Abstract
Cancer recurrence after surgery remains an unresolved clinical problem1,2,3. Myeloid cells derived from bone marrow contribute to the formation of the premetastatic microenvironment, which is required for disseminating tumour cells to engraft distant sites4,5,6. There are currently no effective interventions that prevent the formation of the premetastatic microenvironment6,7. Here we show that, after surgical removal of primary lung, breast and oesophageal cancers, low-dose adjuvant epigenetic therapy disrupts the premetastatic microenvironment and inhibits both the formation and growth of lung metastases through its selective effect on myeloid-derived suppressor cells (MDSCs). In mouse models of pulmonary metastases, MDSCs are key factors in the formation of the premetastatic microenvironment after resection of primary tumours. Adjuvant epigenetic therapy that uses low-dose DNA methyltransferase and histone deacetylase inhibitors, 5-azacytidine and entinostat, disrupts the premetastatic niche by inhibiting the trafficking of MDSCs through the downregulation of CCR2 and CXCR2, and by promoting MDSC differentiation into a more-interstitial macrophage-like phenotype. A decreased accumulation of MDSCs in the premetastatic lung produces longer periods of disease-free survival and increased overall survival, compared with chemotherapy. Our data demonstrate that, even after removal of the primary tumour, MDSCs contribute to the development of premetastatic niches and settlement of residual tumour cells. A combination of low-dose adjuvant epigenetic modifiers that disrupts this premetastatic microenvironment and inhibits metastases may permit an adjuvant approach to cancer therapy.
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Data availability
All data generated are included in the Article and in its Supplementary Information. Source Data for Figs. 1–4, Extended Data Figs. 1, 3–9 are provided with the paper. Gene-expression data that support the findings of this study have been deposited in the Gene Expression Omnibus under accession number GSE124539. All data are also available from the corresponding authors on reasonable request.
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Acknowledgements
This work was supported by grants from the Brockman Foundation, the Skalka-Kronsberg family as well as the Banks Family Foundation, Bermuda. Research funding was provided by the Van Andel Institute through the Van Andel Institute–Stand Up To Cancer Epigenetics Dream Team. Stand Up To Cancer is a division of the Entertainment Industry Foundation, administered by AACR. We acknowledge G. Merlino for providing the LLC tissue (P3 working stock); S. Singhal, who provided HNM007, a p53-null mouse oesophageal squamous cell carcinoma cell line transformed by HrasG12V; S. A. McGrath-Morrow, who provided B6.129S4 Ccr2tm1Ifc/J mice; L. W. Kwak, who provided synthetic, complementary double-stranded oligonucleotides encoding H6 peptide (TIK), and an irrelevant control peptibody (Irr-pep) (D1); A. Tam and R. L. Blosser for their help with flow cytometry; P. Ordentlich from Syndax Pharmaceuticals for providing entinostat; S. Zhou for advice and consultation; L. Bois and J. Murphy for expert technical help; Y. Lai for histological analysis with H&E staining; and W. Zhu for data analysis.
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Z.L., J.Z., S.L. and M.V.B. conceptualized, designed and performed the experiments and wrote the manuscript. S.B.B., L.S., F.H. and D.M.P. designed experiments and wrote the manuscript. M.J.T., Y.T. and M.V. helped with the design the experiments and reviewing the manuscript. K.R. and B.L. helped with the sample collection. C.-P.D. provided LLC tissue in the study. H.Z., W.X., X.K., H.L., X.J., Yanni Wang, Yujiao Wang, R.-W.C.Y., W.Z., Y. Cai, H.E.,Y. Cui, L.X., A.H., J.B.R. and Y.M. performed experiments and assisted in acquisition of data. C.M.R. and R.A.J. designed the clinical trial. K.K., S.C.Y., R.J.B., E.L.B., S.R.B., S.M.C., J.R.B., J.W., Y.J.K. and P.M.F. enrolled patients and assisted in acquisition of data. P.H. helped with the statistical analyses. B.Z. and K.K.-H.W. helped with the CBCT imaging. C.A.Z. assisted with the mouse experiments. J.B.M. and B.D.N. assisted with discussions and reviewing the manuscript.
