0022-5347/01/1666-2472/0

THE JOURNAL OF UROLOGY® Vol. 166, 2472–2483, December 2001

Copyright © 2001 by AMERICAN UROLOGICAL ASSOCIATION, INC.® Printed in U.S.A.

Review Article

REACTIVE STROMA IN PROSTATE CANCER PROGRESSION

JENNIFER A. TUXHORN, GUSTAVO E. AYALA AND DAVID R. ROWLEY
From the Departments of Molecular and Cellular Biology and Pathology, Baylor College of Medicine, Houston, Texas

ABSTRACT

Purpose: The development of an altered stromal microenvironment in response to carcinoma is a

common feature of many tumors. We reviewed the literature describing characteristics of reactive
stroma, how reactive stroma affects cancer progression and how carcinoma regulates reactive stroma.
Moreover, we present a hypothesis of reactive stroma in prostate cancer and discuss how the biology
of reactive stroma may be used in novel diagnostic and therapeutic approaches.
Materials and Methods: An extensive literature search was performed to review reports of the
general features of wound repair stroma, general stromal responses to carcinoma, and stromal
biology of normal and prostate cancer tissues. These studies were analyzed and a reactive stroma
hypothesis in prostate cancer was developed.
Results: Modifications to the stroma of breast, colon and prostate tumors parallel the gener-
ation of granulation tissue in wound repair. These changes include stromal cell phenotypic
switching, extracellular matrix remodeling and angiogenesis induction. Therefore, it is predicted
that a modified wound healing response induces the formation of reactive stroma in cancer to
create a tumor promoting environment. Based on its role in wound repair and its over expression
in prostate cancer, transforming growth factor-␤ stands out as a potential regulator of reactive
stroma.
Conclusions: Reactive stroma in prostate cancer and granulation tissue in wound repair show
similar biological responses and processes that are predicted to promote cancer progression.
Further identification of specific functional and regulatory mechanisms in prostate cancer
reactive stroma may aid in the use of reactive stroma for novel diagnostic and therapeutic
approaches.
KEY WORDS: prostate, prostatic neoplasms, growth substances, granulation tissue, extracellular matrix

