Review
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Breast cancer as a systemic disease: a view of metastasis
A. J. Redig1 & S. S. McAllister1,2,3
From the 1Division of Hematology, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School; 2Harvard Stem Cell
Institute, Boston; and 3Broad Institute of Harvard and MIT, Cambridge, MA, USA
Abstract. Redig AJ, McAllister SS (Brigham and
Women’s Hospital, Harvard Medical School,
Boston, MA, USA; Harvard Stem Cell Institute,
Boston, MA, USA; Broad Institute of Harvard and
MIT, Cambridge, MA, USA). Breast cancer as a
systemic disease: a view of metastasis. (Review).
J Intern Med 2013; 274: 113–126.
Breast cancer is now the most frequently diag-
nosed cancer and leading cause of cancer death in
women worldwide. Strategies targeting the primary
tumour have markedly improved, but systemic
treatments to prevent metastasis are less effective;
metastatic disease remains the underlying cause of
death in the majority of patients with breast cancer
Introduction
Despite advances in the diagnosis and treatment of
human malignancy, cancer remains amongst the
leading causes of morbidity and mortality world-
wide, with 7.5 million deaths attributed to cancer
in 2008 (http://globocan.iarc.fr/factsheets/popu-
lations/factsheet.asp?uno=900), accessed August
12, 2012. Breast cancer is now the most frequently
diagnosed cancer and the leading global cause
of cancer death in women, accounting for 23% of
cancer diagnoses (1.38 million women) and 14%
of cancer deaths (458 000 women) each year [1].
Although breast cancer has a markedly higher
incidence in developed countries, half of new
breast cancer diagnoses and an estimated 60% of
breast cancer deaths are now thought to occur in
the developing world [1]. Metastatic disease, or the
spread of tumour cells throughout the body, is
responsible for the vast majority of cancer patient
deaths and represents the central clinical chal-
lenge of solid tumour oncology. The importance of
understanding the mechanisms underlying the
metastatic process and the complex interactions
between tumour and host during disease progres-
sion has been widely recognized and was amongst
the subjects discussed at the Nobel Conference
on Breast Cancer: Progress and Challenges in
Prevention, Risk Prediction, Tumor Biology and
ª 2013 The Association for the Publication of the Journal of Internal Medicine
doi: 10.1111/joim.12084
who succumb to their disease. The long latency
period between initial treatment and eventual
recurrence in some patients suggests that a
tumour may both alter and respond to the host
systemic environment to facilitate and sustain
disease progression. Results from studies in ani-
mal models suggest that specific subtypes of breast
cancer may direct metastasis through recruitment
and activation of haematopoietic cells. In this
review, we focus on data implicating breast cancer
as a systemic disease.
Keywords: breast cancer, disseminated tumour cells,
metastasis, tumour dormancy, tumour microenvi-
ronment, systemic instigation.
Treatment in Stockholm, Sweden, 14–17 June
2012. Our own findings using a breast cancer
xenograft model were also presented at this
meeting. The aim of this review is to evaluate
key clinical and laboratory observations surround-
ing our emerging understanding about the
complexity of metastasis and to consider funda-
mental questions about the nature of metastatic
disease.
The clinical challenge of metastasis
The decrease in breast cancer-related deaths that
has been observed in the developed world since the
early 1990s is attributed in part to improved
screening, which allows diagnosis at a stage when
curative therapy is still an option [2]. It is clear that
improving strategies to diagnose and treat breast
cancer should remain a high priority in the 21st
century. Unfortunately, however, some patients
with cancer initially present with distant metasta-
ses; these patients are diagnosed with Stage IV
disease that is nearly always incurable. Other
patients, without detectable metastases at the time
of diagnosis, will eventually recur with disease in
distant organs. Amongst women diagnosed with
breast cancer, only a minority presents with Stage
IV disease. Nevertheless, nearly 30% of women
initially diagnosed with early-stage disease will
113
 A. J. Redig & S. S. McAllister
ultimately develop metastatic
months or even years later [3].
The goal of tumour staging algorithms is to help
determine a patient’s recommended therapy based
on several features of the underlying malignancy
believed to predict the risk of recurrence.
Treatment options reflect the underlying staging
work-up in two critical ways. First, with an initial
diagnosis of Stage IV metastatic disease, multidis-
ciplinary treatment strategies focus on palliation of
symptoms, rather than aggressive surgical or
medical treatments that are associated with a high
degree of morbidity. Second, for patients without
detectable metastatic lesions at the time of diag-
nosis but with features of high-risk disease, the
purpose of adjuvant chemotherapy and/or radia-
tion is to destroy the undetectable, occult microm-
etastases a patient may harbour to prevent future
recurrent disease. Standard staging scans or
surgical lymph node dissection does not detect
individual disseminated tumour cells (DTCs), but
the epidemiology of metastatic disease suggests
that some tumour cells escape the primary tumour
prior to surgical resection. Although strategies for
targeting the primary tumour have markedly
improved, targeted therapies directed against the
elusive micrometastatic population – the cells that
‘escaped’ – have been less effective. Despite multi-
disciplinary treatment algorithms, metastatic dis-
ease remains the underlying cause of death in the
majority of patients with breast cancer who suc-
cumb to their disease [1, 2]. Unfortunately, current
clinical strategies fall short both in accurately
identifying patients at high risk of recurrence and
in treating patients with metastatic disease.
