartate …
Figure 6—figure supplement 2
Pyruvate carboxylase knockout PSCs retain ability to enhance PDAC growth.
(A) Western blot analysis of PC expression levels in single-cell cloned sgPC knockout PSCs compared to sgControl clones using β-actin as a control. (B) Fractional labeling of aspartate M+1 following …
Figure 7 with 2 supplements
Malic enzyme 1 contributes to pyruvate carboxylation activity in PDAC cells and is important for tumor growth.
(A–B) CRISPR/Cas9 was used to disrupt PC and/or ME1 as indicated in AL1376 murine PDAC cells. (A) Fractional labeling of M+1 aspartate following culture of the indicated cells for 24 hr in media …
Figure 7—figure supplement 1
Malic enzyme 1 contributes to pyruvate carboxylation activity in PDAC cells and is important for tumor growth.
(A) Western blot for PC and ME1 expression levels in double knockout AL1376 PDAC cells compared to control and ME1 rescue cells using β-actin as a control. (B–D) CRISPR/Cas9 was used to knockout …
Figure 7—figure supplement 2
Expression of ME1 in a human PDAC tissue microarray and in murine organoids and stroma.
(A) Distribution of ME1 staining intensity scores from a tissue microarray containing sections from 100 human pancreatic tumors. (B) Representative TMA cores containing human pancreatic tumors …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57Bl6/J | Jax | Cat# JAX:000664, RRID:IMSR_JAX:000664 | Used for cell line transplantation |
| Genetic Reagent (M. musculus) | LSL-KrasG12D/+ Trp53flox/flox Pdx-1-Cre LSL-tdTomato; KP-/-CT | Bardeesy et al., 2006 PMID:16585505 | Mice from a mixed 129/Sv and C57Bl6/J genetic background as well as pure C57Bl6/J mice were used. Both sexes of mice were included in experiments. | |
| Genetic Reagent (M. musculus) | LSL-KrasG12D/+ Trp53R172H/+ Pdx-1-Cre LSL-tdTomato; KPCT | Hingorani et al., 2005 PMID:15894267 | Mice from a mixed 129/Sv and C57Bl6/J genetic background as well as pure C57Bl6/J mice were used. Both sexes of mice were included in experiments. | |
| Genetic Reagent (M. musculus) | β-actin-GFP | Jax | Cat# JAX:006567, RRID:IMSR_JAX:006567 | PSCs were isolated from β-actin-GFP mice in a C57Bl6/J background. |
| Cell line (M. musculus) | PSC | Danai et al., 2018 PMID:29925948 | Isolated from β-actin-GFP mice in a C57Bl6/J background (Jax 006567). PSCs were immortalized with TERT and SV40 largeT after several passages. | |
| Cell line (M. musculus) | AL1376 | Sullivan et al., 2018 PMID:29941931 | Isolated from KP-/-CT mouse PDAC tumor in a C57Bl6/J background. | |
| Cell line (M. musculus) | Organoid | This paper | Isolated from KP-/-CT mouse PDAC tumor in a C57Bl6/J background | |
| Biological sample (Homo sapiens) | Human pancreatic cancer tissue microarray | Biomax | PA961e | |
| Antibody | α-SMA (mouse monoclonal) | Sigma | Cat# F3777, RRID:AB_476977 | IF (1:500) |
| Antibody | CK19 (Rabbit monoclonal) | Abcam | Cat# ab133496, RRID:AB_ 11155282 | IF (1:100) |
| Antibody | tdTomato (Rabbit polyclonal) | Rockland | Cat# 600-401-379, RRID:AB_2209751 | IF (1:500) |
| Antibody | Pyruvate Carboxylase (Rabbit polyclonal) | Santa Cruz | Cat# sc-67021, RRID:AB_2283532 | IHC (1:50) |
| Antibody | Pyruvate Carboxylase (Mouse monoclonal) | Santa Cruz | Cat# sc-271493, RRID:AB_10649369 | WB (1:100) |
| Antibody | Malic Enzyme 1 (Rabbit polyclonal) | Proteintech | Cat# 16619-1-AP, RRID:AB_2143821 | IHC (1:200) WB (1:250) |
| Antibody | β-actin (Rabbit monoclonal) | Cell Signaling Technologies | Cat# 8457, RRID:AB_10950489 | WB (1:10,000) |
| Antibody | CD16/CD32 (Rat monoclonal) | Thermo Fisher | Cat# 14-0161-82, RRID:AB_467133 | FACS (10uL) |
| Antibody | CD45-APC-Cy7 (Rat monoclonal) | BD | Cat# 557659, RRID:AB_396774 | FACS (1:100) |
| Antibody | Puromycin (Mouse monoclonal) | Sigma | MABE343 | WB (1:25,000) |
