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Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver

Nature Cell Biology volume 17, pages 816–826 (2015)Cite this article

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Abstract

Pancreatic ductal adenocarcinomas (PDACs) are highly metastatic with poor prognosis, mainly due to delayed detection. We hypothesized that intercellular communication is critical for metastatic progression. Here, we show that PDAC-derived exosomes induce liver pre-metastatic niche formation in naive mice and consequently increase liver metastatic burden. Uptake of PDAC-derived exosomes by Kupffer cells caused transforming growth factor β secretion and upregulation of fibronectin production by hepatic stellate cells. This fibrotic microenvironment enhanced recruitment of bone marrow-derived macrophages. We found that macrophage migration inhibitory factor (MIF) was highly expressed in PDAC-derived exosomes, and its blockade prevented liver pre-metastatic niche formation and metastasis. Compared with patients whose pancreatic tumours did not progress, MIF was markedly higher in exosomes from stage I PDAC patients who later developed liver metastasis. These findings suggest that exosomal MIF primes the liver for metastasis and may be a prognostic marker for the development of PDAC liver metastasis.

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Figure 1: PDAC-derived exosomes target and activate KCs, induce liver fibrosis pathways, and increase liver metastasis.
Figure 2: Role of PDAC-derived exosome education in ECM component expression and liver pre-metastatic niche formation.
Figure 3: PDAC-derived exosomes induce FN expression and migration of bone marrow-derived cells to the liver.
Figure 4: TGFβ signalling induces FN upregulation and macrophage recruitment to the liver pre-metastatic niche.
Figure 5: FN and macrophages play major roles in PAN02 exosome-mediated liver pre-metastatic niche formation.
Figure 6: MIF-expressing PAN02 exosomes induce liver pre-metastatic niche formation.
Figure 7: Liver pre-metastatic niche formation and increased exosomal MIF levels precede PDAC lesion development in PKCY mice.
Figure 8: Model for the sequential steps in liver pre-metastatic niche formation induced by PDAC-derived exosomes.

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Acknowledgements

We thank D. L. Bajor (Vonderheide laboratory, University of Pennsylvania) for the gift of the R6560B cells. We thank L. Bojmar for carefully reviewing the paper. We thank S. Rudchenko and M. Barbu-Stevanovic at the Hospital for Special Surgery Fannie E. Rippel Foundation Flow Cytometry Core Facility for expert flow cytometry. We are supported by grants from the Children’s Cancer and Blood Foundation (H.P., D.L.), Manning Foundation (D.L.), Hartwell Foundation (D.L.), Champalimaud Foundation (D.L.), Fundacao para a Ciencia e a Tecnologia (D.L.), Nancy C and Daniel P Paduano Foundation (H.P., D.L.), Mary Kay Foundation (D.L.), Pediatric Oncology Experimental Therapeutic Investigator Consortium (D.L.), James Paduano Foundation (D.L., H.P.), Melanoma Research Alliance (H.P.), Sohn Conference Foundation (H.P.), Beth Tortolani Foundation (D.L., J.B.), Malcolm Hewitt Weiner Foundation (D.L.), Jose Carreras Leukemia Foundation (B.K.T.), Theodore Rapp Foundation (D.L.), American Hellenic Educational Progressive Association 5th District Cancer Research Foundation (D.L.), Charles and Marjorie Holloway Foundation (J.B.), Sussman Family Fund (J.B.), Lerner Foundation (J.B.), Breast Cancer Alliance (J.B.), and Manhasset Women’s Coalition Against Breast Cancer (J.B.).

Author information

Authors and Affiliations

  1. Departments of Pediatrics, Children’s Cancer and Blood Foundation Laboratories, and Cell and Developmental Biology, Drukier Institute for Children’s Health, Meyer Cancer Center, Weill Cornell Medical College, New York, New York 10021, USA

    Bruno Costa-Silva, Swarnima Singh, Haiying Zhang, Basant Kumar Thakur, Annette Becker, Ayuko Hoshino, Till-Martin Theilen, Guillermo García-Santos, Caitlin Williams, Yonathan Ararso, Yujie Huang, Gonçalo Rodrigues, Saya H. Ebbesen, Irina Matei, Héctor Peinado & David Lyden

  2. Gastroenterology Division, Department of Medicine, Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia 19104, USA

    Nicole M. Aiello & Ben Z. Stanger

  3. Department of Medicine, Division of Hematology and Medical Oncology, New York Presbyterian Hospital, Weill Cornell Medical College, New York, New York 10021, USA

