Carcinoma–astrocyte gap junctions promote brain metastasis by cGAMP transfer

A Corrigendum to this article was published on 29 March 2017

Abstract

Brain metastasis represents a substantial source of morbidity and mortality in various cancers, and is characterized by high resistance to chemotherapy. Here we define the role of the most abundant cell type in the brain, the astrocyte, in promoting brain metastasis. We show that human and mouse breast and lung cancer cells express protocadherin 7 (PCDH7), which promotes the assembly of carcinoma–astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with the astrocyte gap-junctional network, brain metastatic cancer cells use these channels to transfer the second messenger cGAMP to astrocytes, activating the STING pathway and production of inflammatory cytokines such as interferon-α (IFNα) and tumour necrosis factor (TNF). As paracrine signals, these factors activate the STAT1 and NF-κB pathways in brain metastatic cells, thereby supporting tumour growth and chemoresistance. The orally bioavailable modulators of gap junctions meclofenamate and tonabersat break this paracrine loop, and we provide proof-of-principle that these drugs could be used to treat established brain metastasis.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Cx43 and PCDH7 are associated with brain metastasis.
Figure 2: Cx43–PCDH7 carcinoma–astrocyte gap junctions mediate brain metastasis.
Figure 3: Gap junctions activate STAT1 and NF-κB pathways in cancer cells.
Figure 4: Gap junctions induce cytosolic dsDNA response in astrocytes.
Figure 5: Inhibition of gap junction activity controls brain metastatic outgrowth.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

RNA-seq data have been deposited in NCBI Gene Expression Omnibus (GEO) under accession number GSE79256.

References

  1. 1

    Gavrilovic, I. T. & Posner, J. B. Brain metastases: epidemiology and pathophysiology. J. Neurooncol. 75, 5–14 (2005)

    PubMed  Google Scholar 

  2. 2

    Stelzer, K. J. Epidemiology and prognosis of brain metastases. Surg. Neurol. Int. 4, S192–S202 (2013)

    PubMed  PubMed Central  Google Scholar 

  3. 3

    Bos, P. D. et al. Genes that mediate breast cancer metastasis to the brain. Nature 459, 1005–1009 (2009)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Eichler, A. F. et al. The biology of brain metastases-translation to new therapies. Nature Rev. Clin. Oncol. 8, 344–356 (2011)

    CAS  Google Scholar 

  5. 5

    Kienast, Y. et al. Real-time imaging reveals the single steps of brain metastasis formation. Nature Med. 16, 116–122 (2010)

    CAS  PubMed  Google Scholar 

  6. 6

    Valiente, M. et al. Serpins promote cancer cell survival and vascular co-option in brain metastasis. Cell 156, 1002–1016 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Giaume, C., Koulakoff, A., Roux, L., Holcman, D. & Rouach, N. Astroglial networks: a step further in neuroglial and gliovascular interactions. Nature Rev. Neurosci. 11, 87–99 (2010)

    CAS  Google Scholar 

  8. 8

    Sofroniew, M. V. & Vinters, H. V. Astrocytes: biology and pathology. Acta Neuropathol. 119, 7–35 (2010)

    Google Scholar 

  9. 9

    Kim, S. J. et al. Astrocytes upregulate survival genes in tumor cells and induce protection from chemotherapy. Neoplasia 13, 286–298 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013)

    ADS  CAS  Google Scholar 

  11. 11

    Theis, M. & Giaume, C. Connexin-based intercellular communication and astrocyte heterogeneity. Brain Res. 1487, 88–98 (2012)

    CAS  PubMed  Google Scholar 

  12. 12

    Nguyen, D. X. et al. WNT/TCF signaling through LEF1 and HOXB9 mediates lung adenocarcinoma metastasis. Cell 138, 51–62 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Winslow, M. M. et al. Suppression of lung adenocarcinoma progression by Nkx2–1. Nature 473, 101–104 (2011)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. 14

    Oshima, A. Structure and closure of connexin gap junction channels. FEBS Lett. 588, 1230–1237 (2014)

    CAS  PubMed  Google Scholar 

  15. 15

    Yoshida, K., Yoshitomo-Nakagawa, K., Seki, N., Sasaki, M. & Sugano, S. Cloning, expression analysis, and chromosomal localization of BH-protocadherin (PCDH7), a novel member of the cadherin superfamily. Genomics 49, 458–461 (1998)

    CAS  PubMed  Google Scholar 

  16. 16

    Kim, S. Y., Chung, H. S., Sun, W. & Kim, H. Spatiotemporal expression pattern of non-clustered protocadherin family members in the developing rat brain. Neuroscience 147, 996–1021 (2007)

    CAS  PubMed  Google Scholar 

  17. 17

    Gaspar, L. E. et al. Time from treatment to subsequent diagnosis of brain metastases in stage III non-small-cell lung cancer: a retrospective review by the Southwest Oncology Group. J. Clin. Oncol. 23, 2955–2961 (2005)

    PubMed  Google Scholar 

  18. 18

    Gaspar, L. E., Scott, C., Murray, K. & Curran, W. Validation of the RTOG recursive partitioning analysis (RPA) classification for brain metastases. Int. J. Radiat. Oncol. Biol. Phys. 47, 1001–1006 (2000)

    CAS  PubMed  Google Scholar 

  19. 19

    Yagi, T. & Takeichi, M. Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev. 14, 1169–1180 (2000)

    CAS  PubMed  Google Scholar 

  20. 20

    Osswald, M. et al. Brain tumour cells interconnect to a functional and resistant network. Nature 528, 93–98 (2015)

    ADS  CAS  PubMed  Google Scholar 

  21. 21

    Sin, W. C. et al. Astrocytes promote glioma invasion via the gap junction protein connexin43. Oncogene 35, 1504–1516 (2015)

    PubMed  Google Scholar 

  22. 22

    Luker, K. E. et al. Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc. Natl Acad. Sci. USA 101, 12288–12293 (2004)

    ADS  CAS  PubMed  Google Scholar 

  23. 23

    Beahm, D. L. et al. Mutation of a conserved threonine in the third transmembrane helix of α- and β-connexins creates a dominant-negative closed gap junction channel. J. Biol. Chem. 281, 7994–8009 (2006)

    CAS  PubMed  Google Scholar 

  24. 24

    Heiman, M. et al. A translational profiling approach for the molecular characterization of CNS cell types. Cell 135, 738–748 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  25. 25

    Boehm, J. S. et al. Integrative genomic approaches identify IKBKE as a breast cancer oncogene. Cell 129, 1065–1079 (2007)

    CAS  PubMed  Google Scholar 

  26. 26

    Cai, X., Chiu, Y. H. & Chen, Z. J. The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling. Mol. Cell 54, 289–296 (2014)

    CAS  PubMed  Google Scholar 

  27. 27

    Stetson, D. B. & Medzhitov, R. Recognition of cytosolic DNA activates an IRF3-dependent innate immune response. Immunity 24, 93–103 (2006)

    CAS  PubMed  Google Scholar 

  28. 28

    Harks, E. G. et al. Fenamates: a novel class of reversible gap junction blockers. J. Pharmacol. Exp. Ther. 298, 1033–1041 (2001)

    CAS  PubMed  Google Scholar 

  29. 29

    Jin, M. et al. Effects of meclofenamic acid on limbic epileptogenesis in mice kindling models. Neurosci. Lett. 543, 110–114 (2013)

    CAS  PubMed  Google Scholar 

  30. 30

    Chan, W. N. et al. Identification of (−)-cis-6-acetyl-4S-(3-chloro-4-fluoro-benzoylamino)-3,4-dihydro-2,2-dimethyl-2H-benzo[b]pyran-3S-ol as a potential antimigraine agent. Bioorg. Med. Chem. Lett. 9, 285–290 (1999)

    CAS  PubMed  Google Scholar 

  31. 31

    Herdon, H. J. et al. Characterization of the binding of [3H]-SB-204269, a radiolabelled form of the new anticonvulsant SB-204269, to a novel binding site in rat brain membranes. Br. J. Pharmacol. 121, 1687–1691 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32

    Read, S. J., Smith, M. I., Hunter, A. J., Upton, N. & Parsons, A. A. SB-220453, a potential novel antimigraine agent, inhibits nitric oxide release following induction of cortical spreading depression in the anaesthetized cat. Cephalalgia 20, 92–99 (2000)

    CAS  PubMed  Google Scholar 

  33. 33

    Damodaram, S., Thalakoti, S., Freeman, S. E., Garrett, F. G. & Durham, P. L. Tonabersat inhibits trigeminal ganglion neuronal-satellite glial cell signaling. Headache 49, 5–20 (2009)