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Z.L. and M.V.B. have patent applications on epigenetic therapy. J.R.B., C.M.R., P.M.F. and R.A.J. serve on the advisory boards of Bristol-Meyers Squibb (BMS) and AstraZeneca. J.R.B., P.M.F. and R.A.J. are on the advisory board of Merck and receive grant support from BMS. J.R.B., C.M.R. and R.A.J. are on the advisory boards of Amgen and Genentech/Roche. Y.J.K., P.M.F. and R.A.J. serve on the advisory board of Novartis. C.M.R. and J.R.B. are on the advisory board of Celgene. J.R.B. and P.M.F. serve on the advisory boards of Eli Lilly and Jansen. C.M.R. and P.M.F. are on the advisory board of AbbVie. R.A.J. and P.M.F. serve on the advisory board of Boehringer Ingelheim. R.A.J. and Y.L.K. are on the advisory board of Takeda. R.A.J. is on the advisory board of Pfizer. S.R.B. is a consultant to BMS. J.W. is cofounder and chief medical officer of Precision Genetics. J.R.B. serves on the advisory board of Syndax, and receives honoraria from Roche/Genentech. C.M.R. is a consultant to and/or advisory board member for Ascentage, Daiichi Sankyo, Ipsen, Loxo, Pharmamar, Vavotek, Bridge Medicines and Harpoon Therapeutics. Y.J.K. serves on the advisory boards of Dracen, Aduro and Sanofi. P.M.F. is a consultant to and/or advisory board member for EMD Serono and lnivata, and receives grant support from Corvus, Kyowa and Novartis. S.B.B. serves on the advisory boards of Mirati Therapeutics, MDxHealth and Aminex Therapeutics. M.V.B., Y.M., J.R.B., P.M.F. and R.A.J. receive grant support from AstraZeneca. R.A.J. and J.R.B. receive grant support from Merck. All other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Efficacy of low-dose AET on cancer recurrence in patients with stage I (T1-2aN0) NSCLC in a phase-II clinical trial.
a, Schema for a randomized phase-II clinical trial of AET in patients with stage I (T1-2aN0) NSCLC (NCT01207726). b, Postsurgical recurrence rates in the observation and AET groups. c, Kaplan–Meier curves for disease-free survival in the observation and AET groups. P = 0.50 by two-sided log-rank test.
Extended Data Fig. 2 Schema outlining the establishment and characteristics of the mouse models of pulmonary metastasis.
a, Schema for establishing the highly aggressive HNM007 model. Pulmonary metastases were collected and serially subcutaneously implanted in the right flanks of mice for 10 passages. b, Schema for establishing the 4T1 model. c, Characteristics of mouse cell line (nonselective) and spontaneous mouse tissue models of pulmonary metastasis (metastases collected selectively from serial pulmonary metastases to produce a solely pulmonary-metastatic phenotype). d, Longitudinal gross pathological photographs of bilateral pulmonary metastases during the natural history of the LLC model in C57BL/6 mice from day 0 to day 15 after surgery. N1, N2 and N3 depict the experiment performed in triplicate. Two mice were killed at each time point from day 0 to day 15 (n = 36); data for 18 mice are shown here as representative photomicrographs. e, H&E staining of pulmonary metastases in LLC, HNM007 and 4T1 mice. The histology of LLC (day 9), HNM007 (day 12) and 4T1 (day 12) pulmonary metastases were confirmed by a pathologist. Scale bars, 100 μm. Representative data were repeated at least three times with similar results.
Extended Data Fig. 3 CD11b+GR1+ cells persist as the predominant immune cells even after resection in the lung premetastatic microenvironment as functional MDSCs.
a, In LLC mice, lung CD11b+Ly6ChighLy6G− and CD11b+Ly6ClowLy6G+ cells collected at 72 h after resection both have suppressive activity in vitro against CD8a T cells. Freshly isolated CD11b+Ly6ChighLy6G− or CD11b+Ly6ClowLy6G+ cells from both lungs at day 3 after resection were cocultured with CD8a T cells for 72 h at different ratios (0:1, 1:1, 2:1, 4:1 and 1:0). T cell proliferation and IFNγ concentrations in the supernatant were measured by FACS (left) and ELISA (right), respectively (n = 3 biological replicates). Representative data were repeated at least three times with similar results. Two-sample, two-sided t-test was used in the comparison with mock (CD8a T cells alone). b, Immune-cell profiles of liver in LLC mice. Single-cell suspensions from the entire liver were analysed by FACS (n = 3 mice per time point) at different time points after surgery. NC, negative control (normal liver from C57BL/6 mice). c, Immune-cell profiles of both lungs in HNM007 mice at different time points after surgery. Single-cell suspensions from both lungs were analysed by FACS (n = 3 mice per time point). NC, negative control (normal lungs from C57BL/6 mice). In b, c, a two-sample, two-sided t-test was used in comparison with the negative control. All bars show mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 4 Consideration of combined AET dosing on the basis of its effect on mouse models.