A model for metastasis
Using our unique breast cancer xenograft model,
we have gained insight into the processes by which
a malignancy alters and responds to the host
environment in order to facilitate and sustain
disease progression. It is clear that the manner in
which the body responds to certain breast malig-
nancies creates a systemic environment that is
amenable to the outgrowth of microscopic dissem-
inated tumours that would otherwise have
remained indolent. This breast cancer xenograft
model relies on implanting a specific type of
aggressively growing human breast tumour into
one anatomical site (e.g. subcutaneous flank or
orthotopic site) of a nude mouse to elicit a host
response, and injecting indolent human breast
114 ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 113–126
Review: Breast cancer and metastasis
lesions, often tumours into distant anatomical sites (e.g. contra-
lateral flank or lung via tail vein injection) to assess
the response to the host systemic environment
[4–6]. These tumours have no direct physical
contact, so the aggressive ‘instigating’ tumour
must induce systemic changes from a distance to
drive the otherwise indolent ‘responding’ tumour to
grow. We termed this process systemic instigation,
and further mechanistic studies indicated that
haematopoietic cells from the bone marrow are
key drivers of the process [4–6]. Specifically, bone
marrow-derived cells are recruited to the responder
tumours and contribute to the development of the
tumour microenvironment, thereby allowing the
previously indolent tumour cells to grow. Of note,
not all breast tumours are able to act as instiga-
tors, suggesting that specific features of the
primary tumour are required to drive systemic
changes in micrometastatic populations.
Our model holds particular promise for the study of
breast cancer because it represents an in vivo
opportunity to dissect the process responsible for
incurable metastatic disease, that is, the conver-
sion of indolent micrometastases into overt clini-
cally significant metastases. The indolent
responding tumours in this model are analogous
to the clinical scenario in which tumour cells
disseminate from the primary tumour only to
remain dormant in distant tissues for months or
even years before continuing to grow as metastatic
disease. The ‘instigating’ tumours are analogous to
those primary tumours (or possibly dominant
metastatic lesions) that retain the ability to influ-
ence the outgrowth of otherwise indolent cancer
cells. Some but not all patients with breast cancer
will develop recurrent disease, just as some but not
all tumours in the xenograft model possess the
ability to elicit responder tumour growth. Using
this model, the results of mechanistic studies also
suggest the possibility of stratifying breast cancers
on a functional basis; such classification would
directly identify those tumours most likely to drive
future metastatic outgrowth.
Does traditional classification of breast tumours apply to DTCs?
Ongoing research continues to demonstrate the
underlying genetic and molecular diversity of
breast cancer, suggesting that understanding
tumour–host interactions may depend upon
understanding the unique characteristics of the
primary tumour subtype. Traditionally, breast
cancer classification relies greatly on the underly-
 A. J. Redig & S. S. McAllister
ing histopathological features of the primary
tumour, including expression of the oestrogen
(ER) and progesterone receptor (PR) and amplifi-
cation of the HER2/neu oncogene product. Under
current clinical guidelines, pathology-based
review of tumour tissue and determination of
tumour staging provide some information about
prognosis as well as suggesting treatment options,
notably with the use of anti-oestrogen therapies
such as tamoxifen or the anti-HER2 agent
trastuzumab (Herceptin) in appropriate patient
populations [7].
Emerging technology has also been used to develop
more detailed molecular profiles of breast cancer
subtypes, with some clinical application. Several
commercially available methods, including the
Oncotype Dx assay (21-gene signature) and the
Mammaprint assay (70-gene signature), have been
approved for clinical use within the last 10 years to
help predict the chance of recurrence and guide
therapy in defined patient subsets [8, 9]. More
recently, whole-genome profiling of breast cancer
has demonstrated that, from a molecular stand-
point, breast tumours fall into four general
categories: basal-like (which generally corresponds
to triple-negative disease), luminal-A (generally ER
positive and low grade), luminal-B (also ER positive
but high grade) and Her2-positive tumours
[10–13]. This evolving paradigm of the molecular
subclassification of breast cancer suggests exciting
new directions from which to investigate the inter-
actions between primary tumour and systemic
environment during the metastatic cascade. In
many studies, distinct genes or pathways within
a given subtype of breast cancer that appear to play
an important prognostic role, including with regard
to metastatic behaviour, are now being identified
[14, 15].
These approaches have their limitations, and sig-
nificant gaps in our ability to use either histopa-
thology or molecular profiling to effectively predict
recurrence still remain. For instance, it is not yet
clear how to combine traditional histopathology,
results from limited gene assays (which cannot be
used in all patient populations) and whole-genome
profiling into a clinically useful algorithm [16].
Findings from molecular profiling studies support
clinical observations that breast cancer as a dis-
ease entity has several distinct patterns whilst also
demonstrating significant heterogeneity amongst
patients. In particular, how to effectively utilize
molecular classification in the clinical context and
Review: Breast cancer and metastasis
whether the same parameters apply to metastatic
disease remain unclear. However, novel methodol-
ogy may eventually change the way in which breast
cancer is classified and the way in which tradi-
tional biomarkers are evaluated.
It was recently demonstrated that whole-genome
sequencing of circulating cell-free DNA could be
used to identify cancer-associated chromosomal
markers in a subset of women with breast cancer
[17]. Several studies are assessing the utility of
evaluating patient blood samples for circulating
tumour cells (CTCs) during risk stratification in the
diagnosis of early-stage breast cancer [18] and to
predict the likely response in patients undergoing
preoperative chemotherapy [19]. There is also
some retrospective evidence to suggest that the
presence or absence of CTCs in patients with
metastatic breast cancer may be of important
prognostic significance regardless of molecular
subtype [20].
