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Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These
are pathogen-trapping structures generated by expulsion of the neutrophils DNA with associated proteolytic
enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells
can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we
observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found
NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and
INTRODUCTION engulfed and digested; (ii) degranulation of cytotoxic enzymes into the
Breast cancer metastasis is associated with very high mortality rates. extracellular space; and (iii) neutrophil extracellular traps (NETs),
Cancer cells can acquire the ability to metastasize by expressing metastasis- which are DNA meshes with associated cytotoxic enzymes that are
promoting genes, such as epithelial to mesenchymal transitionpromoting released into the extracellular space where they trap microorganisms
transcription factors or metalloproteinases (1, 2). However, cancer (11). NETs form in tissues and have been documented in human pan-
cells can also recruit and activate leukocytes, including macrophages, creatic, liver, and gastric cancers (1214), but whether they participate
to promote metastasis (3). Neutrophils, the most abundant leukocytes in cancer progression remains unclear. NETs can also form intra-
in human blood, similarly promote metastasis (48), although they vascularly, and they can damage vascular cells when they form (15).
can kill disseminated cancer cells under certain conditions (9). Neu- Recently, it was shown that NETs induced within the vasculature by
trophils and their precursors are sensitive to many chemotherapy regi- experimentally induced systemic bacterial infection or surgical stress
mens, causing dangerously low neutrophil numbers (neutropenia) aided in metastatic seeding of cancer cells in the liver (5, 12).
during the course of treatment. Because neutropenia carries a risk of We sought to observe how disseminating cancer cells interacted
life-threatening infections, the American Society of Clinical Oncology with neutrophils upon arrival in the lungs, a major site of metastatic
recommends prophylactic treatment with neutrophil-stimulating factors, colonization in breast cancer. We developed confocal intravital lung
including granulocyte colony-stimulating factor (G-CSF), for certain imaging (CILI), a modification of a lung imaging approach used with
chemotherapeutic regimens (10). It is therefore important to determine two-photon microscopy (16). Here, we show that NET-like structures
the conditions under which neutrophils promote metastatic spread. form around disseminated cancer cells in lungs using CILI. We also
Neutrophils normal function is to kill harmful microorganisms in show that cancer cells stimulate neutrophils to form NETs in the ab-
three ways: (i) phagocytosis, a process whereby bacteria or fungi are sence of pathogens in vitro. Finally, we show that NETs stimulate cancer
cell migration and invasion and that treatment with NET-digesting
1
deoxyribonuclease I (DNase I)coated nanoparticles inhibits metasta-
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA. 2Medical
Scientist Training Program, School of Medicine, Stony Brook University, Stony sis. NET formation is a mechanism by which signals from cancer cells
Brook, NY 11794, USA. 3Graduate Program in Genetics, Stony Brook University, activate host cells to enhance metastasis. Understanding the contribution
Stony Brook, NY 11794, USA. 4Department of Cancer Immunology and Virology, of neutrophils to metastases has pressing clinical implications because
Dana-Farber Cancer Institute, Boston, MA 02215, USA. 5 Watson School of
Biological Sciences, Cold Spring Harbor, NY 11724, USA. 6University of Michigan,
many cancer patients receiving chemotherapy also receive prophylactic
Ann Arbor, MI 48109, USA. 7Department of Pathology, Korea University Anam treatment with neutrophil-stimulating factors.
Hospital, Seoul, South Korea. 8Cold Spring Harbor Laboratory Cancer Center,
NCI Shared Resources and St. Giles Foundation Advanced Microscopy Center,
Cold Spring Harbor, NY 11724, USA. 9University of California, Irvine, Irvine, CA
92697, USA. 10Department of Microbiology and Immunobiology at Harvard Med- RESULTS
ical School, Boston, MA 02115, USA. Metastatic cancer cells induce formation of NETs
*These authors contributed equally to this work. To investigate whether neutrophils play a role in metastasis, we first
Present address: Center for Cardiovascular Research, John A. Burns School of
Medicine, University of Hawaii, Honolulu, HI 96813, USA.
compared neutrophil infiltration into tumors from orthotopically
Present address: Edwin L. Steele Laboratories, Department of Radiation Oncolo- transplanted 4T1 and 4T07 murine breast cancer cells. These cells orig-
gy, Massachusetts General Hospital and Harvard Medical School, Boston, MA inate from the same mammary tumor of a BALB/c mouse, but only the
02114, USA. 4T1 cells metastasize (17). We observed significantly (P = 0.0009) more
Present address: The Genetics Division, Department of Medicine, Brigham and
Womens Hospital and Harvard Medical School, Boston, MA 02115, USA. neutrophils in primary 4T1 tumors than in 4T07 tumors (Fig. 1, A and
Corresponding author. Email: egeblad@cshl.edu B). Because the chemokine CXCL1 can recruit neutrophils (18), we
CXCL1
4T1 tumors than nonme-
1.0
tastatic 4T07 tumors (Ly6G 50
immunostaining; mean 0.5
SEM; n = 4 mice, t test). 0 0.0
Scale bar, 50 mm. (C) CXCL1 Ly6G 4T07 4T1 4T07 4T1
P = 0.0008
protein level was higher in D P < 0.01 E F
4T1 than in 4T07 tumors P < 0.01
1011 3000 4T1-shLuci.1309
P = 0.03
Metastatic burden
t test). (D) Cancer cell
109 2000 1.5
derived CXCL1 promoted
10 8 1.0
metastatic seeding after
4T1
DNA and neutrophil elas-
tase (NE) activity coloca- 1.0
lized near 4T1, but not
4T07, cancer cells (shown
0.5
and quantified 30 to 60 min
after cancer cell injection;
Fishers exact test). Scale DNA Myeloperoxidase DNA Myeloperoxidase Citrullinated histone H3 0.0
bars, 50 mm. (K and L) The Citrullinated histone H3
ys
ys
y
ed
da
da
da
at
7
nt
U
measured its mRNA and protein and found higher amounts in 4T1 tance, we performed a Transwell chamber assay. Specifically, neutro-
cells than in 4T07 cells and in 4T1-derived tumors than in 4T07- phils isolated from the mouse bone marrow were plated in the lower
derived tumors (Fig. 1C and fig. S1). Reduction of CXCL1 (and the wells, and cancer cells were added on top of Matrigel-coated mem-
homologous CXCL2) in 4T1 cells with short hairpin RNAs reduced branes in the upper wells (fig. S3). Neutrophils cocultured in this man-
neutrophil infiltration into tumors and increased primary tumor growth ner with metastatic 4T1 cells formed extensive NETs, whereas
but had no effect on macrophage infiltration or cancer cell prolifera- neutrophils similarly cocultured with nonmetastatic 4T07 cells formed
tion in vitro (fig. S1). Tumors with reduced expression of CXCL1/2 few NETs (Fig. 3, A and B).
had an approximately doubled tumor burden; despite this, metastatic Coculturing with neutrophils increased the invasion of 4T1 cells but
burden was not increased (fig. S1). Instead, metastatic burden from had little effect on the invasion of 4T07 cells (Fig. 3C). To test whether
CXCL1/2 knockdown cells was significantly (P = 0.0008) decreased NETs promoted cancer cell invasion, we digested extracellular DNA by
when equal numbers of cancer cells were injected intravenously into adding DNase I to the cultures (Fig. 3, D and E). The neutrophils ability
mice (Fig. 1D). We speculated that increased tumor burden after to stimulate invasion of 4T1 cells was lost when the DNA of the NETs
CXCL1/2 knockdown was caused by reduced recruitment of tumor- was digested, whereas the addition of DNase I had no effect on fetal calf
reactive T lymphocytes (19). Consistent with this idea, CXCL1/2 knock- serum (FCS)stimulated invasion (Fig. 3F).
down did not increase primary tumor growth in T celldeficient, nude To test whether other metastatic cancer cells also induce NETs, we
mice (Fig. 1E). However, CXCL1/2 knockdown significantly (P = 0.03) isolated primary cancer cells from C3(1)-Tag mice, a genetically engi-
reduced spontaneous metastasis in nude mice (Fig. 1F). This suggests neered mouse model of metastatic basal/triple-negative breast cancer
2.0
ulation with 4T1 cancer cells (fig. S5).
NETs/tissue area (mm2)
3 ns ns
Because we did not observe neutrophils
2 with extruded nuclear DNA that were
1.5
otherwise intact, we conclude that, in
our in vitro setting, cancer cells induce
1.0 NETs through a lytic process.
1
NETs are defined by the association
0.5 of neutrophil proteases with the extra-
cellular neutrophilic histone-bound DNA
0 0.0 (27). Hence, we speculated that the proin-
Tumor Lung metastasis
2+
e
vasive effects of NETs required NET-
al
tiv
in
ER
ga
m
e
Lu
-n
Fig. 2. NETs are present in metastatic, triple-negative human breast cancer. (A) Detection of NETs by immuno- promote invasion with no effect on the
fluorescence in human breast tumors and lung metastases. White arrows point to NETs (defined as colocalized myelo- cancer cells ability to invade toward
peroxidase, citrullinated histone H3, and DNA), and yellow arrows point to intact neutrophils. Scale bars, 20 mm. (B) Number FCS. Surprisingly, inhibition of cathepsin
of NETs in matched primary tumors and lung metastases (paired t test). (C) Number of NETs in primary tumor of different G also reduced the extension of NETs
breast cancer subtypes (ANOVA and Tukeys multiple comparisons test). ns, not significant. (Fig. 5, A and B). This suggests that ca-
thepsin G is required for NET release.
PAD4 inhibitor Cl-amidine reduced NET formation (Fig. 4D) and Cleavage of histones by neutrophil elastase is required for chromatin
blocked the neutrophils ability to promote invasion (Fig. 4E). Thus, decondensation and NET release (28, 29), and cathepsin G may have a
metastatic cancer cells activate signaling pathways that include similar role. The neutrophil elastase inhibitor sivelestat also reduced the
NADPH oxidase and PAD4 in neutrophils, causing the formation extension of cancer cellinduced NETs (Fig. 5C). Although the effect of
of invasion-promoting NETs. sivelestat on neutrophil-induced invasion was not significant in assays
Several types of NET formation can occur. One requires NADPH with murine 4T1 cells (Figs. 5D), inhibition of neutrophil elastase sig-
oxidase activity and generally results in neutrophil lysis 2 to 4 hours nificantly (P = 0.02) blocked the neutrophils ability to stimulate the
after activation (24), whereas another process occurs rapidly (5 to invasion of human BT-549 breast cancer cells, as did the inhibition of
60 min), independent of NADPH oxidase activity, and leaves the neu- NADPH oxidase (Fig. 5E).
trophil plasma membrane intact (26). Cancer cellinduced NET for- The proinvasive effects of NET-forming neutrophils in Transwell
mation was dependent on NADPH oxidase activity, which suggested chamber assays suggested that a factor (or factors) generated during
that it occurred through a lytic process. To address this directly, we NET formation acted on cancer cells. To test this possibility, we gen-
performed electron microscopy. Scanning electron microscopy revealed erated a conditioned medium (CM) from unstimulated neutrophils,
that extracellular meshes extruded from nonintact neutrophils 3 hours from neutrophils induced to form NETs by coculturing with cancer
after plating and stimulation with cancer cells, whereas intact neutro- cells, and from neutrophils cocultured with cancer cells in the presence
phils were rarely discernible (fig. S5). These findings were largely similar of the NADPH oxidase inhibitor apocynin to prevent the formation of
to those observed after stimulation with phorbol 12-myristate 13-acetate NETs. CM from cultures induced to form NETs effectively stimulated
(PMA) (fig. S5), which is known to induce NETs through a lytic mech- cancer cell invasion, whereas CM from cultures where NET formation
A B C
NET extension
3 ns
300
Invasion
2 ns
ns 200
1 100
0 0
+4T07 +4T1 +neut. +neut.
DNA Histone H3 Neutrophil elastase Neutrophils +4T07 +4T1
Neutrophils + 4T1
D Neutrophils alone E F ns
w/o neutrophils)
NET extension
3 200
Invasion
P = 0.01 ns
2
100
le
e
I
I
DNA Histone H3 Neutrophil elastase
Ve e I
I
e
D le
cl
e
e
cl
cl
ic
c
as
as
as
as
as
hi
hi
hi
hi
h
Ve
Ve
N
Ve
Ve
N
N
D
D
G Neutrophils Neutrophils H +4T1 +Neutrophils +10% FCS
Neutrophils alone + C3(1)-Tag + C3(1)-Tag + DNase I [% C3(1)-Tag treated with
vehicle w/o neutrophils]
1200 ns
1000
800
Migration
600
300 P = 0.047 P = 0.003
200
100
0
Ve e I
Ve I
I
le
D le
D le
DNA Histone H3 Neutrophil elastase
e
e
ic
c
as
as
as
hi
hi
h
Ve
N
D
600
Invasion
10
400 P = 0.006 P = 0.006
5 200
0 0
Ve I
I
Ve e
e
D le
e
l
cl
ic
c
as
as
hi
hi
h
Human neutrophils
Ve
N
D
was inhibited by apocynin did not (Fig. 5F). To exclude the possibility its ability to stimulate invasion when DNase I was added after collec-
that invasion was stimulated by factors secreted by cancer cells into tion or upon heat denaturation (fig. S6). This suggests that the proin-
the CM, we also used CM from neutrophils that formed NETs in vasive factor is not simply extracellular DNA, but protein factors
response to PMA stimulation. CM from PMA-induced, NET- associated with the DNA mesh.
forming neutrophils also stimulated cancer cell invasion. CM from both
cancer cellinduced and PMA-induced NET-forming neutrophil DNase I treatment prevents lung metastasis in mice
cultures lost the ability to stimulate invasion when neutrophils were Digesting bacterially induced NETs with systemically administered
cultured in the presence of DNase I (fig. S6). In addition, the CM lost DNase I can reduce experimental metastasis in a mouse model of
SF
e
e
cl
cl
-C
Ve
G
Ve
ti
ti
an
+4T1
with control nanoparticles had macro-
B C scopic or microscopic metastases. A
(% area covered by NETs)
w/o neutrophils)
xi.
xi.
cle
cle
cle
cle
cle
xi
i.
i.
h. x
h. ox
h. o
h. o
h. o
in H o
hi
hi
hi
hi
hi
asation of disseminated cancer cells (5).
in H
in PH
in H
in H
Ve
Ve
Ve
Ve
DP
DP
DP
DP
D
We therefore examined whether DNA di-
NA
NA
NA
NA
NA
h.
h.
h.
h.
e
le
cl
cl
cl
cl
in
in
in
in
in
ic
hi
hi
hi
hi
h
Ve
Ve
Ve
Ve
Ve
D
D
PA
PA
PA
PA
PA
A B
250 ns
P = 0.04 P = 0.0006
w/o neutrophils)
4
NET extension
Invasion
150
3
100
2 ns
1 50
0 0
I
le
le
le
le
I
I
le
h.
h.
h.
h.
h.
ic
ic
ic
ic
ic
in
in
in
in
in
h
h
h
Ve
Ve
Ve
Ve
Ve
G
G
in
in
in
in
in
ps
ps
ps
ps
ps
he
he
he
he
he
at
at
at
at
at
C
C
C
+4T1 +Neutrophils +10% FCS
C D 800
P = 0.009 P = 0.04
w/o neutrophils)
3 ns
NET extension
P = 0.02 P = 0.09
Invasion 400
2 ns
200
1
0 0
el eut e
e
se il
se il
se il
e
h.
h.
h.
se il
se il
h.
h.
ta ph
ta ph
ta ph
cl
cl
cl
cl
cl
ta ph
ta ph
in
in
in
in
in
hi
hi
hi
hi
hi
as ro
as ro
as ro
as ro
as ro
Ve
Ve
Ve
Ve
Ve
el eut
el ut
el eut
el eut
e
N
N
N
P = 0.001 P = 0.03
E F 400 ns
(% BT-549 with vehicle)
ns P = 0.0007 P = 0.01
(% BT-549 treated with vehicle
300
ns
1000
Invasion
w/o neutrophils)
200
Invasion
P = 0.02
500 P = 0.01 P = 0.04
100
0 0
h
ph le s
le
le
as ro .
as ro .
49
in 9
le
le
se il
el eut nh
el ut inh
in
se il
ic il
Ve h.
h.
cl
i. 54
ta ph
ic
PH hic
ta ph
ic
ic
eh ph
T5
in
hi
in
N xi. i
i.
h
h.
ox T
N xi.
ox
+v utro
Ve
AD Ve
Ve
Ve
+B
+B
o
PH
ils
PH ils
e
e
PH
AD oph
AD
AD
ro
N
+N utr
N
t
eu
e
N
observed that breast cancer cells can induce neutrophils to form digesting NETs with DNase I blocks invasion. Our data also suggest
NETs after they arrive in the lungs. We also documented the pres- that cathepsin G is not just an effector of NET functions but is also
ence of NETs in the aggressive triple-negative subtype of human involved in the release of NETs, similar to what was reported for
breast cancer. Using in vitro models, we further demonstrated that neutrophil elastase (28). Finally, we demonstrate that treatment
inhibiting the signaling pathways that promote NET formation or with DNase Icoated nanoparticles effectively reduces metastasis
A B C
P = 0.01 DNase Icoated
Vehicle Control nanoparticles nanoparticles
(% 4T1 treated with vehicle
P = 0.006 600
400
P = 0.003 DNase I
w/o neutrophils)
400
200
100
200
0
2U 15U 2U 15U
Medium DNase I DNase I
coated 0
0 10 20
nanoparticles Time after injection (hours)
+Neutrophils
D E F
0.3
Average metastatic
Metastatic burden
20 2
0.1
0 0.0 0
es
rti l
cl ed
cl ed
es
es
pa tro
cl d
rti l
rti l
pa tro
pa tro
rti ate
cl
rti at
cl
cl
rti at
es
no on
es
es
no on
no on
pa co
pa co
pa co
C
C
no I
no I
no I
na ase
na ase
na se
na
na
na
a
N
N
D
D
G H
0.8 P = 0.05
Metastatic burden
3
600
0.4
400
Treatment initiated
200 0.2
0 0.0
cl d
0 5 10 15 20
es
rti l
pa tro
rti ate
cl
es
no on
pa co
no I
na se
a
N
D
Fig. 6. Targeting NETs in vivo reduces metastasis. (A) DNase Icoated nanoparticles reduced neutrophil-stimulated cancer cell invasion in vitro (mean SEM; n = 4, t test).
(B) Injection of DNase Icoated 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 Icoated 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 Icoated 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 Icoated nanoparticles did not affect primary tumor growth. Nanoparticle treatment was
initiated 7 days after tumor cell transplantation (mean SEM; n = 6 mice). (H) DNase Icoated 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].
in vivo. Unexpectedly, cancer cellinduced NETs did not promote ex- it is not simply free DNA that stimulates invasion. Possibly, a chemo-
travasation but did influence the number of histologically detectable tactic factor(s) is associated with NETs. HMGB1 released during NET
metastatic foci. This suggests that NETs mediate the expansion of dis- formation may drive proliferation and migration of cancer cells (12).
seminated cells. Our findings, together with a previous report that Because we found NETs in primary human tumors as well as
NETs induced by infection aid in metastatic seeding of the liver (5), metastases, another important future direction will be to elucidate
raise the exciting possibility of targeting NETs to prevent metastasis. whether NETs also participate in cancer cell migration and invasion
Neutrophils and their precursors are sensitive to chemotherapy, in the primary tumor.
and cancer patients can thus develop life-threatening neutropenia. No- Here, we show that three different types of cancer cells can induce
tably, long-term survival of breast cancer patients receiving chemo- promigratory or proinvasive NETs, but cancer cells can acquire the
therapy is higher for patients with mild chemotherapy-induced ability to invade and migrate through many other means. Therefore,
neutropenia (32). It has been proposed that this is because neutrope- assays to identify patients who may benefit from NET-targeted treat-
nia represents a surrogate marker of adequate chemotherapy dosing ments are needed. Detection of NETs in tumor biopsies may be the
for a given patient (33). However, our findings suggest an alternative most accurate method of identifying patients who might benefit from
hypothesis: mild neutropenia is associated with better survival because NET-targeting approaches. However, because we identified G-CSF as a
neutrophils play a functional role in metastasis. It could therefore be critical factor in the induction of NETs by cancer cells, it is possible
important to develop approaches to prevent or dissolve prometastatic that cancer cell expression of G-CSF could identify patients at risk of
NETs while simultaneously leaving the life-saving degranulating and developing NET-promoted metastasis. Both 4T1 cells and C3(1)-Tag
plates for 15 to 30 min before adding antiG-CSF (R&D Systems), intraperitoneally. Two hours later, 1 105 4T1-luciferase (experi-
human recombinant G-CSF (R&D Systems), apocynin (Abcam), ca- ment 1) or 4T1 cells (experiment 2) were injected intravenously.
thepsin G inhibitor I (EMD Millipore), Cl-amidine (Cayman Chem- Daily nanoparticle treatment was continued for 2 weeks, and the
ical), sivelestat (Tocris Bioscience), DNase I (AM2222, Invitrogen or mice were euthanized when the first mouse showed >10% weight
10104159001, Roche), or DNase Icoated nanoparticles. Cancer cells loss (33 and 26 days after cancer cell injection for experiments
(1 105 4T1, 4T07, or BT-549 cells) in serum-free medium were then 1 and 2, respectively). Metastatic burden was quantified from lung
added to rehydrated Matrigel inserts (BD Biosciences). After 22 hours sections as described above, and results from the two experiments
at 37C, the noninvading cancer cells from the upper surface of the were pooled. Notably, nanoparticles were not given intravenously
membrane were wiped off, and cells on the bottom side of the mem- because the repeated injections would damage the tail veins, making
brane were fixed with 4% paraformaldehyde (PFA) and stained with it difficult to continue treatment through the entire period. The bio-
hematoxylin. The number of invading cells was counted under a light distribution of intraperitoneally and intravenously administrated na-
microscope in six fields of view, and the average number of cells was noparticles is similar (42).
calculated. To test the effect of DNase Icoated nanoparticles on spontaneous
CM was prepared from neutrophil cultures grown alone or with lung metastasis, 4T1 cells (2.5 105) were injected into both inguinal
cancer cells (4T1 or BT-549) present in the upper chamber of the mammary glands of female BALB/c mice. One week later, daily intra-
Transwells, the latter with or without DNase I or apocynin added peritoneal injections with DNase Icoated nanoparticles (75 U per
to the bottom well. The CM from cocultures of neutrophils and cancer mouse) or uncoated nanoparticles were initiated and continued until
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(LMN) vector, M. Monestier (Temple University) for the antibodies to nucleosomes, and performed flow cytometry; J.P., L.M., R.W.W., and K.K. analyzed the NET formation in tissues.
M. Long and V. Vaghela for technical assistance. Funding: This work was supported by the J.J., A.F.S., Y.L., and N.H.W. collected breast cancer samples and were responsible for
CSHL Cancer Center Support Grant 5P30CA045508, funds to M.E. from the U.S. Department of obtaining clinical information from the patients. S.A.H. performed electron microscopy. V.K.
Defense (W81XWH-14-1-0078), the Long Island 2-Day Walk to Fight Breast Cancer, and the Joni designed the experiments using CM. J.P., R.W.W., L.M., and M.E. wrote the manuscript.
Gladowsky Breast Cancer Foundation. M.E., J.J., and A.F.S. were supported by funds from NIH Competing interests: M.E. has stocks valued at under $15,000 in Novo Nordisk, Agios, Pfizer,
(5U01CA180944-02) and the Hope Foundation. M.S.G. was supported by the Cancer Research and Bristol-Myers Squibb, which were not involved with this study. The other authors
Institute CLIP (Clinic and Laboratory Integration Program) Grant; R.W.W. was supported by the declare that they have no competing interests.
National Institute of General Medical Sciences Medical Scientist Training Program Training Award
(T32-GM008444); Z.A. was supported by Aid for Cancer Research; L.M. is a George A. and Submitted 18 May 2016
Marjorie H. Anderson fellow and is supported by a fellowship from the Boehringer Ingelheim Accepted 23 September 2016
Fonds; N.P. was supported by a Formacin de Profesorado Universitario fellowship (AP2010-2197); Published 19 October 2016
K.K. was supported by the National Cancer Institute (K99 CA181490); and V.K. was supported 10.1126/scitranslmed.aag1711
by the Deutsche Forschungsgemeinschaft research fellowship (KU 3264/1-1). Author
contributions: J.P., R.W.W., L.M., and M.E. designed the experiments. J.P. and Y.K.-K. performed Citation: J. Park, R. W. Wysocki, Z. Amoozgar, L. Maiorino, M. R. Fein, J. Jorns, A. F. Schott,
the coculture experiments; R.W.W. and L.M. isolated human neutrophils. R.W.W., J.P., L.M., Y. Kinugasa-Katayama, Y. Lee, N. H. Won, E. S. Nakasone, S. A. Hearn, V. Kttner, J. Qiu,
E.S.N., and J.Q. performed the animal experiments; R.W.W. and M.R.F. performed CILI. Z.A. A. S. Almeida, N. Perurena, K. Kessenbrock, M. S. Goldberg, M. Egeblad, Cancer cells induce
and M.S.G. designed and engineered the DNase Icoated nanoparticles. A.S.A. and N.P. metastasis-supporting neutrophil extracellular DNA traps. Sci. Transl. Med. 8, 361ra138 (2016).
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