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Methotrexate-loaded tumour-cell-derived microvesicles can relieve biliary obstruction in patients with extrahepatic cholangiocarcinoma

Abstract

Most patients with cholangiocarcinoma (CCA) develop extrahepatic malignant biliary obstructions, which require palliative drainage to normalize bilirubin levels and to improve the patients’ overall survival. Here, we report that the infusion of methotrexate-containing plasma-membrane microvesicles derived from apoptotic human tumour cells into the bile-duct lumen of patients with extrahepatic CCA mobilized and activated neutrophils and relieved biliary obstruction in 25% of the patients. Neutrophil recruitment by the microvesicles was associated with an increase in uridine diphosphate glucose and complement C5, and led to the degradation of the stromal barrier of CCA. The microvesicles induced pyroptosis of CCA cells through a gasdermin E-dependent pathway, and their intracellular contents released upon CCA-cell death activated patient-derived macrophages into producing proinflammatory cytokines, which attracted a secondary wave of neutrophils to the tumour site. Our findings suggest a possible treatment for the alleviation of obstructive extrahepatic CCA with few adverse effects, and highlight the potential of tumour-cell-derived microvesicles as drug carriers for antitumour therapies.

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Fig. 1: Effective treatment of obstructive CCA with MTX–TMPs.
Fig. 2: MTX–TMP perfusion attracts antitumor neutrophils.
Fig. 3: Direct attraction of neutrophils by UDPG in MTX–TMPs.
Fig. 4: MTX–TMPs induce CCA-cell pyroptosis.
Fig. 5: MTX–TMP perfusion triggers a secondary wave of recruiting neutrophils.
Fig. 6: MTX–TMP perfusion-recruited neutrophils display an antitumour phenotype.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too large to be publicly shared, but are available for research purposes from the corresponding authors on reasonable request.

References

  1. 1.

    Razumilava, N. & Gores, G. J. Cholangiocarcinoma. Lancet 383, 2168–2179 (2014).

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Doherty, B., Nambudiri, V. E. & Palmer, W. C. Update on the diagnosis and treatment of cholangiocarcinoma. Curr. Gastroenterol. Rep. 19, 2 (2017).

    PubMed  Google Scholar 

  3. 3.

    Wakai, T. et al. Surgical management of carcinoma in situ at ductal resection margins in patients with extrahepatic cholangiocarcinoma. Ann. Gastroenterol. Surg. 2, 359–366 (2018).

    PubMed  PubMed Central  Google Scholar 

  4. 4.

    Ortner, M.-A. & Dorta, G. Technology insight: photodynamic therapy for cholangiocarcinoma. Nat. Rev. Gastroenterol. Hepatol. 3, 459–467 (2006).

    CAS  Google Scholar 

  5. 5.

    Blechacz, B., Komuta, M., Roskams, T. & Gores, G. J. Clinical diagnosis and staging of cholangiocarcinoma. Nat Rev. Gastroenterol. Hepatol. 8, 512–522 (2011).

    PubMed  PubMed Central  Google Scholar 

  6. 6.

    Patel, T. Cholangiocarcinoma. Nat. Clin. Pract. Gastroenterol. Hepatol. 3, 33–42 (2006).

    PubMed  Google Scholar 

  7. 7.

    Banales, J. M. et al. Cholangiocarcinoma: current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA). Nat. Rev. Gastroenterol. Hepatol. 13, 261–280 (2016).

    PubMed  Google Scholar 

  8. 8.

    Artifon, E. L. A., Ferreira, F. C. & Sakai, P. Endoscopic ultrasound-guided biliary drainage. Korean J. Radiol. 13, S74–S82 (2012).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Ma, J. et al. Reversing drug resistance of soft tumor-repopulating cells by tumor cell-derived chemotherapeutic microparticles. Cell Res. 26, 713–727 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Ma, J. et al. Mechanisms by which dendritic cells present tumor microparticle antigens to CD8+ T cells. Cancer Immunol. Res. 6, 1057–1068 (2018).

    CAS  PubMed  Google Scholar 

  11. 11.

    Dong, W. et al. Oral delivery of tumor microparticle vaccines activates NOD2 signaling pathway in ileac epithelium rendering potent antitumor T cell immunity. Oncoimmunology. 6, e1282589 (2017).

    PubMed  PubMed Central  Google Scholar 

  12. 12.

    Jin, X. et al. Pre-instillation of tumor microparticles enhances intravesical chemotherapy of nonmuscle-invasive bladder cancer through a lysosomal pathway. Biomaterials 113, 93–104 (2017).

    CAS  PubMed  Google Scholar 

  13. 13.

    Zhang, H. et al. Cell-free tumor microparticle vaccines stimulate dendritic cells via cGAS/STING signaling. Cancer Immunol. Res. 3, 196–205 (2015).

    CAS  PubMed  Google Scholar 

  14. 14.

    Mause, S. F. & Weber, C. Microparticles: protagonists of a novel communication network for intercellular information exchange. Circ. Res. 107, 1047–1057 (2010).

    CAS  PubMed  Google Scholar 

  15. 15.

    Tang, K. et al. Delivery of chemotherapeutic drugs in tumour cell-derived microparticles. Nat. Commun. 3, 1282 (2012).

    PubMed  Google Scholar 

  16. 16.

    Harada, K. & Nakanuma, Y. Biliary innate immunity: function and modulation. Mediators Inflamm. 2010, 373878 (2010).

    PubMed  PubMed Central  Google Scholar 

  17. 17.

    Zhang, Q. et al. Circulating mitochondrial DAMPs cause inflammatory responses to injury. Nature 464, 104–107 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Albrengues, J. et al. Neutrophil extracellular traps produced during inflammation awaken dormant cancer cells in mice. Science 361, eaao4227 (2018).

    PubMed  PubMed Central  Google Scholar 

  19. 19.

    Sesma, J. I. et al. UDP-glucose promotes neutrophil recruitment in the lung. Purinergic Signal. 12, 627–635 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20.

    Arase, T. et al. The UDP-glucose receptor P2RY14 triggers innate mucosal immunity in the female reproductive tract by inducing IL-8. J. Immunol. 182, 7074–7084 (2009).

    CAS  PubMed  Google Scholar 

  21. 21.

    Sesma, J. I. et al. The UDP-sugar-sensing P2Y14 receptor promotes Rho-mediated signaling and chemotaxis in human neutrophils. Am. J. Physiol. Cell Physiol. 303, C490–C498 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Fairbanks, L. D. et al. Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis? Biochem. J. 342, 143–152 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Sadik, C. D., Miyabe, Y., Sezin, T. & Luster, A. D. The critical role of C5a as an initiator of neutrophil-mediated autoimmune inflammation of the joint and skin. Semin. Immunol. 37, 21–29 (2018).

    CAS  PubMed  Google Scholar 

  24. 24.

    Kew, R. R. & Webster, R. O. Gc-globulin (vitamin D-binding protein) enhances the neutrophil chemotactic activity of C5a and C5a des Arg. J Clin Invest. 82, 364–369 (1988).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25.

    Wang, Y. et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 547, 99–103 (2017).

    CAS  PubMed  Google Scholar 

  26. 26.

    Shi, J. et al. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526, 660–665 (2015).

    CAS  Google Scholar 

  27. 27.

    Ding, J. et al. Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535, 111–116 (2016).

    CAS  PubMed  Google Scholar 

  28. 28.

    Ruhl, S. et al. ESCRT-dependent membrane repair negatively regulates pyroptosis downstream of GSDMD activation. Science 362, 956–960 (2018).

    PubMed  Google Scholar 

  29. 29.

    Yuan, J., Amin, P. & Ofengheim, D. Necroptosis and RIPK1-mediated neuroinflammation in CNS diseases. Nat. Rev. Neurosci. 20, 19–33 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Lin, J. et al. RIPK1 counteracts ZBP1-mediated necroptosis to inhibit inflammation. Nature 540, 124–128 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31.

    Vande Walle, L. & Lamkanfi, M. Pyroptosis. Curr. Biol. 26, R568–R572 (2016).

    CAS  PubMed  Google Scholar 

  32. 32.

