Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis3,4,5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial–mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.
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The clustering analysis used here is described fully in its source manuscript6.
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This work was supported by an HFSP postdoctoral fellowship (LT000110/2015-L/1) and an AACR–AstraZeneca Immuno-oncology Research Fellowship (20-40-12-CASA) to M.C.-A.; T32 CA078207 to A.L.; F30 CA243210 to S.T.C.; and ANR-10-IDEX-0001-02 PSL and ANR-11-LABX-0043 and Fondation ARC pour la recherche sur le cancer to P.B. This research was supported in part by the Tisch Cancer Institute at Mount Sinai P30 CA196521-Cancer Center Support Grant. We thank the Human Immune Monitoring Center for all the single-cell profiling and epigenetic studies; the Merad laboratory and A. Lujambio for discussions and reagents; the Flow Cytometry and the Imaging Core at Mount Sinai; and the Cancer Biorepository at MSSM for sample acquisition. This work was also supported by National Institutes of Health (NIH)–National Cancer Institute grants CA257195, CA254104, AT011326, AI128949 and R56AI137244 to M.M., who is also a Samuel Waxman Cancer Research Foundation Investigator; CA109182, CA216248 and CA218024 to J.A.A.-G., who is also a Samuel Waxman Cancer Research Foundation Investigator; CA257195, CA254104, AT011326 and AI128949 to B.D.B.; NCI grant T32 CA078207 to E.D.; and NIH grant AG049074 to B.R.
J.A.A.-G. is a scientific co-founder of, scientific advisory board member and equity owner in HiberCell and receives financial compensation as a consultant for HiberCell, a Mount Sinai spin-off company focused on therapeutics that prevent or delay cancer recurrence. M.M. serves on the scientific advisory boards of Compugen Inc., Innate Pharma Inc., Morphic Therapeutic, Myeloid Therapeutics, Celsius Therapeutics and Genenta. M.M. receives funding from Genentech Inc., Regeneron Inc., Boerhinger Ingelheim Inc. and Takeda Inc. M.M. has ownership interest of less than 5% in Compugen Inc., Celsius Therapeutics Inc., Dren Bio Inc., and Asher Bio Inc. B.M.M., M.B. and C.M. declare that they are Genentech/Roche employees.
Peer review information Nature thanks the anonymous reviewers 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
a, Gating strategy for sorting of myeloid cells from naive and KP tumour-bearing lungs of Map17creER/+R26tdTom mice for scRNA-seq analysis. Monocytes and macrophages in the lung were gated as singlets, DAPI−CD45+Lin−CD11b−/+Ly6G−CD11clo-int-+SIGLECF+ and Tom− or Tom+. b, Confocal imaging of tdTomato+ bone-marrow-derived leukocytes and TRMs (CD206+, yellow). Images are representative of a single experiment and three tumours imaged. c, Gating strategy for sorting of myeloid cells from naive and KP tumour-bearing lungs of Cx3cr1creER/+R26YFP mice. Monocytes and macrophages were gated as in a and further separated based on YFP expression. d, Confocal imaging of YFP+ bone-marrow-derived leukocytes (red) and TRMs (CD206+, yellow). Scale bar, 50 μm. Images are representative of a single experiment and three tumours imaged. e, Spearman correlation of variable gene expression between the human monocyte and macrophage clusters detected in ref. 6 and those in ref. 10. f, The log2-transformed fold change (FC) between human TRM expression and the maximum cluster expression of non-TRM monocytes and macrophages, determined from data in ref. 6 (x axis) or ref. 10 (y axis). The human alveolar macrophage genes published previously11 are highlighted in red. g, Spearman correlation of variable gene expression between the mouse monocyte and macrophage clusters detected in the present study and in ref. 10. h, Average expression of selected mouse genes from scRNA-seq data in cluster groups I to IV (see Supplementary Table 2). i, Average expression of selected human genes from scRNA-seq data (see Supplementary Table 1). j, Confocal imaging of CD169cre/+R26tdTom KP lesions (day 30, KP-GFP, green) with CD206 (yellow). Scale bar, 100 μm. Images are representative of a single experiment and three tumours imaged. k, Gating strategy for the identification of TRMs and MDMs in naive lungs. TRMs were gated as live/dead−CD45+Ly6G−CD64+MERTK+CD2+CD169+CD206+SIGLECF+; MDMs were gated as singlets, live/dead−CD45+Ly6G−CD64+Mertk+CD2−CD11bhiCD169−CD206−SIGLECF−.
