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Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment

Nature Metabolism volume 3, pages 1484–1499 (2021)Cite this article

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Abstract

The aberrant production of collagen by fibroblasts is a hallmark of many solid tumours and can influence cancer progression. How the mesenchymal cells in the tumour microenvironment maintain their production of extracellular matrix proteins as the vascular delivery of glutamine and glucose becomes compromised remains unclear. Here we show that pyruvate carboxylase (PC)-mediated anaplerosis in tumour-associated fibroblasts contributes to tumour fibrosis and growth. Using cultured mesenchymal and cancer cells, as well as mouse allograft models, we provide evidence that extracellular lactate can be utilized by fibroblasts to maintain tricarboxylic acid (TCA) cycle anaplerosis and non-essential amino acid biosynthesis through PC activity. Furthermore, we show that fibroblast PC is required for collagen production in the tumour microenvironment. These results establish TCA cycle anaplerosis as a determinant of extracellular matrix collagen production, and identify PC as a potential target to inhibit tumour desmoplasia.

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Fig. 1: TGFβ-induced collagen synthesis is linked to Gln-dependent TCA cycle anaplerosis.
Fig. 2: Glutamine de novo synthesis can maintain translation and collagen production when extracellular glutamine is limited.
Fig. 3: PC suppression by TGFβ impairs TCA cycle anaplerosis, translation and collagen production in low glutamine.
Fig. 4: PC anaplerosis is required for collagen production when extracellular glutamine levels are low.
Fig. 5: Pyruvate carboxylase anaplerosis supports collagen transcription when extracellular glutamine levels are low.
Fig. 6: Lactate supports collagen production via PC when glucose and glutamine are limiting.
Fig. 7: Fibroblast PC supports pancreatic and mammary tumour growth and fibrosis.

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Data availability

RNA sequencing data that support the findings of this study have been deposited into the NCBI Gene Expression Omnibus (GEO) with the accession code GSE169588. Human SMAD2/3/4 motif position frequency matrices can be found on JASPAR with the accession code MA0513.1. USCS genome browser tracks are accessible via the following link: http://genome.ucsc.edu/cgi-bin/hgTracks?db=hg38&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=chr11%3A66847371%2D66973751&hgsid=1152675631_ClzjvmWXzEUYlG9AcUArh1LlTkiY. Additional information can be found in the Nature Research Reporting Summary. Further information and requests for reagents may be directed to, and will be fulfilled by, the corresponding author. All data supporting the findings of this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank the members of the Thompson laboratory for helpful discussions. We are thankful to T. Lindsten for help with planning of and protocol preparation for mouse experiments, and to J. Zhu for help with optimization of ChIP experiments. We also thank E. De Stanchina and O. Hayatt from the MSKCC Antitumor Assessment Core for help with tumour allograft experiments. S.S. was supported by postdoctoral fellowships from the Human Frontier Science Program (LT000854/2018) and the European Molecular Biology Organization (ALTF 467-2018). S.S. also received support from the NCI (1K99CA259224) and the Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center. X.C. was supported by the NCI (1K99CA256505). G.M.S. was supported by the NCI (K22CA218472) and the Herbert and Maxine Block Memorial Lectureship Fund. C.B.T. was supported by the NCI (R01CA201318). This work was also supported by the cancer centre support grant (P30CA008748) to MSKCC.

Author information

Author notes

  1. These authors contributed equally: Natalya N. Pavlova, Francesco V. Cimino.

Authors and Affiliations

  1. Cancer Biology and Genetics Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Simon Schwörer, Natalya N. Pavlova, Francesco V. Cimino, Bryan King, Xin Cai & Craig B. Thompson

  2. The Comprehensive Cancer Center, The Ohio State University, Columbus, OH, USA

    Gina M. Sizemore

  3. Department of Radiation Oncology, The Ohio State University, Columbus, OH, USA

    Gina M. Sizemore

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Contributions

S.S. conceived the project, performed most experiments, analysed data, interpreted results, and wrote and edited the manuscript. N.N.P. performed and analysed tRNA charging experiments. F.V.C. provided technical assistance. B.K. provided support for PSC isolation and characterization and experiments with KPC cells. X.C. provided key knowledge and optimized conditions for experiments with lactate. G.M.S. provided cell lines, interpreted results and edited the manuscript. C.B.T. conceived the project, interpreted results, and wrote and edited the manuscript. All authors participated in discussing and finalizing the manuscript.

