Abstract
Animals constantly receive various sensory stimuli, such as odours, sounds, light and touch, from the surrounding environment. These sensory inputs are essential for animals to search for food and avoid predators, but they also affect their physiological status, and may cause diseases such as cancer. Malignant gliomas—the most lethal form of brain tumour1—are known to intimately communicate with neurons at the cellular level2,3. However, it remains unclear whether external sensory stimuli can directly affect the development of malignant glioma under normal living conditions. Here we show that olfaction can directly regulate gliomagenesis. In an autochthonous mouse model that recapitulates adult gliomagenesis4,5,6 originating in oligodendrocyte precursor cells (OPCs), gliomas preferentially emerge in the olfactory bulb—the first relay of brain olfactory circuitry. Manipulating the activity of olfactory receptor neurons (ORNs) affects the development of glioma. Mechanistically, olfaction excites mitral and tufted (M/T) cells, which receive sensory information from ORNs and release insulin-like growth factor 1 (IGF1) in an activity-dependent manner. Specific knockout of Igf1 in M/T cells suppresses gliomagenesis. In addition, knocking out the IGF1 receptor in pre-cancerous mutant OPCs abolishes the ORN-activity-dependent mitogenic effects. Our findings establish a link between sensory experience and gliomagenesis through their corresponding sensory neuronal circuits.
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Acknowledgements
We thank X. Bian, J. Eisen, W. Mo, H. Zong, Y. Zhu, W. Hong, H. Hu, Y.-J. Wang and P. Shi for their critical comments on the work. We also thank the Core Facilities at Zhejiang University School of Medicine and the Core Facility for Large-Scale Tissue Clearing and Data Analysis of the Department of Integrative Medicine and Neurobiology at Fudan University School of Basic Medical Sciences for technical support. This work was supported by the National Natural Science Foundation of China (82192890, 81972915 and 81673035), the National Key Research and Development Program of China (2021YFF1201005 and 2016YFA0101201), the Science Foundation for Distinguished Young Scientists of Zhejiang Province (LR17H160001) and the Thousand Talent Program for Young Outstanding Scientists, China. C.L. is a scholar of the K. C. Wong Education Foundation.
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P.C. and C.L. contributed to experimental design. C.L. wrote the manuscript with input from all authors. P.C. performed the data analysis for all histology, western blotting and PCR, did all quantification and made all figures, with input from B.Q. W.W. designed and performed naris occlusion experiments. R.L., W.W. and Q.W. made all transgenes. J.L. performed brain clearing, light-sheet imaging and FACS experiments with input from P.C. L. Zhang performed RNAscope experiments. B.L. performed the pathological analysis of all samples. P.C. and H.Y. performed behaviour experiments. R.J. analysed single-cell RNA-seq data. P.C. performed statistical analysis with advice from R.B and L. Zeng. C.L. conceived of the project and supervised all aspects of the work.
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Extended data figures and tables
Extended Data Fig. 1 The methodology of tumour mapping.
(a) Mice were dissected at specific timepoint as indicated for analysis. Representative images of whole brain after dissection were shown. The brain boundary is demarcated by the dashed line. The asterisk indicates the tumour region in the OB. (b) Each brain was serially sectioned. For tumour position analysation, every 14 slices were analysed. For OB tumour volume quantification in Figs. 2, 4, every 4 slices were analysed. Tumour area is demarcated by the dashed line. (c) Tumours were confirmed by co-localization of tdT and Ki67 staining. tdT-labelling efficiency of tumour cells was confirmed by quantification of tdT+ cell percentage in Ki67+ cells (n = 5 mice). Representative images show the co-localization of tdT and Ki67 staining in the OB rather than other regions such as the subventricular zone (SVZ), the corpus callosum (CC) and the subgranular zone (SGZ) in the hippocampus. (d) Representative haematoxylin and eosin staining (H&E) images of tumours in the OB, cortex and the bottom of the brain (BoB) at different magnifications. Adjacent slices of OB tumours were used for H&E staining, or Ki67 and tdT staining to validate the feasibility of using tdTomato as the surrogate marker to define tumour mass. T, tumour; N, normal; GL, glomerular layer; EPL, external plexiform layer; ML, mitral cell layer; GRL, granule cell layer; LV, lateral ventricle. Scale bars: (b), 10 mm; (c), 300 μm. Data are mean ± s.e.m.
Extended Data Fig. 2 Mapping tumour locations in the CKO model by histology and three-dimensional reconstruction.
(a) Tumour positions of a cohort of the CKO model at P90 (n = 5), P130 (n = 5), P160 (n = 18), P210 (n = 18) and moribund stage (n = 15). Each row represents one mouse brain. Red blocks indicate the existence of detectable tumour mass in the corresponding brain region. OB, olfactory bulb; AON, anterior olfactory nucleus; OT, olfactory tubercle; Piri/Amy, piriform and amygdala; EC, entorhinal cortex; Hippo, hippocampus; Ctx, cortex; BoB, bottom of the brain. (b) Representative 3D reconstruction images of the CKO model with tumour. Videos were provided in Supplementary Videos 1–4. Red, tumour area; Cyan, corpus callosum (CC); Grey, brain boundary.
Extended Data Fig. 3 Further characterization of tumour distributions.
(a) Cleared brain of the CKO model at different tumour stage. Ms19127 contained no detectable tumour; Ms18980 carried a small tumour in the glomerular layer of the OB; Ms19119 harboured obvious tumour in the glomerular layer; and Ms18905 carried obvious tumour in both the glomerular layer and the GRL of the OB. Stri, striatum. THA, thalamus. Arrows point to tumours in the OB. (b) WES and tumour phylogenies for CKO mice (n = 2 mice) at the moribund stage. Three specimens were analysed in each mouse brain as indicated: N (normal brain tissue), T1 (tumour tissue in the OB) and T2 (tumour tissue in the cerebrum away from the OB). The phylogenetic trees illustrate the evolutionary relationship among the three specimens in each brain. For each branch, the number and the gene names of non-synonymous SNVs that were attributed to the branch are shown. Red signal indicates tdT. (c) Representative image of the coronal section from one mouse OB. Different layers within the OB are visualized with corresponding markers as indicated. Of note, M/T cells were labelled by the Igf1-EGFP transgene (see also Fig. 3), the GAD2 expression was visualized by using Gad2-Cre crossing with the reporter line Rosa26LSL-tdTomato (Ai9). Therefore, the final genotype of this mouse is Igf1-EGFP+/−; Gad2-Cre+/−; Rosa26CAG-LSL-TdTomato +/−. (d) Quantification of tumour incidence of the CKO model within the glomerular layer. (e) Normalized density of FOS+ cells in the glomerular layer (n = 3 mice). Scale bar: (c), 500 μm.
Extended Data Fig. 4 Characterization of the DREADD transgenes.
(a) Configurations of the Omp-hM4Di and the Omp-hM3Dq BAC transgene. “mem”, membrane-tagged. (b) Whole sagittal brain sections and high magnification images from the wild type, Omp-hM4Di and Omp-hM3Dq mouse, respectively (n = 3 mice for each group were examined). (c–d) Schematic diagram of the buried food test (c) and the time for wild type, Omp-hM4Di and Omp-hM3Dq mice to find the buried food (d). To test the olfaction performance, food was buried at random corner and the time that mice spent to find the food was recorded. The time for mice without clozapine injection to find buried food was recorded on day 3. On day 5, test 2 was performed to record the time for mice with clozapine injection to find the buried food (n = 13 mice for WT mice, n = 12 mice for the Omp-hM4Di transgene, n = 19 mice for the Omp-hM3Dq transgene). (e) Schematic diagram to evaluate odorant-stimulated neuronal activity of Omp-hM4Di and Omp-hM3Dq transgene with clozapine administration. Mice were tested in the cage supplied with fresh air and water underneath to maximally remove baseline odorants from the environment. (f and g) Quantification of FOS+ cell density in the glomerular layer (f) and ML (g) from Omp-hM4Di and wild type mice (n = 6 mice for the WT, WT clozapine and Omp-hM4Di clozapine group, n = 5 mice for the Omp-hM4Di group). (h and i) Quantification of FOS+ cell density in the glomerular layer (h) and ML (i) from Omp-hM3Dq and wild type mice (n = 6 mice for the WT and WT clozapine group, n = 5 mice for the Omp-hM3Dq group, n = 4 mice for the Omp-hM3Dq clozapine group). (j) Treatment scheme of the CKO_Omp-hM4Di model analysed at the pre-transforming stage. (k) Proliferation rate of mutant OPCs in the OB of CKO and CKO_Omp-hM4Di mice as treated in (j). Relative proliferation rate was calculated by the proliferation rate of mice received clozapine normalized to the average proliferation rate of mice received vehicle (n = 8 OBs for CKO with vehicle, n = 14 OBs for CKO with clozapine, n = 6 OBs for CKO_Omp-hM4Di with vehicle, n = 12 OBs for CKO_Omp-hM4Di with clozapine). (l) Treatment schedule of the CKO_Omp-hM3Dq model analysed at the pre-transforming stage. (m) Proliferation rate of mutant OPCs in the OB of CKO and CKO_Omp-hM3Dq mice as treated in (l). Relative proliferation rate was calculated by the proliferation rate of mice received clozapine normalized to the average proliferation rate of mice received vehicle (n = 6 OBs for CKO with vehicle, n = 10 OBs for CKO with clozapine, n = 6 OBs for CKO_Omp-hM3Dq with vehicle, n = 8 OBs for CKO_Omp-hM3Dq with clozapine). Of note, the same dataset of CKO and the CKO_Omp-hM3Dq model in (m) were also presented in Fig. 4k as the control for the CKO_Omp-hM3Dq_IGF1R model. Scale bars: (b), 2 mm, 200 μm. One-sided t-test in (d), (f), (g), (h), (i), (k) and (m). NS, not significance, ****p < 0.0001. Data are mean ± s.e.m.
Extended Data Fig. 5 Clozapine does not affect the tumour immune microenvironment, and ORN activity affects glioma progression.
(a) Proliferation rates of two CKO model-derived glioma cell lines treated with clozapine as indicated. The cells were maintained in complete medium. The proliferation rates of culture cells were measured by EdU incorporation assay (n = 4 biologically independent samples for each group). (b–d) Quantification of tumour invasiveness (c) and tumour proliferation (d) in the CKO model under clozapine treatment at the tumour stage. Tumour invasiveness was defined as the distance from the core tumour area (with the highest density in the tumour) to the normal area (where the density of tumour cells declined to the same as that in the region away from the tumour), as the length of white rectangular (b). Five random areas were calculated for each mouse (n = 5 mice for each group). The tumour mice were from the same group of mice used in the first two columns of Fig. 2d. (e) Percentage of granulocytes, macrophage, dendritic cells, B lymphocytes and T lymphocytes in the peripheral blood of the WT mice (at the age of 8 weeks) under clozapine treatment. Clozapine injection, clozapine was administered by i.p. (two injections per week for three weeks). Clozapine drinking, mice were access to clozapine containing water for three weeks (n = 4 mice for each group). For FACS gating strategy, see Supplementary Fig. 2. (f) Percentage of granulocytes, macrophage, dendritic cells, B lymphocytes and T lymphocytes in the peripheral blood of the WT, Omp-hM3Dq and Omp-hM4Di transgenic mice (at the age of 8 weeks) under clozapine treatment. The clozapine treatment was the same as that of injection paradigm in (e). Clozapine was administered by i.p. (two injections per week for three weeks). w/ clozapine, with clozapine. The values are presented as the ratio to the WT mice (n = 4 mice for each group). (g) The blood-brain barrier integrity was evaluated by Evans Blue dye. w/o Evans Blue, without Evans Blue treatment. w/ Evans Blue, with Evans Blue treatment. WT mice at the age of 8 weeks were used. The schemes of clozapine treatment by drinking water or i.p. injection are the same as that in (e). See Methods section for the details (n = 4 mice for each group). (h) Immunochemistry staining of CD15 in the brain of WT, Omp-hM3Dq, Omp-hM4Di and the CKO tumour mice with clozapine treatment. Spleen was used as the positive control. Red arrows point to CD15+ granulocytes in the spleen. WT mice treated with vehicle was used as negative control. T, tumour area. The brain samples of the WT, Omp-hM3Dq, Omp-hM4Di mice were from the mice analysed in (f). The brain samples of CKO tumour mice were from the same group of clozapine-treated CKO mice used in Fig. 2d. (i) Quantification of Iba1+ cell density in CKO_Omp-hM4Di model at the pre-transforming stage as indicated in Extended Data Fig. 4j (n = 6 OBs for CKO, n = 6 OBs for CKO with clozapine drinking, n = 4 OBs for CKO_Omp-hM4Di, n = 6 OBs for CKO_Omp-hM4Di with clozapine drinking). P value: column A vs B, 0.9932; A vs C, 0.3648; A vs D, 0.9984; B vs C, 0.4936; B vs D, 0.9728; C vs D, 0.2964. (j) Quantification of Iba1+ cell density in CKO_Omp-hM3Dq model at the pre-transforming stage as indicated in Extended Data Fig. 4l (n = 6 OBs for CKO with vehicle, n = 6 OBs for CKO with clozapine, n = 4 OBs for CKO_Omp-hM3Dq with vehicle, n = 6 OBs for CKO_Omp-hM3Dq with clozapine). P value: column A vs B, 0.7722; A vs C, 0.6589; A vs D, 0.8465; B vs C, 0.2175; B vs D, 0.3158; C vs D, 0.9725. (k–m) Quantification the density of total macrophage/microglia (marked by Iba1+) (k) and those activated macrophage/microglia (CD68+Iba1+) (l) in the non-tumour region (k, l) or the tumour region (m, n) of the CKO tumour brain. Non-tumour region is defined as the brain region where no obvious tumour lesion was included (usually far away from the tumour core). Clozapine was administered by drinking water from P30 to P210. Mice were analysed at P210 (n = 6 OBs for CKO and n = 4 OBs for CKO with clozapine drinking in (k). n = 4 OBs for each group in (l–n)). (o) Representative images of the macrophage/microglia of the CKO model analysed in (k–n). Low-power and high-power images were both provided. The MERGE channel in low-power images including DAPI, tdT and Iba1 channels. The MERGE channel in high-power images including DAPI, CD68, tdT and Iba1 channels. N, non-tumour region. T, tumour region. Of note, the samples analysed in (k–o) were from the same group of CKO mice used in Fig.2d (the first two columns). (p) Representative images of mTq2 and tdT expression in glomeruli of CKO_Omp-hM4Di mice administered with clozapine. (q and r) Quantification of mTq2 (assumed also hM4Di) and tdTomato intensity among glomeruli of CKO_Omp-hM4Di mice with (q) and without (r) clozapine treatment. Each dot represents one glomerulus. Two-sided Pearson correlation coefficients were calculated. (s and t) Immunofluorescence staining (s) and Western blots (t) of tyrosine hydroxylase (TH) in control and left-naris occlusion mice. Mice were plugged at P31 and analysed after two months. For gel source data, see Supplementary Fig. 1. (u–w) Scheme of the experimental procedure (u) and for single naris occlusion analysis at the pre-transforming stage. Proliferation cell density (v) and proliferation rate of mutant cells (w) were analysed (n = 4 mice for each group). (x–z) Scheme of the experimental procedure (x), representative whole brain images (y) and quantification (z) of L-naris occlusion mice grafted with mouse glioma cell lines. Scale bars: (b), 100 μm; (o), 500 μm and 50 μm; (p), 400 μm; (s), 1000 μm. One-way ANOVA with Dunnett’s multiple comparison post-hoc test in (a), (e), (f), (g), (i) and (j). One-sided t-test in (c), (d), (k), (l), (m), (n), (v), (w) and (z). NS, not significant, ****p < 0.0001. Data are mean ± s.e.m. The box plot marks the median, upper and lower quartiles and whiskers are presented min to max in (v) and (w). See also Methods section for details of each part of the experiment.
Extended Data Fig. 6 Characterization of the Igf1-EGFP transgene.
(a and b) smFISH coupled with immunofluorescence confirms the co-localization of GFP protein and Igf1 mRNA in cells (M/T cells) from the OB (a) and the cerebellum (presumably Purkinje cells) (b) from the Igf1-EGFP transgene. (c) Representative image of EGFP expression in different layers of the OB from the Igf1-EGFP transgene (n = 5 mice were examined). (d) The genetic configurations of the CKO_IGF1R model. (e) The proliferation rate of pre-transforming mutant OPCs in the glomerular layer, EPL and GRL layer of the CKO model after IGF1R was knocked out (n = 6 OBs for the CKO and the CKO_IGF1R model respectively). (f and g) Quantification (f) and histological analysis (g) of GFP+ cells among all known cell types in the OB of the adult mice (at the age of P30). Markers used to identify distinct cell types were indicated (n = 3 mice). Scale bars: (a), 100 μm and 10 μm; (b), 100 μm and 20 μm; (c), 100 μm; (g), 50 μm. One-sided t-test in (e). Data are mean ± s.e.m.
Extended Data Fig. 7 Characterization of IGF1 expression in microglia.
(a and b) Igf1-EGFP BAC transgene reveals the colocalization of GFP (presumably also Igf1) and the microglia marker IbaI in the cortex and the cerebellum at P6 (n = 2 mice were examined). (c) Genotype configuration of the CKO_Igf1-EGFP model, which was used to examine the cellular source of IGF1 during gliomagenesis. (d) The colocalization of Igf1-EGFP with the marks of M/T cells or microglia in the tumour from the OB of the CKO_Igf1-EGFP model. H&E staining was provided to confirm tumour pathology (n = 3 mice were examined). (e) Percentage and average intensity of Igf1-expression microglia and M/T cells in the CKO_Igf1-EGFP model grouped by endpoints (n = 10 mice). Two-sided Pearson correlation coefficients were calculated. (f and g) Percentage and intensity (f), as well as representative images (g) of Igf1-expression microglia and M/T cells in the CKO_Igf1-EGFP model from (e) grouped by tumour volume (n = 6 mice for small group and n = 4 mice for big group). (h) The proximal position of the terminals from GFP+ M/T cells with tdT+ glioma cells in the OB of CKO_Igf1-EGFP model. Orthogonal views are provided to further validate the interactions between two types of cells (n = 2 tumours were examined). Scale bars: (b), 200 μm, 50 μm and 100 μm; (d), H&E staining, 400μm, immunofluorescence, 50 μm; (g), 200 μm; (h), 10 μm. One-sided t-test in (f). NS, not significant. Data are mean ± s.e.m.
Extended Data Fig. 8 Establishment and characterization of the dual-recombinase MADM model.
(a) Genotype and the representative images of the MADM_hGFAP-FlpO tumour model. Brain boundaries are demarcated by dashed lines. The asterisk indicates the tumour region. Of note, tumours preferentially appeared in the olfactory brain structures (n = 5 mice were examined). (b) For Pcdh21+ M/T cells, Cre recombinase induces the knockout of Igf1 alleles and simultaneously turns the cells into red. Notably, if the Cre does not induce the inter-chromosomal recombination between the MADM cassettes, tdT+GFP−MYC− cells will be generate (left branch). If Cre recombinase also conducts the inter-chromosomal recombination between the MADM cassettes, tdT+GFP+MYC+ will be generated (right branch). Notably, Pcdh21-Cre never expresses in OPCs or tumour cells. (c) The breeding scheme to generate the dual-recombinase MADM_IGF1 model. (d–g) Orthogonal analysis of the dual-recombinase MADM model to validate the independent labelling of the Cre-LoxP and the FlpO-FRT recombination systems in the same mouse. The configuration for each genetic composition is provided on the left (n = 2 mice were examined for each genotype). Scale bars: (a), 40 μm; (d–g), 40 μm.
Extended Data Fig. 9 Further validation of M/T cells as the cellular source for IGF1.
(a–c) The analysis scheme (a), representative images (b) and the proliferation rate of mutant cells (c) in the OB of the control and the experiment group at the pre-transforming stage (P60). Arrows point to the proliferating mutant cells (n = 6 OBs for the Control group and n = 8 OBs for the Experiment group). (d) smFISH coupled with immunofluorescence confirms that, after knockout of Igf1 in M/T cells, no other cell types such as microglia regained the abnormal expression of Igf1 to play redundant function as the cellular resource for tumour OPCs (n = 988 Iba1+ cells for WT group, n = 1207 Iba1+ cells for IGF1flox; Pcdh21-Cre-P40 group, n = 1167 Iba1+ cells for IGF1flox; Pcdh21-Cre-P100 group. n = 2 mice for each group). (e) Igf1 transcript from OBs of the IGF1flox and IGF1flox; Pcdh21-Cre mice. IGF1flox; Pcdh21-Cre mice show prominent Igf1 KO bands. P-F, primer Igf1-Full-Forward; P-R, primer Igf1-Full-Reversed. For primers, see Supplementary Table 1 (n = 2 mice for each group). (f and g) Validation of the intersectional genetic manipulation of tumour cells and microglia in the dual-recombinase genetic system. Genotype (f) and characterization (g) of MADM_Cx3cr1 model. In MADM model, tdT+ cells are all MYC+, which are generated by FlpO-dependent inter- chromosomal recombination. In MADM_Cx3cr1 model, MYC−tdT+ cells can be easily found and all of them were Iba1+ microglia generated by Cre-dependent recombination. (h–k) Genotypes (h), representative images (i), the treatment scheme (j) and tumour volume quantification (k) of MADM_Cx3cr1 and MADM_Cx3cr1_IGF1 model (n = 5 mice for MADM_Cx3cr1 model and n = 6 mice for MADM_Cx3cr1_IGF1 model). Scale bars: (b), 40 μm; (d), 100 μm; (g), 50 μm; (i), 50 μm. One-sided t-test in (c) and (k). NS, not significant. Data are mean ± s.e.m.
Extended Data Fig. 10 IGF1 functions independently of the neurotransmitter–receptor-mediated signals.
(a) The t-SNE map of single cell sequencing data of a CKO tumour from previous published dataset6. (b) Expression pattern of neurotransmitter receptor genes in all cell clusters defined above. Tumour cell clusters include astrocytes-like tumour cells and OPC-like tumour cells. Same colour code is used in (a) and (b). (c) Glioma cells from the CKO model co-cultured with primary OB neurons were collected following the flow chart. For FACS gating strategy, see Supplementary Fig. 2. (d) Confirmation of activation of IGF1R signalling upon IGF1 (20 ng ml−1) stimulation. Two independent experiments. For gel source data, see Supplementary Fig. 1. (e) Representative up-regulated genes and neurotransmitter receptor genes of glioma cells which were co-cultured with OB neurons and stimulated by IGF1 compared to co-cultured glioma cells without IGF1 stimulation. Full list of gene expression level can be found in Supplementary Table 2 (n = 2 biologically independent samples without stimulation and n = 3 biologically independent samples with stimulation). (f) Proliferation rate of two independent CKO model derived glioma cell lines treated with 20 ng ml−1 IGF1, 20 ng ml−1 IGF1 and 10 μM AMPAR antagonist DNQX, 10 μM DNQX, 200 μM glutamate, 200 μM GABA or 15 mM KCl (n = 4 biologically independent repeats for each group). (g) The working model in this study. One-way ANOVA with Dunnett’s multiple comparison post-hoc test in (f). NS, not significant, ****p < 0.0001. Data are mean in (e) and mean ± s.e.m. in (f).
Supplementary information
Supplementary Figure 1
This file contains the uncropped western blots.
Supplementary Figure 2
This file contains representative FACS gating strategy.
Supplementary Tables
This file contains primers for real-time qPCR and a full list of the gene expression levels of glioma cells co-cultured with OB neurons.
Supplementary Video 1
This file contains 3D reconstruction videos of Ms1083.
Supplementary Video 2
This file contains 3D reconstruction videos of Ms1610.
Supplementary Video 3
This file contains 3D reconstruction videos of Ms1876.
Supplementary Video 4
This file contains 3D reconstruction videos of Ms1482.
Supplementary Video 5
This file contains brain clearing videos of Ms19127.
Supplementary Video 6
This file contains brain clearing videos of Ms18980.
Supplementary Video 7
This file contains brain clearing videos of Ms19119.
Supplementary Video 8
This file contains brain clearing videos of Ms18905.
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Chen, P., Wang, W., Liu, R. et al. Olfactory sensory experience regulates gliomagenesis via neuronal IGF1. Nature 606, 550–556 (2022). https://doi.org/10.1038/s41586-022-04719-9
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DOI: https://doi.org/10.1038/s41586-022-04719-9
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