a, Lineage tracing experiment in Map17creER/+R26tdTom mice. b, Fraction of labelled (red, tdTom+) cells in the peripheral blood and lung of non-tumour-bearing Map17creER/+R26tdTom mice 6 months after tamoxifen injection (n = 6 blood, n = 5 lung; two independent experiments). Blood Ly6Chi monocytes were identified as singlets, DAPI−CD45+CD11b+CD115+Ly6Chi or Ly6Clo monocytes as CD45+CD11b+CD115+Ly6Clo. Lung monocytes were gated as CD45+CD11b+CX3CR1+Ly6Chi or Ly6Clo. Neutrophils in blood and lungs were identified as singlets, DAPI−CD45+CD11b+Ly6G+. TRMs were identified as singlets, DAPI−CD45+Ly6G−CD11blo/−SIGLECFhiCD11chiCD206hiCD169hi. Data are mean ± s.e.m. c, Frequencies of labelled (red, tdTom+) or not labelled (grey, tdTom−) cells within each cluster groups as defined in b in tumour-bearing mice. d, Lineage tracing experiment in Cx3cr1creER/+R26YFP mice. e, Fraction of labelled (green, YFP+) cells in the peripheral blood and lung of non-tumour-bearing mice (n= 8, pool of two independent experiments). Data are mean ± s.e.m. f, Frequency of labelled (green, YFP+) and non-labelled (grey, YFP–) cells within each cluster groups as defined in b in KP tumour-bearing mice.
a, TRMs (red, CD206+) distribution in KP-GEMMs (green) 14 weeks (wks) after injection of adenovirus-SPC5Cre in KP mice. White dotted lines delimit the tumour area. Unpaired two-tailed Student’s t-test; *P = 0.020. CD206+ macrophage distribution was analysed in n = 4 tumour-bearing mice from one experiment. Data are mean ± s.e.m. b, Gene Ontology categories for ATAC–seq significant (P < 0.05) open (day 15) and closed (day 30) chromatin regions identified in KP-associated TRMs. c, Longitudinal analysis of CD45+ leukocytes, TRMs and Ly6Chi and Ly6Clo monocytes in naive (n = 5) and in day-15 (n = 5) and day-30 (n = 4) KP-bearing mice. One-way ANOVA with Tukey’s test. Data are mean ± s.e.m. TRMs were gated as singlets, live/dead−GFP−CD45+Ly6G−CD64+MERTK+CD2+CD169+CD206+Siglecf+; MDMs were gated as singlets, live/dead−GFP−CD45+Ly6G−CD64+MERTK+CD2−CD11bhiCD169−SIGLECF−. Lung monocytes were gated as CD45+CD11b+CX3CR1+Ly6Chi or Ly6Clo. d, Longitudinal imaging analysis of TRMs identified by the co-expression of CD206 (red) and SIGLECF (yellow) in the KP-GFP orthotopic model. KP tumour cells, green. White asterisks indicate CD206+SIGLECF− macrophages. Yellow asterisks depict SIGLECF+CD206− leukocytes, which are also found in overt tumours and are most probably SIGLECF+ eosinophils. Scale bars, 50 μm (D5, D10 and D15); 100 μm (D25–30). Images are representative of one experiment; n = 3–5 mice; 2–3 tumours analysed per time point. e, Longitudinal imaging analysis of TRMs in the KP-GEMM model. Tumour cells are identified by positive staining with pan-cytokeratin in green. Images are representative of one experiment; 2–3 tumours analysed per mouse; 3 mice per time point. Scale bars, 50 μm. f, Immunohistochemistry converted to pseudofluorescence image of CD206 (red), CD10 (yellow) and cytokeratin (CK, green) staining in non-involved lung and NSCLC tissue. White asterisks indicate CD206+CD10+ TRMs. Scale bars, 250 μm (bottom images); 250 μm (top images). Images are representative of one experiment. g, Immunohistochemistry converted to pseudofluorescence image of CD206 (red), FABP4 (yellow) and cytokeratin (CK, green) staining in non-involved lung and NSCLC tissue. White asterisks indicate CD206+FABP4+ TRMs. Scale bars, 500 μm (left); 400 μm (right). Dotted lines delineate tumour border. Representative images from two non-involved lung and two NSCLC tumours. Images are representative of one experiment.
