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Tumour extracellular vesicles and particles induce liver metabolic dysfunction

Nature volume 618, pages 374–382 (2023)Cite this article

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Abstract

Cancer alters the function of multiple organs beyond those targeted by metastasis1,2. Here we show that inflammation, fatty liver and dysregulated metabolism are hallmarks of systemically affected livers in mouse models and in patients with extrahepatic metastasis. We identified tumour-derived extracellular vesicles and particles (EVPs) as crucial mediators of cancer-induced hepatic reprogramming, which could be reversed by reducing tumour EVP secretion via depletion of Rab27a. All EVP subpopulations, exosomes and principally exomeres, could dysregulate hepatic function. The fatty acid cargo of tumour EVPs—particularly palmitic acid—induced secretion of tumour necrosis factor (TNF) by Kupffer cells, generating a pro-inflammatory microenvironment, suppressing fatty acid metabolism and oxidative phosphorylation, and promoting fatty liver formation. Notably, Kupffer cell ablation or TNF blockade markedly decreased tumour-induced fatty liver generation. Tumour implantation or pre-treatment with tumour EVPs diminished cytochrome P450 gene expression and attenuated drug metabolism in a TNF-dependent manner. We also observed fatty liver and decreased cytochrome P450 expression at diagnosis in tumour-free livers of patients with pancreatic cancer who later developed extrahepatic metastasis, highlighting the clinical relevance of our findings. Notably, tumour EVP education enhanced side effects of chemotherapy, including bone marrow suppression and cardiotoxicity, suggesting that metabolic reprogramming of the liver by tumour-derived EVPs may limit chemotherapy tolerance in patients with cancer. Our results reveal how tumour-derived EVPs dysregulate hepatic function and their targetable potential, alongside TNF inhibition, for preventing fatty liver formation and enhancing the efficacy of chemotherapy.

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Fig. 1: Remote primary tumours induce metabolic dysfunction in the liver.
Fig. 2: Tumour-derived EVPs induce liver metabolic dysfunction.
Fig. 3: Uptake of tumour-derived EVPs by Kupffer cells induces fatty liver formation.
Fig. 4: Palmitic acid in tumour EVPs induces TNF secretion from Kupffer cells and promotes fatty liver generation.
Fig. 5: Tumour EVPs suppress liver drug metabolism and enhance chemotoxicity.

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Data availability

RNA-seq raw data and associated processed data files that support the findings of this study have been deposited in the Gene Expression Omnibus under accession codes GSE199863 and GSE220446. Metabolomics data derived from livers of PBS-injected control and B16F10 tumour-bearing mice (Supplementary Table 1) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233187). Metabolomics data derived from livers of PBS-injected control and K7M2 tumour-bearing mice (Supplementary Table 2) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233253). Lipidomics data derived from livers of PBS-injected control and B16F10 tumour-bearing mice (Supplementary Table 3) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233265). Lipidomics data derived from livers of PBS-injected control and K7M2 tumour-bearing mice (Supplementary Table 4) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233274). Metabolomics data derived from livers of PBS- and B16F10-TE-EVP-educated mice (Supplementary Table 8) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233289). Metabolomics data derived from livers of PBS- and K7M2-TE-EVP-educated mice (Supplementary Table 9) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233298). Lipidomics data derived from livers of PBS- and B16F10-TE-EVP-educated mice (Supplementary Table 10) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233307). Lipidomics data derived from livers of PBS- and K7M2-TE-EVP-educated mice (Supplementary Table 11) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233316). Metabolomics data derived from skin-TE-EVPs and B16F10-TE-EVPs (Supplementary Table 14) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233352). Metabolomics data derived from livers of PBS- and B16F10-CL-EVP-educated mice (Supplementary Table 29) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233379). Lipidomics data derived from livers of PBS- and B16F10-CL-EVP-educated mice (Supplementary Table 30) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233403). Metabolomics data derived from livers of PBS- and K7M2-CL-EVP-educated mice (Supplementary Table 31) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233424). Lipidomics data derived from livers of PBS- and K7M2-CL-EVP-educated mice (Supplementary Table 32) have been deposited at Figshare (https://doi.org/10.6084/m9.figshare.22233445). Source data are provided with this paper.

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Acknowledgements

The authors acknowledge the Genomics Resource Core Facility (Weill Cornell Medicine), Electron Microscopy and Histology Core Facility (Weill Cornell Medicine), Molecular Cytology Core Facility (Memorial Sloan Kettering Cancer Center, MSKCC) and Laboratory of Comparative Pathology (MSKCC) for their high-quality service. The authors also acknowledge technical support from Wyatt Technology. The authors thank members of the Lyden laboratory for insightful discussions. The authors gratefully acknowledge support from the National Cancer Institute (CA232093, CA163117 and CA207983 to D.L. and CA218513 to D.L. and H.Z.), the Thompson Family Foundation (to D.L. and D.K.), the Tortolani Foundation (to D.L. and J.B.), the Pediatric Oncology Experimental Therapeutics Investigator’s Consortium, the Malcolm Hewitt Weiner Foundation, the Manning Foundation, the Sohn Foundation, the AHEPA Vth District Cancer Research Foundation, the Children’s Cancer and Blood Foundation, the Hartwell Foundation (to D.L.), the National Institutes of Health (R01CA234614, 2R01AI107301, R01CA234614 and R01DK121072 to R.E.S.), the United States Department of Defense (W81XWH-21-1-0978 to R.E.S.), the Paul G. Allen Family Foundation UWSC13448 (to R.E.S.), the National Natural Science Foundation of China (81902730 to J.L.), Guangdong Foundation of Medical Science and Technology (A2019213 to J.L.), China Scholarship Council (CSC No. 202008440567 to J.L.), the Swedish Cancer Society Pancreatic Cancer Fellowship (to L.B.), the Lions International Postdoctoral fellowship (to L.B.), the Sweden-America stipend (to L.B.), and the fellowship from Alan and Sandra Gerry Metastasis and Tumor Ecosystems Center of Memorial Sloan Kettering Cancer Center (to C.P.Z.). The part of the research involved in developing osteosarcoma PDXs and tumour imaging was funded in part through the NIH/NCI Cancer Center Support Grant P30 CA008748 to MSKCC, the National Institutes of Health (R01CA237213 to C.E.B and V.P., and R01CA254036 to S.K.B.). R.E.S. is an Irma Hirschl Trust Research Award Scholar. Schematic models were generated in part using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license, with further modifications.

Author information

Author notes

  1. These authors contributed equally: Gang Wang, Jianlong Li

Authors and Affiliations

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

    Gang Wang, Jianlong Li, Linda Bojmar, Haiyan Chen, Gabriel C. Tobias, Mengying Hu, Serena Lucotti, Fengbo Zhao, Michele Cioffi, Han Sang Kim, Huajuan Wang, Pernille Lauritzen, Nancy Boudreau, Irina R. Matei, Haiying Zhang & David Lyden

  2. Department of Orthopedic Surgery, Nanfang Hospital, Southern Medical University, Guangzhou, China

    Jianlong Li & Zhanjun Shi

  3. Department of Biomedical and Clinical Sciences, Linköping University, Linköping, Sweden

    Linda Bojmar

  4. Department of Radiation Oncology, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Haiyan Chen

  5. Key Laboratory of Cancer Prevention and Intervention, China National Ministry of Education, Key Laboratory of Molecular Biology in Medical Sciences, Hangzhou, China

    Haiyan Chen

  6. Duke Proteomics and Metabolomics Shared Resource, Duke University School of Medicine, Durham, NC, USA

    Zhong Li

  7. Cardiovascular Research Institute and Department of Medicine, Weill Cornell Medicine, New York, NY, USA

    Edwin A. Homan, James C. Lo & Geoffrey S. Pitt

  8. Basic Medical Research Center, Medical School of Nantong University, Nantong, China

    Fengbo Zhao

  9. Departments of Molecular Genetics, Cancer Biology and Genetics, The Ohio State University, Columbus, OH, USA

    Valentina Posada & Christin E. Burd

  10. Samuel J. Wood Library, Weill Cornell Medicine, New York, NY, USA

    Peter R. Oxley

  11. Yonsei Cancer Center, Division of Medical Oncology, Department of Internal Medicine, Brain Korea 21 FOUR Project for Medical Science, Severance Biomedical Science Institute, Yonsei University College of Medicine, Seoul, Korea

    Han Sang Kim

  12. Department of Dermatology, Weill Cornell Medical College of Cornell University, New York, NY, USA

    Jonathan H. Zippin

  13. Hepatopancreatobiliary Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Jonathan Hernandez, Constantinos P. Zambirinis & William R. Jarnagin

  14. Thoracic and Gastrointestinal Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Jonathan Hernandez

  15. Division of Surgical Oncology, Rutgers Cancer Institute of New Jersey, New Brunswick, NJ, USA

    Constantinos P. Zambirinis

  16. Fred and Pamela Buffett Cancer Center, University of Nebraska Medical Center, Omaha, NE, USA

    Michael A. Hollingsworth, Paul M. Grandgenett, Maneesh Jain & Surinder K. Batra

  17. Department of Pathology and Microbiology, College of Medicine, University of Nebraska Medical Center, Omaha, NE, USA

    Dominick J. DiMaio

  18. Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE, USA

    Jean L. Grem & Kelsey A. Klute

  19. Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Tanya M. Trippett

  20. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

    Mikala Egeblad

  21. Division of Hematology and Medical Oncology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA

    Doru Paul

  22. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Jacqueline Bromberg

  23. Gastrointestinal Oncology Service, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    David Kelsen

  24. Orthopedic Service, Department of Surgery, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Vinagolu K. Rajasekhar & John H. Healey

  25. Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA

    Robert E. Schwartz

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  1. Gang Wang
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Contributions

D.L., H.Z., G.W. and R.E.S conceived the hypothesis. G.W. coordinated the project, designed the experimental approach, performed most of the experiments, analysed and interpreted the data, and wrote the manuscript. J.L. performed experiments and analysed the data. L.B. coordinated the patient sample preparation and data collection. H.C., G.C.T., M.H., S.L., F.Z., M.C., H.S.K., H.W. and P.L. performed experiments. E.A.H. and J.C.L. performed echocardiography on mice. V.P. and C.E.B. provided livers of genetically engineered mouse model of melanoma. P.R.O. provided help with RNA-seq data analysis. Z.L. conducted metabolomic, lipidomic, free fatty acid, drug metabolism and TPA mass spectrometry. V.K.R. and J.H.H. generated osteosarcoma PDX models. J.H., C.P.Z., M.A.H., P.M.G., D.J.D., J.L.G., K.A.K., M.J., S.K.B., D.K. and W.R.J. provided human samples. J.H.Z., G.S.P., T.M.T., M.E., N.B., Z.S., D.P. and J.B. read the manuscript and provided feedback. I.R.M. edited the manuscript, coordinated sourcing of samples and provided feedback. D.L. and H.Z. interpreted the data and wrote the manuscript. R.E.S. interpreted the data. D.L. led the project.

Corresponding authors

Correspondence to Robert E. Schwartz, Haiying Zhang or David Lyden.

Ethics declarations

Competing interests

D.L. is on the scientific advisory board of Aufbau Holdings. R.E.S. is on the scientific advisory board of Miromatrix and is a speaker and consultant for Alnylam. M.E. is a member of the research advisory board for brensocatib for Insmed, a member of the scientific advisory board for Vividion Therapeutics and a consultant for Protalix BioTherapeutics, and holds shares in Agios. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks Ekihiro Seki and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer review reports are available.

