Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

A bioorthogonal system reveals antitumour immune function of pyroptosis

Abstract

Bioorthogonal chemistry capable of operating in live animals is needed to investigate biological processes such as cell death and immunity. Recent studies have identified a gasdermin family of pore-forming proteins that executes inflammasome-dependent and -independent pyroptosis1,2,3,4,5. Pyroptosis is proinflammatory, but its effect on antitumour immunity is unknown. Here we establish a bioorthogonal chemical system, in which a cancer-imaging probe phenylalanine trifluoroborate (Phe-BF3) that can enter cells desilylates and ‘cleaves’ a designed linker that contains a silyl ether. This system enabled the controlled release of a drug from an antibody–drug conjugate in mice. When combined with nanoparticle-mediated delivery, desilylation catalysed by Phe-BF3 could release a client protein—including an active gasdermin—from a nanoparticle conjugate, selectively into tumour cells in mice. We applied this bioorthogonal system to gasdermin, which revealed that pyroptosis of less than 15% of tumour cells was sufficient to clear the entire 4T1 mammary tumour graft. The tumour regression was absent in immune-deficient mice or upon T cell depletion, and was correlated with augmented antitumour immune responses. The injection of a reduced, ineffective dose of nanoparticle-conjugated gasdermin along with Phe-BF3 sensitized 4T1 tumours to anti-PD1 therapy. Our bioorthogonal system based on Phe-BF3 desilylation is therefore a powerful tool for chemical biology; our application of this system suggests that pyroptosis-induced inflammation triggers robust antitumour immunity and can synergize with checkpoint blockade.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Desilylation bioorthogonal chemistry and its application to an antibody–drug conjugate.
Fig. 2: Phe-BF3 desilylation releases GFP from TES-linked NP–GFP with tumour selectivity in mice.
Fig. 3: Phe-BF3 desilylation releases gasdermin from NP–GSDMA3 to induce pyroptosis.
Fig. 4: Treatment with NP–GSDMA3 and Phe-BF3 causes tumour regression in mice.
Fig. 5: A low level of tumour-cell pyroptosis induces effective antitumour immunity and synergizes with anti-PD1 blockade.

Similar content being viewed by others

Data availability

All data supporting the findings of this study are included in the Article and its Supplementary Information. Single-cell RNA sequencing of tumour-infiltrating immune cells were also deposited at the National Genomics Data Center (NGDC) under accession number PRJCA002149 (https://bigd.big.ac.cn/bioproject/browse/PRJCA002149). Source Data for Figs. 15, Extended Data Figs. 27, 10 are available with the paper.

References

  1. Kayagaki, N. et al. Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526, 666–671 (2015).

    Article  CAS  ADS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  3. Shi, J., Gao, W. & Shao, F. Pyroptosis: gasdermin-mediated programmed necrotic cell death. Trends Biochem. Sci. 42, 245–254 (2017).

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  5. Broz, P., Pelegrín, P. & Shao, F. The gasdermins, a protein family executing cell death and inflammation. Nat. Rev. Immunol. https://doi.org/10.1038/s41577-019-0228-2 (2019).

  6. Liu, Z. et al. Preclinical evaluation of a high-affinity 18F-trifluoroborate octreotate derivative for somatostatin receptor imaging. J. Nucl. Med. 55, 1499–1505 (2014).

    Article  CAS  Google Scholar 

  7. Liu, Z. et al. Boramino acid as a marker for amino acid transporters. Sci. Adv. 1, e1500694 (2015).

    Article  ADS  Google Scholar 

  8. Liu, Z. et al. An organotrifluoroborate for broadly applicable one-step 18F-labeling. Angew. Chem. Int. Edn Engl. 53, 11876–11880 (2014).

    Article  CAS  Google Scholar 

  9. David Crouch, R. Selective monodeprotection of bis-silyl ethers. Tetrahedron 60, 5833–5871 (2004).

    Article  Google Scholar 

  10. Chau, C. H., Steeg, P. S. & Figg, W. D. Antibody–drug conjugates for cancer. Lancet 394, 793–804 (2019).

    Article  CAS  Google Scholar 

  11. Perrin, D. M. [18F]-Organotrifluoroborates as radioprosthetic groups for PET imaging: from design principles to preclinical applications. Acc. Chem. Res. 49, 1333–1343 (2016).

    Article  CAS  Google Scholar 

  12. Nelson, T. D. & Crouch, R. D. Selective deprotection of silyl ethers. Synthesis 1996, 1031–1069 (1996).

    Article  Google Scholar 

  13. Handy, C. J., Lam, Y. F. & DeShong, P. On the synthesis and NMR analysis of tetrabutylammonium triphenyldifluorosilicate. J. Org. Chem. 65, 3542–3543 (2000).

    Article  CAS  Google Scholar 

  14. Liu, Z. et al. From minutes to years: predicting organotrifluoroborate solvolysis rates. Chemistry 21, 3924–3928 (2015).

    Article  CAS  Google Scholar 

  15. Papasani, M. R., Wang, G. & Hill, R. A. Gold nanoparticles: the importance of physiological principles to devise strategies for targeted drug delivery. Nanomedicine 8, 804–814 (2012).

    Article  CAS  Google Scholar 

  16. Wilhelm, S. et al. Analysis of nanoparticle delivery to tumours. Nat. Rev. Mater. 1, 16014 (2016).

    Article  CAS  ADS  Google Scholar 

  17. Blanco, E., Shen, H. & Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 33, 941–951 (2015).

    Article  CAS  Google Scholar 

  18. Swierczewska, M., Lee, S. & Chen, X. The design and application of fluorophore–gold nanoparticle activatable probes. Phys. Chem. Chem. Phys. 13, 9929–9941 (2011).

    Article  CAS  Google Scholar 

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

    Article  CAS  ADS  Google Scholar 

  20. Ruan, J., Xia, S., Liu, X., Lieberman, J. & Wu, H. Cryo-EM structure of the gasdermin A3 membrane pore. Nature 557, 62–67 (2018).

    Article  CAS  ADS  Google Scholar 

  21. Ghiringhelli, F. et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β-dependent adaptive immunity against tumors. Nat. Med. 15, 1170–1178 (2009).

    Article  CAS  Google Scholar 

  22. Lee, P. H. et al. Host conditioning with IL-1β improves the antitumor function of adoptively transferred T cells. J. Exp. Med. 216, 2619–2634 (2019).

    Article  CAS  Google Scholar 

  23. Zou, W., Wolchok, J. D. & Chen, L. PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: mechanisms, response biomarkers, and combinations. Sci. Transl. Med. 8, 328rv4 (2016).

    Article  Google Scholar 

  24. Ribas, A. & Wolchok, J. D. Cancer immunotherapy using checkpoint blockade. Science 359, 1350–1355 (2018).

    Article  CAS  ADS  Google Scholar 

  25. Sharma, P. & Allison, J. P. The future of immune checkpoint therapy. Science 348, 56–61 (2015).

    Article  CAS  ADS  Google Scholar 

  26. Schwartz, H. S. & Grindey, G. B. Adriamycin and daunorubicin: a comparison of antitumor activities and tissue uptake in mice following immunosuppression. Cancer Res. 33, 1837–1844 (1973).

    CAS  PubMed  Google Scholar 

  27. Casares, N. et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J. Exp. Med. 202, 1691–1701 (2005).

    Article  CAS  Google Scholar 

  28. Tu, J., Xu, M., Parvez, S., Peterson, R. T. & Franzini, R. M. Bioorthogonal removal of 3-isocyanopropyl groups enables the controlled release of fluorophores and drugs in vivo. J. Am. Chem. Soc. 140, 8410–8414 (2018).

    Article  CAS  Google Scholar 

  29. Rossin, R. et al. Chemically triggered drug release from an antibody–drug conjugate leads to potent antitumour activity in mice. Nat. Commun. 9, 1484 (2018).

    Article  ADS  Google Scholar 

  30. Zheng, Y. et al. Enrichment-triggered prodrug activation demonstrated through mitochondria-targeted delivery of doxorubicin and carbon monoxide. Nat. Chem. 10, 787–794 (2018).

    Article  CAS  Google Scholar 

  31. Lyon, R. P. et al. Reducing hydrophobicity of homogeneous antibody–drug conjugates improves pharmacokinetics and therapeutic index. Nat. Biotechnol. 33, 733–735 (2015).

    Article  CAS  Google Scholar 

  32. Liu, Z. et al. One-step 18F labeling of biomolecules using organotrifluoroborates. Nat. Protocols 10, 1423–1432 (2015).

    Article  CAS  Google Scholar 

  33. Cheng, Y. et al. Highly efficient drug delivery with gold nanoparticle vectors for in vivo photodynamic therapy of cancer. J. Am. Chem. Soc. 130, 10643–10647 (2008).

    Article  CAS  Google Scholar 

  34. Duncan, B., Kim, C. & Rotello, V. M. Gold nanoparticle platforms as drug and biomacromolecule delivery systems. J. Control. Release 148, 122–127 (2010).

    Article  CAS  Google Scholar 

  35. De, M. et al. Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein. Nat. Chem. 1, 461–465 (2009).

    Article  CAS  Google Scholar 

  36. Rana, S., Yeh, Y. C. & Rotello, V. M. Engineering the nanoparticle–protein interface: applications and possibilities. Curr. Opin. Chem. Biol. 14, 828–834 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Sui, Z. Shen, F. Zheng and P. Xu for reagents; J. Chen, F. Wang, X. Jia, Z. Jiang and W. He for technical assistance; M. Shi for the cartoon illustration; and X. Liu, P.R. Chen, T. Luo and W. Wei for advice. The work was supported by NSFC grants U1867209 and 21778003 to Z.L., NSFC Basic Science Center Project (81788104), the National Key Research and Development Program of China (2017YFA0505900 and 2016YFA0501500), Chinese Academy of Medical Sciences Initiative for Innovative Medicine (2019-I2M-5-084) to F.S.

Author information

Authors and Affiliations

Authors

Contributions

Z.L. and F.S. conceived the study; Q.W., assisted by C.W. and X.Z., performed material synthesis, characterization and chemical analysis; Y.W. and Q.W. performed most of the experiments; C.W. performed radiosynthesis and PET imaging; J.D., W.G. and H.H. provided technical assistance and suggestions. Q.W., Y.W., F.S. and Z.L. analysed the data. F.S. and Z.L. wrote the manuscript with input from all authors. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Feng Shao or Zhibo Liu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Dmitri Krysko, Andreas Linkermann and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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

Extended data figures and tables

Extended Data Fig. 1 Phe-BF3 catalyses desilylation of carbamate linkers containing silyl ether, and synthetic routes of related compounds.

a, Chemical structures of silyl-phenolic-ether-conjugated coumarin derivatives. b, Proposed mechanism for Phe-BF3-catalysed desilylation of the silyl ether that triggers decarboxylation on the carbamate and consequent release of the coumarin. c, Synthetic route of TESO–coumarin. TBSO–coumarin and TIPSO–coumarin were synthesized via a similar strategy. d, Synthetic route of the silyl-ether-containing carbamate linker used for nanoparticle conjugation. e, Synthetic route of Phe-BF3-responsive SEC–MMAE for antibody conjugation.

Extended Data Fig. 2 Establishment of bioorthogonal chemistry based on desilylation that can achieve efficient and controlled MMAE release from the trastuzumab–SEC–MMAE antibody–drug conjugate system.

a, Liquid chromatography (LC) assay of cleavage of TBSO–coumarin induced by Phe-BF3 desilylation. Red and blue mark the maximum absorbance wavelength of free coumarin (365 nm) and the mixtures of Phe-BF3 and TBSO–coumarin (254 nm), respectively. b, Assay of possible desilylation of TBSO–coumarin (10 μM) by various biologically relevant nucleophilic anions (5 mM for GSH, 20 mM for H2O2 and 10 mM for others). The fluorescence intensity of coumarin (mean ± s.e.m., n = 4 independent replicates) was determined at λex = 370 nm and λem = 435 nm. c, Determination of the rate constant of desilylation mediated by Phe-BF3, by chromatography assay. Data shown are mean ± s.e.m. n = 3 independent replicates. d, Purity of the trastuzumab–SEC–MMAE conjugate determined by size-exclusion HPLC. e, f, UV–visible (e) and MALDI–TOF (f) analyses reveal that the drug-to-antibody ratio of trastuzumab–SEC–MMAE is approximately 4. g, h, HPLC–QTOF mass spectrometry analysis of MMAE released from trastuzumab–SEC–MMAE after incubation with Phe-BF3 for 3 h (magenta), 15 h (blue), 20 h (brown) or 24 h (orange) in 50% serum. h, Quantification of the released MMAE using calibration curves. Data shown are mean ± s.e.m. n = 3 independent replicates. il, Nu/Nu mice were implanted subcutaneously with human BGC823 cancer cells. i, Dynamic PET computed tomography 3D projection images of mice bearing BGC823 tumours at the indicated time points after intravenous injection of [89Zr]trastuzumab–SEC–MMAE. j, Time–activity curve (TAC) of [89Zr]trastuzumab–SEC–MMAE in the blood, liver and tumour. k, Local concentration of Phe-BF3 and trastuzumab–SEC–MMAE in BGC823 tumours after tail-vein injection of the corresponding agents. l, Ex vivo biodistribution of [18F]Phe-BF3 in mice bearing BGC823 tumours at 75 min after injection. Mean ± s.d., n = 4 mice. Data shown are representative of three (ai) or two (l) independent experiments.

Source Data

Extended Data Fig. 3 Optimization of the silyl-ether-containing carbamate linker for higher sensitivity to Phe-BF3.

a, Schematic of Phe-BF3-catalysed desilylation that can release the ‘caged’ coumarin from a designed silyl-ether-containing carbamate linker. bd, ‘Decaging’ and liquid chromatography assays of Phe-BF3-catalysed desilylation of TESO–coumarin (TESO–C), TBSO–coumarin (TBSO–C) or TIPSO–coumarin (TIPSO–C). b, c, Photographs (b) and quantification (c) of coumarin fluorescence. d, Blue and magenta mark the maximum absorbance wavelength of TESO–coumarin or TBSO–coumarin (254 nm) and free coumarin (365 nm), respectively. e, Summary of the desilylation efficiency of Phe-BF3 and NaF towards TESO–coumarin, TBSO–coumarin or TIPSO–coumarin. f, Fluorescence-emission assay of possible desilylation of TESO–coumarin (10 μM) by various biologically relevant nucleophilic anions (5 mM for GSH, 20 mM for H2O2 and 10 mM for others). The coumarin fluorescence intensity was determined at λex = 370 nm and λem = 435 nm. g, Summary of desilylation efficiency of TESO–coumarin (10 μM) by FDA-approved organofluorines. e, g, Reaction efficiency was determined by HPLC. ND, not detected. Data shown are representative of two (c, f) or three (b, d, e, g) independent experiments.

Source Data

Extended Data Fig. 4 Release of GFP from NP–GFP induced by Phe-BF3 desilylation, and biodistribution of [89Zr]GFP and NP–[89Zr]GFP in mice.

a, Design of Phe-BF3 desilylation of the silyl-ether-carbamate-linked NP–GFP for releasing GFP from the nanoparticle. GFP fluorescence is quenched in NP–GFP; desilylation-induced cleavage of the linker releases the GFP. b, Workflow of assaying release of GFP from NP–GFP induced by Phe-BF3 desilylation in vitro. The samples were subjected to immunoblotting in c and Fig. 2a. c, Comparison of desilylation-induced release of GFP from TESO- or TBSO-linked NP–GFP by Phe-BF3 and NaF. The loading control (*) was sampled before centrifugation. d, Expression of LAT1 transporter in the four cell types assayed in Extended Data Fig. 4e. e, Assays of release of GFP from NP–GFP induced by Phe-BF3 desilylation in cells. HeLa, EMT6 or 4T1 cells, or primary BMDMs, were treated with NP–GFP (1 mg ml−1) for 24 h and then with Phe-BF3 (100 μM) or NaF for another 24 h. Centrifuged lysates were subjected to anti-GFP and anti-GAPDH immunoblotting. f, g, BALB/c mice were implanted subcutaneously with 4T1 cancer cells. f, Representative PET computed tomography 3D projection images of mice bearing 4T1 tumours at 1, 6, 12 and 18 h after intravenous injection of [89Zr]GFP or NP–[89Zr]GFP. g, Time–activity curve of [89Zr]GFP and NP–[89Zr]GFP in the blood, liver and tumour of mice. h, Representative confocal images of HeLa cells transfected with a plasmid expressing mNeonGreen–NLS. Scale bars, 20 μm. i, Assay of release of mNeonGreen–NLS from the NP–mNeonGreen–NLS conjugates induced by Phe-BF3 desilylation in mice bearing 4T1 tumours. Representative confocal images of tumour sections are shown. Scale bars, 20 μm. Image in the box is magnified in Fig. 2d. Data shown in ci are representative of two independent experiments.

Source Data

Extended Data Fig. 5 Phe-BF3 desilylation of NP–GSDMA3 releases the gasdermin N domain that is capable of forming pores and inducing pyroptosis.

a, b, Preparation of the GSDMA3(N + C) used for conjugation onto the nanoparticle. Purified GSDMA3 containing a engineered PPase cleavage site between the gasdermin N and C domain was cleaved in vitro to obtain GSDMA3(N + C). a, Coomassie blue staining of the prepared GSDMA3 proteins. b, ATP-based viability of CT26 cells electroporated with the prepared GSDMA3 protein. Mean ± s.d., n = 3 independent replicates. c, d, Pore-forming activity of the gasdermin N domain released from NP–GSDMA3 by desilylation catalysed by Phe-BF3. c, Representative negative-stain electron microscopy images of the gasdermin pores formed by purified native GSDMA3(N + C) protein or the GSDMA3(N + C) protein released from NP–GSDMA3. Scale bars, 100 nm. d, Representative 2D averages of the gasdermin pores viewed from the top (left) and the side (right). Scale bar, 10 nm. e, f, HeLa or EMT6 cells were treated with NP–GSDMA3 alone or in combination with Phe-BF3. e, Total cell lysates or their membrane fractions were immunoblotted with indicated antibodies. Flag–GSDMA3(N + C) proteins (the Flag tag was fused C-terminal to the gasdermin N domain, before the PPase cleavage site) were conjugated to the nanoparticle. The GSDMA3 N domain was probed by anti-Flag immunoblotting. f, LDH assay of the effect of various inhibitors on cell death induced by treatment with NP–GSDMA3 and Phe-BF3. Mean ± s.d., n = 3 independent replicates. Fer1, ferrostatin 1; Nec-1, necrostatin 1. g, HeLa, EMT6 or 4T1 cells, or primary BMDMs, were treated as indicated. NP + GSDMA3, nanoparticles mixed with the noncovalent GSDMA3(N + C) complex. GSDMA3(Mut), the pore forming-deficient E14K/L184D mutant version of GSDMA3(N + C). Flow-cytometry plots of PI- and annexin V–-FITC-stained cells are shown. h, Effect of phagocytosis inhibitor cytochalasin D on cell death induced by treatment with NP–GSDMA3 and Phe-BF3 in primary BMDMs. Flow-cytometry plots of PI- and annexin V–Alexa-647-stained cells are shown. Data are representative of two (c–f, h) or three (a, b, g) independent experiments.

Source Data

Extended Data Fig. 6 Biodistribution and cytodistribution of NP–[89Zr]GSDMA3, and effect of treatment with NP–GSDMA3 and Phe-BF3 on mice bearing 4T1 tumours.

ae, BALB/c mice were implanted subcutaneously with 4T1 cancer cells, and intravenously injected with [89Zr]GSDMA3 or NP–[89Zr]GSDMA3. a, b, PET computed tomography 3D projection images of mice bearing 4T1 tumours at the indicated time points after injection of [89Zr]GSDMA3 or NP–[89Zr]GSDMA3. c, Time–activity curve of NP–[89Zr]GSDMA3 in the blood, liver and tumour of injected mice. d, Systemic biodistribution of NP–[89Zr]GSDMA3 in mouse tissues. Data shown are mean ± s.e.m. n = 4 mice. e, Representative transmission electron microscopy images showing the cytodistribution of NP–GSDMA3 in the 4T1 tumour tissue. Nu, nucleus; M, mitochondrion; NP, nanoparticle; RBC, red blood cell. Scale bars, 5 μm (A1), 500 nm (A2–A4). f, g, Immunoblotting assay of endogenous IL-1β, GSDMD and GSDME expression. f, 4T1 or MH-S cells were stimulated with LPS (2 μg ml−1) for 12 h and the cell lysates were subjected to anti-IL-1β or anti-tubulin immunoblotting. g, Lysates of EMT6 and 4T1 cells were blotted with anti-GSDMD, anti-GSDME or anti-tubulin antibodies. h, i, Mice bearing 4T1 tumours were treated with NP–GSDMA3 and Phe-BF3, as depicted in Fig. 4b (n = 7 mice for each group). Average tumour volumes of each group of mice at the indicated time points after the injection are shown as mean ± s.e.m. (in h) with P values in i (two-tailed unpaired Student’s t-test). j, Propidium iodide staining of tumour-cell pyroptosis induced by treatment with NP–GSDMA3 and Phe-BF3 in mice. An independent experiment from that shown in Fig. 5a. Scale bars, 100 μm. All data shown are representative of two (ag, j) or three (h, i) independent experiments.

Source Data

Extended Data Fig. 7 Evaluation of the possible side effects of treatment with NP–GSDMA3 and Phe-BF3 in mice.

a, Records of mouse body weight after the indicated treatments. Data shown are mean ± s.e.m. n = 8 mice for PBS, NP–GSDMA3, Phe-BF3 and intratumourally injected NP–GSDMA3 + Phe-BF3, 9 mice for NP–GSDMA3 + Phe-BF3 and NP–GSDMA3(mut) + Phe-BF3. b, Representative haematoxylin and eosin (H & E) staining of liver and kidney tissues from mice with indicated treatments. Three mice per group. ce, Assay of serum concentrations of alkaline phosphatase, calcium and phosphate in mice treated with PBS (n = 4 mice) or Phe-BF3 (n = 5 mice). Data shown are mean ± s.e.m. Two-tailed unpaired Student’s t test. f, Normal-resolution (left) and high-resolution (right) computed tomography scans of the joint from mice treated with Phe-BF3 or PBS. For the normal-resolution scan, a 3D reconstruction image of the whole joint is shown on the left; representative 2D images of the sagittal plane (top) and axial plane near the femur growth plate (bottom) are shown on the right. For the high-resolution computed tomography scan that images a voxel near the femur growth plate, the whole voxel is shown on the left and representative images of the sagittal plane (top) and the axial plane (bottom) are shown on the right. Data are representative of three (a) or two (bf) independent experiments.

Source Data

Extended Data Fig. 8 Pyroptosis induced by NP–GSDMA3 and Phe-BF3 stimulates inflammation and increases the tumour-infiltrating lymphocytes.

a, d, e, Gating strategy (a) and representative flow-cytometry plots for assessing 4T1 tumour-infiltrating CD3+ T cells (d) or FOXP3+CD4+ regulatory T cells (e) following the indicated treatments. b, c, Representative fluorescence images of CD3–PE-stained 4T1 tumours following treatment with PBS (n = 4 mice), NP–GSDMA3 and Phe-BF3 (n = 4 mice) or NP–GSDMA3(mut) and Phe-BF3 (n = 6 mice). Scale bars, 200 μm (b), 50 μm (c, left), 20 μm (c, right). f, Flow-cytometry analysis of CD4+ or CD8+ T cells upon depletion by their corresponding antibody. Data shown are representative of two (b, c, f) or three (a, d, e) independent experiments.

Extended Data Fig. 9 Tumour-infiltrating immune-cell-subtype analysis by single-cell RNA sequencing.

a, Gating strategy and representative flow-cytometry plots for the enrichment of 4T1 tumour-infiltrating single CD45+ immune cells. b, Heat map of ten immune-cell clusters with unique signature genes. Colours on top of the map indicate the immune-cell clusters. The three or four marker genes used for each cluster are listed alongside the cluster. c, Signature gene-expression patterns for the corresponding cell clusters on the t-SNE plot (n = 4, 18,069 cells). d, t-SNE plots of tumour-infiltrating single CD45+ immune cells of 4T1 tumours from mice treated with PBS (n = 2 mice, 10,171 cells) or NP–GSDMA3 and Phe-BF3 (n = 2 mice, 7,898 cells) (left), and the relative frequencies of different clusters (right). eg, Expression levels of protumoural and immunosuppressive genes (e), proinflammatory chemokine (f) and T and/or natural killer cell activation or effector (g) genes in immune cells. Paired quantile–quantile (QQ) plots were used to compare the gene-expression levels in CD45+ immune cells between 4T1 tumours treated with PBS or NP–GSDMA3 and Phe-BF3. P values were calculated using a two-sided Wilcoxon rank-sum test. All data are representative of two independent experiments.

Extended Data Fig. 10 Antitumour immunity activated by NP–GSDMA3 and Phe-BF3 requires IL-1β, and can synergize with the anti-PD1 therapy.

a, Enzyme-linked immunosorbent assay measurements of IL-1β, IL-18 and HMGB1 concentration in the serum (top) and the tumour homogenates (bottom) of mice treated with PBS, NP–GSDMA3 and Phe-BF3 or NP–GSDMA3(mut) and Phe-BF3 (n = 5 or 7, as shown in the figure for each group). b, Anti-IL-1β, IL-18 or HMGB1 antibodies were intraperitoneally injected into mice bearing 4T1 tumours, before treatment with NP–GSDMA3 and Phe-BF3 (n = 7 mice each group). Mice bearing 4T1 tumours were also treated with PBS alone as the control group (n = 8). Average tumour volumes at the indicated time points after implantation are shown. c, Mice bearing 4T1 tumours were treated with PBS (n = 7 mice), anti-PD1 (n = 8 mice) or NP–GSDMA3 and Phe-BF3 (n = 7 mice) alone, or in combination (n = 8 mice) as shown in Fig. 5i. All data are shown as mean ± s.e.m. (two-tailed unpaired Student’s t-test) and are representative of two independent experiments.

Source Data

Supplementary information

Supplementary Information

This file contains Supplementary Methods, Supplementary Figures 1-28 and figures of the uncropped immunoblots for key data presented in the main text and extended data section of the manuscript.

Reporting Summary

41586_2020_2079_MOESM3_ESM.mp4

Video 1: NP_GA3 accesses the cytosol through the endocytosis pathway. mCherry-tagged human Galectin-3 (mCherry-Gal3) was stably expressed in HeLa cells. The cells were left untreated (Video 1) or treated with NP_GA3 (Video 2) for 1 h and then recorded for about 1.5 h (the exact time duration, h:min:s:ms). Formation of mCherry-Gal3 foci indicates the damage of the endosome containing the NP_GA3. The video was taken using a PerkinElmer UltraVIEW spinning disk confocal microscope and processed using Volocity software. Shown is the video of a representative field in each experimental group. All data are representative of two independent experime.

41586_2020_2079_MOESM4_ESM.mp4

Video 2: NP_GA3 accesses the cytosol through the endocytosis pathway. mCherry-tagged human Galectin-3 (mCherry-Gal3) was stably expressed in HeLa cells. The cells were left untreated (Video 1) or treated with NP_GA3 (Video 2) for 1 h and then recorded for about 1.5 h (the exact time duration, h:min:s:ms). Formation of mCherry-Gal3 foci indicates the damage of the endosome containing the NP_GA3. The video was taken using a PerkinElmer UltraVIEW spinning disk confocal microscope and processed using Volocity software. Shown is the video of a representative field in each experimental group. All data are representative of two independent experiments.

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, Q., Wang, Y., Ding, J. et al. A bioorthogonal system reveals antitumour immune function of pyroptosis. Nature 579, 421–426 (2020). https://doi.org/10.1038/s41586-020-2079-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-020-2079-1

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer