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CANCER 2016 The Authors,


some rights reserved;

Cancer cells induce metastasis-supporting neutrophil exclusive licensee


American Association
extracellular DNA traps for the Advancement
of Science.

Juwon Park,1* Robert W. Wysocki,1,2,3* Zohreh Amoozgar,4* Laura Maiorino,1,5*


Miriam R. Fein,1,3 Julie Jorns,6 Anne F. Schott,6 Yumi Kinugasa-Katayama,1 Youngseok Lee,7
Nam Hee Won,7 Elizabeth S. Nakasone,1,5 Stephen A. Hearn,8 Victoria Kttner,1 Jing Qiu,1
Ana S. Almeida,1 Naiara Perurena,1 Kai Kessenbrock,9 Michael S. Goldberg,4,10 Mikala Egeblad1

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

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migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase
I) blocked these processes. Treatment with NET-digesting, DNase Icoated nanoparticles markedly reduced lung
metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-
promoting tumor-host interaction and a potential therapeutic target.

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

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 1 of 12


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Fig. 1. NETs form during A B C


metastasis of breast can- Non metastatic 4T07 Metastatic 4T1 150 P = 0.0009 P = 0.04

Ly6G+ cells/1.1 mm2


2.0

(ng/mg total protein)


cer. (A and B) More neutro-
phils infiltrated metastatic 100 1.5

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

Tumor volume (mm3)

(% of total lung area)


(mean SEM; n = 5 mice, 4T1-shCxcl1.559 2.0
1010
Radiance (p/s)

Metastatic burden
t test). (D) Cancer cell
109 2000 1.5
derived CXCL1 promoted
10 8 1.0
metastatic seeding after

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107 1000
intravenous injection of 0.5
luciferase-expressing cells 106
0.0
[bioluminescence radiance, 105 0
0 5 10 15 20 shLuci.1309 shCxcl1.559
19 days after cell injection; shLuci.1309 shCxcl1.559 shCxcl1.914 Days after cells injection
individual mice and means
indicated; P = 0.0008, one-way G 4T07 4T1 4T1 + DNase I H P = 0.002
analysis of variance (ANOVA); P = 0.005 P = 0.002

NET-like structures per FOV


P < 0.01, Dunnetts multiple 1.0
comparison]. (E) CXCL1 se-
cretion by 4T1 cells did not
affect primary tumor growth
0.5
in nude mice (mean SEM;
n = 5 mice). (F) Knockdown
of Cxcl1 in 4T1 cells re-
0.0
duced lung metastasis DNA Neutrophil Cancer cell 4T07 4T1 4T1 + DNase I
in nude mice (individual
I
mice and means SD in- 4T07
dicated; t test). (G and H)
J
NET-like structures of ex-
tracellular DNA, sensitive NE activity colocolized
w. extracellular DNA
to intravenous DNase I, were No NE activity colocalized
found around 4T1 cancer w. extracellular DNA
cells in LysM-EGFP mice
20 P = 0.0008
using CILI. Grayscale insert
shows DNA channel (shown 4T1
15
and quantified 30 to 60 min
FOV

after cancer cell injection; 10


values from individual mice
5
and mean SD are indi-
cated; P = 0.002, one-way 0
ANOVA; P < 0.01, Sidaks 4T1 4T07
multiple comparison). Scale DNA Neutrophil DNA Neutrophil Cancer cell NE activity
bar, 50 mm. FOV, field of Cancer cell NE activity
1.5
view. (I and J) Extracellular K L Saline
NETs/lung area (mm2)

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

number of NETs in the lungs


re

7
nt
U

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 mm.

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 2 of 12


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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

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that reduced metastasis is not caused by reduced T cell recruitment (21). Tumors in this model have high numbers of infiltrating neutro-
and that neutrophils mediate this effect. phils and high expression of CXCL1 (22). Like 4T1 cells, C3(1)-Tag
The results of assays with CXCL1/2 knockdown cells suggest that primary cancer cells induced NETs when cocultured with mouse neu-
neutrophils support metastasis upon their recruitment by cancer cells. trophils (Fig. 3G). C3(1)-Tag cells have a limited ability to invade
To investigate the functions of neutrophils at the metastatic site, we through Matrigel, but neutrophils significantly (P = 0.047) increased
performed CILI of mCherry-expressing 4T07 and 4T1 cells injected the migration of C3(1)-Tag cells across uncoated membranes. This
intravenously into LysM-EGFP mice, which express EGFP (enhanced increased migration was blocked by DNase I treatment (Fig. 3H). Ex-
green fluorescent protein) predominantly in neutrophils (20). Greater tensive NETs were also formed when triple-negative human BT-549
numbers of extracellular DNA structures sensitive to intravenously breast cancer cells were cultured with neutrophils from healthy female
injected DNase I were found around 4T1 than around 4T07 cancer volunteers (Fig. 3, I and J, and fig. S3). As observed with 4T1 cells,
cells (Fig. 1, G and H, and movie S1). To explore whether these digesting NETs with DNase I blocked the neutrophils ability to stim-
structures could be NETs, we next used CILI to determine whether ulate BT-549 invasion through Matrigel (Fig. 3K). Thus, both human
the extracellular DNA structures colocalized with the NET-associated and mouse triple-negative breast cancer cells can promote the for-
protease neutrophil elastase using a probe that produces a fluores- mation of promigratory/invasive NETs, whereas treatment with
cent signal after specific cleavage by neutrophil elastase. Neutrophil NET-digesting DNase I blocks migration and invasion induced by
elastase activity colocalized with extracellular DNA in 14 of 17 im- species-matched neutrophils.
aged fields of view after the injection of 4T1 cancer cells but not
after the injection of 4T07 cells (P = 0.0008; Fig. 1, I and J). To further Cancer cells induce lytic NETs, and blocking NET formation
explore whether NETs are formed after the injection of cancer cells, reduces invasion
we performed immunofluorescence staining for NETs in tissue sections Next, we wanted to determine how tumor-induced NETs were
from lungs of mice injected with 4T1 cancer cells. This analysis con- formed. G-CSF can prime neutrophils for NET formation and is se-
firmed that NETs formed in lungs shortly after tail vein injection of creted by 4T1 cells (23). AntiG-CSF blocking antibodies significantly
4T1 cells and that the amounts of NETs were elevated for days after (P = 0.03) reduced the ability of 4T1 cells to induce NETs in the Trans-
the injection (Fig. 1, K and L, and fig. S2). These data demonstrate that well chamber assay (Fig. 4A). Conversely, human recombinant G-CSF
metastatic cancer cells stimulate NET formation at sites of dissemina- induced NET formation by human neutrophils (fig. S4A). Together, this
tion in the absence of infection. suggests that G-CSF secreted by cancer cells induces NETs.
To determine whether NETs could also be found in metastatic An initial step in the intracellular signaling cascade in neutrophils
human breast cancer, we performed immunofluorescence staining that results in pathogen-induced NET formation is the activation of
on a small panel of clinical samples of primary tumor and matched the phagocytic NADPH (reduced form of nicotinamide adenine di-
metastatic lung lesions from breast cancer patients (Fig. 2A). We de- nucleotide phosphate) oxidase (NOX2) enzyme complex (24). In
tected NETs in 16 of 20 primary tumors and in 13 of 19 metastatic our Transwell assay, apocynin, a pharmacologic inhibitor of NOX2,
lung lesions (Fig. 2B). The number of NETs varied between tumors, reduced NET formation and neutrophil-promoted cancer cell invasion
with the highest numbers in triple-negative tumors (six of six pri- (Fig. 4, B and C). The phox47 subunit is a critical component of
mary tumors and seven of seven metastases had detectable NETs) NOX2 in neutrophils. The extension of NETs was greatly reduced
and an absence or very low numbers in luminal breast cancers (Fig. for neutrophils isolated from p47phox/ compared to those isolated
2C). Thus, we detected NETs in human breast cancer and found from p47phox+/+ mice, and p47phox/ neutrophils did not promote
the presence of NETs to be associated with an aggressive subtype cancer cell invasion (fig. S4). Unfortunately, the p47phox/ strain is
of breast cancer. on the C57BL/6 background, precluding in vivo experiments with the
BALB/c-derived 4T1 cells.
NETs promote cancer cell migration and invasion Next, we determined the importance of peptidylarginine deiminase
To determine whether cancer cellinduced NET formation required type 4 (PAD4), the enzyme responsible for histone modifications re-
direct interaction with neutrophils or could be induced over a dis- quired for decondensing neutrophil DNA before expulsion (25). The

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 3 of 12


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A anism (24). We next analyzed the integ-


Breast tumor Lung metastasis
rity of the neutrophils by transmission
electron microscopy. Three hours after
stimulation with cancer cells, neutrophils
typically had decondensed chromatin
dispersed in the cytosol and showed
plasma membrane breakdown (fig. S5).
This morphology was identical to the
morphology of the neutrophils induced
to form NETs by PMA. Of more than
50 evaluated neutrophils, none showed
evidence of DNA-containing vesicles
budding from the nucleus, which is the
morphology described as typical for
nonlytic NET-forming neutrophils (26).
Finally, to demonstrate that the extra-
cellular meshes observed by scanning

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electron microscopy contained DNA,
DNA Myeloperoxidase Citrullinated histone H3
we performed immunogold labeling
with anti-DNA antibodies and detected
B 4
ns C P = 0.04 expelled DNA in the meshes after stim-
NETs/tissue area (mm2)

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

associated protease activity. Inhibition


H

e
Lu

-n

of cathepsin G, a protease associated with


le
ip

NETs, blocked the neutrophils ability to


Tr

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

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 4 of 12


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A B C

(% area covered by NETs)

(% 4T07 w/o neutrophils)


Neutrophils alone Neutrophils + 4T07 Neutrophils + 4T1 4 P = 0.007
400 P = 0.047

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

(% 4T1 treated with vehicle


(% area covered by NETs)
Neutrophils + 4T1 + DNase I
P = 0.006 P = 0.001 300 P = 0.004 P = 0.002
4

w/o neutrophils)
NET extension
3 200

Invasion
P = 0.01 ns
2
100

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1
0 0

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

+Neutrophils +10% FCS


I J K
(% BT-549 treated with vehicle
(% area covered by NETs)

Neutrophils alone Neutrophils + BT-549


ns
15 P = 0.003 800
w/o neutrophils)
NET extension

600
Invasion

10
400 P = 0.006 P = 0.006
5 200

0 0
Ve I

I
Ve e
e

D le

DNA Histone H1 Myeloperoxidase +BT-549


e

e
l
cl
ic

c
as

as
hi

hi
h

Human neutrophils
Ve

N
D

+Neutrophils +10% FCS


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 mm. (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 mm. (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 mm. (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 mm. (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).

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

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 5 of 12


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A DNase I (Fig. 6A). In vivo, a higher con-


(% area covered by NETs)

5 centration of DNase I was found in plas-


P = 0.03 P = 0.03
ma when mice were treated with DNase
4
NET extension

Icoated nanoparticles than with free


3 DNase I (Fig. 6B). Daily intraperitoneal
2 injection of DNase Icoated nanoparticles
significantly (P = 0.002) reduced meta-
1
static burden after intravenous injection
0 of 4T1 cells (Fig. 6, C and D). Three of
9 mice treated with DNase Icoated na-
SF

SF
e
e

cl
cl

-C

noparticles had no detectable metastasis


-C
hi
hi

Ve

G
Ve

ti

ti

by histology, whereas all 10 mice treated


an

an

+4T1
with control nanoparticles had macro-
B C scopic or microscopic metastases. A
(% area covered by NETs)

(% 4T1 treated with vehicle


4 detailed analysis of the tissue revealed that
P = 0.002 P = 0.003 200 P = 0.01 P = 0.004 there was a significant reduction in the
NET extension

w/o neutrophils)

3 number of detectable, individual meta-

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150 ns
static foci (P = 0.0003; Fig. 6E) and a re-
Invasion

2 duction in the average size of the foci (P =


100
0.01; Fig. 6F) after treatment with DNase
1 50
Icoated nanoparticles. This suggests that
NETs are critical for metastatic coloniza-
0 0 tion. NETs that form intravascularly in
response to infection can promote extrav-
Ve .

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

+4T1 +Neutrophils +10% FCS gestion with DNase Icoated nanoparti-


cles or inhibition of NET formation
D E 400
(% 4T1 treated with vehicle

with the PAD4 inhibitor Cl-amidine in-


(% area covered by NETs)

P = 0.02 P = 0.04 P = 0.06 ns fluenced the number of cancer cells that


5 P = 0.001
w/o neutrophils)

300 made it out of the vasculature in our me-


4
NET extension

tastasis model, which does not involve in-


Invasion

3 200 fection. Despite a significant (P = 0.02)


reduction in the number of NETs using
2 ns
100 either approach (fig. S7A), the number
1 of cells that extravasated into the lung
0 0 tissue 24 hours after injection was not
altered (fig. S7B).
h.

h.

h.
h.

h.

e
le

cl

cl

cl
cl

in

in

in
in

in
ic

hi

hi

hi
hi
h

To determine whether DNase Icoated


4

Ve

Ve

Ve
Ve

Ve

D
D

PA

PA

PA
PA

PA

nanoparticles also reduced spontaneous


+4T1 +Neutrophils +10% FCS
metastasis, we injected 4T1 cells into both
Fig. 4. Cancer cells induce NET formation through G-CSF, and neutrophil-stimulated invasion requires NADPH inguinal mammary glands of female mice,
oxidase and PAD4 activity. (A) Blocking antiG-CSF antibodies (1.6 mg/ml) decreased 4T1-induced NET extension (mean and after 7 days, daily treatment with
SEM; neutrophils with vehicle or antiG-CSF, n = 3; neutrophils with 4T1 cells and vehicle or antiG-CSF, n = 5; t test). DNase Icoated nanoparticles began.
(B) The NADPH oxidase inhibitor apocynin (10 mM) inhibited NET formation (mean SEM; neutrophils and vehicle or The DNase Icoated nanoparticles had
NAPDH oxidase inhibitor, n = 3; neutrophils with cancer cells and vehicle or apocynin, n = 5; t test). (C) Apocynin (10 mM)
no effect on primary tumor growth
inhibited neutrophil-stimulated invasion (mean SEM; 4T1 cells only or 4T1 cells with neutrophils and vehicle or apoc-
ynin, n = 4; 4T1 cells only and vehicle or apocynin in 10% FCS, n = 1; t test). (D) PAD4 inhibition (200 mM Cl-amidine)
(Fig. 6G), but the lung metastatic burden
reduced cancer cellinduced NET formation (mean SEM; neutrophils and vehicle or PAD4 inhibitor, n = 6; neutrophils was reduced: all mice treated with con-
with 4T1 cells and vehicle or PAD4 inhibitor, n = 4; t test). (E) PAD4 inhibition (200 mM Cl-amidine) blocked neutrophil- trol nanoparticles had micrometastases,
stimulated cancer cell invasion (mean SEM; n = 3, t test). but three of six mice treated with DNase
Icoated nanoparticles had no detectable
metastatic lung cancer, but the effects are relatively modest (5). Because metastases by histology (Fig. 6H). Together, these data suggest that
we found that DNase I prevented NET-mediated invasion and migra- therapeutic targeting of NETs can prevent lung metastasis.
tion in vitro with three different sources of cancer cells (Fig. 3, F, H, and
K), we speculated that the modest in vivo effects of DNase I were due to
its short half-life in blood (30). Immobilization of an enzyme on the DISCUSSION
surface of nanoparticles can increase enzyme stability (31); therefore, Here, we show that cancer cells can hijack neutrophils, so that the
we developed DNase Icoated nanoparticles. In vitro, DNase Icoated neutrophils ability to eradicate pathogens through formation of
nanoparticles digested NETs and blocked invasion as effectively as free NETs instead aids metastatic spread. By imaging in live mice, we

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 6 of 12


SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

A B
250 ns
P = 0.04 P = 0.0006

(% 4T1 treated with vehicle


P = 0.02

(% area covered by NETs)


5 P = 0.03 200

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

(% 4T1 treated with vehicle


4
(% area covered by NETs)

Downloaded from http://stm.sciencemag.org/ by guest on September 7, 2017


600

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

+4T1 +Neutrophils +10% FCS

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

+Neutrophils +10% FCS


+Neutrophils +CM
Fig. 5. NET formation and neutrophil-stimulated invasion require neutrophil protease activity. (A) Cathepsin G inhibitor I (2 mM) reduced cancer cellinduced
NET formation (mean SEM; n = 4, t test). (B) Cathepsin G inhibitor I (2 mM) 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 mM) reduced cancer cellinduced NET formation (mean SEM; n = 3,
t test). (D) Neutrophil elastase inhibitor sivelestat (10 mM) 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 mM 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 mM apocynin; mean SEM; n = 3 to 6, t tests).

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

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 7 of 12


SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

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)

P = 0.003 P = 0.005 Control nanoparticles

Plasma DNase I (ng/ml)


300
DNase Icoated nanoparticles
Invasion

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

Downloaded from http://stm.sciencemag.org/ by guest on September 7, 2017


P = 0.002 P = 0.0003 P = 0.01
0.4
60 6
Metastatic foci/lung area (mm2)
(% of total lung area)

0.3

Average metastatic
Metastatic burden

foci size (mm2)


40 4
0.2

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

800 Control nanoparticles


( % of total lung area)

DNase Icoated nanoparticles 0.6


Tumor volume (mm )

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

Time after cell injection (days)


na C

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].

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 8 of 12


SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

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

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phagocytic activities of neutrophils intact. Blocking antibodies against tumors express high levels of G-CSF (22, 35), and they are both models
G-CSF reduced cancer cellinduced NET formation in our study and of triple-negative breast cancer. Higher amounts of G-CSF are found in
can reduce metastases in mice (34, 35). However, G-CSF is used clini- human patient samples of triple-negative breast cancers than of other
cally to prevent mortality associated with neutropenic infections and subtypes (34). Our analysis of clinical samples also shows that NETs
cannot be easily targeted. Inhibitors against NOX2 and neutrophil are found in highest amounts in this breast cancer subtype.
proteases also blocked cancer cellinduced NET formation. Un- In conclusion, we demonstrated that cancer cells can hijack the
fortunately, NOX2 and the neutrophil proteases are not good targets physiological processes of microorganism killing through NET for-
because NOX2 is critical for bacterial killing and neutrophil proteases mation to promote metastasis. This concept for how cancer cells exploit
are required to eliminate pathogens by phagocytosis and degranulation. host cells represents a therapeutic opportunity to prevent metastasis,
However, the extracellular DNA mesh of NETs is an exciting and fea- which accounts for the great majority of cancer-associated deaths.
sible target. Treatment with nucleases was reported to reduce metasta-
sis decades ago, but with no insights as to why (36). DNase I treatment
is approved by the U.S. Food and Drug Administration for the treat- MATERIALS AND METHODS
ment of cystic fibrosis, for which it is used to decrease mucus viscosity Study design
resulting from NET accumulation triggered by persistent infections. The objective of this research was to test the hypothesis that cancer
Our results with DNase Icoated nanoparticles serve as a proof of prin- cellinduced NETs promote invasion and metastasis in in vitro and
ciple that NETs are drug targets to reduce metastasis. Another strategy in vivo models of cancer. Animals were excluded from the study when
may be to prevent NETs from forming, for example, by targeting PAD4. the initial intravenous injection of the cancer cells missed the tail vein.
Although we were able to prevent NET formation using PAD4 in- Endpoints for animal experiments were selected before the conduction
hibitors in vivo in short-term assays, we could not test the effect on of the experiments as the time when the first animals in any experi-
metastasis because long-term treatment would be needed. Commer- mental group had a weight loss of >10%. The number of replicates for
cially available PAD4 inhibitors have serum half-lives of ~15 min to specific experiments is listed in the figure legends. The experiments
4 hours (37, 38). It was therefore impossible to compare NET diges- testing the effects of DNase I on invasion in vitro were repeated by
tion by DNase Icoated nanoparticles with inhibition of NET forma- three different coauthors. The investigators who assessed, measured,
tion using PAD4 inhibition and establish which approach is the best or quantified the results of invasion through Matrigel, NET-like
strategy for preventing metastasis. structures by CILI, or metastatic burden after treatment with DNase
Results of our experimental metastasis assays suggest that NETs play Icoated nanoparticles were blinded to the specific intervention.
a role during the establishment of metastases. They may do so through
multiple mechanisms. The DNA mesh can trap circulating cancer cells Animals
at the site of dissemination (5). In addition, intravascular NETs can in- BALB/c mice were purchased from Charles River Laboratories,
crease local vascular permeability (15), which would permit cancer cells p47phox/ (39) and ACTB-ECFP mice (40) were purchased from
to extravasate more easily. This role has been proposed for systemically the Jackson Laboratory, and C3(1)-Tag mice (21) were purchased from
formed NETs. However, our data do not support a role for cancer cell the National Institutes of Health (NIH) mouse repository. LysM-EGFP
induced NETs in enhancing extravasation. Instead, our data suggest mice (20) were provided by M. Looney (University of California, San
that NETs may stimulate further invasion of the cells into the tissue Francisco). All procedures were approved by the Cold Spring Harbor
and the expansion of the colonizing cells. The DNA mesh of NETs Laboratory Institutional Animal Care and Use Committee and were
may promote invasion by acting to concentrate NET-associated pro- conducted in accordance with the NIH Guide for the Care and Use
teases or protecting proteases from the endogenous inhibitors abundant of Laboratory Animals.
in blood. We found that CM from NET-forming cultures can promote
invasion but that both DNase I digestion and heat denaturation can In vitro invasion, migration, and NET formation assays
abolish this effect. This suggests that the DNA mesh is important for Neutrophils (2.5 105 cells) in 750 ml of serum-free medium were
mediating the effect, but because denaturation does not degrade DNA, seeded on poly-L-lysinecoated coverslips (BD Biosciences) in 24-well

Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 9 of 12


SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

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

Downloaded from http://stm.sciencemag.org/ by guest on September 7, 2017


cells was also heat-denatured (65C for 15 min or 95C for 5 min), or 19 days after cancer cell transplantation. Tumor growth and lung
DNase I was added to the collected CM. The CM was added to the metastatic burden were quantified as described above.
lower chamber, and cancer cells were seeded in serum-free medium in
the upper chamber of Matrigel-coated cell culture inserts. CM was also Statistical analysis
prepared from neutrophil cultures treated with 20 nM PMA (Sigma) All statistical analyses were performed using GraphPad Prism
with or without DNase I (Invitrogen). The CM was added to the lower software versions 5 and 6. Data were analyzed using two-sided t tests
chamber, and 4T1 cells were seeded in serum-free medium in the or one-way ANOVA, as indicated in the figure legends with an a value
upper chamber of Matrigel-coated FluoroBlok cell culture inserts. Af- of 0.05. For statistical analyses of the metastatic burden from orthoto-
ter 22 hours, the cells were stained with 1 mM SYTO13 (Molecular pically growing tumors after treatment with DNase Icoated nanopar-
Probes), and the number of invading cells was counted in 10 random ticles and of the effect of DNase Icoated nanoparticles on the size of
fields of view using a fluorescence microscope. metastatic foci, the data were first transformed by taking the square
To test the effect of NETs on C3(1)-Tag primary cancer cell mi- root because the variance of the untransformed data was significantly
gration, tumors (~10 to 12 mm in diameter) were resected from (P = 0.007 and P = 0.0003, respectively) different between the two groups.
C3(1)-Tag mice intercrossed with ACTB-ECFP, and cancer cells were The number of sampled units, n, is indicated in the figure legends.
isolated as described (41). After growth to subconfluency, 5 104 cells
in Dulbeccos modified Eagles medium (DMEM) containing 0.5%
SUPPLEMENTARY MATERIALS
FCS were added to a FluoroBlok cell culture insert with an 8-mm www.sciencetranslationalmedicine.org/cgi/content/full/8/361/361ra138/DC1
pore size (Corning). The plate was incubated for 2 hours to allow Materials and Methods
cells to adhere, and then neutrophils (2.5 105 cells) were seeded Fig. S1. CXCL1 promotes neutrophil recruitment to tumors.
on poly-L-lysinecoated coverslips (BD Biosciences) in the lower 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.
chamber with serum-free DMEM. The cancer cell medium was then
Fig. S4. G-CSF and p47phox promote NET formation and cancer cell invasion.
replaced by serum-free DMEM. After 22 hours, the number of mi- Fig. S5. Breast cancer cells induce the formation of lytic NETs.
grating cells was quantified similarly to the experiments performed Fig. S6. CM from neutrophils induced to undergo NET formation promotes invasion.
with neutrophil CM. Fig. S7. Cancer cellinduced NETs do not promote extravasation.
Neutrophils grown on coverslips in the lower chamber were Movie S1. NET-like structures form in vivo around 4T1 cancer cells in the lung.
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Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 11 of 12


SCIENCE TRANSLATIONAL MEDICINE | RESEARCH ARTICLE

(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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Park et al., Sci. Transl. Med. 8, 361ra138 (2016) 19 October 2016 12 of 12


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 Kttner, Jing
Qiu, Ana S. Almeida, Naiara Perurena, Kai Kessenbrock, Michael S. Goldberg and Mikala Egeblad

Sci Transl Med 8, 361ra138361ra138.


DOI: 10.1126/scitranslmed.aag1711

Metastasis caught in a NET

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Neutrophil extracellular traps, or NETs, are DNA structures that are produced by neutrophils in response to
infection and can promote the spread of cancer in the presence of infection. Park et al. discovered that even in the
absence of infection, metastatic breast cancer cells can stimulate neutrophils to form NETs, which further support
the spread of metastasis. The authors also demonstrated an approach to breaking this vicious cycle using
nanoparticles coated with DNase I, an enzyme that breaks down DNA NETs. This treatment was effective in
reducing lung metastases in mice, demonstrating the potential of NETs as a therapeutic target.

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