Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Burst release of encapsulated annexin A5 in tumours boosts cytotoxic T-cell responses by blocking the phagocytosis of apoptotic cells


Cancer immunotherapies, particularly therapeutic vaccination, do not typically generate robust anti-tumour immune responses. Here, we show that the intratumoral burst release of the protein annexin A5 from intravenously injected hollow mesoporous nanoparticles made of diselenide-bridged organosilica generates robust anti-tumour immunity by exploiting the capacity of primary tumours to act as antigen depots. Annexin A5 blocks immunosuppressive apoptosis and promotes immunostimulatory secondary necrosis by binding to the phagocytic marker phosphatidylserine on dying tumour cells. In mice bearing large established tumours, the burst release of annexin A5 owing to diselenide-bond cleavage under the oxidizing conditions of the tumour microenvironment and the reducing intracellular conditions of tumour cells induced systemic cytotoxic T-cell responses and immunological memory associated with tumour regression and the prevention of relapse, and led to complete tumour eradication in about 50% of mice with orthotopic breast tumours. Reducing apoptosis signalling via in situ vaccination could be a versatile strategy for the generation of adaptive anti-tumour immune responses.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Schematic and characterization of HMSeN–ANX5@HOMV.
Fig. 2: PS blockade by HMSeN–ANX5@HOMV skews the phagocytic clearance of dying tumour cells in vitro.
Fig. 3: PS blockade by HMSeN–ANX5@HOMV causes changes in the transcriptome and cytokine profile in tumours in vivo.
Fig. 4: HMSeN–ANX5@HOMV inflames the TME and potentiates systemic anti-tumour immune responses.
Fig. 5: HMSeN–ANX5@HOMV elicits robust anti-tumour efficacy and durable immunological memory against orthotopic 4T1 breast tumours.
Fig. 6: HMSeN–ANX5@HOMV triggers anti-tumour immunity and therapeutic efficacy against B16F10 melanoma.

Data availability

The main data supporting the results in this study are available within the paper and its Supplementary Information. All data generated in this study, including source data and the data used to make the figures, are available from figshare with the identifier


  1. 1.

    Ying, Z. et al. A safe and potent anti-CD19 CAR T cell therapy. Nat. Med. 1, 947–953 (2019).

    Google Scholar 

  2. 2.

    Rafiq, S. et al. Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumour efficacy in vivo. Nat. Biotechnol. 36, 847–856 (2018).

    CAS  Google Scholar 

  3. 3.

    Chen, Q. et al. In situ sprayed bioresponsive immunotherapeutic gel for post-surgical cancer treatment. Nat. Nanotechnol. 14, 89–97 (2019).

    CAS  Google Scholar 

  4. 4.

    Wang, C. et al. In situ activation of platelets with checkpoint inhibitors for post-surgical cancer immunotherapy. Nat. Biomed. Eng. 1, 0011 (2017).

    CAS  Google Scholar 

  5. 5.

    Gao, S. et al. Engineering nanoparticles for targeted remodeling of the tumour microenvironment to improve cancer immunotherapy. Theranostics 9, 126–151 (2019).

    CAS  Google Scholar 

  6. 6.

    Balachandran, V. P. et al. Identification of unique neoantigen qualities in long-term survivors of pancreatic cancer. Nature 551, 512–516 (2017).

    CAS  Google Scholar 

  7. 7.

    Zhang, L. et al. Peptide-based materials for cancer immunotherapy. Theranostics 9, 7807–7825 (2019).

    CAS  Google Scholar 

  8. 8.

    De Palma, M., Biziato, D. & Petrova, T. V. Microenvironmental regulation of tumour angiogenesis. Nat. Rev. Cancer 17, 457–474 (2017).

    Google Scholar 

  9. 9.

    Smyth, M. J., Ngiow, S. F., Ribas, A. & Teng, M. W. Combination cancer immunotherapies tailored to the tumour microenvironment. Nat. Rev. Clin. Oncol. 13, 143–158 (2016).

    CAS  Google Scholar 

  10. 10.

    Beatty, G. L. & Gladney, W. L. Immune escape mechanisms as a guide for cancer immunotherapy. Clin. Cancer Res. 21, 687–692 (2015).

    CAS  Google Scholar 

  11. 11.

    Lim, S. et al. Recent advances and challenges of repurposing nanoparticle-based drug delivery systems to enhance cancer immunotherapy. Theranostics 9, 7906–7923 (2019).

    CAS  Google Scholar 

  12. 12.

    Guo, C. et al. Therapeutic cancer vaccines: past, present, and future. Adv. Cancer Res. 119, 421–475 (2013).

    CAS  Google Scholar 

  13. 13.

    Wu, G. et al. A Mucin1 C-terminal subunit-directed monoclonal antibody targets overexpressed Mucin1 in breast cancer. Theranostics 8, 78–91 (2018).

    CAS  Google Scholar 

  14. 14.

    Hu, Z., Ott, P. A. & Wu, C. J. Towards personalized, tumour-specific, therapeutic vaccines for cancer. Nat. Rev. Immunol. 18, 168–182 (2018).