    PubMed  PubMed Central  Google Scholar 

  34. 34

    Deeken, J. F. & Loscher, W. The blood-brain barrier and cancer: transporters, treatment, and Trojan horses. Clin. Cancer Res. 13, 1663–1674 (2007)

    CAS  PubMed  Google Scholar 

  35. 35

    Pitz, M. W., Desai, A., Grossman, S. A. & Blakeley, J. O. Tissue concentration of systemically administered antineoplastic agents in human brain tumors. J. Neurooncol. 104, 629–638 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Lim, E. & Lin, N. U. Updates on the management of breast cancer brain metastases. Oncology 28, 572–578 (2014)

    PubMed  Google Scholar 

  37. 37

    Taimur, S. & Edelman, M. J. Treatment options for brain metastases in patients with non-small-cell lung cancer. Curr. Oncol. Rep. 5, 342–346 (2003)

    PubMed  Google Scholar 

  38. 38

    Hirano, S., Suzuki, S. T. & Redies, C. The cadherin superfamily in neural development: diversity, function and interaction with other molecules. Front. Biosci. 8, d306–d355 (2003)

    CAS  PubMed  Google Scholar 

  39. 39

    Patel, S. J., King, K. R., Casali, M. & Yarmush, M. L. DNA-triggered innate immune responses are propagated by gap junction communication. Proc. Natl Acad. Sci. USA 106, 12867–12872 (2009)

    ADS  CAS  PubMed  Google Scholar 

  40. 40

    Ablasser, A. et al. Cell intrinsic immunity spreads to bystander cells via the intercellular transfer of cGAMP. Nature 503, 530–534 (2013)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Demaria, O. et al. STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity. Proc. Natl Acad. Sci. USA 112, 15408–15413 (2015)

    ADS  CAS  PubMed  Google Scholar 

  42. 42

    Woo, S. R. et al. STING-dependent cytosolic DNA sensing mediates innate immune recognition of immunogenic tumors. Immunity 41, 830–842 (2014)

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43

    Sauer, J. D. et al. The N-ethyl-N-nitrosourea-induced Goldenticket mouse mutant reveals an essential function of Sting in the in vivo interferon response to Listeria monocytogenes and cyclic dinucleotides. Infect. Immun. 79, 688–694 (2011)

    CAS  PubMed  Google Scholar 

  44. 44

    Zhang, X. H. et al. Selection of bone metastasis seeds by mesenchymal signals in the primary tumor stroma. Cell 154, 1060–1073 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Wilson, A. A. et al. Lentiviral delivery of RNAi for in vivo lineage-specific modulation of gene expression in mouse lung macrophages. Mol. Ther. 21, 825–833 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Anders, S. et al. Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nature Protocols 8, 1765–1786 (2013)

    PubMed  Google Scholar 

  47. 47

    Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

    PubMed  PubMed Central  Google Scholar 

  48. 48

    Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014)

    PubMed  PubMed Central  Google Scholar 

  49. 49

    Gatza, M. L. et al. A pathway-based classification of human breast cancer. Proc. Natl Acad. Sci. USA 107, 6994–6999 (2010)

    ADS  CAS  PubMed  Google Scholar 

  50. 50

    Minn, A. J. et al. Genes that mediate breast cancer metastasis to lung. Nature 436, 518–524 (2005)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Kang, Y. et al. A multigenic program mediating breast cancer metastasis to bone. Cancer Cell 3, 537–549 (2003)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank D. Macalinao and other members of the Massagué laboratory for discussions. This work was supported by NIH grants P01-CA129243, U54-163167 and P30 CA008748, DOD Innovator award W81XWH-12-0074, the Alan and Sandra Gerry Metastasis Research Initiative (J.M.), the MSKCC Clinical Scholars Training Program (A.B.), the Solomon R. and Rebecca D. Baker Foundation (A.B), and by the Susan G. Komen Organization (X.J.).

Author information

Affiliations

Authors

Contributions

Q.C., A.B. and J.M. conceptualized the project and designed the experiments. Q.C. and A.B. performed the experiments. X.J., M.V., E.E.E., A.L.-S., L.J. and R.P. assisted with the experiments and bioinformatics analysis. H.S. and J.R.C. performed the LC–MS/MS analysis, and K.X. the time-lapse confocal imaging. A.B., Q.C. and J.M. wrote the paper.

Corresponding author

Correspondence to Joan Massagué.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks R. Hynes and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Cancer cell–astrocyte interactions.

a, Representative images and quantification of Cx43 immunostaining in matched primary and brain metastatic samples from patients with NSCLC. Scale bar, 100 μm (n = 8 patients). b, Cancer cells used in this study. The following references are cited in the table: 3, 6, 12, 13, 50 and 51. c, Astrocyte co-culture protects cancer cells. As illustrated in schema (left), cleaved caspase 3+ GFP+ apoptotic BrM cells were quantified by flow cytometery after sFasL addition or chemo-treatments (3 independent experiments). d, Flow cytometric quantification of dye transfer from astrocytes to MDA231-BrM2 cells over time (3 independent experiments).

Extended Data Figure 2 Increased expression of Cx43 and PCDH7 in brain metastatic cancer cells and astrocytes.

a, CX43 and PCDH7 mRNA in parental and BrM cells. Data are mean ± s.e.m. (n = 3 independent experiments in triplicate). b, Cx43 and PCDH7 western blotting in ErbB2 parental and brain metastatic cells, as well as Kras/p53 cell lines (n = 3 independent experiments). c, CX26 and CX30 mRNA in MDA231 parental cell lines and the metastatic derivatives of brain (BrM2), lung (LM) and bone (BoM). d, CX43 and PCDH7 mRNA in BrM cells compared to brain cells (n = 3 independent experiments). e, Kaplan–Meier plot illustrates the probability of cumulative metastasis-free survival in 63 cases (GSE8893) of lung adenocarcinoma based on CX43 and PCDH7 expression in the primary tumour. f, g, Knockdown of Cx43 and PCDH7 with shRNAs as assessed by reverse transcriptase PCR (RT–PCR) (f) and western blotting (g). Data are mean ± s.e.m. (n = 3 independent experiments in triplicate).

Extended Data Figure 3 PCDH7 facilitates gap junction communication.

a, b, Histograms and quantification of dye transfer from astrocytes to control and Cx43- or PCDH7-depleted Kras/p53-393N1 cells (a), and from astrocytes to control or Cx43-depleted MDA231-BrM2 cells, in comparison to carbenoxolone (50 μM) treatment (b). c, d, PCDH7 in astrocytes facilitate gap junctions. c, PCDH7 immunoblotting of control or PCDH7-depleted astrocytes. d, Quantification of dye transfer from MDA231-BrM2 cells to PCDH7-depleted astrocytes (d). e, Quantification of dye transfer from HBMECs to control, Cx43- or PCDH7-depleted MDA231-BrM2 cells. f, Dye transfer from MDA231-BrM2 cells to a mixed population of astrocytes and HBMECs. g, Quantification of dye transfer from control or Cx43-depleted MDA231-BrM2 cells to human microglia. For dye transfer assays, values are mean ± s.e.m. (n = ≥2 independent experiments in triplicate).

Extended Data Figure 4 Cx43 directly interacts with PCDH7, but not with E-cadherin or N-cadherin.

a, Schema illustrating split luciferase assay. Fusion constructs of PCDH7 and Cx43 were created with either NLuc or CLuc. When these proteins are brought into proximity, luciferase is functionally reconstituted, producing photons of light. b, Cx43 and PCDH7 constructs fused with NLuc and CLuc were expressed in parental cell lines. The table (top) numerically identifies the cell line combinations used in the assays (bottom), and BLI of a representative plate. c, Cx43 and PCDH7 western immunoblotting in cancer cells overexpressing fusion proteins. d, Quantification of BLI after co-culture of Cx43-CLuc and PCDH7-NLuc cancer cells and astrocytes for 15 min (3 independent experiments) eg, Luciferase split assay to detect Cx43–E-cadherin or Cx43–N-cadherin interactions. Cell line combinations used in the assays are numerically identified in the table (e), and confirmed by western immunoblotting (f). g, BLI of a representative assay plate; cell line combinations are indicated numerically (n ≥ 2 independent experiments in eg).

Extended Data Figure 5 Inhibition of gap junction activity prevents brain metastatic outgrowth.

a, BLI quantification of brain metastatic lesions formed by control, Cx43- or PCDH7-depleted H2030-BrM3 cells (n = 2 independent experiments with 9 mice total per group). b, Representative images of GFP+ brain metastatic lesions formed by control, Cx43- or PCDH7-depleted MDA231-BrM2 cells. Brain sections or brain metastatic lesions are delineated by dotted white or red lines, respectively. Scale bars, 1,000 μm. c, BLI (images) and quantification (bar graph) of lung metastatic lesions formed by MDA231-BrM2 cells. Values are mean ± s.e.m. (n = 2 independent experiments with 5 mice total in each group). d, e, Gap-junction-mediated brain metastasis requires channel function of Cx43. Wild-type or T154A mutant Cx43 was re-expressed in Cx43-depleted (CX43 sh2) MDA231-BrM2 cells. Cx43 expression was detected by western blotting (d), and brain metastasis formed by these cells was quantified by BLI (e) (n = 2 independent experiments with 10 mice total per group). Source data

Extended Data Figure 6 Cx43 and PCDH7 do not mediate early events of extravasation and vascular co-option in brain metastasis.

a, Cx43 and PCDH7 do not mediate trans-BBB migration. Quantification of control, Cx43- or PCDH7-depleted MDA231-BrM2 cells in 7-day brain lesions was carried out as follows: at the indicated time point, mice were euthanized, brains were sectioned, 10% of the sections were immunostained, and all GFP+ cells in these sections were counted. Data are mean ± s.e.m. (n = 5 brains in each group). b, Cx43 and PCDH7 mediate cancer cell colonization in 14-day brain lesions. Sectioning and staining were carried out as described in a. Representative images are GFP (green) and Ki67 (red) staining. DAPI, nuclear staining. Scale bar, 20 μm. Bar graph is the proportion of Ki67+ cancer cells. Data are mean ± s.e.m. (n = 5 brains in each group). c, Cx43 and PCDH7 mediate cancer cell survival. MDA231-BrM2 cells expressing CX43 shRNA, PCDH7 shRNA or control shRNA were deposited onto living brain sections, five brain slices were seeded with cancer cells of each type. After 48 h, slices were fixed and stained for GFP (green) and cleaved caspase 3 (Casp3) (red) staining. Representative images are shown. Scale bar, 30 μm. After staining, all GFP+ cells were counted on each slice. GFP+ cells with caspase 3+ staining were scored as ‘apoptotic’. Histogram shows proportion of caspase 3+ apoptotic cancer cells. Data are mean ± s.e.m. (n = 5 brain slices in each group). d, Cx43 and PCDH7 do not affect vascular co-option of cancer cells in 14-day brain lesions. Representative images are GFP (green) staining and vascular structure filled with TRITC dextran (red). Scale bar, 20 μm (n = 2 independent experiments).

Extended Data Figure 7 TRAP after cancer cell astrocyte co-culture.

a, Schematic illustration of TRAP experimental set up to isolate translating mRNA from MDA231-BrM2 cells under three conditions (1, 2 and 3). b, Principle component (PC) analysis of TRAP mRNA sequencing. c, Scatter plot of log2 fold changes regulated by astrocytes and gap junction communications between BrM cells and astrocytes. d, STAT1 and NF-κB p65 phosphorylation in H2030-BrM3 cells after a 2 h incubation with conditioned media from astrocyte co-cultures. Conditioned media samples were collected after 24 h co-culture of astrocytes with control or Cx43-depleted H2030-BrM3 cells (n = 3 independent experiments).

Extended Data Figure 8 Gap-junction-generated signalling activates IFN and NF-κB pathways in cancer cells.

a, Cytokine array analysis of conditioned media collected after 24 h co-culture of human astrocytes with control or Cx43-depleted MDA231-BrM2 cells. The log2 fold changes are plotted. b, Schematic of co-culture conditioned media collection and human astrocyte re-isolation (left) ELISA of IFNα and TNF in conditioned media from astrocyte co-cultures with the indicated MDA231-BrM2 cells (right) Data are mean ± s.e.m. (n ≥ 2 independent experiments with 4 total replicates). c, Relative levels of cleaved caspase 3 in MDA231-BrM2 cells treated with various concentrations of carboplatin in the presence or absence of 10 U ml−1 (39 U ng−1) IFNα or 10 pg ml−1 TNF. Data are mean ± s.e.m. (n = 5 technical replicates over 3 independent experiments). d, STAT1 levels in control and STAT1-knockdown LLC-BrM and 393N1 cells. e, Quantification of BLI signal from brain metastases formed by syngeneic LLC-BrM control, or STAT1-knockdown cells (n = 2 independent experiments with 12–15 mice total per group). f, NF-κB Renilla luciferase reporter assay in MDA231-BrM cells expressing control pBABE or SR-IκBα vector. Data are mean ± s.e.m. (n = 3 technical replicates). Source data