a, Heat map of DEGs of TRMs in naive lungs, day-15 and day-30 KP tumours. Red indicates the most significant upregulated and blue the most significant downregulated gene transcripts (P < 0.05, log2-transformed fold change (log2FC) > 1 and log2FC < 1, respectively). TRMs were sorted as singlets, DAPI−CD45+Ly6G−CD11blo/−CD64+MERTK+CD2+CD169+SIGLECFhiCD206+. b, Gene Ontology analysis of upregulated DEGs between naive and early KP-TRMs (day 15) (P < 0.05 and log2FC > 1). c, Number of peaks and heat map representing average ATAC–seq peaks (pks) unchanged (cluster 1), differentially closed (cluster 2) or opened (cluster 3) in TRMs at different times after KP injection. d, e, Representative tracks of significant TRM DEGs (P < 0.05) showing increased chromatin accessibility (dotted red lines) (d) or lower chromatin accessibility (e) in TRMs. Tracks are representative of three pooled mice examined over one single experiment. Data are mean ± s.e.m.
Extended Data Fig. 5 TRMs promote EMT and a pro-invasive signature in KP spheroids, whereas MDMs favour growth.
a, Venn diagrams for DEGs upregulated and downregulated in spheroids co-cultured with TRMs or bone marrow monocytes (BMMs). The number of DEGs uniquely controlled by TRMs or BMMs is shown in blue (TRMs) and red (BMMs). b, Gene Ontology (GO) analysis of significant DEGs (P < 0.05) with upregulated signature controlled by TRMs (blue) and by BMMs (red), respectively. c, Gating strategy for BALF (bronchoalveolar fluid) TRMs gated as CD45+CD11bloCD11c+SIGLECF+CD206+CD169+ and purity quantification in n = 4 mice. BALF routinely showed around 85% pure TRMs among CD45+ leukocytes. d, Confocal representative images and quantification of E-cadherin (red), TWIST1 (white) and β-catenin (red) in KP spheroids cultured alone or with TRMs or BMMs. Scale bars, 5 μm (inset) and 25 μm. One-way ANOVA with Tukey’s test. Data are mean ± s.e.m. Two independent experiments. e, Bar graphs showing the expression (in transcripts per million, TPM) of EMT-signature selected genes in KP-spheroids alone, with TRMs or BMMs. Data are mean ± s.e.m. Results are representative of one experiment. f, Size quantification for KP oncospheres co-cultured with TRMs or BMMs. Data are mean ± s.e.m. Data are representative of two independent experiments. g, Quantification of the number of KP oncospheres in co-cultures with TRMs (blue) or BMMs (red) compared to KP alone in the presence of GM-CSF (light blue) or M-CSF (light red), respectively. Data are mean ± s.e.m. Data are representative of two independent experiments. h, Quantification of KP 3D-matrigel spheroids with invasive protrusions co-cultured with TRMs or BMMs and their respective controls. Data are mean ± s.e.m. Data are representative of two independent experiments. One-way ANOVA with Tukey’s test (f–h). i, Bright-field microscopy images of the spheroids quantified in h. Scale bars, 100 μm. j, k, Number (j) and size (k) of KP oncospheres cultured alone or co-cultured with tumour-associated tTRMs or tMDMs. Results are representative of two independent experiments analysed using one-way ANOVA. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ****P < 0.0001.
