Research ArticleCancer

Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps

See allHide authors and affiliations

Science Translational Medicine  19 Oct 2016:
Vol. 8, Issue 361, pp. 361ra138
DOI: 10.1126/scitranslmed.aag1711
  • Fig. 1.

    NETs form during metastasis of breast cancer. (A and B) More neutrophils infiltrated metastatic 4T1 tumors than nonmetastatic 4T07 tumors (Ly6G immunostaining; mean ± SEM; n = 4 mice, t test). Scale bar, 50 μm. (C) CXCL1 protein level was higher in 4T1 than in 4T07 tumors (mean ± SEM; n = 5 mice, t test). (D) Cancer cell–derived CXCL1 promoted metastatic seeding after intravenous injection of luciferase-expressing cells [bioluminescence radiance, 19 days after cell injection; individual mice and means indicated; P = 0.0008, one-way analysis of variance (ANOVA); P < 0.01, Dunnett’s multiple comparison]. (E) CXCL1 secretion by 4T1 cells did not affect primary tumor growth in nude mice (mean ± SEM; n = 5 mice). (F) Knockdown of Cxcl1 in 4T1 cells reduced lung metastasis in nude mice (individual mice and means ± SD indicated; t test). (G and H) NET-like structures of extracellular DNA, sensitive to intravenous DNase I, were found around 4T1 cancer cells in LysM-EGFP mice using CILI. Grayscale insert shows DNA channel (shown and quantified 30 to 60 min after cancer cell injection; values from individual mice and mean ± SD are indicated; P = 0.002, one-way ANOVA; P < 0.01, Sidak’s multiple comparison). Scale bar, 50 μm. FOV, field of view. (I and J) Extracellular DNA and neutrophil elastase (NE) activity colocalized near 4T1, but not 4T07, cancer cells (shown and quantified 30 to 60 min after cancer cell injection; Fisher’s exact test). Scale bars, 50 μm. (K and L) The number of NETs in the lungs was higher after 4T1 cell injection than in controls (colocalized myeloperoxidase and citrullinated histone H3 immune staining). White arrow, NET; yellow arrow, intact neutrophil (mean ± SEM; n = 3 mice). Scale bar, 10 μm.

  • Fig. 2.

    NETs are present in metastatic, triple-negative human breast cancer. (A) Detection of NETs by immunofluorescence in human breast tumors and lung metastases. White arrows point to NETs (defined as colocalized myeloperoxidase, citrullinated histone H3, and DNA), and yellow arrows point to intact neutrophils. Scale bars, 20 μm. (B) Number of NETs in matched primary tumors and lung metastases (paired t test). (C) Number of NETs in primary tumor of different breast cancer subtypes (ANOVA and Tukey’s multiple comparisons test). ns, not significant.

  • Fig. 3.

    Formation of NETs by metastatic 4T1 breast cancer cells is associated with cancer cell invasion. (A and B) 4T1 but not 4T07 cells increased the formation of NETs (immunostaining for histone H3 and neutrophil elastase; mean ± SEM; n = 3, t test). Scale bar, 50 μm. (C) Neutrophils promoted invasion of 4T1 but not 4T07 cells (mean ± SEM; n = 5, t test). (D and E) DNase I (1.5 U) digested NETs (mean ± SEM; n = 3, t test). Scale bar, 50 μm. (F) DNase I treatment inhibited neutrophil-stimulated invasion of 4T1 cells (mean ± SEM; n = 5 to 7, t test). (G) Primary C3(1)-Tag cancer cells induced NETs. Scale bar, 50 μm. (H) DNase I treatment inhibited neutrophil-stimulated migration of C3(1)-Tag cancer cells (mean ± SEM; n = 3, t test). (I and J) Human BT-549 breast cancer cells promoted NET formation (immunostaining for histone H3 and myeloperoxidase; mean ± SEM; n = 3, t test). Scale bar, 50 μm. (K) DNase I (1.5 U) treatment blocked neutrophil-stimulated invasion of BT-549 breast cancer cells (mean ± SEM; BT-549 cells only or BT-549 cells with neutrophils and vehicle or DNase I, n = 5; BT-549 cells and vehicle or DNase I in 10% FCS, n = 2).

  • Fig. 4.

    Cancer cells induce NET formation through G-CSF, and neutrophil-stimulated invasion requires NADPH oxidase and PAD4 activity. (A) Blocking anti–G-CSF antibodies (1.6 μg/ml) decreased 4T1-induced NET extension (mean ± SEM; neutrophils with vehicle or anti–G-CSF, n = 3; neutrophils with 4T1 cells and vehicle or anti–G-CSF, n = 5; t test). (B) The NADPH oxidase inhibitor apocynin (10 μM) inhibited NET formation (mean ± SEM; neutrophils and vehicle or NAPDH oxidase inhibitor, n = 3; neutrophils with cancer cells and vehicle or apocynin, n = 5; t test). (C) Apocynin (10 μM) inhibited neutrophil-stimulated invasion (mean ± SEM; 4T1 cells only or 4T1 cells with neutrophils and vehicle or apocynin, n = 4; 4T1 cells only and vehicle or apocynin in 10% FCS, n = 1; t test). (D) PAD4 inhibition (200 μM Cl-amidine) reduced cancer cell–induced NET formation (mean ± SEM; neutrophils and vehicle or PAD4 inhibitor, n = 6; neutrophils with 4T1 cells and vehicle or PAD4 inhibitor, n = 4; t test). (E) PAD4 inhibition (200 μM Cl-amidine) blocked neutrophil-stimulated cancer cell invasion (mean ± SEM; n = 3, t test).

  • Fig. 5.

    NET formation and neutrophil-stimulated invasion require neutrophil protease activity. (A) Cathepsin G inhibitor I (2 μM) reduced cancer cell–induced NET formation (mean ± SEM; n = 4, t test). (B) Cathepsin G inhibitor I (2 μM) inhibited neutrophil-stimulated invasion (mean ± SEM; 4T1 cells only and 4T1 cells with neutrophils, n = 4; 4T1 cells in 10% FCS, n = 3; t test). (C) Neutrophil elastase inhibitor sivelestat (10 μM) reduced cancer cell–induced NET formation (mean ± SEM; n = 3, t test). (D) Neutrophil elastase inhibitor sivelestat (10 μM) weakly reduced neutrophil-stimulated invasion of 4T1 cells (mean ± SEM; 4T1 cells only or 4T1 cells with neutrophils and vehicle or sivelestat, n = 5; 4T1 cells and vehicle or sivelestat in 10% FCS, n = 2). (E) NADPH oxidase or neutrophil elastase inhibition (10 μM apocynin or sivelestat, respectively) blocked neutrophil-stimulated invasion of human breast cancer cells (mean ± SEM; BT-549 cells only or BT-549 cells with neutrophils and vehicle or apocynin or sivelestat, n = 4; BT-549 cells and apocynin or sivelestat in 10% FCS, n = 2). (F) CM from neutrophils induced to form NETs by culturing with cancer cells promoted invasion, but not when NET induction occurred in the presence of NADPH oxidase inhibition (10 μM apocynin; mean ± SEM; n = 3 to 6, t tests).

  • Fig. 6.

    Targeting NETs in vivo reduces metastasis. (A) DNase I–coated nanoparticles reduced neutrophil-stimulated cancer cell invasion in vitro (mean ± SEM; n = 4, t test). (B) Injection of DNase I–coated nanoparticles results in higher plasma nuclease activity than injection of free DNase I (mice injected with 75 U of free DNase I, 75 U of nanoparticle-bound DNase I, or equivalent vehicle; mean ± SEM; n = 3 mice). (C and D) DNase I–coated nanoparticles (75 U per mouse) reduced experimental lung metastasis of 4T1 cells (mean ± SEM; n = 9 to 10 mice, t test). Arrows point to metastases. Scale bars, 4 mm. (E and F) DNase I–coated nanoparticles (75 U per mouse) reduced the number and the size of metastatic foci arising after injection of 4T1 cells [mean ± SEM; n = 9 to 10 mice, t test; data were transformed by taking the square root before performing the t test due to significantly (P = 0.0003) different variances (untransformed data graphed)]. (G) DNase I–coated nanoparticles did not affect primary tumor growth. Nanoparticle treatment was initiated 7 days after tumor cell transplantation (mean ± SEM; n = 6 mice). (H) DNase I–coated nanoparticles (75 U per mouse) reduced spontaneous metastasis of 4T1 cells [mean ± SEM; n = 6 mice, t test; data were transformed by taking the square root before performing the t test due to significantly (P = 0.007) different variances].

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/361/361ra138/DC1

    Materials and Methods

    Fig. S1. CXCL1 promotes neutrophil recruitment to tumors.

    Fig. S2. NET formation is present in lungs of mice after injection of 4T1 cancer cells.

    Fig. S3. Neutrophils were harvested for in vitro assay.

    Fig. S4. G-CSF and p47phox promote NET formation and cancer cell invasion.

    Fig. S5. Breast cancer cells induce the formation of lytic NETs.

    Fig. S6. CM from neutrophils induced to undergo NET formation promotes invasion.

    Fig. S7. Cancer cell–induced NETs do not promote extravasation.

    Movie S1. NET-like structures form in vivo around 4T1 cancer cells in the lung.

    References (4349)

  • Supplementary Material for:

    Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps

    Juwon Park, Robert W. Wysocki, Zohreh Amoozgar, Laura Maiorino, Miriam R. Fein, Julie Jorns, Anne F. Schott, Yumi Kinugasa-Katayama, Youngseok Lee, Nam Hee Won, Elizabeth S. Nakasone, Stephen A. Hearn, Victoria Küttner, Jing Qiu, Ana S. Almeida, Naiara Perurena, Kai Kessenbrock , Michael S. Goldberg, Mikala Egeblad*

    *Corresponding author. Email: egeblad{at}cshl.edu

    Published 19 October 2016, Sci. Transl. Med. 8, 361ra138 (2016)
    DOI: 10.1126/scitranslmed.aag1711

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. CXCL1 promotes neutrophil recruitment to tumors.
    • Fig. S2. NET formation is present in lungs of mice after injection of 4T1 cancer cells.
    • Fig. S3. Neutrophils were harvested for in vitro assay.
    • Fig. S4. G-CSF and p47phox promote NET formation and cancer cell invasion.
    • Fig. S5. Breast cancer cells induce the formation of lytic NETs.
    • Fig. S6. CM from neutrophils induced to undergo NET formation promotes invasion.
    • Fig. S7. Cancer cell–induced NETs do not promote extravasation.
    • Legend for movie S1
    • References (4349)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mov format). NET-like structures form in vivo around 4T1 cancer cells in the lung.

    [Download Movie S1]

Stay Connected to Science Translational Medicine

Navigate This Article