Corresponding author

Correspondence to Craig B. Thompson.

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Competing interests

C.B.T. is a founder of Agios Pharmaceuticals and a member of its scientific advisory board. He is also a former member of the Board of Directors and stockholder of Merck and Charles River Laboratories. He holds patents related to cellular metabolism. All other authors declare no competing interests.

Additional information

Peer review information Nature Metabolism thanks Giuseppe Fiermonte, Deepak Nagrath and W. Rathmell for their contribution to the peer review of this work. Primary Handling Editors: George Caputa; Isabella Samuelson.

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Extended data

Extended Data Fig. 1 TGFβ-induced collagen synthesis is linked to glutamine-dependent TCA cycle anaplerosis.

(A) Growth curves of PSCs cultured in the indicated percentage of Gln and treated with TGFβ (2 ng/mL). n = 3 biologically independent samples. (B) Western Blot of PSCs cultured in 100% or 20% Gln and treated with TGFβ for 48 h. (C) Collagen abundance in ECM derived from confluent PSCs cultured in the indicated percentage of Gln and treated with TGFβ. n = 3 biologically independent samples. (D) Growth curves of MFBs cultured in the indicated percentage of Gln and treated with TGFβ. n = 3 biologically independent samples. (E) Western Blot of MFBs cultured in 100% or 20% Gln and treated with TGFβ for 48 h. (F) Collagen abundance in ECM derived from confluent MFBs cultured in the indicated percentage of Gln and treated with TGFβ. n = 3 biologically independent samples. (G) Relative number of NIH-3T3 cells cultured in 10% Gln and treated with TGFβ and 0.2, 1 or 5 mM of asparagine (Asn) or proline (Pro). n = 3 biologically independent samples. (H) Western Blot of NIH-3T3 cells cultured in 10% Gln and treated with TGFβ and the indicated metabolites and concentrations for 48 h. (I) Relative metabolite abundance in NIH-3T3 cells cultured in 10% Gln and treated with TGFβ and dm-Glu (5 mM) or dm-αKG (5 mM) for 48 h. n = 3 biologically independent samples. (J,K) Western Blot of PSCs (J) or MFBs (K) cultured in 100% or 20% Gln and treated with TGFβ for 48 h. (L,M) tRNA charging in PSCs (L) or MFBs (M) cultured in 20% Gln and treated with TGFβ for 48 h. n = 1 independent experiment. (N,O) Western Blot of PSCs (N) or MFBs (O) cultured in 20% Gln and treated with TGFβ and dm-Glu or dm-αKG for 48 h. MFBs were also treated with aspartate (Asp, 20 mM). Mean±SD (A,C,D,F) or mean+SD (G) are shown. Dashed lines (A,D,G) represent cell number at day 0. Two-way ANOVA (A,C,D,F). Western blots are representative of three (B,E,J,K) or two (H,N,O) independent experiments. tRNA charging analyses (L,M) are representative of two independent experiments. All other experiments were performed at least twice.

Source data

Extended Data Fig. 2 Glutamine de novo synthesis can maintain collagen synthesis and proliferation when glutamine is limiting.

(A) Western Blot of PSCs cultured in 20% Gln in the presence of TGFβ and treated with dm-αKG and MSO. (B) Collagen abundance in ECM derived from confluent PSCs cultured in 100% or 10% Gln in the presence of TGFβ and treated with dm-αKG, dm-Glu and MSO. n = 3 biologically independent samples. (C) Relative number of NIH-3T3 cells expressing Ctrl or Glul sgRNA, cultured in 10% Gln and treated with TGFβ alone and dm-αKG or dm-Glu. n = 2 (Glul sg6 dm-Glu), n = 3 (all others) biologically independent samples. (D,E) Growth curves of NIH-3T3 cells (D) or PSCs (E) expressing Ctrl or Glul sgRNA, cultured in 100% or 10% Gln/20% Gln. n = 3 biologically independent samples. (F) Western Blot of PSCs expressing Ctrl or Glul sgRNA, cultured in 20% Gln for 48 h. (G) Collagen abundance in ECM derived from confluent PSCs expressing Ctrl or Glul sgRNA, cultured in 20% Gln. n = 3 biologically independent samples. Mean+SD (B,C,G) or mean±SD (D,E) are shown. One-way ANOVA with Holm-Sidak correction (B), one-way ANOVA (C,G), two-sided unpaired t-test (C: Glul sg6 dm-Glu), two-way ANOVA (D,E). Western blots (A,F) are representative of three independent experiments. All other experiments were performed at least twice.

Source data

Extended Data Fig. 3 TGFβ suppresses PC expression and reduces PC activity.

(A-C) mRNA expression of the indicated genes in NIH-3T3 cells (A), PSCs (B) or MFBs (C) cultured in 100% Gln and treated with TGFβ for 24 h. n = 3 biologically independent samples. (D) Western Blot of PSCs (left) or MFBs (right) cultured in 100% or 20% Gln and treated with TGFβ for 48 h. (E) UCSC genome browser tracks showing putative SMAD2/SMAD3/SMAD4 binding motifs, SMAD4 ChIP-seq peaks in HepG2 cells, the Genehancer promoter element and the PC transcriptional start site (TSS) at the genomic loci of three human PC isoforms. (F) Pcx expression from RNA-sequencing of quiescent PSCs (qPSC), myofibroblastic CAFs (myCAF) and inflammatory CAFs (iCAFs). Data and p-values are from GSE93313. (G) mRNA expression of myCAF and iCAF markers in iCAFs and myCAFs (left); Pcx mRNA expression in myCAFs and iCAFs, relative to qPSCs (right). n = 3 biologically independent samples. (H) [U-13C]-Glc tracing in PSCs cultured in 100% or 20% Gln and treated with TGFβ for 48 h. n = 3 biologically independent samples. (I) [U-13C]-Glc tracing into indicated amino acid residues of cellular proteins. NIH-3T3 cells were cultured in 10% Gln and treated with TGFβ for 48 h. n = 3 biologically independent samples. (J,K) [3,4-13C]-Glc tracing in PSCs cultured in 100% or 20% Gln and treated with TGFβ for 48 h. M + 1 labeling (J). PC activity (K). n = 3 biologically independent samples. (L) Western Blot of PSCs expressing empty vector or human PC cDNA, cultured in 20% Gln and treated with TGFβ. Mean+SD (A-C,G-K) are shown. Two-sided unpaired t-test (A-C,G,I), one-way ANOVA with Holm-Sidak correction (H,J,K). Western blots (D,L) are representative of two independent experiments. [3,4-13C]-Glc tracing in PC-ko cells (F) was performed once. All other experiments were performed at least twice.

Source data

Extended Data Fig. 4 PC is required for collagen synthesis when extracellular glutamine is low.

(A) Western Blot of NIH-3T3 cells expressing Ctrl or PC sgRNA, cultured in 100% Gln for 48 h. (B) Western Blot of PSCs expressing Ctrl or PC sgRNA, cultured in 20% Gln for 48 h. (C) Western Blot of parental MFBs and MFBs expressing Ctrl or PC sgRNA, cultured in 100% or 20% Gln for 48 h. (D) Collagen abundance in ECM derived from confluent PSCs (left) or MFBs (right) expressing Ctrl or PC sgRNA, cultured in 20% Gln. n = 3 biologically independent samples. (E) Western Blot of MFBs expressing Ctrl or PC sgRNA, cultured in 20% Gln and treated with dm-αKG for 48 h. (F) [3,4-13C]-Glc tracing in NIH-3T3 cells expressing Ctrl or PC sgRNA cultured in 100% or 10% Gln for 48 h. n = 3 biologically independent samples. (G-I) Growth curves of NIH-3T3 cells (G), PSCs (H) or MFBs (I) expressing Ctrl or PC sgRNA, cultured in 100% or 10%/20% Gln. n = 3 biologically independent samples. (J) tRNA charging in PSCs (left) or MFBs (right) expressing Ctrl or PC sgRNA, cultured in 20% Gln for 48 h. n = 1 independent experiment. (K) Col1a1 mRNA expression in PSCs (left) or MFBs (right) expressing Ctrl or PC sgRNA, cultured in 20% Gln for 48 h. n = 3 biologically independent samples. (L,M) H3K27ac (L) or H3K27me3 enrichment (M) in NIH-3T3 cells expressing Ctrl or PC sgRNA, cultured in 10% Gln for 48 h. n = 3 (L), n = 4 (M) independent experiments. (N,O) Col1a1 mRNA expression in NIH-3T3 cells expressing Ctrl or PC sgRNA, cultured in 10% Gln for 48 h. Cells were treated with dm-Glu (N) or dm-αKG and MSO (O). n = 3 biologically independent samples. Mean SD (G-I), mean+SD (D, F, K-O) are shown. Dashed lines (G-I) represent cell number at day 0. Two-way ANOVA (F-I), one-way ANOVA (D,K), two-way ANOVA (L,M) analyzing the effects of PC-ko on H3K27ac or H3K27me3 across the analyzed genomic regions, one-way ANOVA with Holm-Sidak correction (N,O). Western blots are representative of two (A,E) or three (B,C) independent experiments. tRNA charging analysis (J) is representative of two independent experiments. All other experiments were performed at least twice.

Source data

Extended Data Fig. 5 Fibroblasts take up and use lactate for TCA cycle anaplerosis via PC.

(A) [U-13C]-Glc and [U-13C]-Lac tracing. NIH-3T3 cells were cultured for 48 h in 100% or 10% Gln in the presence of 10 or 1 mM D-glucose with or without 10 mM Na-lactate. M + 3 isotopologues are shown in Fig. 6b. G, glucose; L, lactate. n = 3 biologically independent samples. (B) [U-13C]-Lac tracing. NIH-3T3 cells were cultured in 10% Gln and treated with AZD3965 (MCT1 inhibitor, 5 µM) or sodium oxamate (LDH inhibitor, 10 mM) for 8 h. [U-13C]-Lac was added in the last 1 h. n = 2 (LDHi), n = 3 (all others) biologically independent samples. (C,D) [1-13C]-Lac tracing in NIH-3T3 cells expressing Ctrl or PC sgRNA cultured in 10% Gln in the presence of 10 mM Na-lactate for 48 h. M + 1 labeling (C). PC activity (D). n = 3 biologically independent samples. (E,F) Collagen abundance in ECM generated by confluent parental (E) or Ctrl or PC sgRNA expressing NIH-3T3 cells (F) cultured in 10% Gln and the indicated concentrations of D-glucose and Na-lactate. n = 3 biologically independent samples. (G) Western Blot of PSCs cultured in 20% Gln and the indicated concentrations of D-glucose for 48 h. (H) [U-13C]-Glc and [U-13C]-Lac tracing into indicated metabolites. PSCs were cultured for 48 h in 20% Gln and 1 mM D-glucose with or without 10 mM Na-lactate. n = 3 biologically independent samples. (I) [U-13C]-Lac tracing in PSCs cultured in 100% or 20% Gln for 48 h. n = 3 biologically independent samples. (J) [1-13C]-Lac tracing. PSCs were cultured for 48 h in 100% or 20% Gln in the presence of 10 or 1 mM D-glucose and 10 mM Na-lactate. n = 3 biologically independent samples. (K) Western Blot of PSCs cultured in 20% Gln and the indicated concentrations of D-glucose and Na-lactate for 48 h. (L) Western Blot of PSCs expressing Ctrl or PC sgRNA, cultured in 20% Gln and the indicated concentrations of D-glucose and Na-lactate for 48 h. Mean+SD (A-F,H-J) are shown. Two-sided unpaired t-test (B), one-way ANOVA (C,D,J), one-way ANOVA with Holm-Sidak correction (E,F), two-sided unpaired t-test with Holm-Sidak correction (I). Western blots (G,K,L) are representative of two independent experiments. [U-13C]-Lac tracing in PSC in low glucose (H) was performed once. All other experiments were performed at least twice.

Source data

Extended Data Fig. 6 Fibroblast PC supports tumor fibrosis and growth.

