Coffelt, S. B., Wellenstein, M. D., and de Visser, K. E. (2016) Neutrophils in
cancer: neutral no more. Nature Reviews Cancer, 16(7), pp. 431-446.
(doi:10.1038/nrc.2016.52)
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1
Neutrophils in cancer: neutral no more
Seth B. Coffelt, Max D. Wellenstein & Karin E. de Visser*
Division of Immunology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX
Amsterdam, The Netherlands,
*corresponding author: Karin E. de Visser ()
Summary
Neutrophils are indispensable antagonists of microbial infection and facilitators of wound
healing. In the cancer setting, a newfound appreciation for neutrophils has come into view.
The traditionally held belief that neutrophils are inert bystanders is being challenged by
recent literature. Emerging evidence indicates that tumors manipulate neutrophils,
sometimes early in their differentiation process, to create diverse phenotypic and functional
polarization states able to alter tumor behavior. In this Review, we discuss the involvement of
neutrophils in cancer initiation and progression, and their potential as clinical biomarkers and
therapeutic targets.
2
The name neutrophil – given to polymorphonuclear, granulocytic cells by Paul Ehrlich in the
late 19th century – is based on the inability of these cells to retain acidic or basic dyes and
for their preferential uptake of pH neutral dyes
1
. Although their neutral staining led to the
identification of these cells, neutrophils in the cancer setting are anything but neutral.
Neutrophils in tumor-bearing hosts can oppose or potentiate cancer progression. These two
types of behavior are controlled by signals emanating from cancer cells or stromal cells
within the tumor microenvironment, which educate neutrophils to execute the demise of the
tumor or facilitate support networks that lead to its expansive spread. These functions can
occur locally in or around the tumor microenvironment, as well as systemically in distant
organs.
Until the past few years, other immune cells such as macrophages have
overshadowed the role of neutrophils in cancer. But recent studies and the development of
new genetic tools have provided the cancer community with new insights into the profound
influence of these dynamic cells by uncovering distinct capabilities for neutrophils throughout
each step of carcinogenesis: from tumor initiation to primary tumor growth to metastasis.
During these processes, neutrophils take on different phenotypes and sometimes opposing
functions. Emerging evidence also indicates that these cells are highly influential, and are
able to change the behavior of other tumor-associated cell types – primarily other immune
cells. In this Review, we focus on how tumors manipulate the generation and release of
neutrophils from the bone marrow. We discuss the mechanisms identified in animal models
by which neutrophils participate in tumor initiation, growth and metastasis. Finally, we
highlight the potential of these cells as clinical biomarkers and therapeutic targets.
Neutrophil origins and life cycle: homeostasis versus cancer [Au: subheading too
long, please shorten to <39 characters; possibly ‘Homeostasis versus cancer’ would
work?]
In humans, neutrophils are the most abundant immune cell population, representing 50-70%
of all leukocytes. Over 10
11
neutrophils may be produced per day
2
, and tumors can increase
this number by even more. Indeed, patients with various cancer types, including but not
limited to breast, lung and colorectal cancer, often exhibit increased numbers of circulating
neutrophils
3,4
. Recent studies have identified key pathways that tumors exploit to disrupt
normal neutrophil homeostasis and these are discussed below.
Granulopoiesis
To accommodate for the notably high production and turnover of neutrophils, the bone
marrow devotes about two-thirds of its space to the formation of neutrophils and monocytes
Comment [SC1]: I don’t like
“Homeostasis versus cancer” The
reader has no idea what we are
referring to until they read the
subheading “Granulopoiesis.” What
about “Neutrophil origins and life
cycle”?
3
in steady-state conditions
5
. During granulopoiesis, neutrophils arise from lymphoid/myeloid-
primed progenitors (LMPPs)
6
, which are derived from hematopoietic stem cells (Figure 1).
LMPPs further differentiate into granulocyte/monocyte myeloid progenitors (GMPs) and
many transcription factors required for this process have been identified (reviewed in
5,7,8
).
Neutrophil maturation then begins, as GMPs differentiate through the following sequence:
myeloblast, promyelocyte, myelocyte, metamyelocyte, banded neutrophil and, finally, a
segmented neutrophil (reviewed in
5,9-11
). The transition from myeloblast to promyelocyte is
marked by the first appearance of primary granules. Secondary and tertiary granules form
sequentially during the myelocyte to metamyelocyte and band cell to segmented cell stage,
respectively
5,12
. These granules compartmentalize an arsenal of defensive factors and
enzymes, such as myeloperoxidase, elastase, defensins, cathelicidins and matrix
metalloproteinases (MMPs), that protect against opportunistic infections and mediate the
resolution of inflammation (reviewed in
12,13
). If large numbers of neutrophils are used up
during infection or cancer, a process called emergency granulopoiesis overtakes steady
state granulopoiesis to rapidly increase neutrophil formation
11
. In tumor-bearing mice and
humans with pancreatic or colon cancer (and most likely other tumor types), the spleen is an
alternative source of neutrophil production
14
.
Granulocyte-colony stimulating factor (G-CSF) is the master regulator of neutrophil
generation and differentiation
15-17
. G-CSF acts at the level of myeloid progenitors to induce
their proliferation and differentiation. Its receptor, G-CSFR, is expressed throughout the
myeloid lineage from early stem and progenitor cells to fully differentiated neutrophils
18,19
,
and G-CSFR-STAT3 (signal transducer and activator of transcription 3) signaling governs
neutrophil formation
20
. The transcription factor RAR-related orphan receptor γ1 (RORC1) is a
recently identified regulator of myelopoiesis in tumor-bearing mice and its expression may be
induced by G-CSF
21
. However, G-CSF is not absolutely required for granulopoiesis, as other
molecules – such as granulocyte-macrophage-colony stimulating factor (GM-CSF),
interleukin 6 (IL-6) and KIT ligand (KITL) – can play a redundant, but lesser role
22-24
. Tumors
in many mouse models of cancer upregulate these cytokines, causing overactive
granulopoiesis and neutrophilia
25-31
.
Neutrophil retention and release from bone marrow
One feature of granulocytes that sets them apart from every other immune cell is their
release from the bone marrow as terminally differentiated, mature cells. Circulating mature
neutrophils account for only 1-2% of all neutrophils throughout the body under homeostatic
conditions
32
. Mature cells are retained in the bone morrow by an interplay between two C-X-
C chemokine receptors, CXCR4 and CXCR2. Constitutive CXCL12 expression by
4
osteoblasts and other bone marrow stromal cells tether CXCR4
+
neutrophils in the bone
marrow, whereas secretion of CXCL1 and CXCL2 by endothelial cells and megakaryocytes
encourage the release of neutrophils into the circulation via CXCR2 signaling
33-38
(Figure 1).
Several adhesion molecules, such as integrin subunit α4 (ITGA4) and vascular cell adhesion
molecule 1 (VCAM1), as well as some proteases are also important in neutrophil retention
39-
41
. In addition to its positive influence on granulopoiesis, G-CSF is a well-known disruptor of
neutrophil retention
42
. G-CSF pressures the bone marrow to release neutrophils through
thrombopoietin (TPO)-induced upregulation of CXCR2 ligands on megakaryocytes
38
,
reduction of CXCL12 expression by bone marrow stromal cells
43,44
and downregulation of
CXCR4 on neutrophils themselves
45
.
Outside the bone marrow, a cascade of other cell types and cytokines, involving IL-
23-expressing phagocytes and IL-17-producing lymphocytes, tightly regulates the production
of G-CSF so that neutrophil numbers are maintained in the circulation. In this feedback
mechanism, macrophages and dendritic cells phagocytose apoptotic neutrophils
47-49
, curbing
the secretion of IL-23
46
– a cytokine that controls IL-17 expression by αβ T cells, γδ T cells,
innate lymphoid cells and other lymphocytes
50,51
. Because IL-17 is upstream of G-CSF
52,53
,
lower levels of IL-17 equate to reduced expression of G-CSF and steady-state release of
neutrophils from the bone marrow
46
. Commensal bacteria and enterocyte-derived CXCL5 in
the gut also play a role in neutrophil homeostasis by increasing or inhibiting IL-17 production,
respectively
54,55
. IL-1β that is released from dying cells or upregulated in response to
inflammatory stimuli is another potent inducer of the IL-17-G-CSF axis
56,57
.
Many of the molecules that control neutrophil release from the bone marrow are
frequently upregulated in tumors or systemically as a result of a tumor
25-28,58
. These factors
override retention signals in the bone marrow, facilitating neutrophil egress and elevated
numbers of circulating neutrophils (Figure 2). Cancer cells themselves produce these
cytokines
27,28,58
, but stromal and immune cells can also contribute to their elevated
expression in tumor-bearing mice. For example, tumor-associated macrophages are a well-
known source of IL-1β
59
. Recently, we showed that neutrophils expand in mammary tumor-
bearing K14-Cre;Cdh1
F/F
;Trp53
F/F
mice because of increased macrophage-derived IL-1β
stimulation of the IL-17-G-CSF axis
26
. Ectopic overexpression of IL-1β in tumors derived from
cancer cell lines or a genetically engineered gastric cancer model also increases the number
of circulating neutrophils
60-63
. As such, aberrant production of cytokines by tumors or stromal
cells can offset the balance of neutrophil retention and release from the bone marrow.
The pressure on the bone marrow to release neutrophils can often be so intense in
tumor-bearing hosts that undifferentiated cells are set free prematurely. Nuclear staining of
circulating neutrophils from mammary and lung tumor models has revealed the existence of