Active Roles of Tumor Stroma in Breast Cancer Metastasis

Department of Chemistry and Biochemistry and Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4390, USA.
International journal of breast cancer 02/2012; 2012(13):574025. DOI: 10.1155/2012/574025
Source: PubMed
ABSTRACT
Metastasis is the major cause of death for breast cancer patients. Tumors are heterogenous cellular entities composed of cancer cells and cells of the microenvironment in which they reside. A reciprocal dynamic interaction occurs between the tumor cells and their surrounding stroma under physiological and pathological conditions. This tumor-host communication interface mediates the escape of tumor cells at the primary site, survival of circulating cancer cells in the vasculature, and growth of metastatic cancer at secondary site. Each step of the metastatic process is accompanied by recruitment of stromal cells from the microenvironment and production of unique array of growth factors and chemokines. Stromal microenvironment may play active roles in breast cancer metastasis. Elucidating the types of cells recruited and signal pathways involved in the crosstalk between tumor cells and stromal cells will help identify novel strategies for cotargeting cancer cells and tumor stromal cells to suppress metastasis and improve patient outcome.

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International Journal of Breast Cancer
Volume 2012, Article ID 574025, 10 pages
doi:10.1155/2012/574025
Review A rticle
Active Roles of Tumor Stroma in Breast Cancer Metastasis
Zahraa I. Khamis,
1
Ziad J. Sahab,
2
and Qing-Xiang Amy Sang
1
1
Department of Chemistry and Biochemistry and Institute of Molecular Biophysics, Florida State University,
Tallahassee, FL 32306-4390, USA
2
Department of Oncology and Lombardi Comprehensive Cancer Center, Georgetown University Medical Center,
Washington, DC 20007, USA
Correspondence should be addressed to Qing-Xiang Amy Sang, qxsang@chem.fsu.edu
Received 15 August 2011; Revised 4 November 2011; Accepted 11 November 2011
Academic Editor: Andra R. Frost
Copyright © 2012 Zahraa I. Khamis et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Metastasis is the major cause of death for breast cancer patients. Tumors are heterogenous cellular entities composed of cancer
cells and cells of the microenvironment in which they reside. A reciprocal dynamic interaction occurs between the tumor cells and
their surrounding stroma under physiological and pathological conditions. This tumor-host communication interface mediates
the escape of tumor cells at the primary site, survival of circulating cancer cells in the vasculature, and growth of metastatic cancer
at secondary site. Each step of the metastatic process is accompanied by recruitment of stromal cells from the microenvironment
and production of unique ar ray of growth factors and chemokines. Stromal microenvironment may play active roles in breast
cancer metastasis. Elucidating the types of cells recruited and signal pathways involved in the crosstalk between tumor cells and
stromal cells will help identify novel strategies for cotargeting cancer cells and tumor stromal cells to suppress metastasis and
improve patient outcome.
1. Introduction
Breast cancer is the most common malignancy and the
second major cause of mortality and morbidity in Western
women [1]. The systemic outgrowth and spread of the cancer
cells through a process known as metastasis is the main cause
of deaths in these patients. Recently, disease-related mortality
and metastasis have declined as a result of early diagnosis and
application of adjuvant therapy. Mammographic screening,
surgery, radiotherapy, chemotherapy, antibody therapy, and
endocrine therapy facilitate the suppression of the metastatic
dissemination of local tumor [2]. However, these treatments
target the tumor cells and disregard the auxiliary cells present
in the surrounding microenvironment that is also referred to
as the stromal cells. These auxiliary cells, including myoep-
ithelial cells, fibroblasts, myofibroblasts, endothelial cells, in-
flammatory cells, and bone-marrow-derived cells (BMDCs)
such as macrophages, mast cells, neutrophils, and lympho-
cytes, are widely recognized to collaborate with cancerous
cells and other host cells to create a tumor-permissive micro-
environment capable of providing continuous support for
tumor growth, progression, angiogenesis, invasion, and me-
tastasis [3, 4].
Metastasis is the systemic dissemination of tumor cells
at sites distinct from the primary lesion. It is a multistep
process that involves detachment of cells from the primary
tumor, followed by survival in the blood vessels or lymphatic
system and finally development of secondary tumor. It is
a poorly understood aspect of carcinogenesis that requires
the clarification of the underlying cellular and molecular
events that control the metastatic cascade from onset to
colonization [5]. It is undisputed that metastasis of tumor
cells is mediated by the reciprocal inter play between tumor
cells and stromal cells and the extracellular mat rix (ECM).
In this paper, we discuss the dierent steps of breast
cancer progression and delineate the importance of the
myoepithelial cell layer disruption for invasion of tumor
cells. We also address the tumor promoting eect of the
stromal cells in each step of the metastatic cascade of breast
cancer.
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2 International Journal of Breast Cancer
Normal breast Ductal carcinoma in situ Invasive ductal carcinoma Advanced breast cancer
Epithelial cells
Myoepithelial cells
Basement membrane
BMDCs
Macrophages
Fibroblasts
Myofibroblasts
Endothelium
Figure 1: Schematic presentation of breast cancer progression accompanied with stromal cells. Normal breast duct is composed of a layer
of epithelial cells and a layer of myoepithelial cells separated from the stroma by a basement membrane. Stromal cells include fibroblasts,
BMDCs, endothelial cells, and other cells. Ductal carcinoma in situ (DCIS) is associated with luminal epithelial cells proliferation, and
recruitment and expansion of stromal cells. In invasive ductal carcinoma, the myoepithelial cell layer is degraded with the underlying
basement membrane and cancerous cells invade the surrounding microenvironment. Advanced breast cancer is associated with complete
loss of myoepithelial cell layer and basement membrane, invasion of epithelial cells, proliferation of stromal cells, and angiogenesis.