The ability to detect isolated tumour cells outside a
primary tumour has led to several attempts to
correlate traditional histopathological markers of
breast cancer with the presence of either DTCs
found in bone marrow or CTCs found in peripheral
blood. However, to date, these studies have yielded
inconclusive results [8, 9]. In one study of surgical
patients, no association was found between the
presence of DTCs or CTCs and other markers
typically used to assess tumour behaviour, such as
tumour size or grade and hormone receptor or
lymph node status, or use of neoadjuvant chemo-
therapy [21]. In a further series of 92 women with
early breast cancer, bone marrow aspirates and
peripheral blood samples were obtained at the time
of surgery, and there was similarly no statistically
significant correlation between the presence of
CTCs or DTCs and tumour size or grade and lymph
node or ER/PR/HER2 status [22]. Nevertheless,
some interesting trends were noted when the
presence of DTCs was compared to ER, PR and
HER2 expression. DTCs were identified in 23% of
patients with ER-positive disease but 37% with ER-
negative disease; 22% of patients with PR-positive
disease but 32% with PR-negative disease; and 0%
of patients with HER2-positive disease but 29%
with HER2-negative disease [22]. Histopathological
subtyping might not provide a basis for predicting
the presence or absence of DTCs, but these data
suggest that the presence of DTCs varies amongst
patients and may reflect underlying functional
differences between primary tumours.
ª 2013 The Association for the Publication of the Journal of Internal Medicine 115
Journal of Internal Medicine, 2013, 274; 113–126
 A. J. Redig & S. S. McAllister
Of particular interest, recent findings challenge
previously held assumptions that the histopatho-
logical biomarkers of the primary tumour are
repeated in metastases. In two recent studies, it
was demonstrated that the ER, PR or HER2 status
of a biopsy-proven metastatic lesion does not
match the pathology of the primary tumour in a
substantial fraction of patients (ranging from 15%
to 40% of the patient cohort) [23, 24]. Such
differences are also seen in the CTC and DTC pools
when breast cancer biomarkers are evaluated. In a
study focusing on developing a multimarker panel
for the characterization of CTCs in patients with
metastatic breast cancer, it was incidentally noted
that the CTCs were HER2 positive in 27% of the
patients with HER2-negative disease [25]. In an
earlier study with 254 patients evaluating ER-
positive DTCs, the concordance between primary
tumour and DTC was only 28% [26]. Furthermore,
within the population of DTCs, ER expression was
heterogeneous in 26% of patients [26].
The findings of a study comparing the evolution of
bone marrow DTCs to that of peripheral blood
CTCs also suggest that these two populations may
be quite different even within an individual patient.
DTCs in the marrow were found with much greater
frequency (24% of 431 patients) than CTCs in the
peripheral blood (13%) [27]. In addition, DTCs in
the bone marrow correlated only with the PR status
of the primary tumour, whereas CTCs in the blood
correlated with ER status, PR status and node
positivity of the primary tumour. A weak (P = 0.05)
correlation was noted between the biomarker
expressions of DTCs and CTCs from the same
patient. However, CTCs in the blood were signifi-
cantly associated with triple-negative primary
tumours and were nearly always triple negative
themselves [27]. Consequently, DTC/CTC survival
and proliferation into overt metastases may be
related to the ability of these cells to regulate
critical signalling pathways already known to drive
breast cancer cell growth, specifically pathways
involving oestrogen, progesterone and HER2. Fur-
thermore, as these studies demonstrate, the histo-
pathology of the primary tumour may not always
direct the best treatment options for targeting
metastatic disease.
From micrometastases to macrometastases
Although CTCs can be found with reproducible
frequency in the peripheral blood of patients with
breast cancer, results from preclinical studies
116 ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 113–126
Review: Breast cancer and metastasis
suggest that the majority of CTCs/DTCs will not
form a clinically detectable overt metastasis [28].
Indeed, results suggest that <1% of all DTCs are
capable of forming an overt tumour [28, 29].
Preclinical findings also indicate that cells from
within a heterogeneous primary tumour intrava-
sate into the vasculature, survive in the circulation
and extravasate into the parenchyma of a distant
tissue. Within this foreign environment, it is
thought that DTCs exist in a state of dormancy
for unknown and varying periods of time. Some
micrometastatic foci will ultimately form overt
tumours, ostensibly through their ability to recruit
the necessary stroma and vasculature, whilst
avoiding detection and elimination by the host
immune system [30, 31]. Metastatic inefficiency is
thought to explain why some patients present with
clinically detectable metastases months or even
years after their initial cancer diagnosis and sur-
gery [32–34]. The ultimate determinants of whether
or not a given tumour cell or population of cells can
give rise to a clinically significant metastasis may
thus include not only the biology of the cancer cells
themselves but also the relationship between
tumour and host.
Differences between signalling pathways required
for invasion and motility, notably those required for
adaptation to a new tissue environment versus
those that drive proliferation of an established
lesion, may in part explain metastatic inefficiency.
Nevertheless, the underlying mechanisms of these
distinct processes are not clearly understood.
Moreover, there is currently no reliable way to
determine which, if any, of the DTCs/CTCs found
in an individual patient with cancer will eventually
result in overt metastatic disease, making it impos-
sible to translate the technical ability to isolate
DTCs/CTCs into a clinically useful algorithm to
guide treatment based on a patient’s likelihood of
recurrence. Determining how to identify which
cells pose the greatest threat of recurrent disease
as well as elucidating the key molecular mecha-
nism(s) required for their survival and development
following dissemination is thus of critical impor-
tance in order to target these cells before they lead
to clinically devastating outcomes.