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Extended data figures and tables

Extended Data Fig. 1 Primary tumours dysregulate the metabolism of metastasis-free livers.

a, Schematic representation of mouse tumour models utilized in this study. B16F10, K7M2, 67NR, B16F1 and 4T1 cells, were orthotopically injected into syngeneic mice. Age and sex-matching mice injected with PBS were used as controls of these experimental mouse models. C57BL/6 TN mice carrying a Cre-inducible NrasQ61R oncogene were employed for generating spontaneous melanoma. Non-tumour bearing control mice carried the NrasQ61R, p16fl/fl and Tyr-CRE-ER(T2) alleles but were not treated to induce CRE activity or tumour formation. Functionally null Mc1r increased melanoma susceptibility. b, Representative H&E staining images of the livers from B16F10-Tb and B16F1-Tb mice (top, left), K7M2-Tb mice (top, right), 67NR-Tb and 4T1-Tb mice (bottom), and their respective controls. This experiment was repeated three times independently with similar results. c, Representative H&E staining images of lung metastases in the PBS control mice and K7M2-Tb mice. This experiment was repeated three times independently with similar results. d, RT-qPCR analysis of Trpm1 and mCherry expression in livers and lungs of mice implanted with B16F10 and mCherry-expressing K7M2 cells, respectively, compared to their controls. n = 5 per group for B16F10-Tb model, n = 4 control and n = 6 K7M2-mCherry-Tb mice. NS, not significant; ND, not detected. e, Principal component analysis (PCA) of gene expression in the livers from mice implanted with B16F10, or K7M2, or 67NR, or B16F1, or 4T1 tumour cells, compared to their respective PBS-injected controls. Results showed that the gene expression profiles of livers from tumour-bearing mice independently segregated from their respective controls. n = 3 mice per group for B16F10-Tb, 67NR-Tb and 4T1-Tb models; n = 5 mice per group for K7M2-Tb and B16F1-Tb models. f-h, GSEA of the gene expression profiles, which were ranked based on the sign of log2FC∗(-log10P value), in the livers from 67NR-Tb mice (f), B16F1-Tb mice (g), or 4T1-Tb mice (h), compared to their respective PBS-injected controls, using hallmark gene sets, and the significantly changed signaling pathways with FDR<0.2 are shown. n = 3 mice per group for 67NR-Tb and 4T1-Tb models; n = 5 mice per group for B16F1-Tb model. Gene lists for signaling pathways are shown in Supplementary Tables 19–21. Scale bars for (b,c), 200 µm. P values were determined by two-tailed, unpaired Student’s t-test (d).

Source Data

Extended Data Fig. 2 Primary tumours induce metabolic dysfunction of the metastasis-free livers.

a,b, PLS-DA plots of metabolites detected in the livers from B16F10-Tb mice (a) and K7M2-Tb mice (b), compared to their PBS-injected controls. Metabolomic mass spectrometry data are shown in Supplementary Tables 1 and 2. n = 5 B16F10-Tb mice and controls; n = 8 K7M2-Tb mice and controls. c,d, Heatmaps showing the significantly changed metabolites, classified into different groups, in the livers from B16F10-Tb mice (c) and K7M2-Tb mice (d), compared to their PBS-injected controls. n = 5 B16F10-Tb mice and controls; n = 8 K7M2-Tb mice and controls. e,f, Metabolite set enrichment analysis of metabolites (shown in c,d) significantly changed in the livers of B16F10-Tb (e) and K7M2-Tb (f) mice, compared to their respective PBS-injected controls. n = 5 B16F10-Tb mice and controls; n = 8 K7M2-Tb mice and controls. P values were determined by the hypergeometric test (e,f).

Extended Data Fig. 3 Primary tumours induce lipid accumulation in metastasis-free livers.

a,b, PLS-DA plots of the lipid species in the livers from B16F10-Tb mice (a) and K7M2-Tb mice (b), compared to their PBS-injected controls. Lipidomic mass spectrometry data are shown in Supplementary Tables 3 and 4. n = 5 mice per group. c,d, Volcano plots showing the significantly (P < 0.05) enriched triglyceride species (red) and cholesteryl ester species (blue) in the livers from B16F10-Tb mice (c) and K7M2-Tb mice (d), compared to their PBS-injected controls. n = 5 mice per group. e, Statistical analysis of BODIPY staining of livers from mouse melanoma B16F1-Tb mice, mouse breast cancer 67NR-Tb and 4T1-Tb mice, genetic melanoma-bearing mice, and their respective controls. n = 5 mice per group for B16F1, 67NR and 4T1 models; n = 14 control and n = 10 tumour-bearing mice for the genetic melanoma model. f, Representative H&E staining images of livers from KPC mice. Liver metastasis was observed in 20-week, but not 14-week tumour bearing mice. g, Statistical analysis of BODIPY staining of the livers from 14-week PDAC tumour-bearing mice, compared to non-PDAC mouse controls. n = 3 mice per group. h, Schematic representation of mouse tumour models utilized in this study. SK-MEL-192 cells were subcutaneously injected into nude (outbred) mice. Patient-derived xenograft (PDX) models of osteosarcoma were generated by transplanting surgically excised primary tumour specimens from clinical patients into NOD/SCID/IL2Rγnull (NSG) mice. Age and sex-matching mice injected with PBS were used as controls of these experimental mouse models. i, Representative H&E staining images of the livers from SK-MEL-192-Tb and control mice. This experiment was repeated three times independently with similar results. j, Representative MRI images showing lung metastasis, but not liver metastasis detected in osteosarcoma-PDX (OS-PDX) mouse models. This experiment was repeated seven times independently with similar results. k, Statistical analysis of BODIPY staining of livers from human melanoma SK-MEL-192-Tb mice, osteosarcoma-PDX tumour-bearing mice, and their respective controls. n = 6 mice per group for SK-MEL-192 model; n = 5 control and n = 7 tumour-bearing mice for osteosarcoma-PDX model. l, Measurement of body mass index (BMI) of PDAC patients and control subjects with benign lesions. n = 14 control subjects and n = 16 PDAC patients. Scale bars, 500 µm for (f) and 200 µm for (i). P values were determined by the two-tailed, unpaired Student’s t-test (c-e,g,k,l). Data are mean ± s.e.m. NS, not significant.

Source Data

Extended Data Fig. 4 Tumour explant (TE)-derived EVPs dysregulate liver metabolism.

a, Representative nanoparticle tracking analysis (NTA) and TEM images (insertions) of B16F10-TE-EVPs (left) and K7M2-TE-EVPs (right). Shown are graphs and images representative of three independent experiments. b, Quantification of the particle numbers in 1 ml of plasma from B16F10-Tb (left) and K7M2-Tb (right) mice, compared to their PBS-injected controls, as well as in 10 µg of EVPs derived from B16F10 (left) and K7M2 (right) cells. n = 15 control and B16F10-Tb mice; n = 8 control and K7M2-Tb mice; n = 5 and n = 3 independent experiments for EVPs derived from B16F10 and K7M2 cells, respectively. c, Schematic illustration of the procedure of syngeneic mouse education with B16F10-TE-EVPs or K7M2-TE-EVPs, and PBS control. d, PCA of gene expression in the livers of mice educated with B16F10-TE-EVPs (left) and K7M2-TE-EVPs (right), compared to PBS-educated controls. n = 5 mice per group for B16F10-TE-EVP education model; n = 3 mice per group for K7M2-TE-EVP education model. e,f, PLS-DA plots of metabolites detected in the livers from B16F10-TE-EVP- (e) and K7M2-TE-EVP- (f) educated mice, compared to PBS-educated controls. Metabolomics data are shown in Supplementary Tables 8 and 9. n = 5 B16F10-TE-EVP-educated mice and controls; n = 6 K7M2-TE-EVP-educated mice and n = 5 controls. g,h, Metabolite set enrichment analysis of the metabolites significantly changed in the livers from B16F10-TE-EVP- (g) and K7M2-TE-EVP- (h) educated mice, compared to PBS-educated controls (see lists of metabolites in Fig. 2e, f). n = 5 B16F10-TE-EVP-educated mice and controls; n = 6 K7M2-TE-EVP-educated mice and n = 5 controls. i,j, PLS-DA plots of the lipid species in the livers from B16F10-TE-EVP- (i) and K7M2-TE-EVP- (j) educated mice, compared to PBS-educated controls. Lipidomics data are shown in Supplementary Tables 10 and 11. n = 7 B16F10-TE-EVP-educated mice and n = 5 controls; n = 5 K7M2-TE-EVP-educated mice and controls. k,l, Volcano plots showing the significantly (P < 0.05) enriched triglyceride species (red) and cholesteryl ester species (blue) in the livers from B16F10-TE-EVP- (k) and K7M2-TE-EVP- (l) educated mice, compared to PBS-educated controls. n = 7 B16F10-TE-EVP-educated mice and n = 5 controls; n = 5 K7M2-TE-EVP-educated mice and controls. m,n, Representative images of BODIPY staining of the livers from B16F10-TE-EVP- (m) and K7M2-TE-EVP- (n) educated mice, and PBS-educated controls. Associated quantification of BODIPY staining was shown in Fig. 2i, j. Scale bars, 200 nm for (a) and 20 µm for (m,n). P values were determined by the two-tailed, unpaired Student’s t-test (b,k,l), or hypergeometric test (g,h). Data are mean ± s.e.m.

Source Data

Extended Data Fig. 5 Tumour cell line (CL)-derived EVPs dysregulate liver metabolism.

a, Representative TEM images of B16F10-CL-EVPs (left) and K7M2-CL-EVPs (right). Scale bar, 200 nm. This experiment was repeated three times independently with similar results. b, NTA of B16F10-CL-EVPs and K7M2-CL-EVPs. Shown are graphs representative of three independent experiments. c,d, Representative images (left) and associated statistical analysis of relative signal intensity (right) of the livers from mice 24 h post intravenous injection of 10 µg of CellVue NIR815-labeled B16F10-CL-EVPs (c) and K7M2-CL-EVPs (d), and their mock PBS-injected controls. n = 6 mice injected with B16F10-CL-EVPs or PBS mock reaction; n = 7 mice injected with K7M2-CL-EVPs and n = 6 mice injected with PBS mock control. e, Schematic illustration of syngeneic mouse education with B16F10-CL-EVPs or K7M2-CL-EVPs, and PBS control. f, PCA of gene expression in the livers from mice educated with B16F10-CL-EVPs (left) and K7M2-CL-EVPs (right), compared to PBS-educated controls. n = 5 mice per group for B16F10-CL-EVP education model; n = 3 mice per group for K7M2-CL-EVP education model. g,h, GSEA of gene expression profiles, ranked based on the sign of log2FC∗(-log10P value), in the livers from B16F10-CL-EVP- (g) and K7M2-CL-EVP- (h) educated mice, compared to PBS-educated controls, using hallmark gene sets. Significantly changed signaling pathways with nominal P < 0.05 are shown. n = 5 B16F10-CL-EVP-educated mice and controls; n = 3 K7M2-CL-EVP-educated mice and controls. Gene lists for signaling pathways are shown in Supplementary Tables 22 and 23. i, Heatmap showing the metabolites significantly changed in the livers of B16F10-CL-EVP-educated mice, compared to PBS-educated controls. n = 5 control and n = 7 B16F10-CL-EVP-educated mice. Metabolomic mass spectrometry data are shown in Supplementary Table 29. j,k, Volcano plots showing the significantly (P < 0.05) enriched lipid classes (red) (j) and the significantly (P < 0.05) enriched triglyceride species (red) (k) in the livers of B16F10-CL-EVP-educated mice, compared to PBS-educated controls. n = 5 control and n = 6 B16F10-CL-EVPs educated mice. Lipidomic mass spectrometry data are shown in Supplementary Table 30. l, Heatmap showing the metabolites significantly changed in the livers of K7M2-CL-EVP-educated mice, compared to PBS-educated controls. n = 5 K7M2-CL-EVP-educated mice and controls. Metabolomics MS data are shown in Supplementary Table 31. m,n, Volcano plots showing the significantly (P < 0.05) changed lipid class (blue) (m), and the significantly (P < 0.05) enriched triglyceride species (red) (n) in the livers from K7M2-CL-EVP-educated mice, compared to PBS-educated controls. n = 5 control and n = 4 K7M2-CL-EVP-educated mice. Lipidomic mass spectrometry data are shown in Supplementary Table 32. o,p, Representative immunofluorescence (IF) images (left) and associated statistical analysis (right) of BODIPY staining of the livers from B16F10-CL-EVP- (o) and K7M2-CL-EVP- (p) educated mice, and PBS-educated controls. n = 4 mice per group for B16F10-CL-EVP-education model; n = 5 mice per group for K7M2-CL-EVP-education model. Scale bars, 20 µm. q, Quantification of BODIPY staining of the livers from B16F10- and K7M2-conditioned media -educated mice, compared to blank DMEM media-educated mice. n = 5 mice per group for B16F10-conditioned media model and n = 4 mice per group for K7M2-conditioned media model. r, Quantification of BODIPY staining of the livers from PBS-educated and B16F10-CL-EVP-educated mice, in the absence or presence of B16F10-conditioned media co-education. n = 4 mice educated with PBS, n = 5 mice educated with B16F10-CL-EVPs alone, and n = 4 mice educated with B16F10-CL-EVPs together with B16F10-conditioned media. P values were determined by the two-tailed, unpaired Student’s t-test for (c,d,j,k,m-p) and one-way ANOVA with post hoc Tukey’s test for (r). Data are mean ± s.e.m. NS, not significant. CM, conditioned media.

