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Fibrosis induced by resident macrophages has divergent roles in pancreas inflammatory injury and PDAC

Nature Immunology volume 24, pages 1443–1457 (2023)Cite this article

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Abstract

Tissue-resident macrophages (TRMs) are long-lived cells that maintain locally and can be phenotypically distinct from monocyte-derived macrophages. Whether TRMs and monocyte-derived macrophages have district roles under differing pathologies is not understood. Here, we showed that a substantial portion of the macrophages that accumulated during pancreatitis and pancreatic cancer in mice had expanded from TRMs. Pancreas TRMs had an extracellular matrix remodeling phenotype that was important for maintaining tissue homeostasis during inflammation. Loss of TRMs led to exacerbation of severe pancreatitis and death, due to impaired acinar cell survival and recovery. During pancreatitis, TRMs elicited protective effects by triggering the accumulation and activation of fibroblasts, which was necessary for initiating fibrosis as a wound healing response. The same TRM-driven fibrosis, however, drove pancreas cancer pathogenesis and progression. Together, these findings indicate that TRMs play divergent roles in the pathogenesis of pancreatitis and cancer through regulation of stromagenesis.

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Fig. 1: Pancreatitis and pancreatic ductal adenocarcinoma display immune rich fibrotic stroma.
Fig. 2: Cerulein treatment increases tissue-resident macrophages.
Fig. 3: Pancreas tissue-resident macrophages have distinct transcriptional phenotypes.
Fig. 4: Human LYVE1+ tissue-resident macrophages display similar phenotype and localization.
Fig. 5: Tissue-resident macrophages maintain tissue integrity during pancreatitis.
Fig. 6: Depletion of tissue-resident macrophages attenuates fibrotic responses.
Fig. 7: Tissue-resident macrophages shape the tissue-protective fibrotic response.
Fig. 8: Tissue-resident macrophages drive fibrosis and pancreatitis-accelerated pancreatic ductal adenocarcinoma progression.

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Data availability

Sequencing data are publicly available under Gene Expression Omnibus repository accession numbers GSE196778 (scRNA-seq) and GSE203005 (bulk RNA-seq). Source data are provided with this paper. All other data are available upon request from the corresponding author.

Code availability

No custom code was generated for this study. Code used to generate figures is available upon request from the corresponding author.

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Acknowledgements

J.M.B. was funded by pre-doctoral fellowship F31 DK122633. D.G.D. and study costs were supported by NCI R01CA273190, R01CA177670, P50CA196510, P30CA09184215, and the BJC Cancer Frontier Fund. Collaborative studies with G.J.R. were supported by RO1AI049653. L.I.K. was funded by 5T32EB021955 2019-2021. K.W.K. was funded by NIDDK R01DK126753. J.C.M. was funded by R01DK105129 and R01CA239645. We thank the Washington University Center for Cellular Imaging, the Flow Cytometry & Fluorescence Activated Cell Sorting Core and Genome Technology Access Center, which are supported by D.G.D. fund P50CA196510.

Author information

Author notes

  1. Jason C. Mills

    Present address: Section of Gastroenterology and Hepatology, Department of Medicine, Baylor College of Medicine, Houston, TX, USA

  2. Jason C. Mills

    Present address: Departments of Medicine, Pathology and Immunology, and Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA

Authors and Affiliations

  1. Department of Medicine, Washington University School of Medicine, St. Louis, MO, USA

    John M. Baer, Chong Zuo, Angela Alarcon de la Lastra, Brett L. Knolhoff, Savannah J. Bogner, Yu Zhu, Mark A. Lewis, Li Ding, Gwendalyn J. Randolph & David G. DeNardo

  2. Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA

    Liang-I Kang, Nicholas C. Borcherding, Nan Zhang, Ki-Wook Kim, Ryan C. Fields, Gwendalyn J. Randolph & David G. DeNardo

  3. Department of Pathology, Stanford University, Palo Alto, CA, USA

    Yu Zhu

  4. Division of Rheumatology, Department of Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO, USA

    Liping Yang, Jennifer Laurent & Wayne M. Yokoyama

  5. Division of Gastroenterology, Department of Medicine, Washington University School of Medicine in St. Louis, St. Louis, MO, USA

    Mark A. Lewis & Jason C. Mills

  6. Department of Pharmacology and Regenerative Medicine, University of Illinois College of Medicine, Chicago, IL, USA

    Ki-Wook Kim

  7. Department of Surgery, Washington University School of Medicine, St. Louis, MO, USA

    Ryan C. Fields

  8. Departments of Pathology and Immunology and Developmental Biology, Washington University School of Medicine in St. Louis, St. Louis, MO, USA

    Jason C. Mills

  9. Siteman Cancer Center, Washington University School of Medicine, St. Louis, MO, USA

    Li Ding & David G. DeNardo

  10. McDonnell Genome Institute, Washington University in St. Louis, St. Louis, MO, USA

    Li Ding

  11. Department of Genetics, Washington University in St. Louis, St. Louis, MO, USA

    Li Ding

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Contributions

J.M.B. and D.G.D. conceived of and designed experiments, and wrote the manuscript with input from all authors. J.M.B., C.Z., L.I.K., A.A.L., N.C.B., B.L.K., S.J.B., Y.Z., L.Y., J.P.L. and D.G.D. performed experiments and analyzed data. N.C.B., Y.Z., M.A.L., N.Z., K.W.K., R.C.F., W.M.Y., L.D., J.C.M. and G.J.R. provided key resources, expertise, input and tissues.

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Correspondence to David G. DeNardo.

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Nature Immunology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editor: Ioana Visan, in collaboration with the Nature Immunology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Pancreatitis and PDAC display immune rich fibrotic stroma.

a. Representative immunohistochemistry images and quantification of Ki67 staining in pancreas tissue of vehicle and cerulein treated mice as in a; n = 4 mice/group, *P = 0.0079. b. Flow cytometry plots of gating strategy used for pancreas F4/80+MHCIIhi/lo macrophages. c. Density of F4/80loMHCII- eosinophils, Ly6G+ granulocytes, and Ly6C+ monocytes in pancreas of mice treated with vehicle or cerulein by 6 hourly i.p. injections every other day for one week; n = 4 mice/group, *P = 0.0286 for eosinophil analysis, *P = 0.0286 for granulocyte analysis, and *P = 0.0286 for monocyte analysis. Data are presented as mean ± SEM unless otherwise indicated. n.s., not significant; *P < 0.05. For comparisons between two groups, Student’s two-tailed t-test was used.

Source data

Extended Data Fig. 2 Cerulein treatment increases tissue-resident macrophages.

a. Flow cytometry of Flt3-YFP+/- cells pre-gated on blood Ly6Clo/hi monocytes. b. Flow cytometry of CSF1R-tdTom mice given tamoxifen by oral-gavage for five consecutive days, then tamoxifen was stopped for 10 weeks, pre-gated on Ly6Chi/lo blood monocytes. c. Quantification of tdTomato+ Ly6Chi/lo blood monocytes after treatment as in b, displayed as percentage of each cell type; n = 8mice/group. d. Schematic of tamoxifen treatment in CX3CR1-CreERT2;LSL-tdTomato mice (hereafter CX3CR1-tdTom mice). e. Flow cytometry of tdTomato−/+ cells in CX3CR1-tdTom mice treated as in d followed by vehicle or cerulein by 6 hourly i.p. injections every other day for one week, pre-gated on Ly6Chi/lo blood monocytes, and F4/80+MHCIIhi/lo pancreas macrophages. f. Representative images of IHC staining of tdTomato in pancreas tissue of CX3CR1-tdTom mice treated as in e, scale bars, 100 µM. g. Quantification of tdTomato+ cells from pancreas tissue of CX3CR1-tdTom mice treated as in e, displayed as percentage of cells; n = 4mice/group, *P = 0.0286. h. Quantification of tdTomato+ blood Ly6Chi/lo monocytes from CX3CR1-tdTom mice treated as in e, displayed as percentage of each cell type; n = 7mice/group. i. Density of tdTomato+ pancreas F4/80+MHCIIhi/lo macrophages from CX3CR1-tdTom mice treated as in e; vehicle, n = 4 mice; cerulein, n = 5 mice, *P = 0.0159. j. Flow cytometry of GFP in CX3CR1-GFP mice, pre-gated on Ly6Chi/lo blood monocytes, and pancreas F4/80+MHCIIhi/lo macrophages. k. Quantification of GFP+ blood Ly6Chi/lo monocytes and pancreas F4/80+MHCIIhi/lo pancreas macrophages from CX3CR1-GFP mice, displayed as percentage of each cell type; n = 3mice/group. l. Representative images of IHC staining of pancreas tissue from CX3CR1-tdTom mice treated as in e and stained for F4/80, tdTomato, and LYVE1, islets of Langerhans outlined in yellow, scale bars, 100 µM, staining was repeated for 8 mice. m. Representative images of multiplex-IHC (mIHC) staining of pancreas tissue from CSF1R-tdTom mice treated with tamoxifen as in b, followed by cerulein treatment as in e, and stained for LYVE1, tdTomato, and F4/80, islet of Langerhans outlined in yellow dash, scale bars, 100 µM, staining was repeated for 4 vehicle- and 4 cerulein-treated mice. Data are presented as mean ± SEM unless otherwise indicated. n.s., not significant; *p < 0.05. For comparisons between two groups, Student’s two-tailed t-test was used.

Source data

Extended Data Fig. 3 Pancreas TRMs have distinct transcriptional phenotypes.

a. Heatmap displaying DEGs in bulk RNAseq data upregulated in Flt3-YFP+ or Flt3-YFP− pancreas macrophages from mice treated with 6 hourly i.p. injections every other day for one week of vehicle (healthy), cerulein (pancreatitis), or orthotopically implanted with the KP1 pancreatic cancer cell line (PDAC). b. Heatmap displaying normalized enrichment score (NES) of significantly enriched gene sets in bulk RNAseq data upregulated in Flt3-YFP+ or Flt3-YFP− pancreas macrophages of mice treated as in a, pathways selected by FDR < 0.05. c. Venn diagram of overlap in number of DEGs upregulated in Flt3-YFP+ or Flt3-YFP− pancreas macrophages from mice treated as in a. d. UMAP plot displaying Clec4f expression in F4/80+MHCIIhi/lo macrophages sorted from livers of Flt3-YFP mice 14 days after implantation with the KP2 pancreatic cancer cell line, and UMAP plot displaying Siglecf expression in CD11bint/hiCD11cint/hiF4/80+ macrophages from lungs of Flt3-YFP mice 15 days after i.v. injection of the KPL86 lung cancer cell line. e. UMAP plot from scRNAseq analysis of CD11bint/hiCD11cint/hiF4/80+ macrophages sorted from Flt3-YFP lungs implanted with KPL86 lung cancer cell line as in a. f. Quantification of Flt3-YFP+ and Flt3-YFP− lung macrophages by cluster from UMAP in e, displayed as percentage of each cluster.

Source data

Extended Data Fig. 4 Pancreas TRMs have distinct transcriptional phenotypes.

a. UMAP plot of scRNAseq analysis of F4/80+MHCIIhi/lo macrophages sorted from pancreas of Flt3-YFP mice treated with vehicle by 6 hourly i.p. injections every other day for one week. b. Quantification of Flt3-YFP+ and Flt3-YFP− pancreas macrophages by cluster from UMAP in a, displayed as percentage of each cluster. c. UMAP plot of scRNAseq analysis of F4/80+MHCIIhi/lo macrophages sorted from pancreas of Flt3-YFP mice orthotopically implanted with the KP1 pancreatic cancer cell line. d. Quantification of Flt3-YFP+ and Flt3-YFP− pancreas macrophages by cluster from UMAP in c, displayed as percentage of each cluster. e. UMAP plots displaying Timd4, Trem2, Folr2, H2-Aa, Cd74, and Cd14 expression in Csf1r and C1qa expressing pancreas macrophages from Flt3-YFP mice treated with cerulein by 6 hourly i.p. injections every other day for one week. f. Violin plots displaying expression of Lyve1 and Cx3cr1 for each cluster of Csf1r and C1qa expressing pancreas macrophages from Flt3-YFP mice treated with cerulein as in a. g. UMAP plots displaying Lyve1, Cx3cr1, and Ccr2 expression in F4/80+MHCIIhi/lo macrophages sorted from pancreas of Flt3-YFP mice treated as in a. h. UMAP plots displaying Lyve1, Cx3cr1, and Ccr2 expression in F4/80+MHCIIhi/lo macrophages sorted from pancreas of Flt3-YFP mice implanted with tumors as in c. i. Bar graph of normalized enrichment score (NES) values of gene sets upregulated in Flt3-YFP+ or Flt3-YFP− pancreas macrophages within pancreatitis cluster 0 (from Fig. 3c). Data are presented as mean ± SEM unless otherwise indicated. n.s., not significant; *p < 0.05. For comparisons between two groups, Student’s two-tailed t-test was used, except i where FDR was used.

Source data

Extended Data Fig. 5 Pancreas TRMs have distinct transcriptional phenotypes.

a. UMAP plots displaying Lyve1, Cx3cr1, Trem2, Ccr2, Mrc1, H2-Ab1, H2-Aa, Cd163, Folr2, and Timd4 expression in Csf1r and C1qa expressing pancreas macrophages from CSF1R- tdTom mice given tamoxifen by oral gavage for five consecutive days, then tamoxifen was stopped for 10 weeks, then cerulein (cer) was given by 6 hourly i.p. injections every other day for one week. b. Bar graph of NES values of gene sets upregulated in tdTomato+ or tdTomato− cells from CSF1R-tdTom mice treated as in a. c. Quantification of LYVE1+F4/80+tdTomato+ pancreas macrophages from CSF1R-tdTom mice treated with tamoxifen as in a, followed by vehicle (veh) or cerulein (cer) treatment as in a, displayed as percentage of LYVE1+F4/80+ macrophages; n = 4 mice/group. d. Schematic of surgical joining of parabiotic pairs of CD45.1+ and CD45.2+ C57BL/6 mice. e. Flow cytometry staining of CD45.1 and CD45.2 in parabiotic pairs of mice, following 6 weeks of surgical joining, pre-gated on blood Ly6C+ monocytes or F4/80+MHCIIhi/lo pancreas macrophages. f. Quantification of percent chimerism (left) and chimerism normalized to blood Ly6Chi monocyte chimerism (relative chimerism, right) for blood Ly6Chi monocytes, LYVE1+CD163+ pancreas macrophages, LYVE1+CD163+ pancreas macrophages, and MHCIIhi pancreas macrophages; n = 4 mice/group, *P = 0.0286 for chimerism analysis and *P = 0.0286 for relative chimerism analysis. Data are presented as mean ± SEM unless otherwise indicated. n.s., not significant; *p < 0.05. For comparisons between two groups, Student’s two-tailed t-test was used, except b where FDR was used.

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Extended Data Fig. 6 Human LYVE1+ TRMs display similar phenotype and localization.

a. UMAP plot displaying mouse LYVE1lo pancreas macrophage scRNA-seq signature using top 100 DEGs from mouse LYVE1lo macrophages mapped into human chronic pancreatitis data set8. b. UMAP plot displaying mouse LYVE1lo pancreas macrophage scRNA-seq signature using top 100 DEGs from mouse LYVE1lo macrophages mapped into published human PDAC data set6. c. UMAP plots displaying CD163, LYVE1, CSF1R, and CD68 expression in human healthy and chronic pancreatitis samples from a, and violin plot showing CD163 gene expression across human pancreatitis macrophage clusters. d. UMAP plots displaying Cd163, Lyve1, Csf1r, and Cd68 expression in scRNA-seq of F4/80+MHCIIhi/lo macrophages sorted from the pancreas of Flt3-YFP mice treated with cerulein by 6 hourly i.p. injections every other day for one week, and violin plot showing Cd163 gene expression across same dataset of mouse pancreas macrophage clusters. e. UMAP plots displaying CD163, LYVE1, CSF1R, and CD68 expression in macrophages from human PDAC data set as in b, and violin plot showing CD163 gene expression across human PDAC macrophage clusters. f. UMAP plots displaying Cd163, Lyve1, Csf1r, and Cd68 expression in mouse F4/80+MHCIIhi/lo macrophages sorted from pancreas of Flt3-YFP mice orthotopically implanted with the KP1 pancreatic cancer cell line, and violin plot showing Cd163 gene expression across same dataset of mouse PDAC macrophage clusters.

Extended Data Fig. 7 TRMs maintain tissue integrity during pancreatitis.

a. Flow cytometry of pancreas F4/80+MHCIIhi/lo macrophages after treatment with CSF1Ab+CLD or IgG+PBS, then a 10-day recovery period (day -1), then cerulein (cer) treatment by 6 hourly i.p. injections every other day for one week (day 7). b. Density of MHCIIhi pancreas macrophages from mice treated as in a; day -1, n = 4mice/group; day 7, n = 3mice/group. c. Density of MHCIIlo pancreas macrophages from mice treated as in a; day -1, n = 4mice/group; day 7, n = 3mice/group, left to right *P = 0.0002 and *P = 0.0002. d. Kaplan-Meier survival curve and body weight measurement of C57BL/6 mice treated with IgG+PBS or CSF1Ab+CLD as in a, followed by cerulein-loaded osmotic pump (10 µg/day cerulein) implantation; IgG+PBS, n = 10mice; CSF1 Ab+CLD, n = 6mice, *P < 0.0001 in survival analysis. e. Relative pancreas weight in C57BL/6 mice treated as in d; IgG+PBS, n = 10mice, CSF1Ab+CLD, n = 4mice, *P < 0.0020. f. IHC images of H&E and amylase stain in pancreas from mice implanted with DMSO-loaded osmotic pump, cerulein treatment as in a, or cerulein-loaded osmotic pump, scale bars, 100 µM. g. Blood glucose concentration at humane survival endpoint in mice treated as in d; IgG+PBS, n = 7mice; CSF1Ab+CLD, n = 6mice, *P = 0.0012. h. Serum amylase level in mice treated as in f; left to right n = 4mice, n = 6mice, and n = 6mice, and *P = 0.0381 and *P = 0.0390. i. H&E-stained liver from mice treated as in f, scale bars, 100 µM. j. Blood Ly6Clo and Ly6Chi monocytes, and pancreas F4/80+, F4/80+MHCIIhi, and F4/80+MHCIIlo macrophages, and flow cytometry of F4/80+MHCIIhi/lo pancreas macrophages in CCR2-WT and CCR2-KO mice treated with cerulein by i.p. injection as in a; n = 4 mice/group and *P = 0.0286 in blood Ly6Clo and Ly6Chi analysis, n = 7 mice in veh CCR2-WT group, n = 5 mice in veh CCR2-KO group, n = 8 mice in cer CCR2-WT group, and n = 6 in cer CCR2-KO group in F4/80+, F4/80+MHCIIhi, and F4/80+MHCIIlo analyses, and *P = 0.0047 in F4/80+MHCIIhi macrophage analysis. k. Kaplan-Meier survival curve and body weight measurement of CCR2-WT and CCR2-KO mice implanted with cerulein-loaded osmotic pumps, as in d; n = 8 mice in CCR2-WT group, n = 10 mice in CCR2-KO group. Data are presented as mean ± SEM unless otherwise indicated. n.s., not significant; *p < 0.05. For comparisons between two groups, Student’s two-tailed t-test was used.

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Extended Data Fig. 8 Depletion of TRMs attenuates fibrotic responses.

a. IHC staining and quantification for podoplanin and fibronectin on pancreas tissue from mice treated with CSF1Ab+CLD or IgG+PBS, then a 10-day recovery period, then cerulein (cer) treatment by 6 hourly i.p. injections every other day for 3, 7, or 17 days, scale bars, 100 µM, IgG+PBS, CSF1Ab+CLD day 3, and IgG+PBS day 7, n = 6mice; CSF1Ab+CLD day 7, n = 7mice; IgG+PBS day 17, n = 5mice; CSF1Ab+CLD day 17, n = 8mice; podoplanin left to right, *P = 0.0022, *P = 0.0012, and *P = 0.0016; fibronectin left to right, *P = 0.0130 and *P = 0.0007. b. H&E and podoplanin stained pancreas tissue from mice treated as in a, with no cerulein treatment, scale bars, 100 µM. c. Podoplanin IHC quantification on pancreas tissue of mice treated as in b; IgG+PBS, n = 7mice; CSF1Ab+CLD, n = 8mice. d. Flow cytometry gating for pancreas fibroblast subsets. e. Density of pancreas PDGFRα-, α-SMA+, and MHCII+ fibroblasts from mice treated with IgG+PBS followed by vehicle (steady-state), IgG+PBS followed by cerulein (IgG+PBS+Cer), and CSF1Ab+CLD followed by cerulein (CSF1 AB+CLD+Cer) as in a; steady-state and IgG+PBS+Cer, n = 7mice, and CSF1Ab+CLD+Cer, n = 9mice. f. Density of pancreas F4/80+MHCIIhi/lo, LYVE1+CD163+, LYVE1−CD163+, LYVE1−CD163− macrophages, and Ly6C+ iFibs in Lyve1-Cre- littermate controls (control), or Lyve1ΔCSF1R mice treated with cerulein by i.p. injections every other day for one week; control, n = 5mice; Lyve1ΔCSF1R, n = 4mice; LYVE1+CD163+, *P = 0.0159, and Ly6C+ iFib, n = 6mice/group. g. Mouse body weight measurement and Kaplan-Meier survival curve following implantation of osmotic pump for delivery of 10 µg/day cerulein in control or Lyve1ΔCSF1R mice; n = 9 mice in control and n = 8 mice in Lyve1ΔCSF1R group. h. Podoplanin and fibronectin IHC staining on pancreas tissue of CCR2-WT and CCR2-KO mice treated with cerulein as in f; scale bars, 100 µM. i. Quantification of podoplanin and fibronectin IHC stains and density of podoplanin+ fibroblasts, Ly6C+ iFibs, and PDGFRα+ fibroblasts in pancreas tissue of CCR2-WT and CCR2-KO mice treated with cerulein as in f; CCR2-WT, n = 8mice; CCR2-KO, n = 7mice for podoplanin and fibronectin IHC analyses, and CCR2-WT, n = 8mice; CCR2-KO, n = 6mice for fibroblast flow cytometry analyses. Data are presented as mean ± SEM unless otherwise indicated. n.s., not significant; *p < 0.05. For comparisons between two groups, Student’s two-tailed t-test was used.

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Extended Data Fig. 9 TRMs shape the tissue-protective fibrotic response.

a. UMAP plot of scRNA-seq analysis of PDPN+ pancreas fibroblasts sorted from mice treated with IgG+PBS followed by vehicle (healthy), IgG+PBS followed by cerulein (IgG+PBS+Cer), and CSF1 Ab+CLD followed by cerulein (CSF1 Ab+CLD+Cer). b. UMAP plots displaying expression of Pdgfra, Ly6c1, and Col8a1 in PDPN+ fibroblasts from a. c. UMAP plot of pancreas PDPN+ fibroblasts separated by healthy, IgG+PBS+Cer, or CSF1 Ab+CLD+Cer samples as in a. d. Violin plot of Col3a1, Col6a5, and Col6a6 expression across samples (healthy, IgG+PBS+Cer, or CSF1 Ab+CLD+Cer); for all comparisons *P < 0.0001. e. UMAP plot of scRNA-seq analysis combining macrophages from Flt3-YFP mice treated with cerulein and fibroblasts from IgG+PBS+Cer and CSF1 Ab+CLD+Cer treated mice. f. UMAP plots displaying Csf1r, Pdgfra, Lyve1, and Cx3cr1 expression in combined macrophage and fibroblast scRNA-seq analysis from e. g. Dot plot showing aggregate score of all incoming (receptor) and outgoing (ligand) interactions summarized for each macrophage and fibroblast cluster from e, as measured by CellChat9. h. Heatmap displaying relative strength of network centrality measures for outgoing (ligands, left heatmap) and incoming (receptors, right heatmap) signaling patterns of each intercellular signaling pathway. i. Heatmap displaying aggregate score across all differentially enriched receptor (receiver) or ligand (sender) pathways between macrophage and fibroblast clusters from e. j. Density of pancreas F4/80+MHCIIhi/lo, LYVE1+CD163+, and LYVE1−CD163− macrophages from mice treated with vehicle (veh) or PDGFRi once per day by i.p. injection along with cerulein treatment by 6 hourly i.p. injections every other day for one week; n = 7 mice in vehicle and n = 8 mice in PDGFRi groups. Data are presented as mean ± SEM unless otherwise indicated. n.s., not significant; *p < 0.05. For comparisons between two groups, Student’s two-tailed t-test was used, except for d where Bonferroni correction was used.

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Extended Data Fig. 10 TRMs drive fibrosis and pancreatitis-accelerated PDAC progression.

a. Genetic loci for p48-Cre;LSL-KRASG12D;p53fl/+ (KPC) model and treatment scheme for IgG+PBS and CSF1 Ab+CLD followed by cerulein by 6 hourly i.p injections every other day for 5 days. b. Genetic loci for p48-Cre;ROSA26-rtTa-IRES-EGFP;TetO-KRASG12D (iKRAS*) model and treatment scheme for IgG+PBS and CSF1 Ab+CLD followed by cerulein by 6 hourly i.p injections on two consecutive days, followed by doxycycline administration in drinking water. c. Representative mIHC images of pancreas tissue from iKRAS* mice treated with IgG+PBS or CSF1 Ab+CLD as in b, stained for hematoxylin, CK19, F4/80, LYVE1, and CD163 (scale bars are 100 µM), and quantification of F4/80+LYVE1+CD163+ macrophages, displayed as the percentage of cells; n = 7 mice in IgG+PBS group, n = 8 mice in CSF1 Ab+CLD group, and *P = 0.0003. d. Representative images of pancreas tissue stained for H&E and CK19 from iKRAS* mice treated with IgG+PBS or CSF1 Ab+CLD as in b, scale bars are 100 µM. e. Quantification of relative pancreas weight and CK19+ cells displayed as percentage of total cells from iKRAS* mice treated with IgG+PBS or CSF1 Ab+CLD as in b; n = 7 mice in IgG+PBS group, n = 10 mice in CSF1 Ab+CLD group, *P = 0.0097 in relative pancreas weight analysis, and *P = 0.0250 in CK19 analysis. f. Tumor weight and size measurements of CCR2-WT and CCR2-KO mice orthotopically implanted with the KP2 pancreatic cancer cell line; n = 9 mice/group. g. Representative images and quantification of pancreas tissue from CCR2-WT and CCR2-KO mice orthotopically implanted with the KP2 pancreatic cancer cell line stained for podoplanin, scale bars are 100 µM; n = 9 mice/group. Data are presented as mean ± SEM unless otherwise indicated. n.s., not significant; *p < 0.05. For comparisons between two groups, Student’s two-tailed t-test was used.

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Supplementary information

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Supplementary Tables 1–4

Supplementary Table 1. scRNA-seq libraries and cell counts. Supplementary Table 2. Key resources. Supplementary Table 3. List of marker genes for each cell type of pancreas PDPN+ fibroblasts sorted from mice treated with IgG + PBS or CSF1Ab + CLD followed by a 10-d recovery period, then cerulein by six hourly i.p. injections every other day for 1 week. Clusters include Ly6C+ iFibs, aSMA+ myFibs and MHCII+ apFibs; ‘p_val_adj column’ displays the adjusted P value using Bonferroni correction. Supplementary Table 4. List of DEGs from each fibroblast cluster (as in Table 3 and Fig. 7c) comparing IgG + PBS to CSF1Ab + CLD samples; ‘p_val_adj column’ displays the adjusted P value using Bonferroni correction.

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Baer, J.M., Zuo, C., Kang, LI. et al. Fibrosis induced by resident macrophages has divergent roles in pancreas inflammatory injury and PDAC. Nat Immunol 24, 1443–1457 (2023). https://doi.org/10.1038/s41590-023-01579-x

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