The presence of DTCs in the bone marrow of women
without evidence of metastatic breast cancer has
recently been shown to be an independent predictor
of decreased recurrence-free survival [35]. The
prognostic significance of DTCs also appears to
extend to women diagnosed with later stages of
 A. J. Redig & S. S. McAllister
breast cancer in whom chemotherapy is incorpo-
rated into treatment modalities. It has recently been
shown that the presence of DTCs correlates with
decreased survival in women receiving neoadjuvant
chemotherapy, irrespective of whether DTCs were
identified prior to chemotherapy [36]. It is signifi-
cant that the women in this study were diagnosed
with locally advanced disease and represent a
subset of patients for whom definitive cure is
possible with systemic chemotherapy. The fact that
DTCs can be identified and have prognostic signif-
icance in breast cancers diagnosed at both early
and later stages suggests that the mobilizing events
enabling these cells to disseminate represent a
pivotal sequence in metastatic progression, and
that DTCs may directly or indirectly support the
outgrowth of metastases.
The relationship between the DTCs in the bone
marrow and the CTCs found in peripheral blood
adds additional complexity to this systemic view of
breast cancer. Are CTCs trafficking directly from
the primary tumour or do they represent DTCs
from the bone marrow niche? Are metastatic
lesions in end organs seeded from the CTC pool
or the DTC pool? Which cohort of tumour cells in
the periphery represents the population most likely
to survive and develop into a metastatic lesion and
are thus the best cells to target with chemotherapy
or assess for prognostic significance? In some
patients, CTCs decrease in number or disappear
altogether with chemotherapy [37], but is this
sufficient to prevent recurrence?
A recent meta-review of 49 different studies collec-
tively enrolling over 6800 patients demonstrated
that, like DTCs, CTCs are a statistically significant
prognostic marker of disease-free survival in
women with early-stage breast cancer as well as
progression-free survival in women with metastatic
disease [38]. However, despite an overall prognos-
tic role for CTCs, their specific function throughout
tumour evolution remains to be determined. In
women with locally advanced disease, compared to
DTCs, peripheral blood CTCs were found with
much lower frequency (~5% of patients before
adjuvant chemotherapy compared to 21% of
patients with DTCs) [36]. Breast cancer-specific
survival was lower for patients with CTCs, but the
presence of CTCs was not as prognostically useful
as that of DTCs in this population [36]. By contrast,
in two studies of women with metastatic disease,
the presence of CTCs was found to be an inverse
predictor of progression-free survival [39, 40].
Review: Breast cancer and metastasis
Interestingly, surgical data have shown that
patients with invasive lobular carcinoma are more
likely than those with invasive ductal carcinoma to
have detectable DTCs or CTCs at the time of
surgery [21]. Together, these findings begin to
suggest that the characteristics and function of
the CTC population, in particular, may change over
time whilst also reflecting both the underlying
biology of the primary tumour and the evolution
of a systemic response in the host during tumour
progression.
Functional stratification: a new way to look at breast cancer?
As mentioned above, current breast cancer staging
is heavily dependent upon the evaluation of pathol-
ogy specimens. However, recent findings suggest
that functional classification of breast tumours
may become an important addition to risk predic-
tion and prognosis. Results from preclinical animal
models suggest that it might be possible to classify
breast cancers on a functional basis, as deter-
mined by their ability to promote outgrowth of
micrometastatic tumour populations at distant
sites. Of note, as presented at the recent Nobel
Conference on Breast Cancer, we have the ability to
use human tumour cell lines and fresh surgical
specimens in our xenograft model to test their
ability to promote systemic instigation and/or
respond to a pro-tumorigenic host systemic envi-
ronment [4–6].
The ability to determine whether or not a given
tumour has the potential to promote the dissemi-
nation of tumour cells from the primary tumour,
support the proliferation of otherwise indolent
disseminated cells or activate systemic signalling
pathways that recruit bone marrow cells to the
developing tumour stroma of a metastatic lesion
would have significant implications for treatment
strategies [41, 42]. The ability to use tumour tissue
in functional assays to predict tumour behaviour
may enable more accurate identification of patients
with a high likelihood of future relapse, thereby
allowing for potentially curative treatment during
the therapeutic window in which the disease can be
controlled.
Systemic promotion of angiogenesis
The significance of angiogenesis in supporting
tumour growth is well established [43], and the
role of the angiogenic switch in facilitating out-
growth of dormant metastases continues to be an
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Journal of Internal Medicine, 2013, 274; 113–126
 A. J. Redig & S. S. McAllister
active area of investigation [44–46]. It is noteworthy
that the vascular endothelial growth factor (VEGF)
inhibitor bevacizumab was initially approved by
the US Food and Drug Administration for the
treatment of metastatic breast cancer in 2008
[47]. However, approval was revoked in late 2011
after further studies failed to show significant
benefit on long-term survival or quality of life
[48]. The results of large-scale studies utilizing
bevacizumab to treat patients with breast cancer
were disappointing given the overwhelming labo-
ratory data pointing to a central role for angiogen-
esis in metastatic growth. Current translational
research efforts are now focusing on studies to
define more precisely the patient population in
which the benefits of anti-angiogenic therapy are
likely to outweigh the risks.
Our xenograft model may help to define a specific
context in which pro-angiogenic signalling is of
critical importance. We recently reported a model
of systemic instigation that relies on promotion of
angiogenesis, whereby cytokines secreted from
certain instigating human luminal breast tumours
lead to recruitment of pro-angiogenic platelets and
VEGF receptor 2 (VEGFR2)-positive bone marrow-
derived cells to the responding tumour with sub-
sequent stimulation of angiogenesis [6] (Fig. 1a).
Importantly, a variety of pro-angiogenic cytokines
were enriched in the platelets from mice bearing
the instigating luminal tumours relative to those
from cancer-free hosts, but VEGF was not one of
these cytokines. Our findings are consistent with
other data, for example, from mouse models using
human breast cancer xenografts showing that
oestrogen signalling helped mobilize and recruit
pro-angiogenic myeloid cells from the bone marrow
to distant tumour sites [49, 50]. The association
between increased angiogenesis in evolving metas-
tases from some subtypes of breast cancers but not
others, as well as the need to target pro-angiogenic
cytokines in addition to VEGF, may begin to
explain the less than remarkable results when
bevacizumab was introduced into clinical proto-
cols. Our findings suggest that the patients most
likely to benefit from targeted anti-angiogenic
therapy may be those with certain luminal breast
cancers whose tumours have been primed to utilize
angiogenic pathways during metastasis.
Hypoxia and the systemic environment
The role of hypoxia in driving tumour growth is well
established in a wide range of tumour types [51,
118 ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 113–126
Review: Breast cancer and metastasis
52], including breast cancer [53]. Emerging data
now suggest that hypoxia within the tumour
microenvironment may also serve as a trigger for
systemic changes in the macroenvironment that
promotes metastasis. In a recent study using a
mouse orthoptic implantation model, activation of
hypoxia-inducible factors (HIFs) within a tumour
led to HIF-dependent recruitment of mesenchymal
stem cells to the site of the primary tumour
followed by subsequent metastasis of breast cancer
cells to lymph nodes and lung [54]. Notably,
downstream growth factors activated by HIFs
facilitated not only the process of metastasis but
also the homing of necessary stromal cells to the
primary tumour [54]. In a separate study using
human breast cancer orthografts in a mouse
model, it was demonstrated that HIF-1 expression
specifically promotes metastasis through lympha-
tic channels by directly transactivating platelet-
derived growth factor B that serves as a chemotac-
tic agent for lymphatic endothelial cells [55]. This
finding raises the intriguing possibility that the
specific route of tumour cell metastasis may reflect
the milieu of the primary tumour.
Molecular changes induced by hypoxia may also be
specific to certain subtypes of breast cancer, with
resulting implications for design and utilization of
targeted therapies. In one study, expression of
CD44, cell-surface glycoprotein and a marker
associated with stem-like behaviour, was
increased under hypoxic conditions specifically in
triple-negative breast cancer cell lines [56]. Fur-
thermore, it was recently reported that the protein
SHARP1 is a crucial negative regulator of HIF-
induced migration in triple-negative breast cancer
cells and in primary tumours [57]. In a cohort of
637 breast cancer tumour samples, including 120
specimens from women with triple-negative breast
cancer, expression of the hypoxia-associated pro-
tein CAIX was significantly associated with triple-
negative tumours; 25% of triple-negative tumour
specimens expressed CAIX compared with only 7%
of the general tumour cohort [58]. In addition,
overall survival was decreased in patients express-
ing high levels of CAIX [58]. In a separate study,
expression of the toll-like receptor-9 (TLR9) was
induced by hypoxia in breast tumours yet with
different functional effect depending upon the
specific tumour subtype [59]. In particular, in ER-
positive tumours, introduction of siRNA targeting
TLR9 did not affect hypoxia-induced invasion, but
siRNA knockdown of TLR9 augmented invasion in
triple-negative tumours [59]. Together, these find-
 A. J. Redig & S. S. McAllister Review: Breast cancer and metastasis
(a)
“Instigating tumour” Pro-angiogenic
e.g., Luminal breast cancer
platelets
primary tumour or growing “Responding tumour”
metastatic lesion Circulation e.g., Indolent disseminated tumour cells

Aspirin
Normal
platelets BV

Unidentified
signal Bone marrow
Bone marrow derived cells CD24+

CD24–

VEGFR1 VEGFR2 Other BMCs

(b)
“Instigating tumour”
e.g., Triple negative breast cancer
primary tumour or growing
metastatic lesion
“Responding tumour ”
e.g., Indolent disseminated tumour cells

Osteopontin and
GRN+ bone marrow
other cytokines
s
derived cells
Bone marrow GRN Myofibroblast/CAF

Fibroblast

GRN– GRN+
Sca+ cKit– CD45+
BMCs

Fig. 1 (a) Model of systemic instigation by luminal breast cancer. Using our preclinical model, we demonstrated that in
hosts with luminal breast cancer, otherwise indolent tumour cells implanted at distant anatomical sites develop into an
actively growing tumour. The responding tumour growth is stimulated by pro-angiogenic platelets and bone marrow-derived
VEGFR2-positive cells that are recruited to the responding tumour site. As a consequence of this systemic cascade, the
responding tumour stroma becomes highly vascularized, and the tumour cells become enriched in the cell-surface marker
CD24, a ligand for platelet-specific p-selectin. Of note, this process is distinct from the growth of responding tumours in hosts
with triple-negative breast cancer (see Fig. 1b). (b) Model of systemic instigation by triple-negative breast cancer. In hosts
with triple-negative breast cancer, otherwise indolent tumour cells implanted at a distant anatomical site develop into an
actively growing tumour. Instigating triple-negative breast tumours secrete osteopontin (OPN) and other cytokines into the
circulation; this subsequently mobilizes a distinct subpopulation of bone marrow cells characterized by Sca1+/cKit /
CD45+ and granulin (GRN) expression. After trafficking to the responding tumour site, these bone marrow-derived cells
activate cancer-associated fibroblasts that support tumour growth.

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Journal of Internal Medicine, 2013, 274; 113–126
 A. J. Redig & S. S. McAllister
ings suggest a more nuanced role for hypoxia in
modulating tumour metastasis that may depend
upon the underlying features of the primary
tumour.
Systemic promotion of stromal desmoplasia
In contrast to luminal instigating tumours that
activate pro-angiogenic pathways, we found that
some triple-negative instigating tumours establish
a tumour-supportive systemic environment that
promotes stromal desmoplasia at sites of dissem-
inated responding tumours (Fig. 1b). This system-
ically induced microenvironment is enriched with
reactive myofibroblasts similar to the situation
observed in pathology specimens from patients
with invasive adenocarcinomas [60, 61]. Under
these instigating conditions, responding tumours
incorporate pro-tumorigenic host bone marrow-
derived cells into the tumour microenvironment;
however, this was not observed in indolent tumours
in control mice [5]. Notably, haematopoietic bone
marrow cells from animals bearing these instigat-
ing tumours are capable of inducing a desmoplastic
reaction when combined with responding tumour
cells, yet further experiments demonstrated that
these haematopoietic cells do not differentiate into
myofibroblasts. Instead, a subset of Sca1+/cKit /
Cd45+ bone marrow-derived cells are selectively
recruited to responder tumours where they subse-
quently activate tissue fibroblasts to form the
tumour-supportive myofibroblasts that enable
tumour growth. These haematopoietic cells act by
secreting granulin, a cytokine known to regulate
cell growth, fibrosis, inflammation and wound
healing [62]. Upon examination of tumour speci-
mens from a cohort of patients with breast cancer,
high granulin expression was correlated with triple-
negative breast cancer and reduced survival [5].
It is noteworthy that bone marrow cells from mice
bearing triple-negative instigating tumours are
rendered pro-tumorigenic even prior to their mobi-
lization into the circulation [4]. This process is
dependent upon secretion of the protein osteopon-
tin by the primary tumour [4]. Osteopontin has
been found at elevated levels in the serum of
patients with metastatic breast cancer and is
associated with both poor prognosis and increased
metastatic burden [63]. Furthermore, the kinetics
of this growth process demonstrate that systemic
instigation takes time to ‘prime’ the host environ-
ment before responding tumours start to grow,
implicating a secreted factor (or factors) in bone
120 ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 113–126
Review: Breast cancer and metastasis
marrow-derived cell recruitment. Osteopontin is
one such factor, but additional components of the
signalling pathways remain to be identified, as
osteopontin was necessary, but not sufficient for
systemic tumour promotion [4].
Integrating histopathological and functional models of tumour
classification
Overall, data concerning the functional behaviour
of different types of breast cancers indicate that the
host systemic response to certain aggressively
growing tumours creates a systemic environment
that is amenable to the outgrowth of tumour cells
that have already disseminated to distant sites but
that would otherwise remain indolent in the
absence of these systemic signals (Fig. 1b). In our
xenograft model, tumours with the ability to
promote systemic instigation and responder
tumour growth have been restricted to either
triple-negative [4, 5] or luminal [6] subtypes.
Importantly, the specific histopathological classifi-
cation of the primary instigating tumour appears to
determine the type of stromal microenvironment
that develops in the distant responding tumour, as
the same population of responding tumour cells
was used in the models of both luminal and triple-
negative instigators (Fig. 1). The response to the
presence of triple-negative instigating tumours
results in a desmoplastic stroma enriched in myo-
fibroblasts [5], whilst the response to luminal
instigating tumours results in increased angiogen-
esis within responding tumour stroma [6].
Although we identified a correlation between the
type of instigating mechanism and tumour recep-
tor status, it remains unclear whether instigating
ability is directly associated with ER/PR/Her2
expression. However, our findings suggest that
integration of data from histopathology, molecular
profiling and determination of the hallmarks of
instigating functional activity may in future
improve diagnostic and therapeutic strategies. At
present, it is clear that patients diagnosed with
metastatic disease have a significantly decreased
overall survival rate compared to those with local-
ized disease (Table 1), but to our knowledge, no
longitudinal, prospective analysis of relevant
tumour classification markers has been conducted
in a large cohort of patients with clinical
end-points, including progression-free and overall
survival.
An expanded perspective on biological functions of
certain carcinomas could help to explain certain
 A. J. Redig & S. S. McAllister
Table 1 A. Relative breast cancer survival by stage at
diagnosis [78]. B. Breast cancer survival rates in patients
with metastatic disease
A It is also clear from preclinical experiments using
Breast cancer stage Five-year relative
at diagnosis survival rate (%)
Localized 98.6
Regional 83.8
Distant 23.4
Unknown 52.4
B responding tumour begins almost immediately [4].
Metastatic breast cancer Median overall
by subtype survival (months)
Her2 positive (n = 113) [79] 38.0
Her2 negative (n = 62) [80] 12.3
Triple negative (n = 53) [81] 32.8
Data shown here are from selected studies illustrating
two significant trends in breast cancer mortality. First,
Table 1(a) shows a clear survival advantage for patients
diagnosed with localized disease with spread limited to
regional nodes. Data are derived from the SEER database
of the National Cancer Institute and are representative of
survival statistics in the developed world. However, whilst
patients with distant metastases do have dramatically
decreased overall survival compared to patients with
more limited disease, the significance of molecular clas-
sification in patients with metastatic disease remains
unclear. Table 1(b) shows data from three small cohort
studies of patients with metastatic disease enrolled in
clinical trials. Variables such as patient age, comorbidi-
ties, initial disease presentation and prior therapy have
not been adjusted between studies. Furthermore, many
clinical trials in patients with metastatic breast cancer do
not include overall survival as a study end-point, limiting
the comparisons that can be made. As functional classi-
fication of breast cancer continues to evolve, future
clinical trials in patients with metastatic disease will
probably include a more rigorous assessment of molec-
ular profiling in an attempt to link expression patterns of
target genes with clinically significant outcome measures.
epidemiological trends in clinical oncology for
which molecular mechanisms remain elusive. For
example, the observed plasticity of disseminated
indolent tumours in response to pro-tumorigenic
systemic environments suggests that the state in
which tumour cells metastasize from a primary
tumour, or the state in which they exist during a
period of dormancy in a foreign tissue, might not be
reflected in the resulting metastatic tumour once it
is detected, thus echoing a recent finding that the
Review: Breast cancer and metastasis
clinical biomarkers of breast cancer appear to be
unstable during disease progression [24].
our xenograft model that the degree of responding
tumour latency depends upon the identity of the
instigating tumour. When responders and triple-
negative instigating tumours are injected into
opposing flanks simultaneously, responding
tumours undergo a considerable period of latency
prior to their growth. Yet if the responding tumour is
injected after the triple-negative instigating tumour
has initiated growth for several weeks, growth of the
By contrast, this reduction in tumour latency is not
seen when the instigating tumour is a luminal
breast cancer [6]. It is interesting to note that the
latency and growth kinetics with which responding
tumours formed in hosts bearing different breast
cancer subtypes reflected the relative rates of
recurrence that are observed in patients with the
respective breast cancer subtype. Specifically,
patients with triple-negative disease are more likely
to experience early relapse than patients with
luminal breast cancers [64]. The characteristics of
the instigating tumour thus seem to influence not
only the tumour microenvironment in the respond-
ing tumour but also the type of systemic signalling
that is induced, perhaps providing a molecular
explanation for the clinical phenomenon in which
women diagnosed with similarly staged or treated
breast cancer can have widely divergent outcomes
with regard to future metastasis.
Our studies also expanded an analysis of systemic
perturbations and responses to other tumour
models and found that colon carcinomas can
promote growth of indolent breast tumours (and
vice versa), whilst aggressively growing breast
tumours can enhance growth of renal cell carcino-
mas [4, 6]. Although these models require much
more investigation, it is well established that
patients diagnosed with breast cancer are at
increased risk of developing a second malignancy
[65]. This trend has been attributed to either
morbidity associated with cancer therapies or to
the underlying genetic factors that led to the
development of the initial malignancy, but it is
also plausible that the systemic changes induced
by one malignancy may contribute in part to the
development of a second.
Consequently, the molecular mechanisms that
control systemic changes induced by a primary
ª 2013 The Association for the Publication of the Journal of Internal Medicine 121
Journal of Internal Medicine, 2013, 274; 113–126
 A. J. Redig & S. S. McAllister
tumour and the ability to accurately define and
characterize instigating and responding tumours
are the subject of intense investigation with the
hope that such information will allow accurate
identification of those patients who will benefit
from specific therapies designed to interrupt these
systemic lines of communication.
Therapeutic applications
Prognosis and diagnosis
Consideration of breast cancer as a systemic
disease leads to a number of possible prognostic
applications. As discussed above, as more becomes
known about primary tumour ‘instigating’ behav-
iour and the hallmarks that can be used to define
tumours as instigators, we might be in a position to
determine whether a given primary tumour is likely
to support the outgrowth of DTCs. Likewise, as
more is learned about disseminated cells that
respond to specific systemic cues, we might be in
a better position to differentiate life threatening
from insignificant DTCs. Such analyses will rely on
improved methods for detecting and analysing
DTCs and CTCs. As technology continues to
develop, it appears that not all methods of assess-
ing CTCs are equally effective. In a prospective trial
of 254 patients with breast cancer, blood samples
were evaluated using two commercially available
methods of detection. Using one assay, the
presence of CTCs was a statistically significant
prognostic marker for overall survival and progres-
sion-free survival, but using the other assay, this
association was not noted [66]. It is also important
to note that whilst the ability to capture DTCs/
CTCs has markedly improved, it remains techni-
cally challenging to analyse these cells once they
are isolated; this step will be critical to incorporate
CTC/DTC biology into treatment strategies.
The use of CTCs/DTCs in predictive algorithms in
the nonresearch clinical setting will also require
additional clarification of exactly which features in
which population of cells should be analysed.
Both CTCs and DTCs can have prognostic signif-
icance in a range of patients, but a clinical
protocol adapted for the use of CTCs obtained
via peripheral blood collection would be more
broadly applicable than the use of DTCs collected
through bone marrow aspiration. Furthermore, it
will also be important to determine whether mon-
itoring simply for the presence or absence of CTCs
is sufficient, or whether specific molecular analy-
sis of this population is more relevant for choosing
122 ª 2013 The Association for the Publication of the Journal of Internal Medicine
Journal of Internal Medicine, 2013, 274; 113–126
Review: Breast cancer and metastasis
therapy. For example, new methodology now
allows isolation of single CTCs in the laboratory
setting with high-fidelity whole-genome amplifica-
tion to search for the epidermal growth factor
receptor (EGFR) copy number amplification [67]. A
gene amplification study revealed enrichment of
genes previously associated with metastasis as
well as a cohort of genes previously linked to the
epithelial mesenchymal transition [68]. Successful
in situ hybridization using CTCs has also been
reported [39]. Currently, gene expression analysis
of primary tumours can help physicians and
patients make informed decisions about the ben-
efits of adjuvant chemotherapy. The next step may
be expanding tumour profiling to the CTC pool,
with a particular emphasis on expression of
markers associated with metastasis. The signifi-
cance of metastatic disease in breast cancer
survival has long been recognized, with the pres-
ence or absence of metastasis, a critical compo-
nent of standard staging algorithms. However,
current staging only enables assessment of metas-
tasis-positive or metastasis-negative clinical pre-
sentation, a binary model that reflects diagnostic
capabilities of several decades ago, rather than
emerging methods to evaluate DTCs or CTCs. With
further technological advances, assessment of
tumour cells that have not yet formed an overt
metastatic lesion may one day be part of staging
algorithms.
Finally, beyond assessment of the CTC/DTC pool,
it may in future be possible to predict the meta-
static potential of a tumour by evaluating either the
serum cytokine profile or, possibly, the presence of
circulating pro-tumorigenic haematopoietic cells in
peripheral blood or bone marrow samples. In
animal models, key steps in the process responsi-
ble for outgrowth of otherwise dormant micromet-
astatic lesions include signalling from the primary
tumour to the bone marrow compartment, followed
by mobilization of pro-tumorigenic haematopoietic
cells to the site of metastatic lesions. Further
elucidation of critical cytokine mediators (one
example of which is osteopontin) or markers of
mobilized haematopoietic cells from the bone mar-
row would allow assessment of these markers
during a diagnostic evaluation with potential
predictive value for determining metastatic risk.
Treatment strategies
The potential therapeutic applications arising
from consideration of breast cancer as a systemic
 A. J. Redig & S. S. McAllister
process are also extensive. First, data demonstrat-
ing the evolution of breast cancer biomarkers
during the process of metastasis suggest that a
more fluid use of drugs targeting the oestrogen
receptor or HER2 may lead to clinical benefit. In an
albeit small cohort of 10 patients with HER2-
positive DTCs detected in bone marrow, the use
of trastuzumab was nonetheless able to clear the
marrow in most patients (8 of 10) [69]. Notably, the
two patients who ultimately presented with meta-
static disease were the patients in whom the
marrow failed to clear. Amongst patients with
metastatic lesions or detectable CTCs/DTCs that
do not match the biomarkers of the primary
tumour, treating the ER or Her2 status of the
metastatic cells may have the potential to improve
disease outcomes.
Second, in view of the finding that a primary
tumour can mobilize pro-tumorigenic bone marrow
cells that aid the outgrowth of disseminated
tumours, specific therapies targeting these long-
range systemic lines of molecular communication
may have clinical significance. In a recent study of
systemic instigation by luminal carcinomas, we
found that the pro-angiogenic role of mobilized
platelets was abrogated by aspirin treatment [6].
There is currently debate regarding whether or not
aspirin is protective against breast cancer. Several
analyses of cohort or case–control studies have
reported modest decreases in breast cancer risk
amongst aspirin users [70, 71], whereas a recent
retrospective analysis of over 80 000 patients
demonstrated no significant association with post-
menopausal breast cancer [72]. However, these
data generally come from vascular biology studies
that are neither powered nor designed to specifi-
cally address the subtleties of a model of breast
cancer in which specific signalling pathways
unique to the biology of a primary tumour and/or
its metastases exert systemic changes that lead to
the mobilization of circulating haematopoietic
cells. Preclinical studies suggest that, in the right
cohort of patients, aspirin therapy may play a
critical role in inhibiting a signalling pathway that
drives metastasis.
However, our preclinical studies suggest that the
secreted proteins osteopontin and granulin may be
superior anti-metastasis targets in some patients
[4, 5]. As previously discussed, certain triple-neg-
ative tumours promote the outgrowth of otherwise
indolent disseminated tumours by secreting osteo-
pontin and inducing mobilization of granulin-
Review: Breast cancer and metastasis
positive haematopoietic cells from the bone mar-
row. Interrupting osteopontin signalling or block-
ing granulin-positive cell mobilization may thus be
effective in preventing the growth of otherwise
indolent tumours in this setting. It is particularly
interesting that elevated plasma levels of osteopon-
tin have been noted in patients with various solid
tumours, thus indicating that osteopontin may be
a potential cancer biomarker or therapeutic target
[73–75]. In a preclinical model of nonsmall-cell
lung cancer, anti-osteopontin antibodies were
shown to inhibit the growth of further lesions in
the lung from the initial primary tumour [76].
Granulin has also been implicated in promoting
tumour progression in preclinical models, and in
one study, it was shown that inhibition of granulin
prevented hepatocellular carcinoma metastasis
[77].
Conclusions
Models of breast cancer as a systemic disease
suggest novel ways to target the process of metas-
tasis, which is responsible for the majority of
treatment failures. However, the complexity inher-
ent in the systemic changes induced by a primary
tumour also highlights a key consideration in
future efforts to develop targeted therapy: selecting
the right systemic therapies for the right patients
based on functional assessment of their primary
disease rather than a static window of tumour
pathology. It is clear that the success of targeting
the processes that lead to clinical metastasis
depends upon appropriate tumour stratification.
Future targeting of the metastatic process is likely
to involve two critical steps. First, it will be
important to identify additional signalling path-
ways that facilitate metastatic tumour growth to
utilize targeted therapies to block progression.
Second, it will be equally important to develop
efficient and reproducible ways to screen individ-
ual patients for the metastasis-promoting path-
ways used by their specific tumours. Basic
experimental and clinical studies have lead to a
large knowledge base regarding the underlying
biology and clinical presentation of metastatic
breast cancer. The next steps will involve integrat-
ing information about the systemic nature of
breast cancer signalling into ongoing development
of individualized therapy.
Conflict of interest statement
No conflicts of interest to declare.
ª 2013 The Association for the Publication of the Journal of Internal Medicine 123
Journal of Internal Medicine, 2013, 274; 113–126
 A. J. Redig & S. S. McAllister
Acknowledgements
SSM is the recipient of a Harvard Stem Cell
Institute Seed Grant, NIH RO1 CA166284-01,
and a Research Scholar Grant from the American
Cancer Society.
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Correspondence: Sandra S. McAllister PhD, Harvard Medical
School, Brigham & Women’s Hospital, 1 Blackfan Circle, Karp
5-214, Boston, MA 02115, USA.
(fax: 617-355-9093; e-mail: smcallister1@partners.org).