Extended Data Figure 9 Gap junctions initiate cytosolic DNA response in astrocytes.

a, Control or Cx43-depleted H2030-BrM3 cells were co-cultured for 18 h with/without astrocytes, and subjected to immunobloting analysis of phosphorylated TBK1 and IRF3 (n = 3 independent experiments). b, Immunoblot of mouse astrocytes depleted of STING with control (non-silencing) or Sting shRNAs. c, Mouse IFNα and TNF were quantified in the conditioned medium after co-culture by ELISA (n = 2 independent experiments with 3 replicates each). d, LLC-BrM growth in syngeneic C57Bl6 mice hosts wild-type (+/+) or knockout (−/−) for Sting. Bottom, diameter of brain metastases. Scale bar, 50 μm. Brains from all mice (n = 22) were sectioned, immunostained, and measured. All GFP+ brain metastases were quantified (2.8 ± 0.67 metastases per Sting+/+ mouse; 1.6 ± 0.55 in Sting−/− mice). e, Quantification of dsDNA in the indicated cellular fractions from 2 × 107 H2030-BrM3, MDA231-BrM2 or human astrocyte cells. Data are mean ± s.e.m. (n = 3 biological replicates; 2 independent experiments). f, Ratio of cytosolic dsDNA and nuclear dsDNA in indicated cancer cells and non-neoplastic cells. g, Representative image of immunofluorescent staining of dsDNA, GFP and CoxIV (a mitochondrial marker) in MDA231-BrM2 cells. h, cGAMP identification. The peak at 4.47 min contains all three selected reaction monitoring (SRM) transitions specific for cGAMP. AA, automatically integrated peak area; RT, retention time. i, j, EdU-labelled MDA231-BrM2 cells were co-cultured with astrocytes for 6 h. Transfer of EdU-labelled DNA from cancer cells to astrocytes was visualized using confocal microscopy (i), or quantified by flow cytometry (j). k, Immunoblot of H2030-BrM3 cells depleted of cGAS with shRNAs or control shRNA. l, Human astrocytes, were cultured for 18 h with/without H2030-BrM cells expressing control or cGAS shRNA. Human IFNα and TNF were quantified in the conditioned medium by ELISA (n = 2 independent experiments in triplicate). m, Quantification of BLI signal from brain metastases formed by H2030-BrM3 cells depleted of cGAS with two independent shRNAs (n = 2 independent experiments with 6 mice total per group). Source data

Extended Data Figure 10 Inhibition of gap junction activity prevents brain metastatic outgrowth.

ad, After treatment with tonabersat or meclofenamate (a), brain metastasis (b), primary tumour growth in mammary fat pads (c), or lung metastasis (d) was quantified by BLI. Data are mean ± s.e.m. (n = 2 independent experiments with 10 mice total in each group). e, Human astrocytes were treated with 200 μM tonabersat or 100 μM meclofenamate for 12 h before transfection with cGAMP (4 μg ml−1) using Lipofectamine 2000 or Lipofectamine alone. Conditioned media was collected 18 h later and assayed for human TNF and IFNα by ELISA (n = 2 biological replicates). f, g, Knockdown of Cx43 and PCDH7 in MDA231-BrM2 cells with tet-on inducible shRNA as assessed by RT–PCR (f) and western blotting (g), after doxycycline treatment in vitro (n = 2 independent experiments). h, Brain ex vivo BLI 14 days after inoculation of MDA231-BrM2 cells (n = 10 mice). Source data

Supplementary information

Supplementary Table 1

The file shows the target sequences of shRNAs. (PDF 3697 kb)

Dye transfer from MDA231-BrM2 cells and astrocytes

MDA231-BrM2 cells with Calcein Red-Orange dye. A single-cell suspension of labeled cancer cells was added to the monolayer cultured astrocytes. Bright field and red fluorescent images in the same region were captured every 20 minutes. (MOV 5272 kb)

PowerPoint slides

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chen, Q., Boire, A., Jin, X. et al. Carcinoma–astrocyte gap junctions promote brain metastasis by cGAMP transfer. Nature 533, 493–498 (2016). https://doi.org/10.1038/nature18268

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing