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The creatine–phosphagen system is mechanoresponsive in pancreatic adenocarcinoma and fuels invasion and metastasis

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Abstract

Pancreatic ductal adenocarcinoma is particularly metastatic, with dismal survival rates and few treatment options. Stiff fibrotic stroma is a hallmark of pancreatic tumours, but how stromal mechanosensing affects metastasis is still unclear. Here, we show that mechanical changes in the pancreatic cancer cell environment affect not only adhesion and migration, but also ATP/ADP and ATP/AMP ratios. Unbiased metabolomic analysis reveals that the creatine–phosphagen ATP-recycling system is a major mechanosensitive target. This system depends on arginine flux through the urea cycle, which is reflected by the increased incorporation of carbon and nitrogen from l-arginine into creatine and phosphocreatine on stiff matrix. We identify that CKB is a mechanosensitive transcriptional target of YAP, and thus it increases phosphocreatine production. We further demonstrate that the creatine–phosphagen system has a role in invasive migration, chemotaxis and liver metastasis of cancer cells.

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Fig. 1: Pancreatic cancer cells are mechanosensitive, and untargeted metabolome profiling revealed major metabolic reprogramming associated with ECM mechanics.
Fig. 2: ECM stiffness directs creatine metabolism in PDAC cells.
Fig. 3: Mitochondrial dynamics and respiratory activity are induced by ECM mechanics in pancreatic cancer cells and support invasive behaviour.
Fig. 4: The phosphocreatine circuit depends on CKB in pancreatic cancer cells, which is regulated by mechanosensing and YAP activity.
Fig. 5: Creatine homeostasis facilitates collective migration of pancreatic cancer cells.
Fig. 6: Creatine homeostasis supports actin dynamics in invasive pseudopods of pancreatic cancer cells, facilitating ECM invasion.
Fig. 7: Creatine homeostasis supports collagen remodelling and invasion of 3D PDAC spheroids and the chemotactic response of pancreatic cancer cells.
Fig. 8: CKB is expressed during PDAC progression and supports metastatic dissemination.

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Data availability

Associated raw data are provided as Source Data Files associated with each main or Extended Data figure. Unprocessed pictures of blots are provided in the Source Data. Original datasets, analyses and methodological details are available from the Source Data or from the corresponding author upon reasonable request. Information regarding experimental design and reagents can also be found in the Reporting Summary.

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Acknowledgements

We acknowledge CRUK Beatson Institute Core Services and Advanced Technologies (C596/A17196), and especially Beatson Advanced Imaging Resource (BAIR). We thank S. Karim for KPC cell lines. We also thank C. Nixon and the Beatson Histology Facility, D. Bryant and his lab for advice, J. Murray for generating fibroblast-derived matrices and E. J. McGhee for assistance with SHG microscopy. We thank M. Neilson for assistance in analysis and statistical advice. We also thank T. Hamilton, C. Baxter and E. Onwubiko for helping with intrasplenic surgery. We thank T. Pompe (University of Leipzig) for providing analysis software for TFM experiments. We thank H. Spence and R.H. Insall for advice and discussion. V.P. is supported by a CRUK Glasgow Centre studentship (A18076) to M.S.S. and L.M.M.; L.M.M. is supported by a CRUK core grant A15673; N.R.P. is supported by an MRC grant to L.M.M. (MR/R017255/1). M.S.S. is funded by an EPSRC Programme Grant (EP/P001114/1). M.C. is funded by an MRC UKRI/Rutherford Fund fellowship (MR/S005412/1). O.M. and T.Z. are funded by a Cancer Research UK Career Development Fellowship (C53309/A19702).

Author information

Author notes

  1. These authors jointly supervised this work: O. Maddocks, M. Salmeron-Sanchez, L. M. Machesky.

Authors and Affiliations

  1. University of Glasgow Centre for the Cellular Microenvironment, Glasgow, UK

    Vassilis Papalazarou, Marco Cantini & Manuel Salmeron-Sanchez

  2. University of Glasgow Institute of Cancer Sciences, Glasgow, UK

    Vassilis Papalazarou, Tong Zhang, Oliver D. K. Maddocks & Laura M. Machesky

  3. CRUK Beatson Institute, Glasgow, UK

    Vassilis Papalazarou, Nikki R. Paul, Amelie Juin & Laura M. Machesky

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  1. Vassilis Papalazarou
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  2. Tong Zhang
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  5. Marco Cantini
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  6. Oliver D. K. Maddocks
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Contributions

V.P. performed most of the experiments and participated in conception and design, analysed data and drafted the manuscript with L.M.M. N.R.P. helped with experiments and offered advice. T.Z. performed LC–MS metabolomics and data analysis. A.J. performed all KPC mice work and provided tissue blocks from the KPC mice. M.C. performed traction-force microscopy experiments. O.M. conceived and designed experiments, analysed data, edited the manuscript and supervised T.Z. M.S.S. supervised V.P., conceived and designed experiments and edited the manuscript. L.M.M. conceived and designed experiments, supervised V.P. and drafted the manuscript with V.P.

Corresponding author

Correspondence to Laura M. Machesky.

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Extended data

Extended Data Fig. 1 Pancreatic cancer cells are mechanosensitive and untargeted metabolomic profiling reveals metabolic reprogramming via ECM mechanics.

Panels b-n, cells atop 0.7–38 kPa fibronectin-coated hydrogels or glass. (a) Polyacrylamide hydrogel elasticity (kPa). Values area mean ± SD from n = 5, 0.7 kPa, n = 7, 7kPa and n = 5, 38kPa hydrogels from 3 independent preparations. (b) Growth curves of KPC cells. Values are mean ± SEM from 3 independent experiments. Statistical significance assessed by two-tailed Mann-Whitney U test, on ‘glass vs 0.7kPa’ p = 0.005 at 48 h and p = 0.014 at 96 h, on ’38kPa vs 0.7kPa’ p = 0.0086 at 48 h and p = 0.094 at 96 h. (c) Representative images of KPC cells. Scale bars, 50 μm. Right panel; Magnification of areas indicated by a dashed box. Scale bars, 25 μm. (d) Immunofluorescence of KPC cells showing F-actin (black) and nuclei (gold). Scale bars, 20μm. Blue arrows lamellipodia and red arrows stress fibres. (e) Immunofluorescence of PANC-1 cells showing F-actin (grey) and nuclei (blue). Scale bars, 50 μm. (f-g) Quantification of (e) showing cell area (μm2) (f) and circularity index (g). Values are mean ± SEM from n = 240, 0.7kPa, n = 268, 7 kPa, n = 276, 38kPa and n = 256 glass cells from 3 independent experiments. Kruskal-Wallis with Dunn’s multiple comparisons test. (h) Immunofluorescence of PANC-1 cells from (e) showing YAP (grey). Scale bars, 50μm. (i) Quantification of (h) showing nuclear to cytosolic YAP ratio. Values are mean ± SD from n = 66, 0.7kPa, n = 41, 7kPa, n = 61, 38kPa and n = 72 on glass. Cells from 3 independent experiments. Kruskal-Wallis test with Dunn’s multiple comparisons test. (j) σ-plot demonstrating metabolite enrichment on soft (0.7kPa, left) and stiff (glass, right) KPC cells. (k) Bar graph pathway enrichment analysis of (j) clustered by -log(p-value). Untargeted analysis on n = 3 ‘0.7kPa’ and n = 3 ‘glass’ independent cultures on same day. Statistics: Fisher’s exact Test. (l) ‘Arginine and Proline’ KEGG pathway from (k). Individual metabolites labelled by KEGG number (https://www.genome.jp/kegg/kegg3.html) and enriched metabolites highlighted (red). Cr: Creatine; pCr: Phosphocreatine. (m-n) AMP, ADP and ATP levels (m) and AMP to ATP ratio (n) of PANC-1 cells. Values are mean ± SD from 3 biological replicates on same day. Two-tailed unpaired t-test with Welch’s correction.

Source data

Extended Data Fig. 2 ECM stiffness directs creatine metabolism in PDAC cells.