2. Evolution of Breast Cancer
The development of breast cancer involves the progression
via a series of intermediate hyperplastic lesions with and
without atypia (atypical ductal hyperplasia, atypical lobular
hyperplasia, and usual ductal hyperplasia) followed by
subsequent evolution into in situ carcinoma, for example,
ductal carcinoma in situ (DCIS) and lobular carcinoma
in situ (LCIS), invasive carcinomas, and metastatic cancers
(Figure 1)[69]. In atypical hyperplasia, the breast cells are
abnormal in number, size, shape, appearance, and growth
pattern that may be seen as an excessive growth of cells of the
ducts (atypical ductal hyperplasia) or the cells of the lobules
(atypical lobular hyperplasia). In usual ductal hyperplasia,
the breast tissue has an increased number of benig n cells
within the duct. DCIS is thought to be a precursor of
invasive ductal carcinoma, in which tumor cells are confined
to the lumen of the mammar y duct. Lobular carcinoma
in situ consists of a noninvasive increase in the cells of
the milk-producing lobules of the breast. Normal breast
ducts are composed of a layer of epithelial cells physically
separated f rom the normal microenvironment by a basement
membrane and myoepithelial cell layer [10, 11]. In situ car-
cinoma is characterized by intact myoepithelial cell layer and
basement membrane, and proliferation of epithelial cells [10,
11]. When the breast tissue undergoes focal disruption of the
myoepithelial cell layer a nd degradation of the underlying
basement membrane, tumor cells i nvade surrounding tissues
and migrate to distant organs, eventually leading to metasta-
sis [1012]. Despite the dramatic improvement in our ability
to detect carcinomas in situ (DCIS), our understanding of
the pathophysiology of this disease and factors involved in its
progression to invasive carcinoma lags far behind.
3. Myoepithelial Cells at a Glance
The normal breast tissue is comprised of two major compart-
ments, the epithelium and the stroma. Myoepithelial cells
together with luminal cells constitute the epithelium of the
ducts and of the lobule of the mammary g l and. The anatomi-
cal position of myoepithelial cells between the stroma and the
luminal epithelial cells from which cancer ar ises facilitates
proper communication between both compartments. They
express a number of tumor suppressor proteins (maspin),
ECM structural proteins (fibronectin, collagen), proteinase
inhibitors (tissue inhibitor of metallopreoteinase-1, TIMP-
1), and angiogenic inhibitors (thrombospondin-1) [13, 14].
They also downregulate the expression of matrix metal-
loproteinases (MMPs) in fibroblasts and tumor cells [15],
contribute significantly to basement membrane production,
and accumulate ECM ra ther than degrade it [14, 16, 17]. The
aforementioned functions suggest that the normal myoep-
ithelial cell layer is a natural paracrine tumor suppressor that
physically and functionally inhibits tumor growth, invasion,
and angiogenesis. This tumor suppressive phenotype was
identified based on the ability of myoepithelial cells to
secrete paracrine factors (such as bFGF, TGF-α,andIL-
6) that inhibit the growth and invasion of breast cancer
cells in coculture assays in vitro [1820]. Collective evidence
suggests that myoepithelial cells also function as autocrine
tumor suppressor that is supported by their resistance to
transformation and their tendency to transform to tumors
of low malignancy [19, 21]. Due to their tumor suppressor
potential, myoepithelial cells have been referred to as the
“Cinderella of the breast [22]. Myoepithelial cells surround
both normal ducts and precancerous lesions of the breast,
for example, DCIS. However, DCIS myoepithelial cells dier
from their normal counterparts in their ability to polarize
luminal cells in three-dimensional collagen assays [16, 23],
which indicates that tumor-derived myoepithelial cells are
unable to transmit the necessary and correct signals to lumi-
nal cells. Moreover, myoepithelial cells isolated from normal
tissue have distinct gene expression pattern as compared to
DCIS myoepithelial cells. The former express high levels of
laminin, tenascin, thrombospondin, cytokeratins, oxytocin
receptor and tropomyosin, whereas DCIS myoepithelial cells
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International Journal of Breast Cancer 3
show overexpression of proteases (cathepsins, MMP-2, and
PRSS11), protease inhibitors (thrombospondin 2, SERP-
ING1,cystatinC,andTIMP3),andcollagens[24]. A com-
mon diagnostic feature of breast cancer progression from in
situ to invasive tumor is the aberration of the fully dierenti-
ated myoepithelial cell layer suggesting that dissolution of the
myoepithelial cell layer is an absolute prerequisite for tumor
invasion. However, it is unknown what mechanisms lead to
focal myoepithelial cell layer disruption and its contr ibution
to tumor progression. Studies by Man and Sang revealed that
focal myoepithelial cell layer disruptions are associated with
higher leukocyte infiltration supporting the release model
which is proposed by Polyak el al. to describe the role of
stromal and myoepithelial cells in invasion onset [11].