    Sun, Y. et al. Chemotherapeutic tumor microparticles combining low-dose irradiation reprogram tumor-promoting macrophages through a tumor-repopulating cell-curtailing pathway. Oncoimmunology 6, e1309487 (2017).

    PubMed  PubMed Central  Google Scholar 

  33. 33.

    Ma, R. et al. Tumor cell-derived microparticles polarize M2 tumor-associated macrophages for tumor progression. Oncoimmunology. 5, e1118599 (2016).

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Pylaeva, E., Lang, S. & Jablonska, J. The essential role of type I interferons in differentiation and activation of tumor-associated neutrophils. Front. Immunol. 7, 629 (2016).

    PubMed  PubMed Central  Google Scholar 

  35. 35.

    Shaul, M. E. & Fridlender, Z. G. Neutrophils as active regulators of the immune system in the tumor microenvironment. J. Leukoc. Biol. 102, 343–349 (2017).

    CAS  PubMed  Google Scholar 

  36. 36.

    El-Benna, J. et al. Priming of the neutrophil respiratory burst: role in host defense and inflammation. Immunol. Rev. 273, 180–193 (2016).

    CAS  PubMed  Google Scholar 

  37. 37.

    Stojkov, D. et al. ROS and glutathionylation balance cytoskeletal dynamics in neutrophil extracellular trap formation. J. Cell Biol. 216, 4073–4090 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Delgado-Rizo, V. et al. Neutrophil extracellular traps and its implications in inflammation: an overview. Front. Immunol. 8, 81 (2017).

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Fridlender, Z. G. & Albelda, S. M. Tumor-associated neutrophils: friend or foe? Carcinogenesis. 33, 949–955 (2012).

    CAS  PubMed  Google Scholar 

  40. 40.

    Kolaczkowska, E. & Kubes, P. Neutrophil recruitment and function in health and inflammation. Nat. Rev. Immunol. 13, 159–175 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Vols, S., Sionov, R. V. & Granot, Z. Always look on the bright side: anti-tumor functions of neutrophils. Curr. Pharm. Des. 23, 4862–4892 (2017).

    CAS  PubMed  Google Scholar 

  42. 42.

    Bergsbaken, T., Fink, S. L. & Cookson, B. T. Pyroptosis: host cell death and inflammation. Nat. Rev. Microbiol. 7, 99–109 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Long, K. B. et al. IFNγ and CCL2 cooperate to redirect tumor-infiltrating monocytes to degrade fibrosis and enhance chemotherapy efficacy in pancreatic carcinoma. Cancer Discov. 6, 400–413 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Mertens, J. C. & Gores, G. J. Targeting tumor stroma: exploiting apoptotic priming. Oncotarget 3, 1501–1502 (2012).

    PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by National Natural Science Foundation of China (81788101, 81661128007, 81530080, 81773062 and 91942314), the Chinese Academy of Medical Sciences Initiative for Innovative Medicine (CAMS-I2M) (2017-I2M-1-001 and 2016-I2M-1-007).

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Contributions

B.H., Y.L. and X.W. conceived the project. Y.L., Yunfeng Gao, Hui Zhang, N.Z., P.X., J.W., Yuan Gao, X.J., X.L., J.L., Y.Z., K.T., J.M., Huafeng Zhang and J.X. performed the experiments. B.H., Y.L., F.Y., W.T. and X.W. developed methodology. B.H., Y.L., Yunfeng Gao, Hui Zhang and X.W. performed data analysis. B.H. and Y.L. wrote the manuscript.

Corresponding authors

Correspondence to Yuying Liu or Ximo Wang or Bo Huang.

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Competing interests

B.H. is the inventor on patent no. ZL201110241369.8, owned by Hubei Soundny (Sheng-Qi-An) Biotech, which covers the preparation and use of drug-packaging tumour-cell-derived microparticles in cancer therapies. The other authors declare no competing interests.

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Clinical research protocol.

Supplementary Methods

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Gao, Y., Zhang, H., Zhou, N. et al. Methotrexate-loaded tumour-cell-derived microvesicles can relieve biliary obstruction in patients with extrahepatic cholangiocarcinoma. Nat Biomed Eng 4, 743–753 (2020). https://doi.org/10.1038/s41551-020-0583-0

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