    Allyson J. Ocean

  4. Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover 30625, Germany

    Basant Kumar Thakur

  5. Proteomics Resource Center, The Rockefeller University, New York, New York 10065, USA

    Milica Tešić Mark & Henrik Molina

  6. Genomics Resources Core Facility, Weill Cornell Medical College, New York, New York 10021, USA

    Jenny Xiang & Tuo Zhang

  7. Graduate Program in Areas of Basic and Applied Biology, Abel Salazar Biomedical Sciences Institute, University of Porto, 4099-003 Porto, Portugal

    Gonçalo Rodrigues

  8. Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 10617, Taiwan

    Tang-Long Shen

  9. Department of Hepato-Pancreato-Biliary Surgery, Oslo University Hospital, Nydalen, Oslo 0424, Norway

    Knut Jørgen Labori

  10. Department of Pathology, Oslo University Hospital, Nydalen, Oslo 0424, Norway

    Inger Marie Bowitz Lothe

  11. Department of Genetics, Institute for Cancer Research, Oslo University Hospital, Nydalen, Oslo 0424, Norway

    Inger Marie Bowitz Lothe & Elin H. Kure

  12. Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    Jonathan Hernandez, Alexandre Doussot & William R. Jarnagin

  13. Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA

    Paul M. Grandgenett & Michael A. Hollingsworth

  14. Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA

    Maneesh Jain, Kavita Mallya & Surinder K. Batra

  15. Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medical College, New York, New York 10021, USA

    Robert E. Schwartz

  16. Department of Molecular Oncology, Microenvironment and Metastasis Laboratory, Spanish National Cancer Research Center (CNIO), Madrid 28029, Spain

    Héctor Peinado

  17. Department of Medicine, Memorial Sloan Kettering Cancer Center and Weill Cornell Medical College, New York, New York 10065, USA

    Jacqueline Bromberg

  18. Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, New York 10065, USA

    David Lyden

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  1. Bruno Costa-Silva
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Contributions

B.C-S. developed the hypothesis, designed the experimental approach, performed experiments, analysed the data, coordinated the project and wrote the manuscript. N.M.A. conducted experiments. S.S., G.R. and T-L.S. performed immunostaining. H.Z. and Y.H. extracted RNA. B.K.T. and A.B. performed western blots. A.H., T-M.T., C.W. and Y.A. maintained mouse colonies. M.T.M. and H.M. performed proteomic analysis. J.X. and T.Z. processed samples and analysed RNA sequencing data. G.G-S. and C.W. processed human samples. P.M.G., M.A.H., K.J.L., I.M.B.L., E.H.K., A.J.O., J.H., A.D., M.J., K.M., S.K.B. and W.R.J. collected patient samples and managed clinical records. S.H.E. contributed to writing the manuscript. R.E.S., I.M., H.P. and B.Z.S. contributed to hypothesis generation, experimental design and data interpretation. J.B. coordinated the project and interpreted data. D.L. conceived the hypothesis, led the project, interpreted data and wrote the manuscript.

Corresponding authors

Correspondence to Ben Z. Stanger, Jacqueline Bromberg or David Lyden.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Morphological and functional characterization of pancreatic ductal adenocarcinoma exosomes.

(a) A representative electron microscope image of exosomes isolated from PAN02 conditioned media. Scale bar, 100 nm. (b) Evaluation of liver metastasis by liver weight (grams) in mice pre-educated with exosomes isolated from a PKCY-mouse model tumor-derived primary cell line (PKCY) or a KPC-mouse model tumor-derived primary cell line (R6560B) before intra-splenic injection with PAN02 cells. Control tumor-bearing mice educated with PBS (TU); n = 4 (TU and PKCYexo + TU) and n = 3 (R6560Bexo + TU) mice from one experiment. ∗∗P < 0.01,∗P < 0.05, N.S. stands for not significant by ANOVA. Scale bar, 1 cm. (c) Flow cytometric quantification of the frequency of liver and lung cells incorporating PKH67-labeled exosomes. Exosomes were isolated from normal pancreas (NP), PAN02, PKCY, and R6560B cells; n = 4 (NP) and n = 5 (R6560B) mice from one experiment, and n = 7 (PAN02) and n = 8 (PKCY) mice pooled from two experiments. Statistical source data can be found in Supplementary Table 4. ∗∗P < 0.01 by ANOVA. (d) Percentage of PKH67-labeled exosome+ liver cells expressing CD11b and F480 markers (KCs). Exosomes were isolated from human (BxPC-3 and HPAF-II) and murine (R6560B) PDAC cell lines; n = 4 (BxPC-3 and HPAF-II) and n = 5 (R6560B) mice from one experiment. (e) Fluorescence microscopy analysis of PKH67-labeled exosomes (green) and αSMA+ cells, S100A4+ fibroblasts, CD31+ endothelial cells, or EpCAM+ epithelial cells (red). Arrows point to green fluorescent signal indicating exosome uptake. Scale bars, 50 μm. (f) Analysis of canonical pathways of genes upregulated by Kupffer cells following in vitro education with BxPC-3 exosomes or PBS treatment. The list is comprised of genes related to liver fibrosis. Data was obtained from one experiment performed in triplicate. Statistical source data can be found in Supplementary Table 2. All data are represented as mean ± s.e.m.

Supplementary Figure 2 Pancreatic ductal adenocarcinoma-derived exosomes induce αSMA and FN expression and increase F4/80+ cell frequency in the liver and migration of BM-derived macrophages to the liver.

(a) Analysis by fluorescence microscopy showing lack of co-localization of FN expression with CD31+ endothelial cells in the livers of mice educated with PAN02 exosomes. Scale bar, 50 μm. (b) Immunofluorescence quantification of αSMA and FN expression in arbitrary units (a.u.) in the livers of mice educated with PBS (CTL), normal pancreas (NP), PKCY, or R6560B exosomes; n = 4 (CTLαSMA; PKCY αSMA; R6560B αSMA and FN) and n = 5 (CTL FN; NP αSMA and FN; PKCY FN) mice from one experiment. Scale bars, 150 μm.∗∗∗P < 0.001; N.S. stands for not significant by ANOVA. (c) Immunofluorescence quantification of the frequency of F4/80+ cell recruited to the livers of mice educated with PBS (CTL), normal pancreas (NP), PKYC, or R6560B exosomes; n = 4 (NP and R6560B) and n = 5 (CTL and PKCY) mice from one experiment. Scale bars, 150 μm.∗∗P < 0.01; N.S. stands for not significant by ANOVA. All data are represented as mean ± s.e.m.

Supplementary Figure 3 Low magnification overview of αSMA, FN and F4/80 expression in the liver of mice educated with PBS (CTL), PAN02 exosomes (Exo), Exo in combination with the TGFβR inhibitor A83-01 (Exo + A83-01), Exo in combination with tamoxifen in the FN conditional knockout mouse model (Exo + TMX (CRE+), Exo in combination with Diphtheria toxin (DT) in the CD11b-DTR (Exo + DT(DTR+), and Exo derived from MIF knockdown pancreatic cancer cells (shMIF Exo).

Scale bars, 500 μm.

Supplementary Figure 4 Metastatic pancreatic cells localize adjacent to liver macrophages.

Fluorescence microscopy analysis of F4/80+ staining and mCherry+ PAN02 cell localization in early metastatic lesions of mice educated with PAN02 exosomes reveals close association between metastatic cells and liver macrophages. Scale bar, 150 μm.

Supplementary Figure 5 Evaluation of MIF knockdown exosome uptake by KCs in vivo and the functional consequences on hStC activation and early metastasis.

(a) Western blot analysis of MIF levels in cells and in exosomes (Exo) of non-infected parental PAN02 cells (WT), PAN02 cells infected with control shRNA (shCTL), MIF knockdown (shMIF), a second construct of MIF knockdown (shMIF(2)), and R6560B cells. Unprocessed scans of blots are presented in in Supplementary Fig. 6. (b) Left panel: flow cytometric quantification of PKH67-labeled exosome incorporation by CD11b+F4/80+ liver cells (KCs), represented as a percentage of all PKH67+ cells; n = 4 (WT and shMIF) and n = 5 (shCTL and shMIF(2)) mice from one experiment. Right panel: exosome protein quantification, represented as microgram (μg) per 106 exosomes; n = 3 (shCTL, shMIF, and shMIF(2)) and n = 4 (WT) independent exosome isolations from in vitro cell culture. N.S. stands for not significant by ANOVA. (c) Size distribution analysis of PAN02 exosomes by NanoSight. (d) Immunofluorescence quantification of mCherry+ PAN02 cells 24 hours after their intra-splenic injection into mice previously educated for three weeks with parental PAN02 exosomes (Exo) or PAN02shMIF exosomes (shMIF exo). Arrows In representative images indicate PAN02 cells. Control animals were educated with PBS (TU); n = 4 mice per cohort from one experiment. ∗∗P < 0.01 by ANOVA. Scale bars, 100 μm. All data are represented as mean ± s.e.m.

Supplementary Figure 6 Unprocessed scans of immunoblots.

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Costa-Silva, B., Aiello, N., Ocean, A. et al. Pancreatic cancer exosomes initiate pre-metastatic niche formation in the liver. Nat Cell Biol 17, 816–826 (2015). https://doi.org/10.1038/ncb3169

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