(A) Outgrowth of KPC spheroids on top of a synthetic ECM (3D gel) Representative images are shown. Scale bar=500 µm. (B) Pearson correlation of total spheroid area from (A) with collagen I concentration used to prepare the synthetic ECM. n = 4 biologically independent samples. (C,D) Western blot of ECM generated by confluent PSCs cultured in 100% or 10% Gln in the presence of TGFβ (C). (D) Outgrowth of KPC spheroids on top of this ECM. Representative images are shown. Scale bar=500 µm. (E) Quantification of spheroid outgrowth from (D). n = 8 biologically independent samples. (F) Survival of KPC-GFP cells after 3 days co-culture with Ctrl or Glul sgRNA expressing PSCs on plastic or PSC-derived ECM in the absence of FBS and Gln. n = 3 biologically independent samples. (G-K) KPC/PSC allograft experiment in nude mice. (B) Representative images of Picrosirius staining of KPC/PSC allografts at day 25 after injection. Scale bar=500 µm. (H) Quantification of Picrosirius staining of KPC/PSC allografts. n = 8 biologically independent tumors. (I) Collagen levels in KPC/PSC allografts at day 25 after injection. n = 5 biologically independent tumors. (J) Representative image of KPC/PSC allograft tumors stained for αSMA, CK8 and DAPI. Scale bar=100 µm. (K) Western Blot of KPC/PSC allografts at day 25 after injection. (L) Volume of DB7 allografts 8 days after injection of DB7 cells alone, with Matrigel or with MFBs. n = 4 (DB7 + Matrigel), n = 8 (DB7, DB7 + MFB) biologically independent tumors. (M) Western Blot of the second batch of DB7/MFB allografts 8 days after injection. The first batch is shown in Fig. 7l. Mean±SD (B,E), median with 25% to 75% percentile box and min/max whiskers (H,I,L), mean+SD (F) are shown. Pearson correlation followed by two-sided unpaired t-test (B), two-way ANOVA (E), two-way ANOVA with Holm-Sidak correction (F), one-way ANOVA (H), two-sided unpaired t-test (I), one-way ANOVA with Holm-Sidak correction (L). Western blots were performed once with 5 (K) or 3-4 (M) biologically independent tumors, or were performed twice (C). Spheroid experiments were performed twice. Tumor growth, staining and hydroxyproline experiments were performed once with multiple biologically independent tumors.

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Extended Data Fig. 7 Gating strategy for flow cytometry.

(A) Gating strategy for OPP staining. Data are from Fig. 1f. (B) Gating strategy for KPC-GFP and PSC co-culture assay. Data are from Extended Data Fig. 6f.

Supplementary information

Reporting Summary

Supplementary Table 1

Primer sequences for RT-qPCR, tRNA-qPCR and ChIP-qPCR.

Source data

Source Data Fig. 1

Images depict uncropped images for western blots in Fig. 1. Red boxes show cropped area.

Source Data Fig. 2

Images depict uncropped images for western blots in Fig. 2. Red boxes show cropped area.

Source Data Fig. 3

Images depict uncropped images for western blots in Fig. 3. Red boxes show cropped area.

Source Data Fig. 4

Images depict uncropped images for western blots in Fig. 4. Red boxes show cropped area.

Source Data Fig. 6

Images depict uncropped images for western blots in Fig. 6. Red boxes show cropped area.

Source Data Fig. 7

Images depict uncropped images for western blots in Fig. 7. Red boxes show cropped area.

Source Data Extended Data Fig. 1

Images depict uncropped images for western blots in Extended Data Fig. 1. Red boxes show cropped area.

Source Data Extended Data Fig. 2

Images depict uncropped images for western blots in Extended Data Fig. 2. Red boxes show cropped area.

Source Data Extended Data Fig. 3

Images depict uncropped images for western blots in Extended Data Fig. 3. Red boxes show cropped area.

Source Data Extended Data Fig. 4

Images depict uncropped images for western blots in Extended Data Fig. 4. Red boxes show cropped area.

Source Data Extended Data Fig. 5

Images depict uncropped images for western blots in Extended Data Fig. 5. Red boxes show cropped area.

Source Data Extended Data Fig. 6

Images depict uncropped images for western blots in Extended Data Fig. 6. Red boxes show cropped area.

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Schwörer, S., Pavlova, N.N., Cimino, F.V. et al. Fibroblast pyruvate carboxylase is required for collagen production in the tumour microenvironment. Nat Metab 3, 1484–1499 (2021). https://doi.org/10.1038/s42255-021-00480-x

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