Source Data

Extended Data Fig. 6 Ablation of Rab27a expression suppresses EVP secretion from tumour cells.

a, Western blot analysis of Rab27a expression in B16F10 cells infected with vector control or Rab27a-CRISPR KO virus. Actin was used as a loading control. Shown is representative data from three independent experiments. b, Western blot analysis of Rab27a expression in K7M2 cells infected with vector control (shCon) or Rab27a-shRNA KD virus. Actin was used as a loading control. Shown is representative data from three independent experiments. c, NTA of the numbers of EVPs secreted from B16F10-control (Vector) or B16F10-Rab27a-KO cells. n = 5 independent experiments per group. d, NTA of the numbers of EVPs secreted from K7M2-control (shCon) or K7M2-Rab27a-KD cells. n = 3 independent experiments per group. e,f, Representative NTA profiles (e) and associated analysis of the diameter mode (f) of EVPs isolated from B16F10-control (Vector) or B16F10-Rab27a-KO cells. n = 6 independent experiments per group. g,h, Representative NTA profiles (g) and associated analysis of the diameter mode (h) of EVPs isolated from K7M2-control (shCon) or K7M2-Rab27a-KD cells. n = 3 independent experiments per group. i, Proliferation of B16F10-control (Vector) and B16F10-Rab27a-KO cells. n = 3 independent experiments. j, Proliferation of K7M2-control (shCon) and K7M2-Rab27a-KD cells. n = 4 independent experiments. k, Statistical analysis of the weights of tumours from mice inoculated with B16F10-control (Vector) or B16F10-Rab27a-KO cells. n = 15 mice per group. l, Statistical analysis of the weights of tumours from mice inoculated with K7M2-control (shCon) or K7M2-Rab27a-KD cells. n = 8 mice per group. m,n, Statistical analysis showing the similar tumour burden from a subset of mice in k and l inoculated with B16F10-Rab27a-KO cells (m), or K7M2-Rab27a-KD cells (n) subjected to BODIPY staining of their livers as shown in (o,p). n = 5 mice per group. o,p, Representative images (left) and associated statistical analysis (right) of BODIPY staining of the livers from B16F10-Rab27a-KO (o) and K7M2-Rab27a-KD (p) tumour bearing mice, and their respective controls. n = 5 mice per group. Scale bars, 20 µm. q, PCA of the gene expression profiling in the livers from mice educated with control PBS, B16F10 exomeres, or B16F10 Exo-S, or B16F10 Exo-L for 4 weeks. n = 4 mice per group. r-t, GSEA of gene expression profiles, which were ranked based on the sign of log2FC∗(-log10P value), in the livers from B16F10 exomere- (r), Exo-S- (s), and Exo-L- (t) educated mice, compared to PBS-educated controls. (n = 4 each). Signaling pathways significantly changed with nominal P < 0.05 are shown. Gene lists for signaling pathways are shown in Supplementary Tables 24–26. u, EVPs isolated from equal numbers of B16F10-Vector control and B16F10-Rab27a-KO cells were resolved via AF4. Shown are real time measurement of UV on a relative scale (right axis), indicating the abundance of fractionated particles. Shaded areas mark the elution time periods for exomeres (red), Exo-S (blue) and Exo-L (green), respectively. As reflected by the UV signal, the production of exomeres, Exo-S (to a less extent) and Exo-L (to the least extent) were reduced in the B16F10-Rab27a-KO cells compared to the B16F10-Vector control cells. P values were determined by the two-tailed, unpaired Student’s t-test (c,d,f,h,k-p), or two-way ANOVA followed with Bonferroni’s multiple comparisons test (i,j). Data are mean ± s.e.m. NS, not significant. KO, knockout. KD, knockdown. For western blotting source data of (a,b), see Supplementary Fig. 1.

