Lack of insight into mechanisms governing breast cancer metastasis has precluded the development of curative therapies. Metastasis-initiating cancer cells (MICs) are uniquely equipped to establish metastases, causing recurrence and therapeutic resistance. Using various metastasis models, we discovered that certain primary tumours elicit a systemic inflammatory response involving interleukin-1β (IL-1β)-expressing innate immune cells that infiltrate distant MIC microenvironments. At the metastatic site, IL-1β maintains MICs in a ZEB1-positive differentiation state, preventing MICs from generating highly proliferative E-cadherin-positive progeny. Thus, when the inherent plasticity of MICs is impeded, overt metastases cannot be established. Ablation of the pro-inflammatory response or inhibition of the IL-1 receptor relieves the differentiation block and results in metastatic colonization. Among patients with lymph node-positive breast cancer, high primary tumour IL-1β expression is associated with better overall survival and distant metastasis-free survival. Our data reveal complex interactions that occur between primary tumours and disseminated MICs that could be exploited to improve patient survival.
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This work was supported by funding from the following: International Mentoring Foundation for the Advancement of Higher Education (IMFAHE) (A.G.R.); Center for Stem Cell Bioinformatics at the Harvard Stem Cell Institute (J.N.H.); Samuel Waxman Cancer Research Foundation, Breast Cancer Research Foundation, and Ludwig Center for Molecular Oncology (R.A.W.); Advanced Medical Research Foundation and Nelune Foundation (C.L.C.); National Institutes of Health (NCI) RO1 CA166284, Presidential Early Career Award for Scientists and Engineers, American Cancer Society Research Scholar award, and Department of Defense BCMRP Era of Hope Scholar Award W81XWH-14-1-0191 (S.S.M.). We thank the following: J. Joyce for the Met1 cell line (with permission from A. Borowsky); J. Sceneay, B. Bierie, Y. Qin and K. Tracy for technical expertise; J. Olive for editorial assistance; E. Frank and the Dana-Farber/Harvard Cancer Center Breast Cancer Advocate group for critical discussion and input; Z. Herbert of the Molecular Biology Core Facilities, Dana-Farber Cancer Institute; G. Bell and B. Rosner for statistical advice; The Specialized Histopathology Core at Dana-Farber/Harvard Cancer Center (supported in part by a NCI Cancer Center Support Grant no. NIH 5 P30 CA06516); the Histology Core Facility at the KI Swanson Biotechnology Center, MIT; and the Flow Cytometry Core Facility at the Whitehead Institute for Biomedical Research. Finally, we thank GG Bernath, A. Bernath, J. Bernath, Z. Corsino-Gupta, P. Nguyen, B. Nguyen and K. Nguyen for their support during the preparation of this manuscript.
The authors declare no competing interest.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Integrated supplementary information
a, Met1 orthotopic primary tumour growth kinetics, FVB mice, represented in Fig. 1b (n = 10 animals). b, Schematic of sequential subcutaneous experimental model (left) and growth kinetics (right) of Met1 secondary tumours (2.5 × 105/mouse; implanted at day 14) in mice with PBS control or Met1 primary tumour (2.5 × 105/mouse; n = 5 animals/cohort). c, Numbers of pulmonary macrometastases following tail vein injection of MT2 or MT3 cells (7.5 × 105cells; n = 3 animals). Macrometastases > 100 microns were quantified on microscope tissue sections from 4 lung lobes per mouse. d, Met1 orthotopic tumour growth kinetics, Nude mice, represented in Fig. 1f (n = 5 animals). e, Schematic of experimental model (left), and number of pulmonary macrometastases following tail vein injection of 2.5 × 105 hMICs into Nude mice with either Matrigel (n = 10 animals) or HMLER primary tumours (n = 9 animals; original injection of 5.0 × 105 cells/mouse) (right). Macrometastases ( > 100 microns) or micrometastases (>5 cells or < 5 cells) were quantified from microscopic whole lung tissue sections. f, Schematic of experimental model (applies to g and h). g, Growth kinetics of HMLER primary tumours, Nude mice, described in Fig. 1h (n = 10 animals). h, 231-MIC tumour growth kinetics, Nude mice, opposite Matrigel control (n = 12 animals) or HMLER primary tumours (n = 5 animals). Representative of 2 experiments. i, Images: representative immunofluorescent images of 231-MIC tumours grown opposite Matrigel control or an HMLER primary tumour (represented in Supplementary Fig. 1h) stained with Ki67 (red), hMIT to identify human mitochondria (green), DAPI (nuclei, blue); Scale bars = 100 µm. Graph: Quantification of Ki67 + hMit + cells as a percentage of the total number of hMit + tumour cells/microscopic field (n = 9 independent images representing 3 tumours/cohort). Source data for a, b, c, d, e, g, h, i in Supplementary Table 1. 2-way ANOVA, followed by Sidak’s multiple comparison test (b, g, h); 1-sided Welch’s t test (e); 2-sided Welch’s t test (c, i)
a, In vitro immunocytochemical flourescence showing E-cadherin (ECAD, red) and DAPI (nuclei, blue) in Met1 parental cell line (mMIC) and Met1-derived clones, MT2 and MT3 (mMIC-MT3). b, Images: Immunofluorescence showing ZEB1 and ECAD expression in cultured hMICs prior to xenotransplantation. Western blot: mesenchymal marker Vimentin (VIM) and epithelial marker ECAD protein in polyclonal HMLER cells and derivative hMIC and HMLER2 cells. GADPH shown as internal control. Positive controls: Ctrl E (epithelial-MCF7Ras); Ctrl M (mesenchymal CD44hi HMLER cells). c, Merged immunofluorescent images of mMIC-MT3 tumours (described in Fig. 1d) stained for basal cytokeratin 14 (CK14, red), luminal CK8 (green) or PyMT antigen (expressed by tumour cells only-green). Arrows - CK14 + tumour cells. d, Images: hMIC tumours (from Fig. 1i) stained with CK14 (red), VIM (green) and DAPI (blue); Graph: quantification of indicated stains on hMIC tumours grown opposite Matrigel (n = 4 tumours) or primary tumour (n = 5 tumours). e, Schematic: modelling early stages of hMIC colonization. Graph: hMIC tumour growth kinetics opposite Matrigel control or HMLER primary tumour (n = 4 tumours/group); differences not statistically significant. f, g, Immunofluorescent images (f) and quantification (g) of hMIC tumours stained for ki67 (red), LgT antigen (tumour cells, green), and DAPI (nuclei, blue) as a percentage of total LgT + cells. Control, n = 10 independent images representing 4 tumours; HMLER cohort, n = 9 independent images representing 4 tumours. h, i, Immunofluorescent images (h) and quantification (i) of staining hMIC tumours for cleaved caspase3 (CASP3, red), human-specific mitochondria (hMIT, green), and DAPI (nuclei, blue) grown in mice with Matrigel control (n = 6 independent images representing 4 tumours) or HMLER primary tumours (n = 5 independent images representing 4 tumours). j, Expression of ZEB1 (ZEB1-GFP construct) or HRAS (HRAS-tomato construct) analyzed by FACS (1.0 × 105 cells) in Control hMIC or ZEB1hi hMIC (from Fig. 2n-p). All scale bars = 100 µm. Source data for d, e, g, i in Supplementary Table 1 and d on Supplementary Figure 9. 2-way ANOVA (e); 2-sided Welch’s t test (d, i); 2-sided Mann-Whitney test (g). Unprocessed blots are available in Supplementary figure 9
a, Experimental schematic for RNA-seq tissue analysis (Fig. 3a-c and Supplementary Fig. 3b-e). b, Met1 primary tumour mass in FVB mice (n = 5 animals). c, d, RNA-seq analysis on lungs from mice with PBS control (n = 4 animals) or a Met1 primary tumour (n = 4 animals). Heatmap (c): top 50 differentially expressed genes (adjusted p-value, DESeq2). Blue = low, green = mean, and yellow = high relative expression levels. PBS control lungs (yellow), Met1 primary tumour-bearing lungs (purple). Volcano plot (d): DESeq2 comparison Single gene with Padj < 0.05 and absolute log2(FoldChange) > 1 (green). e, Experimental schematic and flow cytometric quantification of immune cell populations in lungs of indicated FVB mice at 28-day end point (see Fig. 1a). f, Ratio of genes expressed by pro-metastatic immunosuppressive neutrophils from K14cre;Cdh1F/F;Trp53F/F (KEP) mice to control neutrophils from wild type littermates (KEP:Normal)13 extrapolated onto our signatures from control (blue) primary tumour-bearing lungs (red). Higher ratios indicate higher pro-metastatic KEP signature. Box plot: median, 25th and 75th percentiles, whiskers extend to minimum and maximum values. g, Experimental design to identify optimal anti-Ly6G dose for neutrophil depletion. h, Primary tumour mass in Control anti-IgG2a (n = 3 mice/cohort) and anti-Ly6G (n = 4 mice/cohort). i, Flow cytometric gating strategy for neutrophils (CD45 + CD11b + Gr1 + Ly6Clo) and monocytes (CD45 + CD11b + Gr1 + Ly6Chi). j-l, Flow cytometric analysis of blood at 14d (j), 20d (k), and lungs 20d (l) in experiment described in (g). Control anti-IgG2a n = 3 mice/cohort; anti-Ly6G n = 4 mice/cohort. m, Schematic modelling neutrophil depletion experiment (n-r). n, Primary tumour mass from Fig. 3i (control IgG2a n = 4 mice/cohort; anti-Ly6G n = 8 mice/cohort). o, Immunofluorescent images of Met1 lung metastasis. Scale bars = 200 µm. p, Metastases at higher magnification (indicated in circles in (o) analyzed by ImageJ to measure shortest-length diameter). q, Single channel ECAD or PyMT staining measured by ImageJ analysis (Fig. 3k, l). r, End point flow cytometric analysis of neutrophils and monocytes in lungs of mice treated with control IgG2a (n = 4 mice/cohort) or anti-Ly6G (n = 8 mice/cohort). Source data for b, e, h, j, k, l, n, r in Supplementary Table 1. DESeq2’s Wald test two-sided (c), 2-sided Welch’s t-test (f); 2-sided Welch’s t test (n); 2-way ANOVA followed by Sidak’s multiple comparison test (f, h, j, k, l, o)
a, Some of the predominant cytokines expressed by monocytes, neutrophils or by both. b, ELISA to detect cumulative IL-1β in the conditioned media of Met1 cell line and its derived clones after 3 days in vitro (n = 5 biological replicates/group). c, Met1 parental cells and the MT2 and MT3 subclones stained as indicated (upper panel). Scale bars = 100 µm. Western blot detecting phospho-p65 (p-p65; activated form of NF-kB p65) or total NF-kB p65 (p65) in Met1, MT2 and MT3 treated in vitro with indicated doses of IL-1β at indicated time points (lower panel). d, ELISA to detect cumulative IL-1β in the conditioned media of HMLER and hMIC cell lines over 3 consecutive days of in vitro culture (n = 4 biological replicates/group). e, Experimental schematic and flow cytometric quantification of immune cell populations in digested hMIC tumours from indicated cohorts in Nude mice at the 28-day experimental end point (see Fig. 2i) (n = 3 hMIC tumours in Matrigel control and 4 hMIC tumours in HMLER cohort). Source data for b, d, e in Supplementary Table 1. 2-sided Welch’s t test (b, e); 2-way ANOVA, Sidak’s multiple comparisons test (d). Unprocessed blots are available in Supplementary figure 9
a, qRT-PCR to determine IL-1R1 mRNA levels in hMIC expressing shRNA against IL-1R1 (shA-IL-1R1-MIC and shB-IL-1R1-MIC) compared to hMIC expressing a scrambled shRNA (sh-Ctl-MIC). B-actin (ACTB) served as the internal control (n = 3 PCR technical triplicates). b, Proliferation analysis (CyQuant assay) comparing growth of MIC-control cells to those expressing shRNA against IL-1R1 (shA-IL-1R1-MIC and shB-IL-1R1-MIC; n = 3 in triplicate. Representative of 5 independent experiments. c, hMIC tumour mass (mg) at the 4-week experimental end point (n = 8 animals/cohort, except sh-Ctl-MIC n = 7 animals). d, Met1 tumourspheres generated in 3 independent experiments, treated with IL-1β (10 ng/mL), Anakinra (500 ng/ml) or IL-1β + Anakinra combination (n = 3 replicates, 28 technical replicates) every 48 h for 7d. e, Western blot detecting phospho-p65, total p65, or ECAD in Met1 cells treated with 10 ng/ml of IL-1β and increasing doses of ANAKINRA (IL-1Ra) for 48 h. B-actin (ACTB) was used as a loading control. f, Flow cytometric analysis of monocytes (CD11b+Ly6C+Ly6Glo) presented as % CD11b + cells in blood at 10 days, prior to the injection of contralateral tumours (Matrigel n = 11 animals (combined from Groups 1 and 3); HMLER n = 7 animals). g, Final mass (mg) of hMIC tumours from indicated cohorts (differences not statistically significant, Control n = 7 animals, HMLER n = 6 animals). One experiment performed. Source data from a, b, c, d, f, g in Supplementary Table 1. 2-way ANOVA and Sidak’s multiple comparisons test (a, b); 1-way ANOVA (c, g); 2-sided Student’s t tests (d); 2-sided Mann-Whitney test (f). Unprocessed blots are available in Supplementary figure 9
a, Representative images of F4/80 staining and ImageJ analysis used to quantify area covered by positive F4/80 staining in Fig. 6b. Scale bar = 100 µm. Representative of n = 5 HMLER tumours and n = 6 HMLER2 tumours
a, KEGG IL-17 signalling pathway (mmu04657). The log2 fold change values comparing primary tumour to control lungs. Red, higher expression in primary tumours; green, higher expression in normal lungs (n = 3 lungs from 3 animals for Met1 primary tumours, and n = 4 control lungs from 4 animals). b, Inflammatory cytokine array analysis of conditioned media from HMLER and hMIC cells after 3 days in culture (n = 2 biological replicates, 3 technical replicates). Results are fold change (Log2) HMLER/MIC. c, Mouse inflammatory cytokine array results showing differences between plasma from mice bearing Matrigel control, HMLER, or HMLER + IL-1Ra tumours after 2 weeks in vivo (n = 2, plasma pooled from 3 individual animals per sample). Graph: cytokines statistically different between cohorts. d, hMIC tumour mass (4 weeks) grown opposite Matrigel control (n = 8 tumours), HMLER primary tumour (n = 7 tumours), or HMLER + IL-1Ra antagonist (IL-1Ra) (n = 8 tumours). One experiment was performed. e, Graph: HMLER primary tumour mass embedded in control Matrigel (n = 5 animals) or Matrigel with 100 ng/ml IL-1Ra (n = 6 animals). Incidence (%) marked on graphs. f, Hematoxylin and eosin (H&E) stains from HMLER and HMLER + IL-1Ra tumours (Fig. 7c-g), low and high magnification. g, F4/80 staining and ImageJ analysis to quantify area covered by F4/80 + staining in Fig. 7e. Boxes indicate area shown in Fig. 7e. h, Immunohistochemistry showing macrophage infiltration (F4/80 stain) in hMIC tumours grown opposite Matrigel control, HMLER, or HMLER + IL-1Ra primary tumours. i, j, Photomicrographs of hMIC tumours grown opposite Matrigel control, HMLER primary tumour, or HMLER + IL-1Ra primary tumours. ECAD (red), LgT (green – to detect human tumour cells), DAPI (blue). (h) ZEB1 (red), LgT (green), DAPI (blue). Boxes indicate area shown in Fig. 7f-g. Scale bars = 100 µm. Source data for b, c, d, e in Supplementary Table 1. DESeq2’s Wald test two-sided (a); 2-sided Mann-Whitney test (d); 2-sided Welch’s t test (e)
Supplementary Figure 8 Analysis of IL1 pathway components as predictors of disease outcome in clinical breast cancer specimens.
a, Kaplan-Meier analysis using distant metastasis-free survival (DMFS) as end point with 10-year censoring based on stratification of IL1B gene expression (log2; low, grey line, n = 740; high, red line, n = 639) in tumour tissue from 1,379 breast cancer patients. Logrank p value is shown. Analysis performed using the GOBO database (http://co.bmc.lu.se/gobo/gsa.pl)
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Castaño, Z., San Juan, B.P., Spiegel, A. et al. IL-1β inflammatory response driven by primary breast cancer prevents metastasis-initiating cell colonization. Nat Cell Biol 20, 1084–1097 (2018). https://doi.org/10.1038/s41556-018-0173-5
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