a, Frequency, CTLA-4 and CD73 MFI levels of Treg cells induced by tTRMs and tMDMs with naive CD62L+CD44−CD4+ T cells (one-way ANOVA with Tukey’s test and two-tailed unpaired t-test). Two independent experiments. b, Frequency and phenotype of Treg cells in TRM-depleted mice at day 15 after KP injection. n = 5 mice per group. Two-tailed unpaired t-test; **P = 0.003, *P = 0.025; ***P = 0.0003 and ****P < 0.0001, respectively. c, Expression of Ccl17 and Tgfb1 in TRMs from naive mice (grey) and tumour-bearing mice (day 15 light green, day 30 dark green). Data from DEGs (P < 0.05 likelihood ratio test) list in Fig. 2d (see Supplementary Table 3). One experiment, n = 3 naive, n = 2 KP-TRM day 15 and n = 3 KP-TRM day 30 mice. d, Imaging of TRM-sufficient and deficient mice after instillation of diphtheria toxin. SIGLECF, green; CD206, red. Quantification of Ly6Chi/lo monocytes, neutrophils, MDMs and TRMs in wild-type or CD169-DTR lungs one week after the last dose of diphtheria toxin. n = 5 mice per group. Two-tailed unpaired t-test. TRMs were gated as singlets, live/dead−CD45+Ly6G−CD64+MERTK+CD2+CD169+SIGLECF+CD206+; MDMs as singlets, live/dead−CD45+Ly6G−CD64+MERTK+CD2−CD11bhiCD169−SIGLECF−; monocytes as CD45+CD11b+CX3CR1+Ly6Chi or Ly6Clo (Ly6Chi and Ly6Clo, respectively); and neutrophils as live/dead−CD45+CD11b+Ly6G+. e, Levels of CD169 in Tomato+ monocytes (CD45+CD11b+CX3CR1+) and MDMs (live/dead−CD45+Ly6G−CD64+MERTK+CD2−CD11bhiSIGLECF−) in naive and two-week KP lesions from Ms4a3-tdTom reporter mice. n = 3 mice per group. Two-way ANOVA with Tukey’s multiple comparisons test; ns, not significant. f, Frequencies of MDMs in wild-type and CD169-DTR lungs after DT treatment with diphtheria toxin in naive mice and KP lesions (two weeks). n = 5 per genotype for naive mice and n = 3 per genotype for the KP tumour group. Two-way ANOVA with Tukey’s multiple comparisons test. g, Quantification of Treg cells in spleen and lymph nodes of tumour-bearing mice (day 15) in WT + DT (black) and CD169-DTR + DT mice (red). n = 6 mice per group. Unpaired two-tailed t-test. h, Percentage of KP cells from lungs of wild-type or CD169-DTR mice treated with diphtheria toxin, 24 h after KP injection. n = 6 mice per group. Two-tailed unpaired t-test. i, Image analysis of Ki67+ and CC3+ KP cells in day-15 lesions, and p27+ KP cells in day-5 lesions of WT + DT and CD169-DTR + DT mice. Asterisks show positive KP cells. One-tailed unpaired t-test. n = 3 WT + DT and n = 4 CD169-DTR + DT for Ki67 and CC3; n = 3 WT + DT and n = 4 CD169-DTR + DT for p27. Two to three independent experiments. Scale bars, 25 μm (main images); 10 μm (inset). Data are mean ± s.e.m. (a–i). j, Diphtheria toxin treatment and KP injections in wild-type or CD169-DTR mice. k, Tumour burden in wild-type or CD169-DTR mice that were TRM-depleted after tumour implantation. l, Quantification of Treg cells, IFNγ+TNF+CD8+ effector cells and ratio of CD8/Treg cells in mice from k. Effector T cells were gated as singlets, DAPI−CD45+TCR+CD8+; Treg cells as singlets, DAPI−CD45+TCR+CD4+FOXP3+. n = 5 WT + DT and n = 7 CD169-DTR + DT mice. Three independent experiments. Data are mean ± s.e.m; two-tailed unpaired t-test. (k, l). m, Imaging and quantification of infiltrating FOXP3+ Treg cells (top) and CD3+ T cells (bottom) in WT + DT or CD169-DTR + DT mice at days 12 and 15 after KP injection. Two-tailed unpaired t-test. Data are mean ± s.e.m. Two independent experiments. Scale bar, 100 μm.