(a) Schematic representation of the phosphocreatine circuit. Red indicates metabolite enrichment on soft (0.7kPa) ECM, while blue indicates enrichment on stiff (glass) ECM. (b) Urea cycle and creatine biosynthesis metabolic intermediates of KPC cells cultured on fibronectin-coated 0.7–38 kPa hydrogels and glass coverslips as indicated. Values are mean ± SD from 3 biological replicates within the same day. Statistical significance assessed by one-way ANOVA. (c) Urea cycle and creatine biosynthesis metabolic intermediates of PANC-1 cells cultured as indicated. Values are mean ± SD from 3 biological replicates within the same day. Statistical significance assessed by two-tailed unpaired t-test with Welch’s correction. (d) Arginine-derived labelled carbon and nitrogen incorporation in urea cycle and creatine biosynthesis metabolites of KPC cells cultured as indicated and supplemented with L-arginine-13C615N4 for 1, 3 and 6 hours. Values are mean ± SD from 3 biological replicates within the same day.

Source data

Extended Data Fig. 3 Mitochondrial dynamics and respiratory activity are induced by ECM mechanics in pancreatic cancer cells and support invasive behavior.

In a-i, cells were cultured atop of 0.7-38kPa fibronectin-coated hydrogels and glass coverslips. (a) Glucose-derived labelled carbon incorporation in glucose and TCA cycle intermediates of KPC cells as indicated and treated with U-13C6-glucose for 3 hours. (b) GSH, GSSG levels and GSH/GSSG ratio of KPC cells cultured as indicated. (c) NADPH levels of cells from (b). (d) Glycolysis and TCA cycle metabolite levels of PANC-1 cells cultured as indicated. (e) GSH, GSSG levels and GSH/GSSG ratio of PANC-1 cells from (d). (f) Left; Mitochondrial mass (Mitotracker, MTG) of KPC cells as indicated. Values (gMFI) are mean ± SD relative to control (glass) from 3 independent experiments. Right; Representative histogram from left panel. (g) Left; Mitochondrial membrane potential (TMRE) of KPC cells cultured as indicated. CCCP; negative control. Values (gMFI) are mean ± SD relative to control (glass) from 3 independent experiments. Right; Representative histogram from left panel. (h) Left; Cellular ROS (CellROX) of KPC cells cultured as indicated. H2O2; positive, NAC + H2O2; negative control. Values (gMFI) are mean ± SD relative to control (glass) from 3 independent experiments. Right; Representative histogram from left panel. (i) Top; Maximum intensity projections of z-stack acquisitions of PANC-1 cells cultured as indicated showing labelled mitochondria. Scale bars, 10μm. Bottom; Magnification of areas indicated by a dashed box. Pictures representative of 2 independent experiments. Scale bars, 5μm. (j) Maximum intensity projections of z-stack acquisitions of KPC cells cultured on fibronectin-coated dishes (left) or invading 3D ECM (middle, right) showing labelled mitochondria. Pictures representative of 3 independent experiments. Scale bars, 7μm (left, right) and 8μm (middle). Values in a-e represent mean ± SD from 3 biological replicates performed on the same day. In b, d, e: statistical significance assessed by two-tailed unpaired t-test with Welch’s correction. In f, g, h: statistical significance assessed by two-tailed one-sample t-test on LN transformed values.

Source data

Extended Data Fig. 4 The phosphocreatine circuit depends on CKB in pancreatic cancer cells, which is regulated by mechanosensing and YAP activity.

(a) qRT-PCR of creatine kinases mRNA in KPC cells. Mean ± SD from 3 independent experiments. (b) Ckb expression in KPC cells. Cdk2: normalisation control. Values mean ± SD and relative to control from 3 independent experiments. Two-tailed one-sample t-test on LN transformed values. (c) PANC-1 cells immunoblotted for CKB and ERK1/2 (loading control). (d) Densitometric quantification of (c). Mean ± SD and relative to control from 3 independent experiments. Two-tailed one-sample t-test on LN transformed values. (e) % of EdU positive nuclei in KPC cells treated with aphidicolin. Mean ± SD from n = 6 biological replicates and 2 independent experiments. (f) KPC cells from (e) immunoblotted for CKB and α-Tubulin (loading control). (g) Densitometric quantification of (f). Mean ± SD relative to control from 3 independent experiments. (h) Immunofluorescence of KPC cells on fibronectin or concanavalin A, F-actin (grey) and pPaxillinTyr118 (magenta). Scale bars, 50μm. (i) Immunofluorescence of KPC cells expressing GFP or VD1-GFP, Vinculin (magenta) and GFP (grey). Scale bars, 50μm. (j) Immunofluorescence of cells from (i) showing YAP (Green) and F-actin (magenta). Scale bars, 50μm. In h-j: pictures representative of 3 independent experiments. (k) Densitometric quantification of YAP in YAP-silenced KPC cells. Mean ± SD and relative to control from 4 independent experiments. Two-tailed one-sample t-test on LN transformed values. (l-m) Yap (l) and Ckb (m) expression from (k). Values are mean ± SD and relative to control from 3 independent experiments. Gapdh: normalisation control. (n) Immunofluorescence of KPC cells expressing GFP, GFP-YAP or GFP-YAP5SA showing GFP (grey). Scale bars, 20μm. (o) Cells from (n) were immunoblotted for GFP. Pictures in n-o representative of 3 independent experiments. (p-q) Creatine pathway metabolites (p) and phosphocreatine/creatine ratio (q) of cells from (n) cultured on 0.7 kPa hydrogels. Values are mean ± SD from 3 biological replicates within the same day. Two-tailed unpaired t-test with Welch’s correction.

Source data

Extended Data Fig. 5 Creatine homeostasis facilitates collective migration of pancreatic cancer cells.

(a) CCr activity schematic. (b) Ccr uptake by KPC cells. Values in b and c are mean ± SD from 3 biological replicates within the same day and representative of 2 independent experiments. (c) Phosphocreatine circuit and ATP levels of (b). (d) ADP to ATP ratio of (b). Mean ± SD from 4 independent experiments. (e) Growth curves of (b). Mean ± SD from 3 independent experiments. (f) Left; CCr-treated cells. Scale bars, 100μm. Right; Magnification of dashed boxes. White arrows; protrusions. Scale bars, 50μm. (g) Average protrusion length from (f). Values are mean ± SD from n = 206 control, n = 145 5 mM and n = 142 10 mM CCr-treated cells. (h) Cell speed from (f). Mean ± SD from n = 263 control, n = 185 5 mM and n = 192 10 mM CCr-treated cells from 3 independent experiments. (i) Immunofluorescence of CCr-treated cells showing F-actin (magenta) and nuclei (blue). Scale bars, 20μm. (j, k) Cell area (μm2) (j) and circularity index (k) from (i). Mean ± SD from n = 60 control, n = 78 5 mM and n = 69 10 mM CCr-treated cells from 3 independent experiments. (l–n) Strain energy (l), maximum force (m) of CCr-treated cells and strain energy of blebbistatin treated cells (n). Mean ± SD of n = 33 control, n = 36 CCr-treated and n = 17 blebbistatin-treated cells from 3 independent experiments. Mann-Whitney U test. (o) Creatine and phosphocreatine levels of control or pCr supplemented cells. Values in o and p are mean ± SD from 3 biological replicates within the same day. (p) ADP/ATP ratio of (o). (q) Control (EV) or CKBCRISPR KO cells expressing GFP or CKB-GFP were immunoblotted for GFP and CKB. Pictures representative of 3 independent experiments. (r) Pictures of cells from (q) populating a wounded monolayer. Scale bars, 100μm. (s) Relative wound closure of control or CCr-treated wild-type (EV) cells. Values in s, t, u are mean ± SD from 3 independent experiments. (t–u) Relative wound closure (t) and relative closure at t1/2 of control (u) of control or CCr-treated CKBCRISPR KO cells. In d,s,u: two-tailed one-sample t-test on LN transformed values. In g,j,k: Kruskal-Wallis with Dunn’s multiple comparisons test.

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Extended Data Fig. 6 Creatine homeostasis supports actin dynamics and ECM invasion of pancreatic cancer cells.

(a, b) Wound closure migration (a) and relative closure at t1/2 of control (b) from KPC cells invading 3D ECM as indicated. Mean ± SD from 3 independent experiments. (c, d) pAMPK signal intensity at the ‘back’ and ‘front’ of control (c) and the ‘front’ of control or CCr-treated KPC monolayers (d) invading 3D ECM. Mean ± SEM from 3 independent experiments with n = 84 ‘t = 0,back’; n = 113 ‘t = 0,front’; n = 63 ‘t = 24 h,back’; n = 117 ‘t = 24,front’; n = 62 ‘t = 48,back’; n = 97 ‘t = 48,front’; n = 122 ‘t = 24,CCr’; n = 91 ‘t = 48,CCr’ cells. Two-tailed paired t-test. (e) Control or CCr-treated KPC cells immunoblotted for pAMPKα1T183/ α2T172, AMPKα1/α2 and GAPDH (loading control). (f) Densitometric quantification of pAMPK/AMPK levels from (e). Mean ± SD and relative to control from 3 independent experiments. (g) Pictures of control (nc) or CKB silenced (CKBsi#01, CKBsi#02) cells invading 3D ECM. Scale bars, 100μm. (h) Control (EV) or CKB-depleted (CKB-KO) cells expressing GFP or CKB-GFP, invading 3D ECM. Scale bars, 100μm. (i) Wound closure migration of control (EV), CKB-KO and CKB-KO P-Cr-treated cells invading 3D ECM. Mean ± SD from 2 independent experiments with 5 technical replicates. (j, k) Wound closure over time (j) and relative closure at t1/2 of control (k) from control (EV) cells from (h). Values are mean ± SD from 4 independent experiments. (l) Control or CCr-treated cells invading fibroblast-derived ECM. Scale bars, 200μm. (m, n) Cell speed (m) and Euclidean distance (n) from (l). Values are mean ± SD from n = 317 control, n = 290 5 mM and n = 239 10 mM CCr from 4 independent experiments. Kruskal-Wallis with Dunn’s multiple comparisons test. (o, p) LifeAct-mTagRed signal intensity from actin photoactivation experiments; (o) control or CCr-treated cells. Values are Mean ± SEM derived from n = 31 control, n = 23 5 mM CCr, n = 35 10 mM CCr and n = 28 jasplakinolide-treated cells from 3 independent experiments. (p) control (nc) or CKB-silenced (CKBsi#01, CKBsi#02) cells. Values are mean ± SEM derived from n = 36 n.c., n = 31 CKBsi#01, n = 38 CKBsi#02 and n = 21 jasplakinolide-treated cells from 3 independent experiments. In b,f,k: two-tailed one-sample t-test on LN transformed values.

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Extended Data Fig. 7 Creatine homeostasis supports collagen remodelling, invasion and the chemotactic response of pancreatic cancer cells.

(a) Representative 3D reconstructions of z-stack acquisitions showing nucleic acid labelling (grey) of control or CCr-treated spheroids after 96 hours of invasion within 3D ECM. (b) Protrusion number per spheroid of control or CCr-treated KPC spheroids as indicated. Values are from n = 12 control, n = 13 5 mM and n = 13 10 mM CCr-treated spheroids from 3 independent experiments. (c) Average protrusion length (μm) of control or CCr-treated KPC spheroids as indicated. Values are from n = 12 control, n = 13 5 mM and n = 13 10 mM CCr-treated spheroids from 3 independent experiments. (d) Representative plot profiles of SHG intensity of control or CCr-treated KPC spheroids. Green-shaded area shows Full width half maximum (FWHM) of Collagen I (SHG) intensity. (e) Full width half maximum (FWHM) of Collagen I (SHG) intensity from (d). Each dot represents average value (from 6 plot profiles) per spheroid. Values are mean ± SD of n = 9 t = 0, n = 12 t = 48 h and n = 12 CCr-treated spheroids from 3 independent experiments. Statistical significance assessed by one-way ANOVA. (f) Cell speed of cells treated as indicated chemotaxing towards a 10% FBS gradient. Values are mean ± SEM from n = 262 control, n = 278 5 mM and n = 275 10 mM CCr-treated cells from 4 independent experiments. Statistical significance assessed by Kruskal-Wallis with Dunn’s multiple comparisons test. (g) Cell speed of control (n.c.) or CKB-silenced (CKBsi#01, CKBsi#02) cells chemotaxing towards a 10% FBS gradient. Values are mean ± SEM from n = 241 control, n = 252 CKBsi#01and n = 232 CKBsi#02 cells from 3 independent experiments. Statistical significance assessed by Kruskal-Wallis with Dunn’s multiple comparisons test.

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Extended Data Fig. 8 CKB is expressed during PDAC progression and supports metastatic dissemination.

(a) Quantification of PicroSirius Red positive area per tissue area from normal mice (Pdx1-Cre+;Kraswt/wt;p53wt/wt) and PDAC from KPC (Pdx1-Cre;LSLKrasG12D;LSLp53R172H) mice. Values are mean ± SEM from n = 4 normal and n = 9 PDAC pancreata. Statistical significance assessed by two-tailed Mann-Whitney U test. (b) Quantification of Fibronectin positive area per tissue area from (a). Values are mean ± SEM from n = 5 for normal and n = 11 for PDAC pancreata. Statistical significance assessed by two-tailed Mann-Whitney U test. (c, d) YAP positive cells (%) (c) and nuclear to cytosolic YAP ratio (d) from normal mice (Pdx1-Cre+;Kraswt/wt;p53wt/wt) and PDAC from KPC (Pdx1-Cre;LSLKrasG12D;LSLp53R172H) mice. Values are mean ± SEM from n = 5 normal and n = 10 (c), n = 9 (d) PDAC pancreata. Statistical significance assessed by two-tailed Mann-Whitney U test. (e) High magnification pictures of PDAC tissue sections from KPC mice showing YAP and CKB. Scale bars, 20μm. Representative of n = 9 PDAC pancreata. (f) Weight (gr) of animals at time of sacrifice treated as indicated from intrasplenic transplantation experiment. Values are mean ± SEM from n = 8 control (EV), n = 8 CCr-treated and n = 7 CKB-KO mice.

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Supplementary information

Supplementary Information

Supplementary Tables 1–7

Reporting Summary

Supplementary Video 1

Mitochondria of live KPC cells cultured on glass or 0.7-kPa hydrogels were labelled with Mitotracker Green (MTG), and imaging was performed using a Zeiss 880 confocal with Airyscan. Pictures were acquired at 60-s intervals for 30 min total time. CCCP was administered 10 min before starting the imaging. Scale bars, 20 μm. Representative of three independent experiments.

Supplementary Video 2

KPC cells were plated at low confluency and supplemented with cyclocreatine as indicated for 16 h. Cells were monitored over a period of 16 h with pictures taken at 20-min intervals using a Nikon TE2000-E time-lapse microscope. Scale bar, 100 μm. Representative of three independent experiments.

Supplementary Video 3

KPC cells were plated to form a monolayer and were supplemented with cyclocreatine as indicated, for 16 h. Following monolayer wounding, wound closure was monitored for 24 h with images acquired at 60-min intervals using the Incucyte System (Essen Bioscience). Red mask indicates wound area, and purple line marks the initial wound area. Scale bar, 100 μm. Representative of three independent experiments.

Supplementary Video 4

KPC cells were plated on Matrigel-coated plates to form a monolayer followed by monolayer wounding. Following embedding in Matrigel matrix and cyclocreatine supplementation as indicated, wound closure was monitored for 90 h with images acquired at 60-min intervals using the Incucyte System (Essen Bioscience). A red mask indicates wound area, and purple line marks the initial wound area. Scale bar, 100 μm. Representative of three independent experiments.

Supplementary Video 5

KPC cells were transfected with PA-GFP–actin (green) and LifeAct-mTagRed (red), plated on fibroblast-derived ECM and treated with CCr or jasplakinolide as indicated. Cells were photoactivated (t = 6 s) at the tips of pseudopods invading fibroblast-derived ECM. Images were acquired at 1-s intervals for a period of 60 s. Scale bars, 10 μm. Representative of three independent experiments.

Supplementary Video 6

KPC spheroids were treated with cyclocreatine as indicated, embedded in Matrigel–collagen I matrix and monitored over a period of 96 h, with images acquired at 60-min intervals using the Incucyte System (Essen Bioscience). Scale bar, 200 μm. Representative of three independent experiments.

Supplementary Video 7

Serum starved KPC cells were treated with cyclocreatine as indicated and allowed to chemotax towards a 10% FBS gradient using an Insall chamber set-up. Cell migration was monitored for 48 h, with images acquired at 30-min intervals using a Nikon TE2000-E time-lapse microscope. Colour-coded tracks indicate manual cell tracking quantification using the MTrackJ plugin in Fiji software (ImageJ v2.0.0). Scale bars, 50 μm. Representative of four independent experiments.

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Papalazarou, V., Zhang, T., Paul, N.R. et al. The creatine–phosphagen system is mechanoresponsive in pancreatic adenocarcinoma and fuels invasion and metastasis. Nat Metab 2, 62–80 (2020). https://doi.org/10.1038/s42255-019-0159-z

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