Abstract
Tumour-associated macrophages (TAMs) largely express an alternatively activated (or M2) phenotype, which entails immunosuppressive and tumour-promoting capabilities. Reprogramming TAMs towards a classically activated (M1) phenotype may thwart tumour-associated immunosuppression and unleash anti-tumour immunity. Here we show that conditional deletion of the microRNA (miRNA)-processing enzyme DICER in macrophages prompts M1-like TAM programming, characterized by hyperactive IFN-γ/STAT1 signalling. This rewiring abated the immunosuppressive capacity of TAMs and fostered the recruitment of activated cytotoxic T lymphocytes (CTLs) to the tumours. CTL-derived IFN-γ exacerbated M1 polarization of Dicer1-deficient TAMs and inhibited tumour growth. Remarkably, DICER deficiency in TAMs negated the anti-tumoral effects of macrophage depletion by anti-CSF1R antibodies, and enabled complete tumour eradication by PD1 checkpoint blockade or CD40 agonistic antibodies. Finally, genetic rescue of Let-7 miRNA activity in Dicer1-deficient TAMs partly restored their M2-like phenotype and decreased tumour-infiltrating CTLs. These findings suggest that DICER/Let-7 activity opposes IFN-γ-induced, immunostimulatory M1-like TAM activation, with potential therapeutic implications.
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Acknowledgements
We thank M. Smyth (QIMR Berghofer, Herston, Australia) for providing MC38-OVA cells; F. Schütz (Swiss Institute of Bioinformatics) for advice on statistical methods; C. Maderna, F. Jammes, L. Giesbrecht, S. Takahashi, C. W. Rmili and A. Bellotti (M.D.P’s laboratory) and C. Wolter (Roche) for technical help; the BIOP team at EPFL for advice on image processing and proteome profiler cytokine array analysis; and F. Radtke, T. Petrova and C. Brisken for providing breeding pairs of transgenic mice. This work was supported by grants from the Fonds National Suisse de la Recherche Scientifique (FNS 31003A-143978; FNS 31003A-165963) and Fondation pour la lutte contre le cancer to M.D.P. D.L. was supported by an FNS International Short Visits fellowship (IZK0Z3_160843/1). S.K.H. was supported by the Swiss Federal Commission for Scholarships for Foreign Students (Swiss Government Excellence Scholarship 2015.0430).
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C.B. designed and performed experiments, analysed and interpreted the data, and wrote the manuscript; M.L.S. designed and performed experiments and bioinformatics analyses, generated lentiviral constructs, analysed and interpreted the data, and wrote the manuscript; D.L. designed and performed experiments, and analysed and interpreted data; D.T. and S.K.H. performed experiments; A.K. performed RNA-Seq; S.H. and C.H.R. performed experiments, provided therapeutic antibodies, and advised on pharmacological studies; C.-H.O. analysed RNA-Seq data and performed bioinformatics analyses; M.D.P. designed, interpreted and supervised research, and wrote the manuscript.
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A.K., S.H., C.H.R. and C.-H.O. are Roche employees. No other authors declare any competing financial interests
Integrated supplementary information
Supplementary Figure 5 T helper cytokines modulate macrophage DICER.
(a) qPCR analysis of Dicer1 (normalized to Gapdh; fold-change (FC) versus siCtrl; mean values ± SEM) in immortalized endothelial cells (iECs) either transfected with a control siRNA (siCtrl; n = 4 biologically independent cell cultures) or anti-Dicer1 siRNA (siDicer1; n = 4 biologically independent cell cultures). Statistics by unpaired Student’s t test on ΔCt values. Cultures indicated by red circles/squares were also analyzed by Western blotting, as shown in (b). (b) Western blot analysis of DICER and vinculin (VCN) in the iECs indicated by red circles/squares in (a). The experiments in (a) and (b) were performed to validate the specificity of the anti-DICER polyclonal antibody used in our studies. (c) Western blot analysis of DICER and VCN in iBMMs either not treated (NT) or treated as indicated. The blot was probed with a polyclonal anti-DICER antibody (top panel) and subsequently re-probed with a monoclonal anti-DICER antibody (middle panel). VCN is shown in the bottom panel. (d) qPCR analysis of Dicer1 (normalized to Gapdh; FC versus CD11c+ TAMs; mean values ± SEM) in MRC1+ and CD11c+ TAMs isolated from treatment-naïve MC38 (n = 3 independently sorted TAM samples from 3 mice/group) or LLC (n = 4 independently sorted TAM samples from 4 mice/group). Statistics as in (a). Statistical significance: ∗∗, P < 0.01; ∗∗∗, P < 0.001. Unprocessed Western blots are shown in Supplementary Fig. 8.
Supplementary Figure 6 A LysM-Cre transgene mediates preferential Dicer1 deletion in macrophages.