Source Data

Extended Data Fig. 7 Tumour-derived EVPs are uptaken by Kupffer cells in the liver.

a, Flow cytometry analysis of the percentage of EVP positive cells in the livers from mice 24 h post intravenous injection of 10 µg of CellVue Burgundy-labeled B16F10-CL-EVPs (left) and K7M2-CL-EVPs (right), and PBS controls. n = 7 mice per group. b, Flow cytometry analysis of the percentage of different cell types, including Cd45+ immune cells, Cd31+ vascular endothelial cells, desmin+ stellate cells, albumin+ hepatocytes, Lyve1+ lymphatic and sinusoidal endothelial cells, among the B16F10-CL-EVP and K7M2-CL-EVP positive cells shown in a. n = 4 each. c,d, Representative flow cytometry gating strategy evaluating the percentage of different cell types, including Cd31+ vascular endothelial cells, albumin+ hepatocytes, Lyve1+ lymphatic and sinusoidal endothelial cells, desmin+ stellate cells, Cd45+ immune cells and Cd11b+F4/80+ Kupffer cells in Cd45+ immune cells, among the B16F10-CL-EVP- (c) and K7M2-CL-EVP- (d) positive cells shown in a,b and Fig. 3a. e, Representative images of the immunofluorescence (IF) co-staining of EVPs (indicated by white arrows) with Cd31+ vascular endothelial cells, albumin+ hepatocytes, Lyve1+ lymphatic and sinusoidal endothelial cells, and desmin+ stellate cells 24 h post intravenous injection of B16F10-CL-EVPs (top) or K7M2-CL-EVPs (bottom). DNA in blue. Scale bars, 20 µm. This experiment was repeated three times independently with similar results. P values were determined by the two-tailed, unpaired Student’s t-test (a).

Source Data

Extended Data Fig. 8 Depletion of Kupffer cells alleviates tumour EVP-induced fatty liver formation without impairing tumour growth.

a, Schematic illustration of liposome or clodronate (100 µl of suspension per 10 g of mouse weight) treatment, delivered via intravenous injection to B16F10-Tb and K7M2-Tb mice. The concentration of clodronate in the suspension is 5 mg ml−1. b, Representative flow cytometry gating strategy examining the abundance of Kupffer cells (Cd11b+F4/80+) in the livers from PBS-injected control mice, B16F10-Tb (up) or K7M2-Tb (bottom) mice, and tumour-bearing mice treated with liposome or clodronate. Associated quantification of Kupffer cell abundance was shown in Fig. 3c. c,d, Representative IF images (left) and associated statistical analysis (right) of Kupffer cell staining of the livers from B16F10-Tb (c) and K7M2-Tb (d) mice, and tumour-bearing mice treated with liposome or clodronate. F4/80 staining for Kupffer cells in red, and DNA in blue. For B16F10-Tb model, n = 6 B16F10-Tb mice, n = 5 B16F10-Tb mice treated with liposome or clodronate. For K7M2-Tb model, n = 5 mice per group. e, Measurement of the tumour volumes from B16F10-Tb (left) or K7M2-Tb (right) mice, and tumour-bearing mice treated with liposome or clodronate as shown in (a). n = 5 B16F10-Tb mice and n = 7 B16F10-Tb mice treated with liposome or clodronate; n = 3 K7M2-Tb mice and n = 5 K7M2-Tb mice treated with liposome or clodronate. f, Representative IF images of BODIPY staining of the livers from PBS-injected control mice, B16F10- or K7M2-Tb mice, and the tumour-bearing mice treated with liposome or clodronate as shown in Fig. 3d. g, Representative IF images (left) and associated statistical analysis (right) of the precision-cut liver slices (PCLS) stained with F4/80 (red) and DAPI (blue) to show the Kupffer cell depletion in mouse livers 24 h post treatment with liposome or clodronate (100 µl of suspension per 10 g of mouse weight). n = 3 mice per group. h, Schematic illustration of PCLS from naïve mice 24 h post treatment with liposome or clodronate (100 µl of suspension per 10 g of mouse weight) followed by EVP (10 µg ml−1) treatment ex vivo for 48 h. Liver slices were then subjected to BODIPY staining. i,j, Representative IF images of BODIPY staining of PCLS sectioned from liposome- or clodronate- (100 µl of suspension per 10 g of mouse weight) treated mice (as shown in g) that were further treated with 10 µg ml−1 of B16F10-CL-EVPs or B16F10-TE-EVPs (i), K7M2-CL-EVPs or K7M2-TE-EVPs (j) ex vivo for 48 h (see also Fig. 3e, f). PCLS from naïve mice treated with PBS were used as controls. BODIPY in green and DNA in blue. Scale bars, 20 µm. P values were determined by the one-way ANOVA with post hoc Tukey’s test (c,d), or two-way ANOVA followed with Bonferroni’s multiple comparisons test (e), or two-tailed, unpaired Student’s t-test (g). Data are mean ± s.e.m. NS, not significant.

Source Data

Extended Data Fig. 9 Tumour EVPs promote TNF secretion from Kupffer cells.

a, Representative IF image of the primary Kupffer cells isolated from naïve mice. F4/80 in red and DAPI in blue. Shown is a representative image from three independent experiments. Scale bar, 20 µm. b, ELISA analysis of IL-6 secretion from naïve mice-derived Kupffer cells after treatment with LPS (1 µg ml−1). n = 3 independent experiments per group. c,d, Representative cytokine array blots and associated quantification charts for the cytokines and chemokines in the whole cell lysates of Kupffer cells isolated from B16F10-Tb mice (c) and K7M2-Tb mice (d), and their respective PBS-injected controls. e,f, Representative cytokine array blots and associated quantification charts for the cytokines and chemokines in the conditioned media of Kupffer cells isolated from naïve mice and educated with 10 µg ml−1 of B16F10-CL-EVPs (e) or K7M2-CL-EVPs (f) in vitro for 3 days, and their respective PBS-educated controls. g, TNF ELISA on the conditioned medium of Kupffer cells educated with 10 µg ml−1 of B16F10-CL-EVP- (left), or K7M2-CL-EVP- (right), and their respective PBS controls for 3 days. n = 3 independent experiments per group. h, RT-qPCR analysis of Tnf expression in Kupffer cells isolated from B16F10-Tb (left), or K7M2-Tb (right) mice, and their respective PBS-injected controls. n = 4 mice per group. i, RT-qPCR analysis of Tnf expression in Kupffer cells treated with control PBS, EVPs derived from B16F10 cells or B16F10 tumour explants (left), or EVPs derived from K7M2 cells or K7M2 tumour explants (right) for 4 h. n = 3 independent experiments per group. j, TNF ELISA on EVP-depleted conditioned media from B16F10 and K7M2 cells. n = 2 independent experiments. ND, not detected. CM, conditioned medium. k, GSEA of gene expression profiles, ranked based on the sign of log2FC∗(-log10P value), in hepatocytes 24 h post treatment with recombinant mouse TNF protein (25 ng ml−1), compared to PBS control, using Gene Ontology gene sets. Shown are downregulated lipid catabolism-associated gene sets. NES, normalized enrichment score. NOM p-value, nominal p-value. l, Schematic Illustration of anti-TNF antibody (200 µg per mouse) or IgG1 isotype control (200 µg per mouse) treatment of tumour-bearing mice or tumour EVP-educated mice. I.P., intraperitoneal. m, Weights of tumours from mice inoculated with B16F10 (left) or K7M2 (right) cells and treated with anti-TNF antibody or IgG1 isotype control as shown in (l). n = 5 mice per group for B16F10-Tb model and n = 3 per group for K7M2-Tb model. n,o, Representative images of BODIPY staining of the livers from B16F10- and K7M2-Tb mice (n), and B16F10-CL-EVP- and K7M2-CL-EVP-educated mice (o), treated with anti-TNF antibody or IgG1 isotype control (see also Fig. 4d, e). Scale bars, 20 µm. P values were determined by the one-way ANOVA with post hoc Tukey’s test (b), or two-tailed, unpaired Student’s t-test (g-i,m). Data are mean ± s.e.m. NS, not significant. For cytokine array blot source data of (c-f), see Supplementary Fig. 2.

Source Data

Extended Data Fig. 10 Tumour EVP-packaged palmitic acid (PA) induces TNF secretion from Kupffer cells.

a, Volcano plot of metabolites significantly changed (highlighted in red, P < 0.05) in B16F10-TE-EVPs versus skin tissue explant-derived EVPs (skin-TE-EVPs). n = 5 B16F10-TE-EVPs and n = 4 skin tissue explant-derived EVPs. b, Quantitative mass spectrometry analysis of the long-chain free fatty acids, including saturated and unsaturated fatty acids, in the B16F10-TE-EVPs and skin tissue explant-derived EVPs (skin-TE-EVPs). n = 3 per EVP group. c, RT-qPCR analysis of Tnf expression in Kupffer cells from naïve C57BL/6 mice treated with DMSO or the TLR4 inhibitor TAK (5 µM) for 1 h followed by vehicle (100% ethanol diluted into PA-carrier medium at 1:1000 dilution) or 200 µM of PA with or without TAK (5 µM) for 4 h. n = 3 mice per group. d, FFA content in the EVPs isolated from B16F10 (left) or K7M2 (right) cells treated with DMSO or C75 (40 µM) for 48 h. n = 4 independent experiments for DMSO- or C75-treated B16F10 cells; n = 3 independent experiments for DMSO- or C75-treated K7M2 cells. e, RT-qPCR analysis of Tnf expression in Kupffer cells isolated from naïve C57BL/6 (left and middle) or BALB/c (right) mice then treated with control PBS, Melan-a-CL-EVPs (left), skin tissue explant-derived EVPs (skin-TE-EVPs, middle), osteoblast-EVPs (Ob-EVPs, right), or bone tissue explant-derived EVPs (bone-TE-EVPs, right) in vitro for 4 h. n = 3 independent experiments per group for treatment with Melan-a-CL-EVPs, Ob-EVPs, bone-TE-EVPs, and their PBS controls. n = 4 independent experiments for treatment with skin-TE-EVPs and PBS. f, Quantification of BODIPY staining of precision-cut liver slices treated with Ob-EVPs or K7M2-CL-EVPs ex vivo for 48 h, or livers educated with Ob-EVPs in vivo for 4 weeks, compared to PBS-treated or PBS-educated controls, respectively. n = 4 mice per group for ex vivo EVP treatment and n = 5 mice per group for in vivo EVP education. g, Representative LI-COR Odyssey images (left) and associated statistical analysis of relative signal intensity (right) of the livers from mice 24 h post intravenously injection of 10 µg of CellVue NIR815-labeled Ob-EVPs and mock PBS control. n = 3 mice per group. h, Quantification of BODIPY staining of the PCLS treated with control PBS, bone tissue explant-derived EVPs (bone-TE-EVPs) or K7M2-TE-EVPs (left), skin tissue explant-derived (skin-TE-EVPs) or B16F10-TE-EVPs (right) ex vivo for 48 h. n = 4 mice per group. i, RT-qPCR analysis of Tnf expression in the Kupffer cells isolated from C57BL/6 mice and treated with DMSO or 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.2 µM) in vitro for 4 h. TPA induced Kupffer cell Tnf expression, compared to DMSO control. n = 3 independent experiments. j, Representative LC-MS/MS chromatograms of TPA standard (5 nM, left) and TPA detected in the Mela-a-CL-EVPs (right). The concentration of TPA detected in Mela-a-CL-EVPs was 6.5 nM per 100 µg of EVPs. k, RT-qPCR analysis of Tnf expression in Kupffer cells which were pre-treated with DMSO or TAK (5 μM) for 1 h, and subsequently treated with PBS, 10 µg of B16F10-TE-EVPs or K7M2-TE-EVPs with or without TAK (5 µM) for 4 h. n = 3 independent experiments. P values were determined by the two-tailed, unpaired Student’s t-test (a,b,d-i,k), or one-way ANOVA with post hoc Tukey’s test (c), or Data are mean ± s.e.m. NS, not significant.

Source Data

Extended Data Fig. 11 Tumour EVPs suppress liver drug metabolism and enhance chemotoxicity.

a, Schematic illustration of the procedure of drug-metabolizing activity analysis of the PCLS. Substrates of Cytochrome P450 enzymes (including phenacetin, bupropion, tolbutamide, dextromethorphan and midazolam) were added to the media, and their corresponding metabolites (including acetaminophen, hydroxybupropion, 4-hydroxytolbutamide, dextrorphan and 1-hydroxymidazolam) were analyzed by LC-MS/MS. b, Drug-metabolizing activity of the core CYP enzymes in the PCLS sectioned from PBS-injected mice, and B16F10-Tb mice treated with anti-TNF antibody or IgG1 isotype control. n = 5 mice per group. c, Drug-metabolizing activity of Cyp1a2 (left) or Cyp2b10 (right) in the PCLS pre-treated with DMSO control or mathoxsalen (5 µM), or phenobarbital (0.1 mM) for 24 h. n = 3 mice per group. d, Drug-metabolizing activity of the core CYP enzymes in the PCLS sectioned from naïve mice pre-treated with PBS or 10 µg ml−1 of B16F10-CL-EVPs with co-treatment of anti-TNF antibody (20 µg ml−1) or IgG1 isotype (20 µg ml−1) control for 24 h. n = 5 mice per group. e, Schematic illustration of the procedure of chemotoxicity analysis using EVP-educated mice. For melanoma model (top), C57BL/6 mice were intravenously injected with PBS or 10 µg of B16F10-CL-EVPs every other day for 4 weeks, and then intraperitoneally injected with dacarbazine (60 mg kg−1) or 0.9% NaCl together with intravenous injection of PBS or 10 µg of B16F10-CL-EVPs every other day for 4 times. Retroorbital blood of the mice was then collected for complete blood count. For osteosarcoma model (bottom), BALB/c mice were intravenously injected with PBS, 10 µg of osteoblast-EVPs (Ob-EVPs) or K7M2-CL-EVPs every other day for 4 weeks, and then intraperitoneally injected with doxorubicin (1 mg kg−1) or DMSO every 24 h together with intravenous injection of PBS, 10 µg of Ob-EVPs or K7M2-CL-EVPs every other day for 25 days, and mice were then subjected to echocardiography. f, Statistical analysis of the heart rates of PBS-, Ob-EVP- or K7M2-CL-EVP-educated mice after treatment of DMSO or doxorubicin (cumulative dose of 25 mg kg−1). No difference of heart rates was observed in different groups. n = 6 mice for PBS groups and n = 7 mice for K7M2-CL-EVP groups, n = 5 and n = 6 for DMSO and doxorubicin treated Ob-EVP-educated mice, respectively. g, Representative M-mode images of echocardiography for PBS-, Ob-EVP- or K7M2-CL-EVP-educated mice after treatment of DMSO or doxorubicin as described in (e). LVIDd, left ventricular internal dimension at end diastole. LVIDs, left ventricular internal dimension at end systole. EF, ejection fraction. FS, fractional shortening. P values were determined by the one-way ANOVA with post hoc Tukey’s test (b,d), or two-tailed, unpaired Student’s t-test (c), or two-way ANOVA followed with Fisher’s LSD test (f). Data are mean ± s.e.m. NS, not significant. Ob, osteoblast. I.V., intravenours. I.P., intraperitoneal.

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Supplementary Video 1

Representative video of the echocardiography for PBS-educated mice after treatment with DMSO. BALB/c mice were intravenously injected with PBS every other day for 4 weeks, and then intraperitoneally injected with DMSO every 24 h together with intravenous injection of PBS every other day for 25 days, and mice were then subjected to echocardiography. FujiFilm VisualSonics, Inc. is the copyright holder of the logo that appears in the top left of the video, and permission was obtained for its use in Nature.

Supplementary Video 2

Representative video of the echocardiography for PBS-educated mice after treatment with doxorubicin. BALB/c mice were intravenously injected with PBS every other day for 4 weeks, and then intraperitoneally injected with doxorubicin (1 mg kg−1) every 24 h together with intravenous injection of PBS every other day for 25 days, and mice were then subjected to echocardiography.

Supplementary Video 3

Representative video of the echocardiography for osteoblast-EVP-educated mice after treatment with DMSO. BALB/c mice were intravenously injected with 10 µg of osteoblast-EVPs every other day for 4 weeks, and then intraperitoneally injected with DMSO every 24 h together with intravenous injection of 10 µg of osteoblast-EVPs every other day for 25 days, and mice were then subjected to echocardiography.

Supplementary Video 4

Representative video of the echocardiography for osteoblast-EVP-educated mice after treatment with doxorubicin. BALB/c mice were intravenously injected with 10 µg of osteoblast-EVPs every other day for 4 weeks, and then intraperitoneally injected with doxorubicin (1 mg kg−1) every 24 h together with intravenous injection of 10 µg of osteoblast-EVPs every other day for 25 days, and mice were then subjected to echocardiography.

Supplementary Video 5

Representative video of the echocardiography for K7M2-CL-EVP-educated mice after treatment with DMSO. BALB/c mice were intravenously injected with 10 µg of K7M2-CL-EVPs every other day for 4 weeks, and then intraperitoneally injected with DMSO every 24 h together with intravenous injection of 10 µg of K7M2-CL-EVPs every other day for 25 days, and mice were then subjected to echocardiography.

Supplementary Video 6

Representative video of the echocardiography for K7M2-CL-EVP-educated mice after treatment with doxorubicin. BALB/c mice were intravenously injected with 10 µg of K7M2-CL-EVPs every other day for 4 weeks, and then intraperitoneally injected with doxorubicin (1 mg kg−1) every 24 h together with intravenous injection of 10 µg of K7M2-CL-EVPs every other day for 25 days, and mice were then subjected to echocardiography.

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Wang, G., Li, J., Bojmar, L. et al. Tumour extracellular vesicles and particles induce liver metabolic dysfunction. Nature 618, 374–382 (2023). https://doi.org/10.1038/s41586-023-06114-4

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