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Ferroptosis of tumour neutrophils causes immune suppression in cancer

Nature volume 612, pages 338–346 (2022)Cite this article

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Abstract

Ferroptosis is a non-apoptotic form of regulated cell death that is triggered by the discoordination of regulatory redox mechanisms culminating in massive peroxidation of polyunsaturated phospholipids. Ferroptosis inducers have shown considerable effectiveness in killing tumour cells in vitro, yet there has been no obvious success in experimental animal models, with the notable exception of immunodeficient mice1,2. This suggests that the effect of ferroptosis on immune cells remains poorly understood. Pathologically activated neutrophils (PMNs), termed myeloid-derived suppressor cells (PMN-MDSCs), are major negative regulators of anti-tumour immunity3,4,5. Here we found that PMN-MDSCs in the tumour microenvironment spontaneously die by ferroptosis. Although decreasing the presence of PMN-MDSCs, ferroptosis induces the release of oxygenated lipids and limits the activity of human and mouse T cells. In immunocompetent mice, genetic and pharmacological inhibition of ferroptosis abrogates suppressive activity of PMN-MDSCs, reduces tumour progression and synergizes with immune checkpoint blockade to suppress the tumour growth. By contrast, induction of ferroptosis in immunocompetent mice promotes tumour growth. Thus, ferroptosis is a unique and targetable immunosuppressive mechanism of PMN-MDSCs in the tumour microenvironment that can be pharmacologically modulated to limit tumour progression.

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Fig. 1: Ferroptosis in tumour-associated PMN-MDSC.
Fig. 2: Ferroptosis confers immunosuppressive activity to PMNs.
Fig. 3: Mechanisms of ferroptosis induction in tumour PMN-MDSCs.
Fig. 4: Antitumour effect of ferroptosis inhibition.

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Data availability

All data are available and all figures are supplied with the raw data. RNA-seq data were deposited to the GEO under accession number GSE205371; and scRNA-seq data to the GEO under accession number GSE213861. Metabalomic data were submitted to Metabolomics Workbench (http://dev.metabolomicsworkbench.org:22222/data/DRCCMetadata.php?Mode=Study&StudyID=ST002160&Access=MvrS7201; https://doi.org/10.21228/M87Q56). All data are publicly available. Source data are provided with this paper.

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Acknowledgements

We thank the staff at AstraZeneca Production Informatics and Tempus teams for providing the RNA-seq and clinical datasets; N. Barkas and C. Rand for assistance in performing scRNA-seq experiments; and E. Bonner for technical assistance. This work was supported by National Institute of Health grant AI156924 (to V.E.K.); National Institute of Health grant CA243142 (to V.E.K.); National Institute of Health grant CA165065 (to V.E.K.); National Institute of Health grant 2T32DK007780-21 (to R.K.); National Institute of Health grant R01 CA266342 (to Y.N. and V.E.K.); The University of Pennsylvania Biomedical Graduate Studies Program (to R.K.); The Wistar Institute Animal and Flow Cytometry Core facilities under Cancer Center Support Grant P30 CA010815 (to Y.N.); National Institute of Health grant P30-CA016520 (to R.H.V.); National Institute of Health grant R01-CA-229803-01 (to R.H.V.); National Institute of Health grant P30DK046200 (to A.G.); and National Institute of Health grant DK108722 (to A.G.).

Author information

Author notes

  1. Nune Markosyan & Robert H. Vonderheide

    Present address: Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

  2. Mohit Sehgal

    Present address: Center of Cell and Gene Therapy, Biopharma Division, Intas Pharmaceuticals, Ahmedabad, India

  3. These authors contributed equally: Rina Kim, Ayumi Hashimoto

Authors and Affiliations

  1. Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    Rina Kim, Nune Markosyan, Bereket A. Gebregziabher & Robert H. Vonderheide

  2. Wistar Institute, Philadelphia, PA, USA

    Rina Kim, Shuyu Fu, Mohit Sehgal, Laura Garcia-Gerique, Andrew Kossenkov, Hui Deng, Laxminarasimha Donthireddy & Yulia Nefedova

  3. Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Rina Kim & Robert H. Vonderheide

  4. Early Oncology R&D, ICC, AstraZeneca, Gaithersburg, MD, USA

    Ayumi Hashimoto, Kristin Hicks & Dmitry I. Gabrilovich

  5. Center for Free Radical and Antioxidant Health, Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA, USA

    Vladimir A. Tyurin, Yulia Y. Tyurina & Valerian E. Kagan

  6. Oncology R&D, Research and Early Development, Translational Medicine, AstraZeneca, Cambridge, UK

    Gozde Kar

  7. Penn Genomic Analysis Core, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    John W. Tobias

  8. Early Oncology R&D, AstraZeneca, Gaithersburg, MD, USA

    Rebecca A. Halpin

  9. Oncology Data Science, AstraZeneca, Gaithersburg, MD, USA

    Nevena Cvetesic

  10. Human Nutrition Research Center, Tufts University, Boston, MA, USA

    Andrew Greenberg

  11. Helen F. Graham Cancer Center and Research Institute, Christiana Care, Newark, DE, USA

    Brian Nam

  12. Parker Institute for Cancer Immunotherapy, University of Pennsylvania, Philadelphia, PA, USA

    Robert H. Vonderheide

  13. Department of Chemistry, University of Pittsburgh, Pittsburgh, PA, USA

    Valerian E. Kagan

  14. Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, USA

    Valerian E. Kagan

  15. Department of Radiation Oncology, University of Pittsburgh, Pittsburgh, PA, USA

    Valerian E. Kagan

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Contributions

Conceptualization and experimental design: D.I.G. Methodology: V.E.K. and A.G. Investigation: R.K. (in vitro and in vivo experiments), A.H. (in vitro and in vivo experiments, experiments with human samples), N.M. (in vivo experiments), M.S. (experiments of sensitivity of ferroptosis in vitro), L.D. (in vivo experiments), K.H. (in vitro experiments), V.A.T. (evaluation of lipids), Y.Y.T. (evaluation of lipids), L.G.-G. (experiments with human samples), S.F. (studies of macrophages), J.W.T. (gene expression data analysis), B.A.G. (gene expression data analysis), G.K. (gene expression data analysis), A.K. (gene expression data analysis), H.D. (in vitro experiments), R.A.H. (gene expression experiments) and N.C. (gene expression experiments). Resources: A.G. and B.N. Funding acquisition: V.E.K., R.H.V. and Y.N. Supervision: D.I.G., R.H.V., V.E.K. and Y.N. Writing—original draft: R.K., A.H., N.M., V.E.K. and D.I.G. Writing—review and editing: R.H.V., Y.N., V.E.K. and D.I.G.

Corresponding author

Correspondence to Dmitry I. Gabrilovich.

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A.H., G.K., K.H., R.A.H., N.C. and D.I.G. are employees and stakeholders of AstraZeneca.

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Extended data figures and tables

Extended Data Fig. 1 Gene expression profile of mouse and human PMN-MDSC.

a. RNA seq of PMN-MDSC from matched blood (TBB) and tumour tissue (TBT) of lung cancer patients and peripheral blood of healthy donors (HD). Colour key represents the normalized Z score. b. RNA seq of sorted CD14high and CD14− PMN-MDSC from EL-4 TB mice. Colour key represents the normalized Z score. c. Ferroptosis gene signature used in further studies. P values between tumour and blood PMN samples were calculated in two-sided unpaired Student’s t-test. d. Signature score of 8 ferroptosis genes in indicated populations of cells by single cell RNAseq. Data were captured from (22). Analysis was performed using BBrowser (https://bioturing.com/). T– tumour, B – blood.

Extended Data Fig. 2 Expression of ferroptosis related genes.

a. Expression of ferroptosis related genes in PMN from blood and tumour of cancer patients. qRT-PCR performed in CD11b+CD14−CD66b+ PMN isolated from blood (PB, n = 9) and tumours (Tumour, n = 6) of cancer patients. Ct value of housekeeping gene was subtracted from Ct value of gene of interest for each samples (dCt). The mean of dCt for PB group was calculated. Each PB and tumour sample was normalized to this value. P-values are shown in the graph (Two-sided unpaired Mann-Whitney test), non-significant p values are not shown. b. Expression of ferroptosis related genes in PMN-MDSC from indicated tumour models. qRT-PCR performed in CD11b+L6ClowLy6G+ PMN-MDSC isolated from indicated tissues from implanted (EL4, CT26, and LLC) and autochthonous (KPC) tumour bearing mice. Mean ± SEM are shown. P values were calculated in one way ANOVA with Tukey’s test for correction for multiple comparisons. * p < 0.0001. Other p values are shown on graphs. N = 5 for EL4 model; n = 3 for CT26 model; n = 4 for LLC model; n = 6 for KPC model.

Source data

Extended Data Fig. 3 Ferroptosis in PMN-MDSCS and M-MDSC.

a. Content of oxygenated PE species PE(36:4-OOH) and PE(36:4-OH-OOH) in M-MDSC isolated from spleen and tumour of LLC TB mice. b. Viability of M-MDSC isolated from BM, spleen, and tumour of implanted EL4 and CT26 TB mice and treated with RSL3. Viability of the cells assessed by cell counts and expressed as a percent of untreated cell counts. c. CD71 expression in BM PMN after treatment with DMSO or various inducers of cell death for 18 h: 20µM RSL3 for ferroptosis, 1 µM Shikonin for necroptosis, and 0.25 µM Staurosporine for apoptosis. Mean ± SD are shown. *P < 0.05, in one-way ANOVA. d. CD71 expression in tumour and spleen PMN-MDSC and M-MDSC. e. BM PMN were treated with DMSO or 20 μM RSL3 for 4 h, washed extensively and proportion of live cells was counted by trypan blue exclusion method. The washed cells were then incubated in fresh media at for additional 16 h, and the proportion of viable cells was counted. Proportion of viable cells was calculated based on the number of the DMSO-treated cells. N = 4. P values calculated in two-sided Student’s t-test (a, d middle panels), or One-way ANOVA with Tukey’s HSD post-hoc test (b,c,d) and are shown on graphs. * p < 0.0001. In all panels, mean ± SD are shown. f. Expression of Alox12/15 by qRT-PCR in PMN from Alox12/15flCre− and Alox12/15flCre+ mice. N = 3. g. BM PMN-MDSC isolated from the LLC tumour bearing Alox12/15flCre− and Alox12/15flCre+ mice (n = 6 per group) were treated with 0–10 µM RSL3 for 4 h, and then washed 3 times and followed by further 16 h incubation. Cell number was determined by trypan blue exclusion method. h. BM PMN-MDSC from the LLC TB mice were treated with IKE for 6 h, and then washed 3 times and followed by further 16 h incubation; cell numbers determined by trypan blue exclusion (n = 4). i. PMN cell counts by supernatant generated from BM PMN isolated from WT mice and treated with indicated inhibitors or IKE after pre-treatment with inhibitors (n = 3/group). Representative experiment of four shown. j. BM PMN of EL-4 TB mice were cultured with 10 ng/mL GM-CSF and tumour explant supernatant for 24 h in normoxia or hypoxia (0.3% O2) with or without 1 µM liproxstatin-1 (Lip-1) and then cocultured with CellTrace-labelled PMEL splenocytes in indicated ratios, in the presence ofcognate peptide (n = 3/group). T cell proliferation was evaluated as above. Two experiments with the same results were performed. One-way ANOVA with Tukey’s HSD post-test performed. P values are shown on graphs. k. Suppression of T cell proliferation by supernatants of M-MDSC isolated from EL4 tumours of Alox12/15flCre− and Alox12/15flCre+ mice (n = 6/group). T cell proliferation was determined by flow cytometry as CellTrace dye dilution in CD3+CD8+ cells and expressed as a percent of CD8+ T cells stimulated in the absence of supernatants, l, m. Effect of ACSL4 deletion on suppressive activity of PMN-MDSC. l. Expression of Acsl4 in PMN from Acsl4flCre+ and Acsl4flCre− mice. N = 4. m. Suppression of T cell proliferation by supernatants of PMN-MDSC isolated from tumours and spleens of Acsl4flCre− and Acsl4flCre+ EL4-TB mice. T cell proliferation was determined by flow cytometry as CellTrace dye dilution in CD3+CD8+ cells and expressed as a percent of CD8+ T cells stimulated in the absence of supernatant. N = 4. Mean ± SD are shown. P values were calculated in unpaired two-sided Student’s t-test and shown on graphs. *p < 0.0001.

Source data

Extended Data Fig. 4 Gene changes induced by ferroptosis induction.

a. Expression of ferroptosis related genes measured by qRT-PCR in human BM PMN treated with RSL3. Mean ± SEM shown. P values on the graphs were calculated in unpaired two-sided Mann-Whitney test. N = 8, except HMOX1 where n = 7. b. RNA seq data of mice BM PMN treated for 4 h with DMSO (C), 100 nM staurosporine (S4) or 20 uM RSL3 (R4). Colour key represents the normalized Z score. Table shows functional attributes of the genes. c. Pathways/function changed in RSL3 treated BM PMN vs. control (DMSO) and staurosporine treated PMN; Z-score was calculated by Ingenuity Pathway Analysis where the z-score statistic is based on binomial disturution. http://pages.ingenuity.com/rs/ingenuity/images/0812%20downstream_effects_analysis_whitepaper.pdf. Only pathways that were different between groups with p < 0.01 adjusted for multiple comparisons are shown. d. Expression of indicated genes in DMSO or 20 uM RSL3 treated PMN measured by qRT-PCR. N = 4. e. Concentration of released PGE2 levels by ELISA, in the supernatants of DMSO or 20uM RSL3 treated PMN after 8 h. N = 4. Mean ± SD shown in d,e. P values shown on graphs were calculated in unpaired two-sided Student’s t-test. *p < 0.0001.

Source data

Extended Data Fig. 5 Effect of ALOX12/15 deletion on gene expression and metabolome of tumour PMN-MDSC.

a. GSEA enrichment analysis of ALOX12/15 KO vs control PMN-MDSC. NES, normalized enrichment score. P-value was calculated by GSEA based on permutation analysis. b, c. Transcription changes in Alox12/15 deficient PMN-MDSC. b. Top 25 up and downregulated genes. c. Gene set enrichment analysis using GSEA. NES score is shown. d. Metabolome of control and ALOX12/15 deficient tumour PMN-MDSC was evaluated by LC-MS. Ingenuity Pathway Analysis (IPA) was performed on differentially expressed metabolites - metabolites names were mapped to KEGG identifiers and given as input to IPA with default settings for metabolomics analysis. Pathways significantly up-regulated in ALOX12/15 deficient PMN-MDSC are shown. Changes considered significant if fold change > 2 converted from Log2 ratio and Benjamini-Hochberg q-value corrected for multiple testing < 0.05.

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Extended Data Fig. 6 Ferroptosis in genetically engeneered mice.

a. Ferroptosis related genes in PMN-MDSC from Fatp2flCre+ TB mice. qPCR of tumour PMN-MDSCs from Fatp2flCre− (WT) and Fatp2flCre+ (FATP2 KO) mice. N = 3. Mean ± SD are shown. P values shown on graphs were calculated in unpaired two-sided Student’s t-test. b. Oxidized PE in ALOX12/15 deficient mice PMN-MDSC. Content of oxygenated (PE(38:4+1[O]) PE(38:4+2[O]) PE(36:4+2[O]) and non-oxygenated (PE(38:4) PE species in PMN MDSC isolated from LLC TB Alox12/15flCre− and Alox12/15flCre− mice tumours. N = 3. Mean ± SD are shown. P values shown on graphs were calculated in unpaired two-sided Student’s t-test. c. Content of PE species containing oxygenated AA in PMN MDSC isolated from LLC TB WT and MPO KO mice. N = 3. d. Liproxstatin-1 inhibits MPO (0.05U)/H2O2(50 uM) induced formation of C18:0-Cl (left panel), PE-18:0p/20:4+3[O] (middle panel) and LPE(20:4+3[O]) (right panel) from PE(18:0p/20:4) in 20 mM phosphate buffer containing 100 mM NaCl and 100 uM DTPA, pH = 7.4, after 30 min incubation at 37 °C. The structure of PGE2 containing PE(18:0p/20+3[O]) was verified by three criteria (retention time (34 min), exact mass (m/z 798.5306 + 3.3 ppm) and MS/MS fragments (m/z 798→351, 271, 189, 113). LPE(20:4+3[O]) was identified as PGE2 containing species by exact mass (m/z 548.2636 + 3.4 ppm) and retention time (6 min). N = 4. Mean ± SD are shown. P values shown on graphs were calculated in unpaired two-sided Student’s t-test (a) or one-way ANOVA with correction for multiple comparsons.

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Extended Data Fig. 7 Ferroptosis induced PMN suppression is abrogated by inhibition of MPO.

a. BM PMNs were treated with 5 µM of MPO inhibitor (iMPO, 4-Aminobenzoic Acid Hydrazide, Cayman Chemical) for an hour followed by 6 h treatment with 40 µM IKE. The cells were washed 3 times then incubated with fresh media for 16 h. Supernatant was used for T cell proliferation assay. PMEL splenocytes were labelled with CellTrace dye and stimulated with cognate peptide in the presence of 50% of supernatant for 48 h. Left panel - T cell proliferation measured by flow cytometry N = 3. Four separate experiments are shown. Right panel – number of cells recovered after the incubation with IKE. N = 4. Mean ± SD are shown. P values shown on graphs were calculated by one-way ANOVA with correction for multiple comparisons. * p < 0.0001. b, c. GPX4 expression in tumour PMN-MDSC. b. GPX4 protein expression by Western blotting, in PMN-MDSC from BM, spleen and tumour of CT26 and EL4 TB mice. Results from individual mice are shown. c. GPX4 protein expression in BM PMN treated with increasing concentrations of supernatants obtained from tumour explants (TES) maintained under normoxic or hypoxic conditions. Three experients witt the same results were performed. For gel source data, see Supplementary Fig. 1. d–f. Regulation of ferroptosis and suppressive activity in TAM by PMN-MDSC. TAM (CD11b+Ly6G− Ly6Clow F4/80+) were sorted from EL4 tumour. d. Contents of oxygenated phospholipid ferroptotic cell death signals, PE(38:4+2[O]) and PE (38:5+2[O]), in TAM isolated from WT, ALOX12/15 KO and MPO KO mice. n = 4, p values were calculated in unpaired two-sided Student’s t-test, * - p < 0.05, ** - p < 0.01, *** - p < 0.001. e. PGE2 contents in TAM from WT and ALOX KO mice (left panel) and WT and MPO KO mice (right panel). n = 4, Mean ± SD are shown. P values were calculated in unpaired two-sided Student’s t-test. f. TAM isolated from S100A8-cre x ALOX12/15fl mice and their littermate controls were co-cultured with CellTrace-labelled OT-1 splenocytes in the presence of 0.025 ng/mL SIINFEKL peptide. T cell proliferation was analysed by flow cytometry after 2 days incubation. Proliferation of T cells in the absence of TAM in each experiment was set as 100%. Mean ± SD are shown. P values were calculated in two-sided unpaired Student’s t-test. (n = 4).

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Extended Data Fig. 8 Effect of liproxstatin-1 treatment on PMN-MDSC in vivo.

a. Tumour growth curve of DMSO and 15 mg/kg liproxstatin-1 treated EL-4 and LLC TB mice (n = 4/group). b. Suppression of T cell proliferation by PMN-MDSC isolated from tumours of EL-4 or LLC TB mice treated with liproxstatin-1. T cell proliferation was determined by flow cytometry as CellTrace dye dilution in CD3+CD8+ cells and expressed as a percent of CD8+ T cells stimulated in the absence of PMN-MDSC. N = 4 DMSO group, n = 7 Liproxstatin-1 group. c. PGE2 amount in supernatants after 3 h incubation of PMN-MDSC isolated from CT-26 TB mice treated with DMSO or liproxstatin-1 mice for 8 days. N = 8 d. Numbers of PMN (red staining, some stained cells indicated by white aroowheads) and T cells (green staining, some stained cells indicated by white astersks) by IF in tumours from DMSO and Liproxstatin−1 treated TB mice. N = 5. Mean ± SEM shown in a and d, and mean ± SD in b-c. Unpaired, two-sided Stident’s t test was used in b and c, and two-sided Mann-Whitney test in d. e. Effect of PMN depletion on antitumour activity of liproxstatin-1. CT26 tumour cells were implanted subcutaneously into Balb/c mice. Mice were treated with DMSO or 15 mg/kg Liproxstatin-1 with or without Ly6G depletion starting from day 10 post inoculation (DMSO and Lirpoxstatin-1 groups n = 10; DMSO Ly6G and Lyproxstatin-1 Ly6G groups n = 8). Ly6G depletion was initiated 7 days post inoculation with 200µg/mouse anti-Ly6G (1A8) and 50µg/mouse anti-rat kappa light chain (MAR 18.5) every 3 days. Mean ± SD are shown. P values were calculated in two-way ANOVA.

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Extended Data Fig. 9 Effect of IKE treatment on tumour growth and phenotype of T cells in liproxstatin-1 treated mice.

a. EG7 tumour growth in C57BL/6 mice treated with IKE (n = 5/group). b. LC/MS quantitative assessment of ferroptotic cell death signals (PE(18:0/20:4-OOH), PE(18:1-20:4-OOH) and PE(18:0/22:4-OOH) in tumours from IKE treated CT26 TB mice. N = 7 in DMSO group; N = 8 in IKE group. Mean ± SEM shown in a and mean ± SD in b. P were determined by unpaired two-sided Student’s t-test. c,d. Phenotype of T cells in liproxstatin-1 treated mice. Flow cytometric analysis of percentages of subsets of T cells in CT26 TB mice c. lymph nodes and d. tumours 8 days after treatment with DMSO or liproxstatin-1. (n = 10–20/group). Mean ± SD are shown. P values calculated in two-sided unpaired Student’s t test. Tem – effector memory T cells, Tcm – central memory T cell, and Trm-tissue resident memory T cells.

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Extended Data Fig. 10 Single cell RNAseq of tumour tissues.

CT26 TB mice were treated with liproxstatin-1 for 8 days. Tumours were collected and analysed by scRNAseq. a. UMAP visualization of cell populations; b. Gene set enrichment analysis of differentially expressed genes at adjusted p value < 0.01 for liproxstatin-1 treated vs DMSO treated CD4+ and CD8+ T cells. Enrichment p-values were calculated as described in fgsea R package and p-values were adjusted using Benjamini-Hochberg method.

Extended Data Fig. 11 Induction of ferroptosis in human PMN.

a. qRT-PCR measurements of ferroptosis related genes in DMSO or 20uM RSL3 treated PMN isolated from peripheral blood of healthy individuals. N = 8. b. PGE2 levels measured by ELISA in supernatants of DMSO or 20uM RSL3 treated PMN isolated from peripheral blood of healthy individuals. N = 8 c. PGE2 levels measured by ELISA in supernatants of PMN-MDSC isolated from peripheral blood and tumours of patients with NSCLC. N = 4. Mean ± SD are shown. P values were determined by unpaired two-sided Student’s t-test.

Extended Data Fig. 12 Correlation of ferroptosis signature with PMN-MDSC signature and clinical outcome in cancer patients.

The data were obtained from TCGA database. a. Correlation between ferroptosis and PMN-MDSC signatures in patients with various tumours. Spearman’s correlation coefficient and associated probability (p value) was calculated. b. Association of ferroptosis signature and clinical outcome. Patient survival in top, mid, and bottom thirds of ferroptosis genes expression levels based on TCGA dataset. The number of patients in each group are shown on the graph. P values were calculated between high and low or intermediate third of gene expression using log-rank (Mantel-Cox) test.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2 and Supplementary Tables 1–4. Supplementary Fig. 1: the gating strategy for flow cytometry analysis of T cells (a) and myeloid cells (b). Supplementary Fig. 2: uncut gels corresponding to Extended Data Fig. 7. Supplementary Table 1: a list of antibodies and gating strategies. All antibodies are commercially available and were validated on the basis of information provided by the supplier. Titration experiments were performed before to the study. Supplementary Table 2: a list of inducers, inhibitors, blockers and reagents used in this study. Supplementary Table 3: a list of primers used in this study. Supplementary Table 4: definitions and abbreviations.

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Kim, R., Hashimoto, A., Markosyan, N. et al. Ferroptosis of tumour neutrophils causes immune suppression in cancer. Nature 612, 338–346 (2022). https://doi.org/10.1038/s41586-022-05443-0

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