a, Scheme of OT-I and OT-II adoptive transfer experiments in B16-F10/OVA wild-type and CD169-DTR mice. b, Relative quantification of OT-I T cells in the lungs and tumour-draining lymph nodes (tdLN). OT-I T cells were gated as viable, CD45.1+TCR+CD8+. n = 4—5 mice. Data are representative of two independent experiments. c, Quantification of OT-I cells in the tumours of mice in b. CD45.1+ OT-I T cells were quantified in 7–8 tumours from mice in b. n= 7 WT + DT and N = 8 CD169-DTR + DT from one experiment. Scale bar, 50 μm. d, Quantification of OT-II cells in the tumours of mice in b. OT-II T cells were gated as viable, CD45.1+TCR+CD4+. n = 5–8 mice per group. Data are representative of one experiment. Data are mean ± s.e.m; unpaired two-tailed t-test (b–d). e, Scheme of the contribution of the TRM compartment and MDMs to tumour progression. This scheme was created with BioRender.com.
scRNAseq differential average expression of cells per group over transcript expression in all groups of monocytes and macrophages in human.
scRNAseq differential average expression of cells per group over transcript expression in all groups of monocytes and macrophages in mouse.
Differential expression gene list in bulk RNA-seq of TRMs (alveolar macrophages, AM) in naïve, KP D15 and KP D30 tumours. Significant genes (KP D15 over naïve) are included (p-value<0.05). All data is also included in this table with no further statistical test.
Differential expression ATAC-seq peaks detected in TRMs in naïve, KP D15 and KP D30 tumours. Significant genes (KP D15 over naïve and KP D30 over naïve) are included (p-value<0.05).
Differential expression gene list in bulk RNA-seq of KP-spheroids co-cultured with TRMs and BMMs. Significant genes uniquely found in either KP+TRM over KP alone, and KP+BMM over KP alone are included (p-value<0.05), as well as significant genes found in both conditions.
These tables include the list of antibodies used for flow cytometry (Supplementary Table 6), antibodies used for Multiplexed immunohistochemical consecutive staining on a single slide (Supplementary Table 7) and antibodies used for Clearing imaging (Supplementary Table 8).
This zipped file includes the Supplementary Imaging Macros and a guide.
KP-spheroids co-cultured with TRMs. KP-GFP expressing spheroids were co-cultured with alveolar macrophages and imaged for 4 hours. Invasive protrusions are visualized. Scale bar corresponds to 200 μm.
KP-Spheroids co-cultured with BMMs. KP-GFP expressing spheroids were co-cultured with bone-marrow monocytes and imaged for 4 hours. Note the size of KP-GFP spheroids co-cultured under these conditions. Scale bar corresponds to 200 μm.
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Casanova-Acebes, M., Dalla, E., Leader, A.M. et al. Tissue-resident macrophages provide a pro-tumorigenic niche to early NSCLC cells. Nature 595, 578–584 (2021). https://doi.org/10.1038/s41586-021-03651-8