The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincle-induced immune suppression

Abstract

Neoplastic pancreatic epithelial cells are believed to die through caspase 8-dependent apoptotic cell death, and chemotherapy is thought to promote tumour apoptosis1. Conversely, cancer cells often disrupt apoptosis to survive2,3. Another type of programmed cell death is necroptosis (programmed necrosis), but its role in pancreatic ductal adenocarcinoma (PDA) is unclear. There are many potential inducers of necroptosis in PDA, including ligation of tumour necrosis factor receptor 1 (TNFR1), CD95, TNF-related apoptosis-inducing ligand (TRAIL) receptors, Toll-like receptors, reactive oxygen species, and chemotherapeutic drugs4,5. Here we report that the principal components of the necrosome, receptor-interacting protein (RIP)1 and RIP3, are highly expressed in PDA and are further upregulated by the chemotherapy drug gemcitabine. Blockade of the necrosome in vitro promoted cancer cell proliferation and induced an aggressive oncogenic phenotype. By contrast, in vivo deletion of RIP3 or inhibition of RIP1 protected against oncogenic progression in mice and was associated with the development of a highly immunogenic myeloid and T cell infiltrate. The immune-suppressive tumour microenvironment associated with intact RIP1/RIP3 signalling depended in part on necroptosis-induced expression of the chemokine attractant CXCL1, and CXCL1 blockade protected against PDA. Moreover, cytoplasmic SAP130 (a subunit of the histone deacetylase complex) was expressed in PDA in a RIP1/RIP3-dependent manner, and Mincle—its cognate receptor—was upregulated in tumour-infiltrating myeloid cells. Ligation of Mincle by SAP130 promoted oncogenesis, whereas deletion of Mincle protected against oncogenesis and phenocopied the immunogenic reprogramming of the tumour microenvironment that was induced by RIP3 deletion. Cellular depletion suggested that whereas inhibitory macrophages promote tumorigenesis in PDA, they lose their immune-suppressive effects when RIP3 or Mincle is deleted. Accordingly, T cells, which are not protective against PDA progression in mice with intact RIP3 or Mincle signalling, are reprogrammed into indispensable mediators of anti-tumour immunity in the absence of RIP3 or Mincle. Our work describes parallel networks of necroptosis-induced CXCL1 and Mincle signalling that promote macrophage-induced adaptive immune suppression and thereby enable PDA progression.

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Figure 1: RIP1 and RIP3 expression in PDA.
Figure 2: CXCL1 is expressed in PDA in a RIP1/3-dependent manner.
Figure 3: Deletion of RIP3 or blockade of RIP1 protects against pancreatic oncogenesis.
Figure 4: RIP3 deletion in the epithelial or extra-epithelial compartment protects against PDA and enhances immunogenicity.

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Acknowledgements

This work was supported by grants from the German Research Foundation (L.S.), the National Pancreas Foundation (C.P.Z.), the Pancreatic Cancer Action Network (G.M.), the Lustgarten Foundation (G.M.), and National Institute of Health Awards CA155649 (G.M.), CA168611 (G.M.), and CA193111 (G.M., A.T.-H.). We thank the New York University Langone Medical Center (NYU LMC) Histopathology Core Facility, the NYU LMC Flow Cytometry Core Facility, the NYU LMC Microscopy Core Facility, and the NYU LMC BioRepository Center, each supported in part by the Cancer Center Support Grant P30CA016087 and by grant UL1 TR000038 from the National Center for the Advancement of Translational Science (NCATS).

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Authors

Contributions

L.S. carried out in vivo experiments, flow cytometry, analysis and interpretation, manuscript preparation, and statistical analysis; G.W. carried out in vivo experiments, flow cytometry, analysis and interpretation, manuscript preparation, and statistical analysis; S.T. carried out in vivo experiments and IHC; N.N.G.L. performed western blotting; S.A. carried out IHC; D.A. performed flow cytometry; A.A. performed tissue culture and cell line generation; R.B. provided technical assistance and critical review; D.D. performed flow cytometry and provided critical review; S.H.G. carried out mouse breeding and provided critical review; A.T.-H. provided technical assistance and critical review; M.P. performed western blotting and flow cytometry and provided critical review; A.O. carried out immunoprecipitation; C.P.Z. provided technical advice and performed PCR and flow cytometry; M.P. performed western blotting; M.R. carried out genotyping; D.T. performed animal breeding and in vivo tumour experiments; C.H. carried out histological analysis; M.H. performed FACS and data analysis; V.R.M. performed FACS and data analysis; D.E. created cell lines and performed in vivo experiments; G.M. conceived, designed, supervised, analysed and interpreted the study and provided critical review.

Corresponding author

Correspondence to George Miller.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 RIP3 deletion in PDA induces an aggressive tumour phenotype in vitro but mitigates oncogenesis in vivo.

a, KrasG12D;Rip3+/+ and KrasG12D;Rip3−/− PDEC were cultured at equal densities and tested for proliferation after 24 h using the XTT assay (n = 6 per group). b, Lysate was harvested from KrasG12D;Rip3+/+ and KrasG12D;Rip3−/− PDEC and tested for expression of selected tumour suppressor and oncogenic genes. Representative data and density plots from biological duplicates are shown. Experiments were reproduced three times. c, p48Cre;KrasG12D;Rip3+/+ (n = 11) and p48Cre;KrasG12D;Rip3−/− (n = 9) mice were killed at 3, 6, or 9 months of age. Representative trichrome-stained sections are shown and the fraction of fibrotic pancreatic area was calculated for each cohort. Graphs show mean ± s.e.m. ns, not significant; *P < 0.05, ***P < 0.001, ****P < 0.0001 (unpaired t-test). For gel source data, see Supplementary Fig. 1.

Extended Data Figure 2 RIP3 deletion induces immunogenic reprogramming of the pancreatic TME.

a, p48Cre;KrasG12D;Rip3+/+ and p48Cre;KrasG12D;Rip3−/− mice were killed at 3 months of age. Paraffin-embedded sections were stained using a mAb directed against F4/80. Representative images and quantitative data are shown (n = 5 per group). b–f, The fraction of peri-tumoral CD3+ T cells (b), CD19+ B cells (c), Gr1+CD11b+ MDSC (d), F4/80CD11c+MHC II+ dendritic cells (e), and CD11cGr1CD11b+F4/80+ TAMs (f) were determined by flow cytometry. g, Arg1 expression was determined by IHC. Representative images and quantitative data are shown (n = 5 per group). h, CD206 expression in TAMs was assessed by flow cytometry. i, Correlation between high and low tertiles of RIP1–RIP3 co-expression and CD11b expression was tested in human PDA tissues using the UCSC RNA-seq database. Each point represents data from one patient. Graphs show mean ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001 (unpaired t-test). Flow cytometry experiments were carried out twice.

Extended Data Figure 3 RIP3 deletion in PDA is associated with CD4+ and CD8+ T cell activation but RIP3 deletion does not alter growth of B16 melanomas or subcutaneously implanted pancreatic tumours.

Wild-type and Rip3−/−mice (n = 7 per group) were orthotopically implanted with KPC-derived tumour cells. a–c, Mice were killed three weeks later and intra-tumoral CD4+ and CD8+ T cell expression of IL-10 (a), PD-1 (b), and CD44 (c) was measured by flow cytometry. d, Co-expression of CD25 and FoxP3 on CD4+ T cells was also analysed. *P < 0.05, **P < 0.01 (unpaired t-test). Data were reproduced in two separate experiments. e, Wild-type and Rip3−/−mice (n = 3 per group) were implanted subcutaneously with B16 melanoma cells and tumour size was measured at 4–7-day intervals. P = not significant at all time points. f, Wild-type and Rip3−/−mice (n = 3 per group) were implanted subcutaneously with KPC-derived tumour cells and tumour size was measured at 4–7-day intervals. P values were not significant at all time points (unpaired t-test). Graphs show mean ± s.e.m. Source data

Extended Data Figure 4 CXCL1 blockade protects against pancreatic oncogenesis.

a, b, Pancreases from 6-month-old KC mice were analysed for co-expression of CD45 and CXCR2 (a) and CD68 and CXCR2 (b) by confocal microscopy. c, Wild-type mice were challenged with an orthotopic injection of KPC-derived tumour cells. Cohorts were treated thrice weekly with anti-CXCL1 monoclonal antibodies or isotype control. Pancreatic tumours were removed three weeks after implantation. Representative photographs and quantitative analyses of tumour volume and weight are shown (n = 7 per group). d, Wild-type (n = 5) and Rip3−/−mice were challenged with an orthotopic injection of KPC-derived tumour cells. Rip3−/−mice were serially treated with anti-CXCL1 monoclonal antibodies (n = 3) or isotype control (n = 5). Pancreatic tumours were harvested three weeks after implantation and tumour weight was recorded. ei, Wild-type mice were challenged with an orthotopic injection of KPC-derived tumour cells and cohorts were serially treated with anti-CXCL1 monoclonal antibodies or isotype control. The fraction of peri-tumoral Gr1+CD11b+ MDSC (e) and Gr1CD11b+F4/80+ TAMs (f), the expression of CD44 (g) and TNFα (h) on CD3+ T cells, and the fraction of peri-tumoral CD3+ T cells (i) were determined by flow cytometry. Graphs show mean ± s.e.m. ns, not significant; *P < 0.05, **P < 0.01, ****P < 0.0001 (unpaired t-test). Flow cytometry data were reproduced three times. Source data

Extended Data Figure 5 High SAP130 levels in PDA.

a, Paraffin-embedded sections of human PDA and surrounding normal pancreas tissue were tested for expression of SAP130 by IHC compared with isotype control. Representative images and summary data from ten patients with PDA are shown. b, SAP130 expression was measured by qPCR in human AsPC-1 cells after treatment with PBS or gemcitabine (n = 3 per group). c, SAP130 expression was assayed by IHC in paraffin-embedded pancreases from 6-month-old p48Cre;KrasG12D;Rip3+/+, p48Cre;KrasG12D;Rip3−/−, and wild-type mice (n = 4 per group) compared with respective isotype controls. Representative images and quantitative data are shown. d, Sap130 expression was tested by qPCR in KPC-derived tumour cells treated with PBS or gemcitabine with or without Nec-1s in triplicate. e, f, SAP130 expression was tested by confocal microscopy in CK19+ epithelial cells (e) and CD45+ inflammatory cells (f) in mouse PDA. g, Co-expression of SAP130, RIP1, and RIP3 was tested by confocal microscopy in human PDA. h, Correlation between high and low tertiles of combined RIP1/RIP3 and SAP130 expression was tested using the UCSC RNA-seq database. Graphs show mean ± s.e.m. *P < 0.05, ****P < 0.0001 (unpaired t-test). i, Patients with PDA with high or low tertile levels of SAP130 expression were compared in a Kaplan–Meier survival analysis using the UCSC RNA-seq database. j, Pancreas lysate from 6-month-old wild-type or KC mice was immunoprecipitated using an anti-SAP130 or control antibody and then tested for expression of SAP130 and Mincle. Input controls were similarly probed. Results were reproduced in two separate experiments. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 6 High Mincle signalling in PDA.

a, Mincle expression was tested in paraffin-embedded sections of PDA and surrounding normal pancreas from ten patients with PDA. Representative stromal and ductal areas of PDA tumours and quantitative data are shown. b, CD45+ and pancreas-infiltrating leukocytes and CD45 tumour or parenchymal cells from human PDA and PBMC were tested for Mincle expression and compared. c, PDA-infiltrating and PBMC-derived CD14+CD15+ and CD14+CD15 cells from patients with PDA were gated and tested for Mincle expression compared with isotype control. Representative histograms and quantitative data are shown. d, CD45+ and CD45 cells from PDA and spleens from 6-month-old KC mice were tested for expression of Mincle. Representative histograms are shown. e, Pancreas-infiltrating leukocyte suspensions from 6-month-old KC mice were tested for Mincle expression by immunofluorescence microscopy and compared with isotype control. f, Granulocytes, dendritic cells, and macrophages from PDA and spleens from 3-month-old KC mice were gated by flow cytometry and tested for expression of Mincle, and compared with isotype control. Representative histograms and quantitative data are shown (n = 3). g, Whole pancreas lysates from wild-type, Rip3−/−, p48Cre;KrasG12D;Rip3+/+, and p48Cre;KrasG12D;Rip3−/− mice were probed for CARD9, p-Syk, Syk, p-PLC-γ, and PLC-γ by western blotting. Density analysis was performed in triplicate. h, Pancreases from wild-type, Mincle−/−, Rip3−/−, p48Cre;KrasG12D, p48Cre;KrasG12D;Mincle−/−, and p48Cre;KrasG12D;Rip3−/− mice (n = 3 per group) were stained using a monoclonal antibody directed against p-Syk. Representative images and quantitative data are shown. Graphs show mean ± s.e.m. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired t-test). i, Pancreases from 3-month-old KC and p48Cre;KrasG12D;Mincle−/− mice were tested for CXCL1 expression by PCR in biological duplicates. Data were reproduced twice. For gel source data, see Supplementary Fig. 1.

Extended Data Figure 7 Mincle ligation accelerates pancreatic oncogenesis.

a, Wild-type or Mincle−/− mice (n = 4 per group) were administered a single dose of vehicle or the Mincle ligand TDB and p-Syk expression was tested in Gr1F4/80+CD11b+ splenic macrophages 4 h later by flow cytometry. Representative data are shown. b, Six-week-old KC mice were treated with TDB or vehicle for 8 weeks before being killed (n = 5 per group). Representative H&E-stained sections are shown and the fraction of ducts exhibiting normal morphology, ADM, graded PanIN lesions, or foci of invasive cancer are shown. ci, Rip3−/− mice were orthotopically implanted with KPC-derived tumour cells and treated thrice weekly with TDB (n = 4) or vehicle (n = 5) before being killed 3 weeks after implantation. c, Representative images of tumours and pancreatic weights and tumour volume are shown. di, The fraction of CD3+ T cells (d), Gr1+CD11b+ MDSC (e), and Gr1CD11b+F4/80+ TAMs (f) was determined by flow cytometry. Expression of MHC II (g), CD204 (h), and PD-L1 (i) in TAMs is shown for each cohort. Data were reproduced in two separate experiments. j, Wild-type (n = 4 per group), Rip3−/− (n = 4 per group), and Mincle−/− (n = 3 per group) mice were challenged orthotopically with KPC-derived tumour cells. On days 7 and 14 after implantation, mice underwent mini-laparotomies, tumour volume was measured in situ, and PBS or recombinant SAP130 was injected into the tumours. On day 20, mice were killed and the final tumour volume was recorded. The fold-increase in tumour volume between days 7 and 20 in SAP130- versus PBS-treated tumours is shown for wild-type, Rip3−/−, and Mincle−/− mice. k, l, Wild-type mice were similarly challenged with orthotopic KPC-derived tumour and then given intra-tumoral injections of PBS (n = 4) or recombinant SAP130 (n = 3) on days 7 and 14. On day 20, tumours were removed and the fraction of CD3+ T cells (k) and Gr1+CD11b+ MDSC (l) among CD45+ tumour-infiltrating leukocytes was determined. Graphs show mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t-test). Source data

Extended Data Figure 8 Mincle deletion protects against pancreatic oncogenesis.

a, p48Cre;KrasG12D;Mincle+/+ (n = 11) and p48Cre;KrasG12D;Mincle−/− (n = 9) mice were killed at 3, 6, or 9 months of age. Representative H&E-stained sections of pancreases are shown. The percentage of pancreas occupied by intact acinar structures, and the fractions of ducts exhibiting normal morphology, ADM, or graded PanIN I–III lesions were calculated. b, Weights of pancreases from 3-month-old p48Cre;KrasG12D;Mincle+/+ (n = 11) and p48Cre;KrasG12D;Mincle−/− (n = 9) mice. c, Kaplan–Meier survival analysis was performed for p48Cre;KrasG12D;Mincle+/+ (n = 29) and p48Cre;KrasG12D;Mincle−/− (n = 28) mice (P = 0.06). The controls were the same as for the experiments shown in Fig. 3. d, KPC-derived tumour cells were orthotopically implanted in the pancreases of wild-type or Mincle−/− mice. Animals were killed 3 weeks after implantation (n = 7 per group). Tumour volume was recorded. Representative images of pancreatic tumours are shown. e, KPC-derived tumour cells were orthotopically implanted in the pancreases of wild-type (n = 19), Mincle−/− (n = 19), and Rip3−/− (n = 18) mice. Kaplan–Meier survival analysis was performed (wild-type vs Mincle−/−: P = 0.03; wild-type vs Rip3−/−: P < 0.0001; Mincle−/− vs Rip3−/−: P = 0.03). f, Wild-type and Mincle−/− mice were orthotopically implanted with KrasG12D;Rip3+/+ PDEC or KrasG12D;Rip3−/− PDEC. Mice were treated with a neutralizing anti-CXCL1 monoclonal antibody or isotype control (mean n = 4 per group). Mice were killed 3 weeks after implantation and tumour volume was recorded. Graphs show mean ± s.e.m. ns, not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001 (unpaired t-test). Source data

Extended Data Figure 9 Mincle deletion in PDA enhances the immunogenicity of the inflammatory TME.

a, p48Cre;KrasG12D;Mincle+/+ and p48Cre;KrasG12D;Mincle−/− mice were killed at 3 months of age. Paraffin-embedded sections of pancreas were stained using monoclonal antibodies directed against F4/80 and CD3 (n = 5 per group). Representative images and quantitative data are shown. bd, The fraction of peri-tumoral CD3+ T cells (b) and expression of IFN-γ (c) and IL-10 (d) on CD4+ and CD8+ T cells were determined by flow cytometry. eg, The fraction of tumour-infiltrating Gr1+CD11b+ MDSC (e), F4/80CD11c+MHC II+ dendritic cells (f), and Gr1CD11b+F4/80+ TAMs (g) was also determined by flow cytometry. h, Arg1 expression was determined by IHC. Representative images and quantitative data are shown. i, CD206 expression in TAMs was determined by flow cytometry. Graphs show mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired t-test). Experiments were performed twice with similar results.

Extended Data Figure 10 RIP3 and Mincle signalling are necessary for macrophage-induced suppression of T cell immunity in PDA.

ac, Wild-type, Rip3−/−, and Mincle−/− mice were challenged with orthotopic implantation of PDA cells. Before tumour implantation, mice were treated daily for 3 days with a neutralizing anti-CD90 monoclonal antibody (a), a neutralizing anti-F4/80 monoclonal antibody (b), or isotype control. Antibodies were administered twice weekly for the duration of the experiment. Mice were killed 21 days after implantation and the pancreatic tumours were weighed. Controls were shared for both experiments and are shown twice (n = 4 for Mincle−/− anti-CD90 and anti-F4/80-treated groups; n = 3 for other groups). (c) CD4+ and CD8+ T cell activation was determined by expression of CD44 in wild-type, Rip3−/−, and Mincle−/− mice treated with anti-F4/80 monoclonal antibody or isotype control. Graphs show mean ± s.e.m. ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001 (unpaired t-test). In vivo cellular depletion experiments were performed on two separate occasions with similar results. d, Schematic depicting immunosuppressive implications of RIP1/RIP3-driven CXCL1 expression and Mincle activation. Source data

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This file shows the original gels for western blots for Figures 1c, 1g, 1i and Extended Data Figures 1, 5, 6. (PDF 927 kb)

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Seifert, L., Werba, G., Tiwari, S. et al. The necrosome promotes pancreatic oncogenesis via CXCL1 and Mincle-induced immune suppression. Nature 532, 245–249 (2016). https://doi.org/10.1038/nature17403

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