| Antibody | Vinculin (Mouse monoclonal) | Abcam | Cat# ab18058, RRID:AB_444215 | WB (1:1000) |
| Commercial Assay or Kit | Protein A Antibody Purification Kit | Sigma | PURE1A | |
| Commercial Assay or Kit | RNAqueous-Micro Total RNA Isolation Kit | Life Technologies | AM1931 | |
| Commercial Assay or Kit | iScript cDNA Synthesis Kit | Bio-Rad Laboratories | 1708890 | |
| Commercial Assay or Kit | Pierce BCA Protein Assay Kit | Pierce | 23225 | |
| Commercial Assay or Kit | Amaxa Basic Nucleofector Kit for Primary Mammalian Epithelial Cells | Amaxa | VPI-1005 | |
| Recombinant DNA Reagent | pUSPmNG (plasmid) | Li et al., 2019 PMID:31694929 | U6 sgRNA PGK with mNeonGreen to express sgRNAs for PC and ME1 double knockout cell lines | |
| Recombinant DNA Reagent | LentiCRISPRv2 (plasmid) | Sanjana et al., 2014 PMID:25075903 | Lentiviral construct to create PC and ME1 knockout cells | |
| Recombinant DNA Reagent | Modified LentiCRISPRv2 (plasmid) | Horlbeck et al., 2016 PMID:27661255 | Modified dCas9-KRAB fusion protein to create PC and ME1 knockdown cells | |
| Recombinant DNA Reagent | pLV-Hygro-EFS (plasmid) | Vectorbuilder | Custom lentiviral construct for re-expressing PC or ME1 cDNA under a CMV promoter | |
| Peptide, recombinant protein | EGF | Thermo Fisher | PMG8041 | |
| Peptide, recombinant protein | FGF | Peprotech | 100-26 | |
| Peptide, recombinant protein | Gastrin I | TOCRIS | 3006 | |
| Peptide, recombinant protein | Noggin | Peprotech | 250-38 | |
| Chemical compound, drug | Collagenase I | Worthington Biochemical | LS004194 | |
| Chemical compound, drug | Collagenase XI | Sigma | C9407 | |
| Chemical compound, drug | Collagenase P | Sigma | 11213865001 | |
| Chemical compound, drug | Dispase II | Roche | 04942078001 | |
| Chemical compound, drug | TrypLE Express | Thermo Fisher | 12605-010 | |
| Chemical compound, drug | DNAse I Type II | Sigma | D4527 | |
| Chemical compound, drug | DNAse I Type IV | Sigma | D5025 | |
| Chemical compound, drug | GlutaMAX | Thermo Fisher | 35050 | |
| Chemical compound, drug | HEPES | Thermo Fisher | 15630 | |
| Chemical compound, drug | TGF-b inhibitor A-83-01 | TOCRIS | 2939 | |
| Chemical compound, drug | Rho Kinase Inhibitor Y-27632 | Sigma | Y0503 | |
| Chemical compound, drug | N-Acetylcysteine (NAC) | Sigma | A9165 | |
| Chemical compound, drug | Nicotinamide | Sigma | N0636 | |
| Chemical compound, drug | B-27 supplement | Thermo Fisher | 17504 | |
| Chemical compound, drug | Methoxamine (MOX) reagent | ThermoFisher | TS-45950 | |
| Chemical compound, drug | N–methyl–N–(tert–butyldimethylsilyl)trifluoroacetamide + 1% tert–Butyldimethylchlorosilane | Sigma | 375934 | |
| Chemical compound, drug | Pyridine | Sigma | 270407 | |
| Chemical compound, drug | Nycodenz | VWR | 100356-726 | |
| Chemical compound, drug | Sulforhodamine B | Sigma | 230162 | |
| Chemical compound, drug | SYBR Green Master Mix | Sigma | L6544 | |
| Software, algorithm | Prism | GraphPad | Statistical analysis and graphing | |
| Software, algorithm | FlowJo | BD | Flow cytometry data analysis | |
| Other | SYTOX Red Dead Cell Stain | Life Technologies | S34859 | FACS (1:1000) |
| Other | Advanced DMEM/F12 | Thermo Fisher | 12634 | |
| Other | DMEM without pyruvate | Corning | 10-017-CV | |
| Other | GBSS | Sigma | G9779 | |
| Other | Growth factor reduced (GFR) matrigel | Corning | 356231 | |
| Other | Plastic coverslips | Thermo | 174985 | |
| Other | Flow cytometry staining buffer | Thermo Fisher | 00-4222-57 |
Additional files
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Supplementary file 1
qPCR primer sequences.
Sequences of primers used for qPCR reactions.
- https://cdn.elifesciences.org/articles/56782/elife-56782-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56782/elife-56782-transrepform-v2.pdf