4. Microenvironmental Influences on Breast
Cancer Development
The tumor microenvironment or the stroma is composed
of extracellular and cellular tissue network that surrounds
and interacts with tumor cells. The cellular part includes
fibroblasts, myofibroblasts, endothelial cells, adipocytes, and
various immune cells [25, 26]. These cells are surrounded by
an ECM that is a dynamic three-dimensional structure com-
posed of many components including collagens, laminin,
and fibronectin. The ECM is also a rich source of matrix
metalloproteinases (MMPs) and soluble growth factors that
aect neoplastic dissemination [27]. A specialized ECM,
called basement membrane, is made of several glycoproteins
and proteoglycans and separates the epithelial and endothe-
lial cell layers from the surrounding microenvironment [28].
A well-organized basement membrane acts as a gatekeeper of
invasive phenotype providing a physical support, a signaling
intermediate between dierent compar tments and a regu-
lator of cell behavior. During tumor development, cancer
cells become in direct contact with a remodeled stroma
that was long considered to be a passive responder to the
malignant transformation [4]. The significance of the tumor
microenvironment as an active contributor in promoting
and initiating breast cancer development is proposed. The
dierence in molecular signatures between stromal cells from
tumors and normal tissues bear witness that stromal cells
providecuesfortumorigenesis.
In contrast to normal fibroblasts, cancer-associated fibro-
blasts (CAFs) [29] enhance tumor growth and metastasis
through the production of growth factors and ECM proteins
and modulating immune polarization [30]. The y also have
dierent gene signatures related to paracrine signaling, trans-
criptional regulation, extracellular matrix, cell-cell interac-
tion and cell adhesion/migration such as wnt1 inducible
signaling pathway protein 1 (WISP1), kruppel like factor 4
(KLF4), TGFβ2, fibulin1 (FBLN1), plasminogen activator
inhibitor 2 (PAI2), and tissue plasminogen activator (PLAT)
[29, 31]. Recently, Tyan et al. found that human breast
cancer cells dramatically aected surrounding fibroblasts.
They induced hepatocyte growth factor (HGF) production
by fibroblasts to support their own growth and progression
[32]. It is noteworthy to mention that a model which
delineates the role of tumor microenvironment in breast
cancer initiation and progression has been proposed. This
model or Reverse Warburg eect suggested that tumor
cells can induce an oxidative stress on neighboring fibro-
blasts which promotes stromal autophagy associated with
Caveolin-1 loss and elevated cytokine production. This, in
turn, results in production of nutrients that can nourish
anabolic tumor cells [33, 34].
Tumor stroma also includes myofibroblasts, which are
activated fibroblasts with α-smooth muscle actin (α-SMA)
expression. In human tissue sections with invasive breast
cancer, higher proportion of myofibroblasts were associated
with higher-grade upregulation of Ki-67, VEGF, and bFGF,
and shorter overall survival and relapse-free survival [35].
Tumor invasion and angiogenesis was shown to be promoted
by α-SMA-positive myofibroblasts and not by α-SMA-
negative fibroblasts [36]. Stromal myofibroblasts promote
tumorigenesis of oral squamous cell carcinomas, for exam-
ple, by secreting activin A [37]. In the tumor-stroma inter-
active microenvironment transforming growth factor-beta 1
(TGF-beta 1) promotes stromal fibroblast-to-myofibroblast
transdierentiation by modulating phenotypic and func-
tional genes. For example, MiR-21 was recently shown to
participate in TGF- b1-induced myofibroblast transdieren-
tiation by targeting and downregulating programmed cell
death 4 (PDCD4) gene [38, 39]. Furthermore, cancer-cell-
derived TGF-b release proangiogenic vascular endothelial
growth factor A (VEGFA) from the myofibroblasts in eso-
phageal squamous cell to regulate angiogenesis [40].
Implicated with angiogenesis, endothelial cells are re-
cruited to the tumor microenvironment where they enhance
neovascularization and metastasis. Adipose tissue, composed
of adipocytes, has long been associated with cancer devel-
opment. Coculture of adipocytes with cancer cells resulted
in increased invasiveness of cancer cells and modified phen-
otype of the adipocytes characterized by lower lipid accu-
mulation, decreased expression of adipocyte markers, and
overexpression of proteases (MMP-11) and proinflammatory
cytokines (IL-6, IL-1β)[
41]. Il-6 depletion from adipocytes
inhibited the invasion and migration of breast tumor cells
[42].
The stromal compar tment also contains various bone-
marrow-derived cells (BMDCs) such as macrophages, mast
cells, neutrophils, and lymphocytes that are recruited by
the primary tumor cells to increase tumor cell migration,
angiogenesis, and invasion [3]. At sites of focal disruptions
of the myoepithelial cell layer in breast tissues, immune
infiltrates were obser ved at the invasive front suggesting a
potential role of these cells in malignancy and subsequent
metastatic spread [10, 11].
To metastasize, tumor cells are required to escape the
primary tumor, intravasate the blood stream or lymphatic
circulation, survive in the vasculature, extrude from the
blood vessels or lymphatic system, arrest at distant sites, and
develop into secondary mass. At each step of the metastatic
cascade, stromal cells appear to be crucial players in the
transition from benig n to invasive and finally metastatic
disease (Figure 2)[5, 43]. The metastatic cascade is a quite
inecient process meaning that failure to complete any
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4 International Journal of Breast Cancer
(1)
(2)
(3)
(4)
(5)
(6)
?
Figure 2: St romal cells involved in metastatic cascade. (1) Myofi-
broblasts, fibroblasts, and macrophages and other BMDCs play a
major role in promoting primary tumor growth. (2) Intravasation
is enhanced by the paracrine interactions between tumor cells and
macrophages. (3) The fusion of tumor cells and macrophages is
questionable (?) and may promote the survival of the tumor cells in
vasculature. (4) During extravasation, direct interaction of tumor
cells and macrophages enhance tumor cell egress of the vessels. (5)
BMDCs and myofibroblasts stimulate tumor cell metastatic dissem-
ination. (6) The recruitment of endothelial cells, myofibroblasts,
and BMDCs to the tumor site increases vascularization.
step will quench the whole process. Until now, it is unclear
which step is the key rate-limiting one that contributes
to the ineciency of the metastatic lesion. Metastatic
colonization has been suggested as a major rate-limiting
step because intravenous injection of cancer cells resulted
in about 90% arrest and extravasation with only 0.1% of
cells growing at the secondary site [44, 45]. Extravasation
was also thought to be a key r ate-limiting step in metastasis
with highly metastatic cells extravasating faster than poorly
metastatic ones. However, a study by Koop et al. showed
that extravasation is independent of the metastatic ability
and highly metastatic ras-transformed cells extravasate at the
same rate of control fibroblasts [46]. Similar to extravasation,
intravasation was proposed to be a major rate-limiting step.
In vivo data showed that intravasation can be a major rate-
limiting step in the metastatic cascade. Wyckoet al. showed
that metastatic cells exhibited a faster entry into the vascu-
lature than poorly metastatic cells. Moreover, Zijlstra et al.
quantitatively evaluated the rate limiting steps in HEp-3 and
HT-1080 human tumorigenic cells. The authors found that
HEp-3 cells had higher metastatic rate than HT-1080 cells
due to the lower eciency of the latter cells in intravasation
and metastatic colonization [47]. Collectively, intravasation
and growth at secondary sites represent major rate-limiting
steps in the metastatic cascade. However, the rate-limiting
step may vary depending on the tumor type [45].
4.1. Primary Tumor Growth. Under normal physiological
conditions, the surrounding microenvironment imposes
proper tissue architecture maintained by basement mem-
brane alignment and intercellular communication [3]. This
interplay between epithelial cells and surrounding stroma
maintains organ homeostasis that serves as a protective
constraint against malignant transformation. During tumor
development, cancerous cells circumvent the normal con-
trols regulating the activity of ECM proteases. In response to
these proteolytic enzy mes, the basement membrane under-
goes gradual degradation and structural changes causing
violation of normal tissue boundaries and conduits for
malignant cell egress. To invade, tumor cells should lose
cell-cell and cell-ECM interactions mediated by integrins
and cadherins. Invasion is also accompanied by proteolytic
degradation of surrounding tissue mediated by proteases,
motility of tumor cells mediated by chemokines and growth
factors, and recruitment of stromal cells.
Fibroblasts play an important role in cancer progression.
They are primarily responsible for the synthesis, deposition,
and remodeling of the basement membrane and ECM
through the production of collagen (I, III, IV, and V),
fibronectin, and laminin. They are also an important source
of paracrine growth factors (HGF, EGF, FGF2, and TGFβ),
proteolytic enzymes (MMP-1, MMP-7), and cytokines
(IL6, CXCL12) that can aect cell proliferation, survival,
morphology, and death [48, 49]. During tumorigenesis,
fibroblasts maintain an activated phenotype character ized
by the expression of α-smooth muscle actin (α-SMA) and
are referred to as carcinoma-associated fibroblasts (CAFs)
or myofibroblasts [49]. In breast carcinomas, about 80% of
stromal fibroblasts acquired the CAF-activated phenotype
[48]. During tumor development, CAFs exhibit a higher pro-
liferation index and become the predominant cell population
in the stroma. Once the basement membrane is degraded,
CAFs are accumulated causing expansion of tumor stroma
and increased deposition of ECM through expression of
stress fibers and α-SMA. This phenot ype is termed desmo-
plasia and is associated with recruitment of inflammatory
cells and activation of angiogenesis [50]. Several studies
demonstrated a direct involvement of fibroblasts in initiation
of cancer. In a xenograft mouse model, Kuperwasser et
al. showed that the upregulation of transforming growth
factor-β (TGFβ) or hepatocyte growth factor (HGF) in
mouse fibroblasts stimulated the initiation of benign and
malignant lesions in the breast epithelium [51]. In addition,
a gene expression profiling study of all cell types in normal
and neoplastic breast demonstrated that overexpression of
CXCL12 in myofibroblasts was correlated with epithelial cell
proliferation and invasion [23]. Another study of xenograft
mouse model coinjected with MCF-7 breast cancer cells and
CAFs or normal fibroblasts showed that xenografts infused
with CAFs had enhanced g rowth than xenografts injected
with normal fibroblasts [52]. To reconcile, these data reveal
the major role of CAFs in tumor initiation and progression.
An initial reaction of the host to the tumor development
is the recruitment of leukocytes and subsequent local inflam-
mation [53]. As a physiological response to tissue injur y,
inflammatory cells are recruited to the injured site to support
tissue repair and remodeling through the production of
growth factors and cytokines such as TNF-α, CCL2, CXCL8,
CCL5, TGF-β and so forth. In normal conditions, the
inflammatory response is resolved once the tissue is repaired
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International Journal of Breast Cancer 5
[54, 55]. However, the tight regulation of inflammation is
overridden during malignant transformation recalling the
historic view of tumors as “wounds that never heal” [56].
Persistent inflammatory response characterized by activated
leukocytes and secretion of several cytokines and chemokines
(including tumor necrosis factor (TNF), interleukins, and
interferons) elaborates the formation of tumor-promoting
microenvironment. Such protumor stroma is incentive not
only for primary tumor development but also for metastatic
dissemination into systemic circulation.
Neoplastic transformation is regulated by dynamic reci-
procity between epithelial cells, activated stromal cells and
ECM components. If the changes in the microenvironment
occur prior or concomitant with epithelial cell changes is
still debatable. Recently, a gene expression profiling study
on laser captured breast stroma and epithelia showed 90%
change in the stromal gene expression at the transition from
normal to DCIS, and only 10% stromal alterations in DCIS
compared to invasive disease [57, 58]. Another study used
mRNA in situ hybridization of breast tissue with dierent
stages of cancer to examine the expression of angiogenic
factors and stromal components. A similar expression profile
was observed in carcinoma in situ, invasive cancer, and
metastatic disease [59]. These studies suggest that stromal
changes induced by the emerging epithelial lesions precede
invasion and that cancer cells invade into an abnormal breast
microenvironment with growth-promoting eects.
4.2. Intravasation. Intravasation or penetration of tumor
cells into the vasculature involves the movement of cancer
cells through the ECM, the basement membrane, and finally
through the endothelium of the blood vessel or lymphatic
duct. The underlying molecular mechanisms governing
intravasation are not clear as all studies have focused on
later steps in the metastatic cascade. Detailed evaluation of
all steps in tumor metastasis is crucial to understand the
cellular mechanisms controlling neoplastic dissemination.
In a murine breast cancer model, Yang et al. found the
transcr iption factor, Twist, as a key regulator of metastasis
[60]. It augments epithelial-to-mesenchymal transitions and
promotes the rate of hematogenous intravasation. Another
possible mechanism that executes the migration of tumor
cells across the vessel wall was shown by intravital imag-
ing studies of experimental mammary carcinomas [61,
62]. These studies showed that c arcinoma cells intravasate
through the blood vessels due to chemoattractive gradients
generated by perivascular macrophages that are recruited
by the tumor cells to the injured site [61]. In breast carci-
nomas, macrophages and cancerous cells form a paracrine
loop involving epidermal growth factor (EGF) and colony
stimulating factor-1 (CSF-1) to augment chemotaxis and
intravasation [63]. EGF produced by macrophages promotes
migration of neoplastic cells into hematogenous vasculature
through its interaction with EGF receptor expressed on
breast cancer cells. Tumor cells, in turn, express CSF-1
which acts as a potent chemoattractant for CSF-1 receptor
positive macrophages [64]. This crosstalk lends credence to
the collaborative work between tumor microenvironment
and neoplastic cells at the site of intravasation.
4.3. Survival in Vasculature. Once malignant cells have
invaded the ang iogenic vasculature, they are subject to harsh
microenvironment characterized by hemodynamic shear
forces, surveillance of immune cells, and lack of substratum
[43]. To bypass these perils, tumor cells use platelets as
a shield. Through their tissue factor, tumor cells bind
coagulation factors (VIIA and X) on the platelets creating an
embolus that arrests in the capillaries [65, 66]. These aggre-
gates protect the cancerous cells from immune-cell-mediated
lysis and decrease the shear forces of the blood circulation,
thus increasing their survival, arrest, and extravasation
[67]. Whether macrophages can infer protective eects on
neoplastic cells in the bloodstream as they do during invasion
and intravasation is not yet known. To explain the metastatic
phenotype, Pawelek and Chakraborty proposed the fusion of
macrophages or other BMDCs with cancer cells forming a
hybrid capable of surviving in the circulation and homing to
secondary sites [68]. Should the fusion theory be accepted in
human cancers, BMDCs would have a beneficial role in the
survival of tumor cells in the vasculature.
4.4. Extravasation. After survival and arrest in the circula-
tion, tumor cells must escape out of the blood and lymphatic
vessels in a process known as extravasation. To do so, tumor
cells induce disruptions in the endothelial junctions that
allow tumor cells to bind to the subendothelial ECM and
extrude into target organs. Vascular permeability is mediated
by activated Src kinases in endothelial cells, which once
exposed to vascular endothelial growth factor (VEGF) from
tumor cells promotes endothelial retraction, resulting in
movement of cancer cells towards surrounding tissues [69].
To identify the role of macrophages in metastasis, Qian et
al. used an animal model of breast cancer metastasis and an
intact ex vivo lung imaging system to show that modified host
macrophages are required for proper metastatic seeding and
growth [70]. The authors found that macrophage ablation
dramatically decreased the number of tumor cells observable
in the lungs [70]. Moreover, lung-resident macrophages were
visualized to physically interact with tumor cells as soon as
they extravasate through the vessel walls, thus promoting
the rate of extravasation and metastatic dissemination [70].
Immunophenotyping of metastasis-associated macrophages
showed distinct profile from lung resident macrophages. The
prometastatic macrophages are characterized by cell surface
expression of CSF1R, CD11b, F4/80, with high levels of
CCR2, CX3CR1, and VEGFR, a bsence of Gr1 and low CD11c
[70, 71
].
4.5. Metastatic Site. Metastasis is regulated not only by chan-
ges in tumor cells but also by reciprocal interactions with
the surrounding microenvironment. In his “seed and soil”
hypothesis, Paget proposed that tumor cells or “seeds
can only colonize microenvironments or soils” that are
compatible with their growths [72]. For example, breast
cancers metastasize to lungs, bone, liver, and brain, whereas
advanced prostate cancers colonize the bone as the pre-
dominant site [45]. Since the circulatory patterns provide
only partial explanation for the tissue tropism aspect of
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6 International Journal of Breast Cancer
metastasis, several molecular and cellular mechanisms have
been proposed. An emerging paradigm suggests that primary
tumor cells may secrete fac tors capable of inducing a fer-
tile microenvironment, termed premetastatic niches, that fa-
vors the seeding and proliferation of metastatic cells at
unique sites [73]. For example, (ADAMTS1) and matrix me-
talloproteinase-1 (MMP1) participate in a paracrine signal-
ing cascade that includes the release of metastasis membrane-
bound epidermal-growth-factor (EGF-) like growth factors,
amphiregulin (AREG), heparin-binding EGF (HB-EGF),
and transforming growth factor alpha (TGF alpha) from
tumor cells resulting in a downregulation of osteoprotegerin
expression in osteoblasts and therefore modulating brain
microenvironment in favor of osteoclastogenesis and bone
metastasis [74]. Recent work have identified the following
mediators of extravasation and brain colonization: the cy-
clooxygenase COX2, the epidermal growth f actor receptor
(EGFR) ligand, HBEGF, and the alpha 2,6-sialyltransferase,
ST6GALNAC5, which were found to enhance breast cancer
passage through the blood brain barrier and to facilitate their
adhesion to brain endothelial cells [75]. EGFR ligands and
COX2 are also linked to breast cancer infiltration of the lungs
[76, 77]. Recently, breast c ancer cells infiltrating the lungs
were shown to support their own metastasis-initiating ability
by expressing tenascin C (TNC). TNC is an extracellular
matrix protein of stem cell niches that promotes the survival
and outgrowth of pulmonary micrometastases via upregu-
lation of stem cell sign aling components, musashi homolog
1 (MSI1), and leucine-rich repeat-containing G protein-
coupled receptor 5 ( LGR5) u ntil the tumor stroma takes over
as a source of TNC [78]. Through the production of growth
factors (VEGFA) and chemokines (S100A8, S100A9), tumor
cells induce the recruitment of BMDCs, and endothelial
progenitor cells to the premetastatic niche [79]. The bone
marrow progenitors are VEGF receptor-1 (VEGFR1) positive
and can migrate and proliferate in response to tumor-derived
VEGF [79]. These VEGFR1
+
cells also express integrin
VLA-4 and tend to form clusters induced by integrin-
fibronectin interactions, the latter of which is synthesized
by resident fibroblasts [73]. Several other molecules have
been implicated in preparing the premetastatic niche and
increasing metastasis. Matrix metal loproteinase-9 (MMP-
9) secreted by BMDCs degr a des the basement membrane
and liberates the matrix-sequestered VEGFR1 ligand, the
VEGFA, promoting the homing of more VEGFR1
+
cells into
the niche [80]. Through the production of VEGFA, TGFβ,
and tumor necrosis factor-α (TNFα), tumor cells enhance
the expression of the chemo attractants S100A8 and S100A9
in lung endothelium and myeloid cells, wh ich in turn pro-
mote tumor cell homing and adhesion to the metastatic site
[81, 82]. The role of activated fibroblasts in metastasis has
been revealed in some studies. Fibroblast-specific protein-
1 (FSP-1/S100A4), a fibroblast-specific marker, is highly ex-
pressed on tumor-associated fibroblasts and is released upon
stimulation of fibroblasts by tumor cells. Mice deficient in
FSP-1 exhibited a significant reduction in tumor growth
and metastasis [83]. Injection of FSP-1-positive fibroblasts
into these mice restored the ability of mammary adeno-
carcinoma cells to develop tumors and generate metastasis
suggesting a potential role of tumor-associated fibroblasts
in the metastatic dissemination. Another study found that
only metastasized melanoma cells were aected by fibroblasts
suggesting that fibroblasts might be important in creating the
permissive soil that supports tumor cell growth at distant
sites [84]. Recently, O’Connell and colleagues found that
S100A4
+
fibroblasts provide the proper metastatic niche
to support metastatic colonization. Through production of
VEGFA and tenascin-C, fibroblasts can promote angiogene-
sisaswellasprovideprotectionagainstapoptosis,respective-
ly [85].
Following the implantation of tumor cells, persistent
growth of metastasis is maintained by the establishment of
sucient blood supply capable of providing the necessary
oxygen, growth factors, nutrients, and metabolites. Blood
vessels are composed of vascular basement membrane,
endothelial cells and specialized smooth muscle cells, the per-
icytes [28]. The induction of a tumor vasculature termed the
angiogenic switch requires basement membrane assembly,
recruitment and proliferation of endothelial precursors, and
pericytes attachment [86]. Initially, the vascular basement
membrane is degraded by several MMPs produced by stro-
mal cells, endothelial cells, or tumor cells [87]. This basement
membrane degradation causes the release of endothelial
cells to migrate and proliferate, the liberation of matrix-
sequestered growth factors such as VEGF, basic fibroblast
growth factor (bFGF), and platelet-derived growth factor
(PDGF) and the disassembly of the pericytes that line the
blood vessels [87]. In response to VEGF, VEGFR2
+
endothe-
lial progenitor cells are recruited to the metastatic site
through VEGFA signaling to contribute to vessel formation
[88]. Analysis of these progenitor cells shows upregulation
of several angiogenic molecules (VEGF, FGF, PDGF, CXCL1,
etc.) that further bolster local angiogenesis and subsequent
metastatic colonization [86]. Moreover, V EGFR1
+
BMDCs
has been shown to produce several angiogenic factors and are
required to provide stability to the neovessels [89]. Inhibition
of VEGFR1
+
BMDCs either during primary tumor or after
the formation of premetastatic niche caused the prevention
of endothelial cell migration and metastasis [79]. Thus, the
recruitment of VEGFR2
+
endothelial progenitor cells into
vessels requires the incorporation of VEGFR1
+
BMDCs to
support neovascularization [60, 73]. Tumor angiogenesis is
also regulated by several immune cells [90]. Macrophages,
for example, are a good source of angiogenic fac tors such as
VEGF and MMP-9 [91]. In a mouse model of highly agg res-
sive metastatic mammary carcinoma, Lin and Pollard found
that tumor-associated macrophages may provide essential
cues to press the angiogenic switch [91]. Colony stimulating
factor-1 (CSF-1) deletion caused failure in macrophage
homing to the malignant stroma that was associated with
attenuated angiogenic responses, decreased neoplastic pro-
gression and inhibition of pulmonary metastasis. Moreover,
CAFs have been shown to be actively involved in boosting
tumor angiogenesis. The coinjection of CAFs and MCF-7
breast cancer cells into nude mice resulted in the recruitment
of bone-marrow-derived endothelial progenitors in response
to CAF-derived stromal cell derived factor (SDF1/CXCL12)
stimulating angiogenesis and tumor formation [52]. Another
Page 6
International Journal of Breast Cancer 7
proangiogenic mechanism of CAFs involves the release of
several factors such as VEGF and FGF which can positively
contribute to vascularization [48].
5. Perspectives
It is evident that metastasis is a multistep process where
each stage requires an intricate interplay between cancerous
cells and cells of the microenvironment. This tumor-host
crosstalk suppor ts the notion that cotargeting cancer cells
and tumor stromal cells will be a viable approach for
mammary cancer prevention and treatment. Researchers are
dedicated to explore the stromal cells as an eective target for
anticancer therapeutics. Such host-targeted therapies should
be directed tow ards BMDCs, fibroblasts, and endothelial cells
that home to the metastatic site to support tumor dissemi-
nation and outgrowth. It is important to inhibit the mobi-
lization and proliferation of the stromal cells and to disrupt
tumor-stroma interactions mediated by paracrine factors. To
achieve these goals, several agents have been investigated and
they fall into several categories including protease inhibitors
(e.g., MMP inhibitors), antiadhesive molecules (e.g., anti-
integrin p eptides or antibodies), signal pathway modula-
tors (e.g., tyrosine kinase pathway inhibitors), antifibrotic
drugs (e.g., pirfenidone), and antiangiogenic molecules (e.g.,
VEGF and bFGF antagonists) [92, 93]. Clinical trials using
MMP inhibitors (MMPIs) were disappointing for several
reasons. The trials were conducted only on patients with
advanced disease, and MMPIs used were broad spectrum
MMPIswhichcanblockbadMMPsaswellasgood
ones. Avastin/bevacizumab, a monoclonal antibody targeted
against all isoforms of VEGF-A, has recently been withdrawn
from the FDA list for the treatment of breast cancer [94].
Unlike trastuzumab which is a HER-2-targeted antibody,
avastin delayed tumor progression with no improvement i n
overall survival. This was accompanied by adverse side eects
including hypertension, neuropathy, and infection [95].
Recently, a variety of studies have been conducted to target
the tumor microenvironment. Wu et al. have shown that
targeting Galectin-1 to significantly inhibit CAF-conditioned
medium-induced tumor cell migration and invasion in
oral squamous cancer cells (OSCCs) resulting in a reduced
metastasis in vivo [96]. It is followed that Galectin-1 down-
regulation reduces the production of monocy te chemotactic
protein-1 (MCP-1/CCL2) which promotes the migration
of OSCCs by binding to CCR2 receptor. Blocking the
interaction between MCP-1 and CCR2 abolishes migration.
Moreover, Kim et al. have proposed to target myofibroblasts
overexpressing laminin-332 which caused the formation of
the dense fibrosis via desmoplastic reaction during epithelial
to mesenchymal transition (EMT) [97]. This alteration of the
tumor microenvironment preceded tumor invasion and was
found in invasive ductal carcinoma. Targeting of laminin-332
overexpressing myofibroblasts was supposed to prevent the
formation of the dense fibrosis, thus inhibiting the invasion-
friendly stromal alteration. It is important to note that
Angiotensin-(1–7), an endogenous 7-amino acid peptide
hormone of the renin-angiotensin system, has been shown to
target the tumor microenvironment to inhibit CAF growth
and tumor fibrosis [98]. Additionally, Liu et al. proposed
the targeting of the coagulation cascade that is activated in
the tumor microenvironment and presented preclinical data
targeting tissue factor (TF), an enzyme cofactor in ac tivating
coagulation that plays a critical role in tumor growth [99]. TF
inhibition by TF:FVIIa inhibitor led to growth retardation
in tumor models. Coenegrachts et al. demonstrated that
the selective neutralization of host-derived bone-derived
placental growth fac tor (PlGF) by anti-mouse alphaPlGF
reduced the engraftment of tumor cells in the bone, inhibited
their interaction with matrix components, reduced the inci-
dence, number, and size of bone metastases, and preserved
bone therefore inhibiting both the progression of metastasis
and the settlement of tumor in the bone [100]. Truitt et
al. have shown the role that Eph receptor tyrosine kinase
EphB6 plays in suppressing cancer invasiveness through
c-Cbl-dependent signaling, morphologic changes, and cell
attachment and that its targeting might enable the regulation
of both cell attachment and migration [101]. These stroma-
targeted therapies combined with antitumor approaches will
be translated into a double-edged sword that cancerous cells
will not easily survive. These therapeutic approaches require
a full understanding of the cellular and molecular mecha-
nisms governing the tumor-host interactions, accompanied
with the development of new mouse models and intravital
imaging techniques. Once accomplished, cancer patients will
experience better survival rates and quality of life.
Conflict of Interests
The authors declare that n o conflict of interests exists.
Acknowledgments
This work was supported by Grant BCTR0504465 from the
Susan G. Komen for the Cure Breast Cancer Foundation,
grants from the Florida Breast Cancer Coalition Research
Foundation, and the Florida State University to Professor Q.-
X. Sang. The authors thank Dr. Mark D. Roycik for critical
reading of this paper.
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    • "The dialog established further transforms not only the cancer cell phenotype but also their environment. If the role of immune cells is well acknowledged [32], the function of fibroblasts is beginning to emerge [9,11,16,21,28,343536. Focus on "
    No preview · Article · Mar 2016
    • "In contrast, when cultured without a scaffold, fibroblasts accumulated in the centre of the aggregates forming a core, as shown by us [26] and by others [43,44]. In cultures without scaffolds, the fibroblasts remained viable and maintained collagen expression, however the cellular organization did not resemble the human tumours where epithelial and stromal cells are organized into distinct compart- ments [3]. Imaging and quantification of collagen within the microcapsules demonstrated a significant increase of collagen deposition over the time in the co-cultures, suggesting that fibroblasts were playing their biologic role by actively producing collagen [45]. "
    [Show abstract] [Hide abstract] ABSTRACT: Currently there is an effort towards the development of in vitro cancer models more predictive of clinical efficacy. The onset of advanced analytical tools and imaging technologies has increased the utilization of spheroids in the implementation of high throughput approaches in drug discovery. Agitation-based culture systems are commonly proposed as an alternative method for the production of tumor spheroids, despite the scarce experimental evidence found in the literature. In this study, we demonstrate the robustness and reliability of stirred-tank cultures for the scalable generation of 3D cancer models. We developed standardized protocols to a panel of tumor cell lines from different pathologies and attained efficient tumor cell aggregation by tuning hydrodynamic parameters. Large numbers of spheroids were obtained (typically 1000-1500 spheroids/mL) presenting features of native tumors, namely morphology, proliferation and hypoxia gradients, in a cell line-dependent mode. Heterotypic 3D cancer models, based on co-cultures of tumor cells and fibroblasts, were also established in the absence or presence of additional physical support from an alginate matrix, with maintenance of high cell viability. Altogether, we demonstrate that 3D tumor cell model production in stirred-tank culture systems is a robust and versatile approach, providing reproducible tools for drug screening and target verification in pre-clinical oncology research.
    No preview · Article · Jan 2016 · Journal of Biotechnology
    • "Additionally, the activated stromal cells promote tumour progression by stimulating cancer cell proliferation and migration, and ultimately tumour metastasis [3]. Infiltrating stromal cells in the tumour are the main providers of matrix metalloproteinases (MMPs) that, through remodelling of ECM, release chemotactic agents and loosen the matrix contributing to tumour cell dissemination [4]. "
    [Show abstract] [Hide abstract] ABSTRACT: 3D cell tumour models are generated mainly in non-scalable culture systems, using bioactive scaffolds. Many of these models fail to reflect the complex tumour microenvironment and do not allow long-term monitoring of tumour progression. To overcome these limitations, we have combined alginate microencapsulation with agitation-based culture systems, to recapitulate and monitor key aspects of the tumour microenvironment and disease progression. Aggregates of MCF-7 breast cancer cells were microencapsulated in alginate, either alone or in combination with human fibroblasts, then cultured for 15 days. In co-cultures, the fibroblasts arranged themselves around the tumour aggregates creating distinct epithelial and stromal compartments. The presence of fibroblasts resulted in secretion of pro-inflammatory cytokines and deposition of collagen in the stromal compartment. Tumour cells established cell-cell contacts and polarised around small lumina in the interior of the aggregates. Over the culture period, there was a reduction in oestrogen receptor and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential in co-cultures. These phenotypic alterations, typical of advanced stages of cancer, were not observed in the mono-cultures of MCF-7 cells. The proposed model system constitutes a new tool to study tumour-stroma crosstalk, disease progression and drug resistance mechanisms.
    No preview · Article · Nov 2015 · Biomaterials
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