(a) Flow cytometry analysis of GFP+ immune cells in blood, spleen, liver, lung, and tumors of LLC-bearing LysM-Cre;ROSAmT/mG mice (n = 4 mice), ROSAmT/mG mice (n = 4 mice) or wild-type (WT) control mice (2 mice). Data show percentages (mean values) of GFP+ cells in each of the indicated cell types. (b) GFP (green), Tomato (red), MRC1 (blue) and DAPI (white) immunostaining of LLC tumors from ROSAmT/mG (upper panel) or LysM-Cre;ROSAmT/mG (lower panel) mice. Scale bar, 20 μm. Images are representative of the tumors in (a). (c) Flow cytometry analysis of blood obtained from D+/+ or D−/− mice of either 6 or 15 weeks of age. Data show percentages (mean values ± SEM) of the indicated cell types. n = 4 or 5 (D+/+ non-classical monocytes), 7 (D−/− non-classical monocytes), and 8 (other D−/− cells) or 10 (other D+/+ cells) mice. Statistics by unpaired Student’s t test. (d) PCR analysis of genomic Dicer1 in neutrophils (N), CD11c+ TAMs (M1), MRC1+ TAMs (M2), and tumor-derived cells depleted of endothelial and hematopoietic cells (T), all isolated from LLC tumors of LysM-Cre;Dicer1lox/lox(D−/−), LysM-Cre;Dicer1+/+ (D+/+), or Dicer1lox/lox mice. Arrows indicate (i) genomic deletion of Dicer1 23rd exon (Dicer−/−); (ii) floxed but not deleted Dicer1 23rd exon (Dicer1lox/lox); and (iii) wild-type Dicer1 locus (Dicer1+/+). Statistical significance: ∗∗, P < 0.01; ∗∗∗, P < 0.001. Source data for (a) are reported in Supplementary Table 5.
Supplementary Figure 7 DICER inactivation in TAMs depletes miRNAs and inhibits tumor metastasis without disrupting hematopoiesis.
(a,b) qPCR analysis of miRNAs (normalized to U6; FC values ± SEM) in CD11c+ and MRC1+ TAMs, Ly6G+ neutrophils and Ly6G−Ly6C+ monocytes, isolated from LLC (n = 4 independently sorted cell samples from 4 mice/group for all cell types; n = 3 for M1 TAMs in D−/−) or MC38 (n = 3 independently sorted cell samples from 3 mice/group) tumors of either D−/− or D+/+ mice. Statistics by unpaired Student’s t test on ΔCt values. N.D., not detected. Note that the LLC-bearing mice in Fig. 2a and Supplementary Fig. 3a belong to independent experiments. (c,d) Representative images of lungs showing spontaneous LLC (c) and experimental MMTV-PyMT (d) micrometastases. Arrows indicate selected micrometastases. Scale bar, 1.5 mm. (e) Percentage of the indicated hematopoietic cell types (mean values ± SEM) in MC38 tumor-bearing D−/− (n = 7) or D+/+ (n = 11) mice. Statistics by two-way ANOVA. Statistical significance: ∗∗, P < 0.01; ∗∗∗, P < 0.001.
Supplementary Figure 8 DICER deficiency induces pro- to anti-tumoral TAM conversion.
(a) Flow cytometry analysis of immune cells in MMTV-PyMT tumors, treated as indicated. One representative tumor per condition (of 7 analyzed) is shown. The two mother dot plots on the left (upper and bottom rows) display an equal number of events. MDSC, myeloid-derived suppressor cells comprising Ly6G+ neutrophils, Ly6C+ monocytes and Ly6G−Ly6C− immature myeloid cells. (b) Percentage (mean values ± SEM) of CD11c+ and MRC1+ TAMs in D−/− or D+/+ mice, treated as indicated. MC38: n = 9 (D+/+; IgG), 9 (D+/+; anti-CSF1R), 5 (D−/−; IgG), and 11 (D−/−; anti-CSF1R) mice; LLC: n = 7 (D+/+; IgG), 7 (D+/+; anti-CSF1R), 6 (D−/−; IgG), and 6 (D−/−; anti-CSF1R) mice. Statistics by Mann-Whitney U test. (c) Tumor weight (FC versus D+/+, IgG; mean values ± SEM) in D−/− or D+/+ mice, treated as indicated. MC38: n = 10 (D+/+; IgG), 9 (D+/+; anti-CSF1R), 11 (D−/−; IgG), and 11 (D−/−; anti-CSF1R) mice; LLC: n = 17 (D+/+; IgG), 17 (D+/+; anti-CSF1R), 13 (D−/−; IgG), and 16 (D−/−; anti-CSF1R). Right panel combines the data from 2 independent experiments. Statistics by unpaired Student’s t test. (d) qPCR analysis of miRNAs (normalized to U6, FC versus D+/+; mean values ± SEM) in CD8+ T cells isolated from the spleen of D−/− or D+/+ mice (n = 3 independently sorted T-cell samples from 3 mice/group). Statistics by unpaired Student’s t test on ΔCt values. (e) PCR analysis of genomic Dicer1 in CD8+ T cells isolated from the spleen of D+/+ or D−/− mice (n = 3 independently sorted T-cell samples from 3 mice/group). Arrows indicate (i) genomic deletion of Dicer1 23rd exon (Dicer1−/−); (ii) floxed but not deleted Dicer1 23rd exon (Dicer1lox/lox); and (iii) wild-type Dicer1 locus (Dicer1+/+). CT−, wild-type allele (BMDM macrophages); CT+, deleted allele (Dicer1 knockout iEC clone). Statistical significance: ∗∗, P < 0.01; ∗∗∗, P < 0.001 Source data for (c) are reported in Supplementary Table 5.
Supplementary Figure 9 Anti-CD8 antibodies deplete tumor CTLs.
(a) Immunostaining of MC38 tumors of D+/+ or D−/− mice, treated as indicated. Data show the number of CD8+ cells per field (mean values ± SEM). Only tumor areas with relatively abundant CD8+ T cells were imaged and analyzed. n = 8 (D+/+; IgG), 6 (D+/+; anti-CD8a), 4 (D−/−; IgG), and 6 (D−/−; anti-CD8a) mice. Statistics by unpaired Student’s t test (b) Representative images from (a). CD8 (red) and DAPI (blue). Scale bar 50 μm. Statistical significance: ∗∗, P < 0.01; ∗∗∗, P < 0.001.
Supplementary Figure 10 Bioinformatics analyses identify Let-7 as a candidate miRNA.
(a,c) Volcano plots showing the distribution of miRNAs based on the FC of their top-300 predicted targets (according to TargetScan-assigned score). Data in (a) show miR-155−/− versus miR-155+/+ B-cells after 48h of LPS, IL-4 and α-CD40 treatment (GSM1479580 and GSM1479572; re-analysis in GSE60426); data in (c) show miR-223−/− versus miR-223+/+ neutrophils (GSE60426). P values were obtained by Kolmogorov-Smirnov test (versus whole transcriptome). Confidence interval (CI) was obtained by randomly resampling of ∼104 miRNA:target interactions. (b,d) Cumulative distribution of logFC values of the top-300 predicted targets. Data in (b) show miR-155−/− versus miR-155+/+ B-cells; data in (d) show miR-223−/− versus miR-223+/+ neutrophils. The red line indicates the logFC of targets for the indicated miRNAs. The back line indicates the logFC in the whole transcriptome. Statistics by Kolmogorov-Smirnov test. (e) Number of pre-miRNAs inferred to have (or not) activity affected by Dicer deletion in TAMs. (f) Top-10 miRNAs with activity predicted to be inhibited by Dicer deletion in TAMs. Ranking is based on the activity scores obtained from the AML TCGA signature-based miRNA association study. (g) Genes negatively correlated to hsa-let-7e in the AML TCGA signature-based miRNA association study. Data show genes with significance level of P < 0.05 and RPKM > 1 in at least one sample. Note that 13/18 genes show concordant deregulation in the AML TCGA and D−/− versus D+/+ TAM datasets.
Supplementary Figure 11 Rescue of Let-7d-5p activity in TAMs opposes the effects of DICER inactivation.
(a) Flow cytometry analysis showing D+/+ or D−/− iECs20 transduced with either an LV expressing a GFP transgene with artificial target sequences for miR-142-3p (miRT-142-3p reporter) or a control LV expressing GFP (no miRT). Transduced cells were then superinfected with LVs to overexpress miR-511 (LV-miR-511), miR-142 (LV-miR-142) or a hybrid miR-451/miR-142-3p (LV-miR-142 / Dicer independent), together with OFP. Note that the Dicer-independent miR-451/142-3p LV, but not a LV expressing the wild-type miR-142-3p, efficiently suppressed GFP in D−/− iECs, which do not express miR-142-3p endogenously. (b,c) Percentage (mean values ± SEM) of blood OFP+CD45+ cells (b) or distinct hematopoietic cell types (c) in mice reconstituted with D−/− HS/PCs previously transduced with the indicated LVs, and analyzed at 6 weeks post-HS/PC transplant. n = 4 (LV-Let-7d) and 8 (LV-control) mice. Statistics by unpaired Student’s t test (b) or two-way ANOVA (c). (d,e) Percentage (mean values ± SEM) of hematopoietic cell types in the BM (d) or blood (e) of MC38 tumor-bearing mice that had been reconstituted with HS/PCs transduced with the indicated LVs. n = 4 (d) or 3 (e) mice for LV-Let-7d and 8 for LV-control. Statistics as in (c). (f) Tumor volume (mean values ± SEM) in MC38-bearing mice, previously reconstituted with D−/− HS/PCs transduced as indicated. n = 4 (LV-Let-7d) and 8 (LV-control) mice. Statistics as in (c).
Supplementary Figure 12 Source data for Western blots.
Unprocessed blots are shown for the Western blots of Fig. 1b (a and b), Supplementary Fig. 1b, c (c and d), and Fig. 7k (e).
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Baer, C., Squadrito, M., Laoui, D. et al. Suppression of microRNA activity amplifies IFN-γ-induced macrophage activation and promotes anti-tumour immunity. Nat Cell Biol 18, 790–802 (2016). https://doi.org/10.1038/ncb3371
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DOI: https://doi.org/10.1038/ncb3371
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