    CAS  Google Scholar 

  15. 15.

    Gudkov, A. V. & Komarova, E. A. The role of p53 in determining sensitivity to radiotherapy. Nat. Rev. Cancer 3, 117–129 (2003).

    CAS  Google Scholar 

  16. 16.

    Wu, M. et al. Self-luminescing theranostic nanoreactors with intraparticle relayed energy transfer for tumour microenvironment activated imaging and photodynamic therapy. Theranostics 9, 20–33 (2019).

    CAS  Google Scholar 

  17. 17.

    Johnstone, R. W., Ruefli, A. A. & Lowe, S. W. Apoptosis: a link between cancer genetics and chemotherapy. Cell 108, 153–164 (2002).

    CAS  Google Scholar 

  18. 18.

    Li, M. O., Sarkisian, M. R., Mehal, W. Z., Rakic, P. & Flavell, R. A. Phosphatidylserine receptor is required for clearance of apoptotic cells. Science 302, 1560–1563 (2003).

    CAS  Google Scholar 

  19. 19.

    Yoshida, H. et al. Phosphatidylserine-dependent engulfment by macrophages of nuclei from erythroid precursor cells. Nature 437, 754–758 (2005).

    CAS  Google Scholar 

  20. 20.

    Fadok, V. A. et al. A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 405, 85–90 (2000).

    CAS  Google Scholar 

  21. 21.

    Huynh, M.-L. N., Fadok, V. A. & Henson, P. M. Phosphatidylserine-dependent ingestion of apoptotic cells promotes TGF-β1 secretion and the resolution of inflammation. J. Clin. Invest. 109, 41–50 (2002).

    CAS  Google Scholar 

  22. 22.

    Wu, Z., Ma, H. M., Kukita, T., Nakanishi, Y. & Nakanishi, H. Phosphatidylserine-containing liposomes inhibit the differentiation of osteoclasts and trabecular bone loss. J. Immunol. 184, 3191–3201 (2010).

    CAS  Google Scholar 

  23. 23.

    Proskuryakov, S. Y., Gabai, V., Konoplyannikov, A., Zamulaeva, I. & Kolesnikova, A. Immunology of apoptosis and necrosis. Biochemistry 70, 1310–1320 (2005).

    CAS  Google Scholar 

  24. 24.

    Silva, M. T., Do Vale, A. & dos Santos, N. M. Secondary necrosis in multicellular animals: an outcome of apoptosis with pathogenic implications. Apoptosis 13, 463–482 (2008).

    Google Scholar 

  25. 25.

    Edinger, A. L. & Thompson, C. B. Death by design: apoptosis, necrosis and autophagy. Curr. Opin. Cell Biol. 16, 663–669 (2004).

    CAS  Google Scholar 

  26. 26.

    Ouyang, L. et al. Programmed cell death pathways in cancer: a review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 45, 487–498 (2012).

    CAS  Google Scholar 

  27. 27.

    Krysko, D. V. et al. Immunogenic cell death and DAMPs in cancer therapy. Nat. Rev. Cancer 12, 860–875 (2012).

    CAS  Google Scholar 

  28. 28.

    Davidovich, P., Kearney, C. J. & Martin, S. J. Inflammatory outcomes of apoptosis, necrosis and necroptosis. Biol. Chem. 395, 1163–1171 (2014).

    CAS  Google Scholar 

  29. 29.

    Boersma, H. H. et al. Past, present, and future of annexin A5: from protein discovery to clinical applications. J. Nucl. Med. 46, 2035–2050 (2005).

    CAS  Google Scholar 

  30. 30.

    Narula, J. et al. Annexin-V imaging for noninvasive detection of cardiac allograft rejection. Nat. Med. 7, 1347–1352 (2001).

    CAS  Google Scholar 

  31. 31.

    Logue, S. E., Elgendy, M. & Martin, S. J. Expression, purification and use of recombinant annexin V for the detection of apoptotic cells. Nat. Protoc. 4, 1383–1395 (2009).

    CAS  Google Scholar 

  32. 32.

    van Genderen, H. O. et al. Extracellular annexin A5: functions of phosphatidylserine-binding and two-dimensional crystallization. Biochim. Biophys. Acta 1783, 953–963 (2008).

    Google Scholar 

  33. 33.

    Schutters, K. & Reutelingsperger, C. Phosphatidylserine targeting for diagnosis and treatment of human diseases. Apoptosis 15, 1072–1082 (2010).

    CAS  Google Scholar 

  34. 34.

    Schlegel, R. & Williamson, P. Phosphatidylserine, a death knell. Cell Death Differ. 8, 551–563 (2001).

    CAS  Google Scholar 

  35. 35.

    Martin, S. et al. Immunologic stimulation of mast cells leads to the reversible exposure of phosphatidylserine in the absence of apoptosis. Int. Arch. Allergy Immunol. 123, 249–258 (2000).

    CAS  Google Scholar 

  36. 36.

    Dillon, S. R., Mancini, M., Rosen, A. & Schlissel, M. S. Annexin V binds to viable B cells and colocalizes with a marker of lipid rafts upon B cell receptor activation. J. Immunol. 164, 1322–1332 (2000).

    CAS  Google Scholar 

  37. 37.

    Kroemer, G. et al. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell Death Differ. 16, 3–11 (2009).

    CAS  Google Scholar 

  38. 38.

    Frey, B. & Gaipl, U. S. The immune functions of phosphatidylserine in membranes of dying cells and microvesicles. Semin. Immunopathol. 33, 497–516 (2011).

    CAS  Google Scholar 

  39. 39.

    Barroso, G. et al. Mitochondrial membrane potential integrity and plasma membrane translocation of phosphatidylserine as early apoptotic markers: a comparison of two different sperm subpopulations. Fertil. Steril. 85, 149–154 (2006).

    CAS  Google Scholar 

  40. 40.

    Mura, S., Nicolas, J. & Couvreur, P. Stimuli-responsive nanocarriers for drug delivery. Nat. Mater. 12, 991–1003 (2013).

    CAS  Google Scholar 

  41. 41.

    Cheng, C. A. et al. Supramolecular nanomachines as stimuli-responsive gatekeepers on mesoporous silica nanoparticles for antibiotic and cancer drug delivery. Theranostics 9, 3341–3364 (2019).

    CAS  Google Scholar 

  42. 42.

    Brannon-Peppas, L. & Blanchette, J. O. Nanoparticle and targeted systems for cancer therapy. Adv. Drug Deliv. Rev. 64, 206–212 (2012).

    Google Scholar 

  43. 43.

    Sadauskas, E. et al. Kupffer cells are central in the removal of nanoparticles from the organism. Part. Fibre Toxicol. 4, 10 (2007).

    Google Scholar 

  44. 44.

    Tavares, A. J. et al. Effect of removing Kupffer cells on nanoparticle tumour delivery. Proc. Natl Acad. Sci. USA 114, E10871–E10880 (2017).

    CAS  Google Scholar 

  45. 45.

    Xu, H., Cao, W. & Zhang, X. Selenium-containing polymers: promising biomaterials for controlled release and enzyme mimics. Acc. Chem. Res. 46, 1647–1658 (2013).

    CAS  Google Scholar 

  46. 46.

    Kim, J. H., Lee, J., Park, J. & Gho, Y. S. Gram-negative and Gram-positive bacterial extracellular vesicles. Semin. Cell Dev. Biol. 40, 97–104 (2015).

    CAS  Google Scholar 

  47. 47.

    Fogh-Andersen, N. et al. Composition of interstitial fluid. Clin. Chem. 41, 1522–1525 (1995).

    CAS  Google Scholar 

  48. 48.

    Hoffmann, P. R. et al. Phosphatidylserine (PS) induces PS receptor–mediated macropinocytosis and promotes clearance of apoptotic cells. J. Cell Biol. 155, 649–659 (2001).

    CAS  Google Scholar 

  49. 49.

    Somersan, S. & Bhardwaj, N. Tethering and tickling: a new role for the phosphatidylserine receptor. J. Cell Biol. 155, 501–504 (2001).

    CAS  Google Scholar 

  50. 50.

    Xu, X. et al. Advances in engineering cells for cancer immunotherapy. Theranostics 9, 7889–7905 (2019).

    CAS  Google Scholar 

  51. 51.

    Kim, O. Y. et al. Immunization with Escherichia coli outer membrane vesicles protects bacteria-induced lethality via Th1 and Th17 cell responses. J. Immunol. 190, 4092–4102 (2013).

    CAS  Google Scholar 

  52. 52.

    Castrillo, A., Joseph, S. B., Marathe, C., Mangelsdorf, D. J. & Tontonoz, P. Liver X receptor-dependent repression of matrix metalloproteinase-9 expression in macrophages. J. Biol. Chem. 278, 10443–10449 (2003).

    CAS  Google Scholar 

  53. 53.

    Inaba, K. et al. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. J. Exp. Med. 176, 1693–1702 (1992).

    CAS  Google Scholar 

Download references


This work was supported by the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, the Faculty of Health Sciences, University of Macau, a Start-up Research Grant of the University of Macau (SRG2018-00130-FHS) and the Science and Technology Development Fund (FDCT), Macao SAR (FDCT 0109/2018/A3). We thank V. Schram for help with confocal microscopy imaging, Henry S. Eden for proof-reading the manuscript and Guangzhou Sagene Biotech for help with making the pattern diagrams.

Author information




X.C., L.L., W.F. and Z.Y. conceived the study, designed the experiments and wrote the manuscript. L.L., J.Z., W.F. and Z.Y. performed the experiments and analysed data. Y.D. and G.N. re-evaluated the anti-tumour activity.

Corresponding authors

Correspondence to Wenpei Fan or Zhen Yang or Xiaoyuan Chen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, L., Zou, J., Dai, Y. et al. Burst release of encapsulated annexin A5 in tumours boosts cytotoxic T-cell responses by blocking the phagocytosis of apoptotic cells. Nat Biomed Eng 4, 1102–1116 (2020).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing