Data availability
Datasets generated in this study are available at the Gene Expression Omnibus repository (GSE192482). Any additional information required to reanalyse the data reported in this paper is available on request. A GitHub repository (https://github.com/KochInstitute-Bioinformatics/Imada_FastingCancer/tree/master) is available for this study. Source data are provided with this paper.
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Acknowledgements
We thank the Swanson Biotechnology Center at the Koch Institute including the Flow Cytometry, Histology, and Genomics & Bioinformatics Core facilities (NCI P30-CA14051); the Department of Comparative Medicine for mouse husbandry support; S. Holder and members of the Hope Babette Tang (1983) Histology Facility for substantial histological support; H. Ohdan, T. Matozaki and the members of the Yilmaz laboratory for discussions; K. Kelley for laboratory management; and L. Galoyan for administrative assistance. S.I. is supported by the Japan Society for Promotion of Science (JSPS) overseas fellowship and the Kanzawa Medical Research Foundation. Ö.H.Y. is supported by R01CA211184, R01CA034992, R01CA257523, R01 DK126545, U01CA250554 and U54CA224068; a Pew-Stewart Trust scholar award; the Kathy and Curt Marble cancer research award; a Koch Institute–Dana-Farber/Harvard Cancer Center Bridge project grant; and AFAR. Ö.H.Y. receives support from the MIT Stem Cell Initiative. A.T. was funded by an Emmy Noether Award from the German Research Foundation (DFG; 467788900) and the Ministry of Culture and Science of the State of North Rhine-Westphalia (NRW-Nachwuchsgruppenprogramm), and holds the Peter Hans Hofschneider of Molecular Medicine endowed professorship by the Stiftung Experimentelle Biomedizin. C.-W.C. is supported by K99/R00DK123407. R.O.C. was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2016/23142-3 and 2019/02640-3). M.A.R.V. is funded by FAPESP (2018/15313-8) and R01 DK126969-01. G.C.-K. is supported by a TUBITAK2219 international postdoctoral research fellowship. C.A. was supported by an Italian Association for Cancer Research (AIRC) IG Investigator.
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Extended data figures and tables
Extended Data Fig. 1 Refeeding does not alter the intestinal morphology, the number of ISCs, or differentiated cells.
a, Normalized mice weight ratio of AL (left) and fasted 24h- refed 24 h (right). n = 10–12 mice group, pooled from 3 independent experiments. b, Quantification (left) and representative images of phospho-H3+ cells by IHC per jejunal crypt (right). n = 5 mice per group. Scale bar, 25 µm. c, Length of crypt (left) and villi (right) in the jejunum. n = 4 mice per group. d, Quantification (left) and representative images of IHC for OLFM4 per jejunal crypt (right). n = 3 mice per group. Scale bar, 25 µm. e, Quantification (left) and representative images of in situ hybridization (ISH, red) for Lgr5 mRNA (right). n = 4 mice per group. Scale bar, 50 µm. f,g, Quantification (left) and representative images (right) of Cleaved caspase3+ cells in the villi (f) and crypt (g). n = 5 mice per group. Scale bar, 25 µm. h, Quantification (left) and representative images (right) of Paneth cells by IHC for Lysozyme. n = 4 mice per group. Scale bar, 25 µm. i, Quantification (left) and representative images (right) of jejunal Alician Blue staining. n = 5 mice per group. Scale bar in main images, 50 µm. j, Organoid-forming assay for intestinal crypts isolated from AL, Fasted, Refed 1d, and Refed 3d mice. Quantification (left) and representative images of day 3 organoids (right). n = 3 mice per group, pooled from 3 independent experiments. Scale bar, 200 µm. k, Quantification (left) and representative images of IHC for tdTomato (orange arrows, right) in the colon. n = 20 crypts per measurement, n = 5 mice per group, pooled from 3 independent experiments. Scale bar, 25 µm. One-way ANOVA (a–k). Data are mean ±s.d, *p < 0.05, **p < 0.01, ****p < 0.0001, ns = not significant.
Extended Data Fig. 2 Circadian cycle does not have an impact on refeeding-mediated crypt proliferation and organoid-forming capacity.
a, Schematic of BrdU assay with different fasting time. b, Quantification (left) and representative images of BrdU+ cells (4 h after BrdU administration) by IHC per jejunal crypt (right). n = 25–30 crypts per mouse for measurement, n = 5 mice per group. c, Quantification (left) and representative images of BrdU+ cells (4 h after BrdU administration) by IHC per jejunal crypt (right). n = 25–30 crypts per measurement, n = 5 mice per group. Scale bar, 50 µm. d, Organoid-forming assay for intestinal crypts isolated from AL, Fasted, Refed 1d mice. Quantification (left) and representative images of day 3 organoids (right). n = 3 mice per group, Scale bar, 500 µm. One-way ANOVA (b–d). Data are mean ± s.d. *p < 0.05, ***p < 0.001, ****p < 0.0001.
Extended Data Fig. 3 Insulin-PI3K signal is a trigger to activate mTORC1 signal after refeeding.
a, Time course of blood glucose levels measured from tail-tip samples in AL (red) and refed (pre and post refeeding, blue) mice. n = 4 mice per group. b, Immunoblots for phospho-AKT and mTORC1 downstream targets in crypts from AL, Refed 1 h and Refed 1 h treated with OSI-906 (left) or BKM120 mice (right). c, Immunoblots for pS6 and total S6 in crypts from AL, Fasted, and Refed mice (started fasting and refeeding at 9PM). d, Immunoblots for pS6 and total S6 in crypts from AL, Refed 1d with or without rapamycin treatment. e, Quantification of BrdU+ cells per jejunal crypt from AL and Refed 1d with or without rapamycin treatment (left), and representative images of IHC for BrdU (right). n = 4–5 mice per group, pooled from 3 independent experiments. Scale bar, 25 µm. f, Immunoblots for pS6 and total S6 in crypts from AL and Refed 1d Tsc1 WT or KO mice. g, Immunoblots for pS6 and total S6 in crypts from AL and Refed 1d Raptor WT or KO mice. Two-way ANOVA (a). One-way ANOVA (e). Data are mean ±s.d. **p < 0.01, ***p < 0.001.
Extended Data Fig. 4 Refeeding stimulates proliferation and stemness in primitive ISC subsets.
a, Feature heatmaps for genes encoding enterocyte, secretory lineage, and stem cell markers. b, GSEA for Biton-I, II, III gene signatures among ISC subsets (clusters 5, 2 and 10) from AL (al) mouse. NES, normalized enrichment score; FDR, false discovery rate. c, Cell-cycle marker gene analysis for each ISC subset (5, 2,10) from all dietary condition. d, Frequency of cells expressing S phase or G2/M phase cell-cycle marker genes in clusters 5, 2, 10, and non-ISC subsets in the different dietary conditions with or without rapamycin treatment (rf). e, Violin plots for the mean expression of Biton-I, -II, and III gene signatures within each cluster across the different dietary conditions with or without rapamycin treatment (rf). f,g, Gkn3 (f) or Pdgfa (g) gene expression level in cluster 5 (left) and representative images of in situ hybridization (ISH, red) (right). n = 4–5 mice per group. Scale bar, 10 µm. h, qPCR for Oat on FACS-sorted ISCs. n = 4–9 mice per group, pooled from 4 independent experiments. Duplicate measurements were taken from each mouse. i, Immunoblots for OAT, pS6 and total S6 in crypts from AL Tsc1 WT or KO mice. j,k, Ornithine level in the crypts from AL or refed Tsc1loxp/loxp;Villin-CreERT2 (j) or Raptorloxp/loxp;Villin-CreERT2 (k) mice. n = 5–6 mice per group, pooled from 4 independent experiments. l, Ornithine level in the crypts from refed 4 h mice with or without OAT inhibitor (5-FMO) treatment. n = 6–7 mice per group. pooled from 3 independent experiments. One-way ANOVA (c, e–h, j). Fisher’s exact two-sided test (d). Unpaired two-tailed t-tests (k, l). Data are mean ±s.d. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Extended Data Fig. 5 mTORC1 activity regulates the polyamine level and hypusinatin of eIF5A in crypts.
a, qPCR on FACS sorted ISCs from Lgr5-EGFP-IRES-creERT2 mice. n = 5 per group, pooled from 3 independent experiments. b,c, Polyamine level in the crypts from refed 4 h mice treated with or without OAT inhibitor (5-FMO) (b) or ODC1 inhibitor (DFMO) (c). n = 5–7 mice per group, pooled from 3 independent experiments. d,e, Polyamine level in the crypts from AL or refed 24 h Tsc1loxp/loxp;Villin-CreERT2 (d) or Raptorloxp/loxp;Villin-CreERT2 (e) mice. n = 4–6 mice per group, pooled from 4 independent experiments. f, Schematic of isotope tracing experiments. g, The proportion of isotope-labeled putrescine in the crypt samples from mice under different dietary conditions at various incubation times (0/1 h/2 h/4 h). n = 1–4 mice per group. h, Immunoblots for hypusinated elF5A and total elF5A in crypts from AL or refed 24 h Raptor WT or KO mice. i, Immunoblots for hypusinated elF5A and total elF5A in crypts from AL or refed 24 h mice with or without DFMO treatment. DFMO 40, 200: DFMO 40 mg/kg, 200 mg/kg. j, Immunoblots for of phospho-AKT and mTORC1 downstream targets in crypts from 1 h or 4 h refed mice with or without BKM120 treatment. k, Immunoblots for hypusinated elF5A and total elF5A in crypts from AL or refed 24 h Tsc1 WT or KO mice. l, Immunoblots for mTORC1 downstream targets in crypts from AL or refed 24 h mice with or without DFMO treatment. DFMO 40, 200: DFMO 40 mg/kg, 200 mg/kg. m, Immunoblots for puromycin in crypts from AL, fasted 24 h or refed 24 h mice (started fasting and refeeding at 9 PM). n, Polyamine level in crypts from AL or refed 24 h Odc1 WT or KO mice. n = 4–6 mice per group, pooled from 4 independent experiments. o, Representative images of day 3 organoids of crypts from AL Odc1 WT or KO mice. DFMO (1.5 mM) or/and Spermidine (50 uM) were added to the culture medium for the treatment group. Scale bar, 500 µm. p, Schematic of the irradiation mouse model, including the timeline of ODC1 inhibitor (DFMO) administration. q, Immunoblots for DHPS in crypts from AL or refed 4 h mice. r, Schematic of in vivo GC7 treatment in AL or Refed 24 h mice. s, Organoid assay for crypts from AL or refed 24 h mice treated with or without GC7 treatment (75 µM). Quantification (left) and representative images of day 3 organoids (right). n = 3 mice per group, pooled from 3 independent experiments. Scale bar, 500 µm. t, Immunoblots for puromycin and hypusinated elF5A in crypts from AL or refed 24 h mice with or without GC7 treatment. u, Schematic of the irradiation mouse model including the timeline of rapamycin and polyamine administration. v, Immunoblots for puromycin in crypts from refed 24 h+rapa C57BL/6 mice with or without polyamines administration. Unpaired, two-tailed, Mann-Whitney test (b, c), One-way ANOVA (a, d, e, n, s). Data are mean ± s.d. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Extended Data Fig. 6 Post-fast refeeding augments tumorigenicity.
a, Schematic of Cre recombinase activity in Lgr5-IRES-CreERT2; Rosa26LSL-tdTomato reporter mice. b, Quantification of tdTomato+ crypts in jejunum (left) and ileum (right) samples. n = 2 mice per group, pooled from 2 independent experiments. c, Representative images of tdTomato+ crypts in jejunum (left) and ileum (right). Scale bar, 50 µm. d, Schematic of tumour burden calculation. Scale bar, 50 µm. e, Quantification of β-catenin+ Apc-null tumours in colon from Apcloxp/loxp; Lgr5-EGFP-IRES- creERT2 mice (left), and representative images of Apc-null tumour lesion by IHC for β-catenin (black dot circle, right). Scale bar, 50 µm. n = 9 mice per group, pooled from 3 independent experiments. f, Ratio of β-catenin+ Apc-null tumour length to colon length in colon of Apcloxp/loxp; Villin-creERT2 mice (left), and representative images of Apc-null tumours by IHC for β-catenin (right). Tumours are surrounded by a yellow dotted line (right). n = 11–21 mice per group, pooled from 3 independent experiments. Scale bar, 50 µm. g, Representative images of Apc- null tumour lesions by IHC for β-catenin from IF experiments. Tumours are surrounded by a black dotted line. Scale bar, 50 µm. One-way ANOVA (e, f). Data are mean ±s.d. *p < 0.05, ***p < 0.001, ns; not significant.
Extended Data Fig. 7 mTORC1-Polyamine-protein synthesis axis boosts tumorigenicity.
a, Schematic of Apc tumour model with Apcloxp/loxp;Villin-CreERT2 mice with or without rapamycin. b, Schematic of Apcloxp/loxp; Lgr5-EGFP-IRES creERT2 tumor model with or without rapamycin. c, Quantification of tumour burden in the small intestine (left) and representative tumour images by IHC for β-catenin (right) in Apcloxp/loxp;Villin-CreERT2. n = 5–8 mice per group, pooled from 3 independent experiments. Scale bar, 100 µm. d, Quantification of tumour burden in small intestine (left) and colon (right) in Apcloxp/loxp; Lgr5-EGFP-IRES creERT2 mice. n = 8–22 mice per group, pooled from 3 independent experiments. e, Representative tumour images by H&E (small intestine) or IHC for β-catenin (colon) from Apcloxp/loxp; Lgr5-EGFP-IRES creERT2 mice. Tumours are surrounded by white or black dotted lines. Scale bar, 100 µm. f, Schematic of Apcloxp/loxp; Lgr5-EGFP-IRES-creERT2 tumour model with or without the Tsc1loxp/loxp allele (WT or KO). g, Ratio of tumour length to intestinal length (left) and representative images of tumour lesions by H&E and IHC for pS6 (right). n = 5–6 mice per group, pooled from 3 independent experiments. Scale bar, 100 µm. h, Schematic of Apc tumour model with Apcloxp/loxp;Villin-CreERT2 mice with or without DFMO. i, Quantification of tumour burden in the small intestine (left) and representative tumour images by IHC for β-catenin (right) with or without DFMO. n = 4–7 mice per group, pooled from 3 independent experiments. Scale bar, 100 µm. j, Quantification of tumour burden in colon treated with ODC inhibitor (DFMO) (left), and representative tumour images by IHC for β-catenin (right). Tumours are surrounded by yellow dotted lines. n = 5–7 mice per group, pooled from 3 independent experiments. Scale bar, 100 µm. k, Schematic of Apc tumour model with Apcloxp/loxp;Villin-CreERT2 mice with or without protein synthesis inhibitor (cycloheximide). l, Schematic of assessing the effect of cycloheximide on protein synthesis. m, Immunoblots for puromycin in crypts labelled with puromycin. CHX 5, 15: CHX 5 mg/kg, 15 mg/kg. n, Quantification of tumour burden in the small intestine (left) and representative tumour images by IHC for β-catenin (right) with or without protein synthesis inhibitor (cycloheximide). n = 5–6 mice per group, pooled from 3 independent experiments. Scale bar, 100 µm. o, Immunoblots for puromycin in isolated crypts from Refed 1d Apcloxp/loxp; Lgr5-EGFP-IRES-creERT2 or Apcloxp/loxp; Rpl24Bst/+; Lgr5-EGFP-IRES-creERT2 mice without tamoxifen administration. p, Quantification of tumour burden in the small intestine of Apcloxp/loxp; Rpl24Bst/+; Lgr5-EGFP-IRES-creERT2 mouse model (left) and representative images of tumours by IHC for β-catenin. Tumours are surrounded by yellow dotted lines (right). n = 9–16 mice per group, pooled from 3 independent experiments. Scale bar, 100 µm. One-way ANOVA (c, d, i, j, n). Unpaired two-tailed t-tests (g, p). Data are mean ±s.d. *p < 0.05, **p < 0.01, ****p < 0.0001.
Extended Data Fig. 8 Chronic calorie restriction and acute 24 h fast-refeeding regimen enhance ISC function through a distinct mechanism.
a, qPCR for Bst1 on FACS-sorted ISCs from AL, Fasted 24 h, Refed 24 h, and CR (fasted state) mice. n = 6–7 mice per group, pooled from 5 independent experiments. Duplicate measurements were taken from each mouse. b, IHC for Lysozyme in the small intestine of Atoh1 WT and KO mice. Scale bar, 100 um (left) and 50 um (right). c, Organoid assay for crypts from AL or refed 24 h Atoh1 KO mice. Quantification (left) and representative images of day 3 organoids (right). n = 6 mice per group, pooled from 2 independent experiments. Scale bar, 500 µm. d, Levels of polyamines in crypts from AL, CR (fasted or refed state), or rapamycin-treated mice. n = 7–8 mice per group, pooled from 4 independent experiments. e, Organoid assay for crypts from AL, CR (fasted or refed state), or rapamycin-treated mice. Quantification (left) and representative images of day 3 organoids (right). n = 5–13 mice per group, pooled from 4 independent experiments. Scale bar, 500 µm. One-way ANOVA (a, d, e). Unpaired two-tailed t-tests (c). Data are mean ±s.d. *p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.
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This file contains Supplementary Figures 1 and 2. Supplementary Figure 1 contains the source data of the Western Blots shown in main and extended figures. Supplementary Figure 2 contains the gating strategy for GFP+ cells from Lgr5-IRES-GFP-Cre mice.
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Supplementary Table 1 (download DOCX )
This file contains tables showing the top differentially expressed genes in clusters 5, 2, and 10 from different dietary conditions.
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This file contains a complete list of resources used in this manuscript including antibodies, chemical, peptides, recombinant proteins, deposited data, experimental mouse models, oligonucleotides, software and algorithms, and RNA probes.
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Transition setting and internal standard for the analysis of polyamines
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Transition setting and internal standard for the analysis of amino acids
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Imada, S., Khawaled, S., Shin, H. et al. Short-term post-fast refeeding enhances intestinal stemness via polyamines. Nature 633, 895–904 (2024). https://doi.org/10.1038/s41586-024-07840-z
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DOI: https://doi.org/10.1038/s41586-024-07840-z