a, Top, effect of different dosages of epigenetic modifiers on the viability of LLC1, HNM007 and 4T1 cells in vitro (72 h, Cell Counting Kit-8). Graphs show the mean of 3 independent experiments; two-sample, two-sided t-tests compared with mock. Bottom, effect of low-dose 5-azacytidine (100 nM) plus entinostat (50 nM) on the proliferation of LLC1, HNM007 and 4T1 cells in vitro. A total of 1 × 105 viable cells was plated per well. Cells were collected at 24, 48 and 72 h and counted using a cell counter (Bio-Rad) after Trypan blue exclusion. Graphs show the mean of 3 independent experiments; significance at 72 h was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. b, The effect of low-dose 5-azacytidine (100 nM) plus entinostat (50 nM) on the viability of MDSCs from bone marrow (day 3) of LLC mice (top) and HNM007 mice (bottom) in vitro (Cell Counting Kit-8). Graphs show the mean of 3 independent experiments. c, The effect of low-dose 5-azacytidine (100 nM) plus entinostat (50 nM) on the apoptosis of MDSCs from bone marrow (day 3) of LLC and HNM007 mice in vitro. Cell apoptosis was measured by FACS at 48 h. The bottom right quadrant (annexin-V+/7-AAD−) and top right quadrant (annexin-V+/7-AAD+) represent early and late apoptotic cells, respectively. Graphs show the percentage of total apoptosis (early and late apoptosis) in mock and treatment groups (n = 3 biological replicates). d, Tumour growth and body weight of NSG mice bearing LLC tissue that were treated with different doses of entinostat plus 5-azacytidine. Significance at day 12 (top) and day 14 (bottom) was determined by one-way ANOVA followed by Tukey’s test for multiple comparisons. e, Summary table of tumour growth, body weight and treatment-related death of NSG mice bearing LLC tissue. Regimens in red indicate dosages with no effect on tumour growth, weight loss or treatment-related death. f, Tumour growth and body weight of NSG mice bearing HNM007 tissue that were treated with 5-azacytidine at 0.5 mg kg−1 d−1 plus entinostat at 5 mg kg−1 d−1 or vehicle. Significance at day 14 was determined by two-sample, two-sided t-test. g, h, The effect of low-dose AET on the proliferation (g) and apoptosis (h) of donor-derived CD45.1+ MDSCs from bone marrow in CD45.2 LLC mice. Proliferation and apoptosis of immature (MHC-II−) and mature (MHC-II+) CD45.1+ cells were measured by FACS at 36 h after transfusion (day 2). Graphs in g show the percentage of Ki67+ cells (n = 3 mice per group). Graphs in h show the percentage of total apoptosis (early and late apoptosis) in mock-treated and low-dose-AET groups (n = 3 mice per group). In b, c, g, h, two-sample, two-sided t-tests were used. All bars show mean ± s.e.m. *P < 0.05.
Extended Data Fig. 5 Low-dose AET disrupts the lung premetastatic microenvironment, mainly by affecting MDSCs.
a, The effect of low-dose AET (5-azacytidine 0.5 mg kg−1 d−1 plus entinostat 2.5 mg kg−1 d−1) on MDSCs from the lung at day 3 after resection in 4T1 mice (n = 3 mice per group). b, c, Immunofluorescence staining of CD4+ and CD8+ T cells (b) or GR1+ cells (c) from the lung premetastatic microenvironment (day 3) in LLC mice with or without low-dose AET. Negative control was normal lungs from tumour-free C57BL/6 mice. Immunofluorescence staining was performed using CD4 (green) and CD8 (red) antibodies, or GR1 (red) antibodies. Merged images contain DAPI staining for cell nuclei (blue). Original magnification 20×. Representative data were repeated at least three times with similar results. d, e, The mRNA (d) and protein (e) levels of representative molecular factors known to promote premetastatic microenvironment formation from both lungs of normal mice, and mock- or low-dose-AET-treated LLC mice (day 3) were measured by quantitative PCR and western blot. Two-sample, two-sided t-test for quantitative PCR experiments (n = 3 biological replicates). For gel source data, see Supplementary Fig. 1. All the experiments were performed in triplicate and similar results were obtained. f, Top, graph showing the percentages of donor-derived cell subsets (CD45.1+ MDSC cells) in the lungs of LLC mice or of sham-surgery mice (tumour-naive recipient mice) 36 h after surgery. Bottom, graph showing the percentages of donor-derived cell subsets (CD45.1+ MDSC cells) in the lungs of low-dose-AET- or vehicle-treated sham-surgery mice (tumour-naive recipient mice) 36 h after surgery. Purified 5 × 106 MDSCs from bone marrow of CD45.1 mice bearing LLC tumours (day 0) were adoptively transferred into CD45.2 recipient mice in the sham-surgery tumour-naive model or LLC model (n = 3 mice per group). In a, f, two-sample, two-sided t-tests were used. All bars show mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 6 Low-dose AET induces substantial changes in immune-cell chemotaxis and migration in MDSCs in LLC mice.
a, Left, schema showing the effect of low-dose AET on monocytic or polymorphonuclear MDSCs transferred from CD45.1 to CD45.2 C57BL/6 mice in the LLC model. CD45.1+ cells (transferred polymorphonuclear MDSCs) were identified in the lungs of the recipient mice and analysed by flow cytometry (right, n = 3 mice per group). b, Left, schema showing trafficking ability of adoptively transferred monocytic or polymorphonuclear MDSCs from low-dose-AET-treated or untreated CD45.1 mice in the LLC model. CD45.1+ cells (transferred polymorphonuclear MDSCs) were identified in the lungs of the recipient CD45.2 mice and analysed by flow cytometry at 18 h after transfer (right, n = 3 mice per group). c, Left, top 10 upregulated gene sets from GSEA of lung monocytic MDSCs after 72 h of treatment with low-dose AET. Middle and right, representative upregulated GSEA plots of immune-cell chemotaxis and migration. Colour gradation is representative of log2-transformed fold change over mock (n = 3 biologically replicates). Gene-set enrichment P values, NES values and FDR values reported are calculated with 1,000 permutations in the GSEA software. FDR q < 0.25 was deemed significant. d, DAVID analyses of significantly downregulated genes using the KEGG Gene Ontology in MDSCs from bone marrow of LLC mice, treated or untreated with low-dose AET. Top 20 downregulated pathways are presented (n = 3 biologically replicates). Hypergeometric test (FDR-adjusted P < 0.05). e, Low-dose AET significantly decreases the nuclear activation of p52 and RELB (OD450 nm (mock versus low-dose AET), p52: 0.95 ± 0.035 versus 0.721 ± 0.011, P = 0.0034; RELB, 0.251 ± 0.012 versus 0.1 ± 0.003, P = 0.0002), but not p50 and p65 in monocytic MDSCs from bone marrow of LLC mice in vivo. Nuclear lysates were incubated with oligonucleotides containing the NF-κB-binding consensus sequence, and specific antibodies were used to detect the different subunits within the bound complexes (n = 3 biological replicates). f, FACS shows the effect of 30 mg kg−1 d−1 and 75 mg kg−1 d−1 of BMS-345541 (a highly selective IKB kinase inhibitor) on CCR2 expression in monocytic MDSCs from bone marrow of LLC mice on day 3 after surgery. The experiments were performed in triplicate, and similar results were obtained. g, CXCR1 and CXCR2 expression of polymorphonuclear MDSCs collected on day 3 from the bone marrow or lung detected by quantitative PCR (top) and FACS (bottom) in LLC mice treated with vehicle or with 72 h of low-dose AET (n = 3 biological replicates). h, Transwell migration assay of sorted polymorphonuclear MDSCs from bone marrow of low-dose-AET- (72 h) or vehicle-treated LLC mice, induced by CXCL1 (20 ng ml−1 and 50 ng ml−1) for 120 min (n = 3 biological replicates). In a, b, e, g, h, two-sample, two-sided t-tests were used. All bars show mean ± s.e.m.
Extended Data Fig. 7 Low-dose AET promotes the differentiation of monocytic MDSCs towards macrophages in the LLC model.
a, DAVID analyses of the significantly downregulated and upregulated genes using the KEGG Gene Ontology in LLC mice, treated or untreated with low-dose AET (n = 3 biological replicates). Hypergeometric test (FDR-adjusted P < 0.05). b, c, The mRNA (b) and protein (c) levels of representative transcription factors were measured by quantitative PCR and western blot, respectively. In vitro, splenic monocytic MDSCs from LLC mice were cultured for 3 days with tumour-conditioned medium. In vivo, monocytic MDSCs from both lungs of mock- (day 3) and low-dose-AET-treated LLC mice (day 3) were sorted for analysis. For gel source data, see Supplementary Fig. 1. Representative data were repeated at least three times with similar results. In b, a two-sample two-sided t-test was used, n = 3 biological replicates. All bars show mean ± s.e.m.
Extended Data Fig. 8 Low-dose AET promotes the differentiation of monocytic MDSCs towards an interstitial macrophage-like population in the lung premetastatic microenvironment.
a, Gating strategy used to identify and analyse lung interstitial macrophages in the lung premetastatic microenvironment by FACS. b, The effect of low-dose AET on lung interstitial macrophages from LLC mice. The percentage and cell counts of interstitial macrophages from both lungs in mock- and low-dose-AET-treated mice were analysed by FACS at day 3 after surgery (n = 3 mice per group). Two-sample, two-sided t-test. All bars show mean ± s.e.m. c, Gating strategy used to identify and analyse CD45.1+ lung interstitial macrophages from the lungs of recipient CD45.2 mice after the transfusion of CD45.1+ monocytic MDSCs. d, Kaplan–Meier curves showing the disease-free survival and overall survival of Ccr2-knockout LLC mice after transfusion of wild-type monocytic MDSCs (5 × 106), low-dose-AET-treated (in vivo) wild-type monocytic MDSCs (5 × 106) or vehicle at day 1 and day 4, respectively. e, Kaplan–Meier curves showing the disease-free survival and overall survival of the Ccr2-knockout LLC mice after transfusion of wild-type polymorphonuclear MDSCs (5 × 106), low-dose-AET-treated (in vivo) wild-type polymorphonuclear MDSCs (5 × 106) or vehicle at day 1 and day 4, respectively. In d, e, two-sided log-rank tests were used.
Extended Data Fig. 9 Low-dose AET inhibits pulmonary metastases and prolongs overall survival in mouse models.
a, Representative photographs showing lungs treated with vehicle or low-dose AET in LLC (day 6) and HNM007 (day 10) mice. The red arrows indicate the metastases. b, Representative CBCT images of lung metastases on day 6 after resection in LLC mice treated with vehicle or low-dose AET. The red arrows indicate the metastases. c, Representative H&E-stained images of lung sections from HNM007 (top) and 4T1 (bottom) mice treated with low-dose AET or vehicle at different time points after surgery. Scale bars, 2 mm. Graph shows area and numbers of metastatic nodules. At each time point, three mice were killed for analysis. For each sample, sections from three levels were analysed. Two-sample, two-sided t-test. d, Kaplan–Meier curves showing the disease-free survival and overall survival of HNM007 and 4T1 mice, treated with low-dose AET (for the 4T1 model, 5-azacytidine 0.5 mg kg−1 d−1 plus entinostat 2.5 mg kg−1 d−1) or vehicle after surgery. e, FACS showing the effect of T-cell-depleting antibodies on CD4+ and CD8+ T cells in the peripheral blood of LLC mice. n = 3 mice per group. Two-sample, two-sided t-test. f, Kaplan–Meier curves showing the disease-free survival and overall survival of LLC mice treated with vehicle, CCR2 antagonist (RS102895) (Sigma), low-dose AET and RS102895 plus low-dose AET after surgery. g, Kaplan–Meier curves showing the disease-free survival and overall survival of HNM007 mice treated with vehicle, CCR2 antagonist (RS504393) (Sigma), low-dose AET and RS504393 in combination with low-dose AET. In d, f, g, two-sided log-rank tests were used. Representative data in a, b were repeated at least three times with similar results. All bars show mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001.
Extended Data Fig. 10 Representation of the effect of low-dose adjuvant epigenetic modifiers on lung metastases in the tumour pulmonary-metastasis models.
Graphic model showing the inhibition of pulmonary metastases by low-dose AET via its effect on MDSCs. First, low-dose AET can inhibit the trafficking of monocytic and polymorphonuclear MDSCs from the bone marrow to the premetastatic microenvironment by downregulating the expression of CCR2 and CXCR2, respectively. Second, even if MDSCs migrate to the lung, low-dose AET can skew the differentiation of monocytic MDSCs towards an interstitial macrophage-like phenotype in the lung premetastatic microenvironment. Therefore, low-dose AET can disrupt the lung premetastatic microenvironment, ultimately inhibiting pulmonary metastases.
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Lu, Z., Zou, J., Li, S. et al. Epigenetic therapy inhibits metastases by disrupting premetastatic niches. Nature 579, 284–290 (2020). https://doi.org/10.1038/s41586-020-2054-x
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DOI: https://doi.org/10.1038/s41586-020-2054-x
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