Cancer progression is a complex multistep process that
begins with the transformation of normal cells and continues
with tumor growth, invasion and metastasis. In addition to
transformed carcinoma cells, tumors also contain stromal
cells, extracellular matrix, newly formed blood vessels and
immune components. However, there is considerable evi-
dence that tumor stroma is different than the stroma in
normal tissue. To maintain tissue homeostasis the host stro-
mal compartment reacts to carcinoma in a process similar to
the general wound repair pathway. The result of this re-
sponse is the creation of a new stromal microenvironment,
into which carcinoma grows and invades. Studies indicate
that this new reactive stroma environment enhances tumor-
igenesis by supporting cancer cell survival, proliferation and
migration, and by inducing angiogenesis. Therefore, it is
becoming more clear that cancer progression is not exclu-
sively regulated by the disruption of oncogene and tumor
suppressor pathways in cancer cells. Much evidence now
suggests that cancer progression also co-depends on the stro-
mal compartment to create a more tumor promoting micro-
environment. In this review we discuss the role of stroma in
maintaining tissue homeostasis and present mechanisms by
which carcinoma may induce the host programmed wound
healing response. In addition, we consider how the genera-
tion of this reactive stroma environment may promote cancer
progression. We also propose that the stromal compartment
Supported by National Institutes of Health Grants RO1 CA58093,
RO1 DK45909, SPORE CA58204 and UO1 CA84296.
2472
may be an attractive target for additional diagnostic and
therapeutic approaches to prostate cancer.
BIOLOGY OF THE STROMA
Structure and function. The stroma was once viewed as a
passive support structure that contributed little to the over-
all biological function of tissues. However, it is now under-
stood that the stroma is a dynamic environment that directly
influences epithelial cell behavior and performs tissue repair
in response to injury. The stromal compartment is a complex
arrangement of stromal cells and extracellular matrix plus
associated growth factors, regulatory molecules and remod-
eling enzymes. Blood vessels, nerves and immune cells are
also integral parts of the stroma (fig. 1, A). These compo-
nents act in a coordinated manner to regulate cell function
and maintain overall tissue homeostasis.
Fibroblasts and smooth muscle cells are the principal stro-
mal cell types in the human prostate gland. A primary func-
tion of these cells is to synthesize the structural and regula-
tory components of the extracellular matrix. The
extracellular matrix is a meshwork of fibrillar proteins, ad-
hesive glycoproteins and proteoglycans.1 Moreover, it is a
reservoir of active and latent growth factors.2 Structural
components, such as collagen and elastic fibers, provide me-
chanical strength and flexibility to the tissue. These compo-
nents also serve as a substrate for cell attachment and mi-
gration, which are mediated by adhesive glycoproteins, such
 REACTIVE STROMA IN PROSTATE CANCER PROGRESSION
FIG. 1. A, normal human prostate gland is composed of secretory
epithelium surrounded by stroma containing fibroblasts, smooth
muscle cells (SMC), structural and regulatory components of extra-
cellular matrix (ECM), blood vessels, nerves and immune cells. Re-
ciprocal stromal-epithelial interactions regulate normal tissue struc-
ture and function. B, prostate carcinoma cells proliferate and invade
through basement membrane into stroma, which responds to repair
disruption in tissue homeostasis, creating new stromal microenvi-
ronment termed reactive stroma. Reactive stroma shows stromal cell
phenotypic switching to myofibroblasts, extracellular matrix remod-
eling, elevated angiogenesis and influx of tumor associated macro-
phages (TAM).
as fibronectin and laminin. Proteoglycans regulate extracel-
lular matrix structure and permeability.3 They also bind to
and modulate the activity of growth factors, proteases and
protease inhibitors.1, 2
Together stromal cells and the extracellular matrix create
a microenvironment that regulates the growth and func-
tional differentiation of adjacent cells.4 For example, it is well
established that prostate smooth muscle cells mediate
stromal-epithelial interactions that are important for the
development and maintenance of tissue differentiation.5 The
inductive capability of extracellular matrix is also evident
when studied in vitro. Culturing primary human prostate
epithelium on reconstituted basement membrane extracellu-
lar matrix induced cells to show a clustered morphology and
increase the secretion of prostate specific antigen and pros-
tatic acid phosphatase.6 In contrast, when grown on tissue
culture plastic, these cells showed elevated proliferation and
reduced synthesis of prostate specific secretory proteins. It is
clear that activation of growth factor receptors and extracel-
lular matrix interactions through integrins function to initi-
ate intracellular signaling cascades that control cell motility,
proliferation and differentiation. Accordingly stromal regu-
lation of cell behavior likely occurs by mobilizing extracellu-
lar matrix bound growth factors and by modulating direct
cell-extracellular matrix interactions.7
In addition to producing the extracellular matrix compo-
nents and growth factors necessary to regulate normal tissue
function, the stromal compartment is also responsible for
tissue repair in response to injury. Thus, the stroma must be
poised to respond rapidly to changes in homeostasis. Two key
characteristics of the stromal compartment enable this to
occur, namely stromal cell plasticity and extracellular matrix
organization. The storage of latent growth factors, proteases
and protease inhibitors in the extracellular matrix allows for
immediate mobilization of these factors to regulate extracel-
lular matrix and tissue remodeling.2 Therefore, the stroma is
designed to effect a timely response to wounding and to
maintain tissue homeostasis.
Stromal response in wound repair. The process of wound
healing exemplifies the complexity of stromal biology, in that
it integrates all components of the stromal compartment in
response to disruptions in tissue homeostasis. The wound
repair response is typically initiated when injury to the skin
or internal tissues results in the rupture of blood vessels.
Spillage of blood into the interstitial matrix leads to platelet
aggregation and clot formation. The initial clot, which is
composed of fibrin, fibronectin and platelets, serves as a
provisional matrix for cell migration. Neutrophils, macro-
phages and stromal cells are recruited to the wound site by
2473
growth factors and cytokines released by degranulating
platelets. Activated macrophages also secrete growth factors,
such as transforming growth factor-␤ (TGF-␤), platelet de-
rived growth factor (PDGF), fibroblast growth factor-2 and
vascular endothelial growth factor (VEGF), which induce
angiogenesis and fibroplasia. Quiescent fibroblasts from sur-
rounding tissue migrate to the wound site, where they pro-
liferate and produce extracellular matrix components, such
as collagen types I and III, fibronectin and proteoglycans.
This collection of macrophages, fibroblasts and new blood
vessels embedded within a loose matrix is called granulation
tissue. Epithelial cells are also stimulated to proliferate and
migrate through the granulation tissue to reestablish the
epithelial layer. As the wound continues to heal, granulation
tissue fibroblasts differentiate into myofibroblasts, which de-
velop characteristics of smooth muscle to assist in wound
closure.8 Fibroblasts and myofibroblasts continue to secrete
large amounts of extracellular matrix, especially collagen
type I, as well as proteases, which degrade extracellular
matrix components. The balance of extracellular matrix pro-
duction and degradation results in remodeling of the granu-
lation tissue to form a scar.3, 9 –11
A similar repair process is observed in vascular injury.3, 12
Platelets adhere to damaged endothelium in vessels and
aggregate to form a clot, which recruits infiltration of inflam-
matory cells. Growth factors and cytokines released from
activated platelets and macrophages induce migration of
smooth muscle cells from the media of the vessel wall to the
intima. Smooth muscle cells that migrate to the vessel intima
appear to dedifferentiate from a quiescent contractile pheno-
type to a synthetic proliferative phenotype.13, 14 In the intima
the smooth muscle cells are induced to proliferate and secrete
extracellular matrix components, including collagen, fi-
bronectin and glycosaminoglycans, to generate a modified
granulation tissue. During the final phase extracellular ma-
trix remodeling occurs and the endothelium grows to cover
the healed area.
As these examples illustrate, the stromal compartment
responds to injury by creating a microenvironment that is
fundamentally different from normal stroma. The granula-
tion tissue environment drives the repair process by recruit-
ing immune cells, inducing stromal cell activation and secre-
tion of extracellular matrix, growth factors and remodeling
enzymes, stimulating angiogenesis and promoting epithelial
cell proliferation and migration. These growth promoting
conditions are necessary to repair the wound rapidly and
reestablish tissue homeostasis.
Stromal cell phenotypic switching: the myofibroblast. As
described, a key feature of the stromal response in wound
repair is the ability of stromal cells to show plasticity or
phenotypic switching.13, 15 Fibroblasts and smooth muscle
cells can reversibly regulate their proliferation rate and phe-
notype in response to the state of tissue homeostasis. Under
normal conditions these cells have a low proliferative index
and likely secrete only the factors necessary to maintain
normal tissue function.13 However, in response to pathologi-
cal situations stromal cells rapidly modulate to a reactive
phenotype, which proliferates and synthesizes extracellular
matrix, growth factors and remodeling enzymes necessary
for tissue repair.
It appears that fibroblasts and vascular smooth muscle
cells modulate to a common myofibroblast phenotype during
wound repair.16 The myofibroblast is described as an inter-
mediate between fibroblasts and smooth muscle cells based
on cytoskeletal protein expression and ultrastructural fea-
tures.8 Fibroblasts in granulation tissue switch to a myofi-
broblast phenotype, which is identified by vimentin and
smooth muscle ␣-actin expression. These cells are also char-
acterized by formation of contractile filaments, a well devel-
oped rough endoplasmic reticulum and Golgi apparatus, cell
attachments to the extracellular matrix, and gap junctions.15
2474 REACTIVE STROMA IN PROSTATE CANCER PROGRESSION
In response to vascular injury smooth muscle cells switch
from a quiescent contractile phenotype to a proliferative syn-
thetic phenotype, which also shows the features of myofibro-
blasts.14 Cells with the myofibroblast phenotype increase
vimentin expression and decrease expression of markers that
identify smooth muscle cells, such as calponin and smooth
muscle myosin heavy chain. In addition, a reduction in mi-
crofilaments is accompanied by an increase in “synthetic
cellular organelles, such as Golgi and rough endoplasmic
reticulum.”13 Gap junctions and focal tight junctions have
also been reported.17 That these cells switch to a common
phenotype is further supported by the observation that fibro-
blasts and arterial smooth muscle cells show myofibroblast
characteristics when cultured in vitro.15, 18
Together these studies suggest that stromal cells show
plasticity via a continuum of phenotypes ranging from fibro-
blast to myofibroblast to smooth muscle (fig. 2). Furthermore,
stromal cells appear to modulate between the phenotypes
based on physiological and pathological stimuli13, 15 with the
myofibroblast as the principal cell type in pathological con-
ditions. Cells characterized as myofibroblasts have been re-
ported in numerous pathological processes, including wound
healing granulation tissue, hypertrophic scars, fibromatoses
and stromal response to neoplasia.19 Accordingly it is pre-
dicted that the stromal phenotypic switch to an activated
myofibroblast cell type is a generic host response to disrup-
tions in tissue homeostasis that require repair. Therefore,
myofibroblasts would be expected to be present in almost any
physical or biological wound, ranging from a laceration to
carcinogenesis. Moreover, activation of stromal cells to the
reactive myofibroblast phenotype and their subsequent con-
tribution of extracellular matrix components, growth factors
and proteases to the stromal microenvironment likely have
an important role in the progression of these pathological
conditions.
ALTERED STROMAL BIOLOGY IN CANCER: REACTIVE STROMA
Carcinoma cell proliferation and invasion through the
basement membrane into the surrounding stroma would be
classified as a disruption in normal tissue homeostasis. Due
to its primary role in wound repair it is expected that the
stroma would respond in an effort to repair the damage.
Modifications in stromal cell phenotype and extracellular
matrix composition have been reported in many types of
human cancer, particularly breast and colon cancer.20 –22
These changes have previously been referred to as desmopla-
sia (the formation of abundant collagenous stroma) or the
stromal response/stromal reaction. However, we use the term
reactive stroma to describe the genesis of a new stromal
microenvironment that is created in response to carcinoma
and accompanies tumor progression. The reactive stroma in
cancer is typified by stromal cell phenotypic switching, ex-
tracellular matrix remodeling, increased growth factor bio-
availability, elevated protease activity, increased angiogene-
FIG. 2. Stromal cells show phenotypic switching or plasticity.
Continuum of phenotypes from fibroblast to myofibroblast to smooth
muscle cells is observed based on physiological and pathological
conditions. Cell types may be identified histologically based on ex-
pression pattern of vimentin, ␣-actin and late stage markers of
smooth muscle differentiation, such as calponin and smooth muscle
myosin heavy chain (SM-MHC). Identifying specific cell types may
be helpful for identifying reactive stroma in prostate cancer.
sis and an influx of inflammatory cells (fig. 1, B). It has been
suggested that the stromal response to cancer is a modified
wound repair response10, 23–25 and, therefore, the reactive
stromal environment is likely to mimic granulation tissue.
These conditions would be predicted to promote tumorigen-
esis. Although the concept of reactive stroma in human pros-
tate cancer is just emerging,24 several studies to date have
described aspects of reactive stroma and shown that they are
associated with prostate cancer progression.
Stromal cell phenotype. Perhaps the most common marker
of reactive stroma in cancer is the appearance of activated
stromal cells with myofibroblastic characteristics. Cells with
the ultrastructural features of myofibroblasts have been ob-
served in the stroma of various types of invasive and meta-
static carcinoma, including that of the breast, lung, colon,
stomach and prostate.26 Smooth muscle ␣-actin has become
the marker most often used to identify myofibroblasts by
immunohistochemistry.15 This test is effective in tissues in
which normal interstitial stromal cells do not express smooth
muscle ␣-actin, as in the breast.27 However, smooth muscle
cells in normal prostate stroma express ␣-actin. Thus, it is
necessary to examine multiple markers to distinguish myo-
fibroblasts from mature smooth muscle cells in the prostate.
We have previously examined the expression of vimentin,
smooth muscle ␣-actin and calponin, a later stage marker of
smooth muscle differentiation,13 in the reactive stroma of
Gleason 3 prostate cancer by immunohistochemistry. Our
preliminary results showed an increase in vimentin staining
and a decrease in calponin staining, while the level of ␣-actin
remained essentially the same.28 These observations suggest
that the primary stromal cell type in prostate cancer reactive
stroma is the myofibroblast. These data are consistent with
observations by Hayward et al that “zones immediately sur-
rounding foci of prostatic carcinoma cells contained a fibro-
blastic stroma devoid of smooth muscle cells.”5 The loss of
smooth muscle in human prostate cancer is clearly illus-
trated in specimens stained with Masson’s trichrome. Fig-
ure 3 shows that normal prostate stroma is composed of
red staining smooth muscle cells surrounded by blue stain-
ing collagen fibers, while the reactive stoma surrounding
Gleason 3 prostate cancer is predominantly blue with rel-
atively few scattered smooth muscle cells. Together these
data suggest that human prostate cancer reactive stroma
is characterized by a switch in stromal cell phenotype and
reactive stroma is composed primarily of myofibroblasts.
Since fibroblasts and vascular smooth muscle cells have
the potential to switch to the myofibroblast phenotype during
wound repair, it is possible that myofibroblasts in cancer
reactive stroma may be derived from either cell type. A study
in which breast cancer cells were co-cultured with fibroblasts
or vascular smooth muscle cells in 3-dimensional collagen
gels explored this issue.27 In fibroblast and cancer cell co-
cultures the cells showed morphological organization similar
to that of in vivo tumors and the fibroblasts “readily con-
verted to myofibroblasts,”27 as assayed by ␣-actin expression.
Although vascular smooth muscle cell and cancer cell co-
cultures resulted in morphology similar to that of the fibro-
blast/cancer cell co-cultures, smooth muscle cells maintained
the expression of ␣-actin, calponin, and smooth muscle myo-
sin. These results indicate that fibroblasts are the primary
source of myofibroblasts in the reactive stroma of breast
tumors. To our knowledge it is not known whether myofibro-
blasts in human prostate tumors originate from fibroblasts,
smooth muscle cells or both cell types. In vitro studies have
shown that prostate fibroblasts can modulate to the myofibro-
blast phenotype.29, 30 In addition, Hayward et al suggested that
loss of appropriate paracrine signals from carcinoma cells may
result in dedifferentiation of prostate smooth muscle cells.5
Although it may potentially lead to the myofibroblast pheno-
type, direct evidence supporting carcinoma induced dedifferen-
tiation of smooth muscle is lacking. Therefore, it is predicted
 REACTIVE STROMA IN PROSTATE CANCER PROGRESSION 2475

FIG. 3. Human prostate cancer specimens. A, normal human prostate epithelial acini surrounded by collagenous stroma (blue areas)
containing smooth muscle cells (red areas) and fibroblasts. B, Gleason 3 human prostate cancer in reactive stroma with some remaining
smooth muscle cells. C, Gleason 3 human prostate cancer in reactive stroma lacking smooth muscle cells. D, Gleason 4 human prostate
cancer progressing to stromal independent state with little stroma visible except around blood vessels. Reactive stroma composition in
prostate cancer may potentially be used to predict tumor progression rate. Masson’s trichrome stain, reduced from ⫻400.

that myofibroblasts in human prostate cancer reactive stroma
are derived from fibroblasts adjacent to carcinoma cells.
In wound repair activated fibroblasts/myofibroblasts syn-
thesize the key extracellular matrix components of granula-
tion tissue, namely fibronectin, collagen types I and III, and
many other glycoproteins and proteoglycans. They also re-
lease growth factors that stimulate angiogenesis and pro-
teases that function to remodel the granulation tissue.9
Therefore, the presence of myofibroblasts in reactive stroma
indicates that there may be increased production of extracel-
lular matrix components and overall extracellular matrix
remodeling during prostate cancer progression.
Extracellular matrix synthesis and remodeling. Examination
of collagen fibers in human prostate stroma by scanning elec-
tron microscopy indicated a disruption of the normal connective
tissue framework in diseased states.31 In normal prostate it has
been reported that the “stromal network of collagen fibers is
loosely woven, fine and smooth in texture,” while in Gleason
score 7 adenocarcinoma the collagen fibers “appeared swollen in
diameter” and there was “no regularity in the spatial relation-
ship of the fibers.”31 Some areas of collagen destruction were
also observed. These data support the concept that remodeling
of the extracellular matrix is a key feature of reactive stroma in
prostate cancer. Changes in collagen synthesis are not well
characterized in prostate carcinoma but Masson’s trichrome
staining suggests that there may be an increase in collagen
fibers in reactive stroma (fig. 3). Using an antibody to procolla-
gen I we observed collagen synthesis by reactive stromal cells in
human prostate cancer,28 which is consistent with breast can-
cer. In situ hybridization studies showed that messenger
(m)RNA for types I and III procollagen are elevated in the
stromal cells of invasive breast carcinoma,32, 33 and an increase
in collagen types I and III protein has been observed by immu-
nohistochemistry.34 These data confirm that alterations in the
collagen component of the extracellular matrix framework oc-
cur during cancer progression.
Another important component of the extracellular matrix
is fibronectin, which is a high molecular weight glycoprotein
that mediates cell adhesion and migration.1 Alternative
splicing of pre-mRNA produces multiple isoforms of this pro-
tein that differentially include cell type specific adhesion
domains.35 Fibronectins are widely expressed, especially at
sites of active morphogenesis and inflammation, but the ex-
pression of specific isoforms is regulated temporally and spa-
tially.1 Interestingly fibronectin molecules containing the
ED-A and ED-B domains are primarily found during devel-
opment, although ED-A⫹ and ED-B⫹ fibronectin expression
has also been reported during wound healing and in the
reactive stroma of many types of cancer.35, 36 Sites of wound
repair have been shown to have increased local expression of
total fibronectin, including isoforms containing the ED-A and
ED-B domains, which were expressed by macrophages and
activated granulation tissue fibroblasts.37 Moreover, stromal
cells in human breast32 and colorectal38 tumors also showed
increased expression of total fibronectin, and ED-A⫹ and
ED-B⫹ isoforms. In addition, double staining experiments
using ED-B⫹ fibronectin in situ hybridization probes and
smooth muscle ␣-actin antibodies identified myofibroblasts
as the source of ED-B⫹ fibronectin in oral squamous cell
carcinoma.39 In human prostate cancer the level of fibronec-
tin protein was elevated compared with that in normal tissue
and benign prostatic hyperplasia,40 and reverse
transcriptase-polymerase chain reaction using mRNA iso-
lated from human prostate cancer tissue revealed increased
expression of ED-B⫹ fibronectin.41 Also, these studies indi-
cated that fibronectin may regulate prostate cancer cell ad-
hesion and migration. Incubation with a polyclonal fibronec-
tin antibody acted to disrupt adhesion of LNCaP human
2476 REACTIVE STROMA IN PROSTATE CANCER PROGRESSION
prostate cancer cells.41 Another study showed that the pres-
ence of plasma fibronectin induced invasion of DU145 human
prostate cancer cells through serum-free basement mem-
branes in an in vitro invasion assay.42 It has been suggested
that since ED-A⫹ and ED-B⫹ fibronectins are associated
with cell migration during development, they may have a
similar function in wound healing.37 If it is correct, expres-
sion of these embryonic fibronectins in reactive stroma may
create an extracellular matrix microenvironment that sup-
ports cancer cell migration and invasion.
Myofibroblasts in the stroma of breast and colon carcinoma
were also shown to increase the expression of other extracel-
lular matrix glycoproteins, such as tenascin-C and
thrombospondin-1.20, 22 These proteins are expressed at sites
of tissue remodeling, where they act to influence cell adhe-
sion and migration.35 Thrombospondin-1 has been shown to
stimulate tumor cell motility and may mediate tumor cell
adhesion to endothelial cells.43 The role of thrombospondin-1
in prostate cancer is not well documented but expression in
reactive stroma is likely.
Tenascin-C is closely associated with sites of stromal-
epithelial interactions during development and tissue remod-
eling.35 In the prostate tenascin was highly expressed around
developing glands but only weak expression was observed in
normal adult tissue.44 Examination of high grade prostatic
intraepithelial neoplasia revealed periglandular expression
of tenascin-C and low/moderate grade human prostate cancer
showed strong tenascin-C expression in the peritumoral stro-
ma.45 Interestingly high molecular weight splice variants of
tenascin-C, which contain additional fibronectin type III-like
repeats, have been reported in breast,46 colorectal47 and pros-
tate44 cancer reactive stroma. It has been suggested that
expression of the high molecular weight isoform may favor
cancer cell migration.47
Alterations in proteoglycan and glycosaminoglycan synthe-
sis have also been noted in wound repair and tumor reactive
stroma.9, 48 An increase in proteoglycans with chondroitin
sulfate side chains has been reported in the stroma of colon47
and breast cancer as well as in healing skin wounds.48 Ele-
vated levels of chondroitin sulfate in prostate cancer reactive
stroma have been associated with prostate specific antigen
failure.49 In addition, expression of versican, a chondroitin
sulfate proteoglycan, was shown to be higher in early stage
prostate cancer reactive stroma than in normal prostate tis-
sue.50 Increased versican expression was also reported in
breast carcinoma reactive stroma51 and versican was shown
to be expressed by smooth muscle cells at sites of vascular
repair.52
Together these studies indicate that the extracellular ma-
trix surrounding prostate carcinoma is significantly modified
in comparison to the extracellular matrix microenvironment
of normal prostate tissue, and most extracellular matrix syn-
thesis results from phenotypically switched reactive stromal
cells. It has been suggested that the alterations in extracel-
lular matrix composition are induced by carcinoma cells.32, 53
Co-culture of human fibroblasts with MCF-7 human breast
cancer cells provides data to support this theory. Production
of collagen types I and III, and fibronectin was increased in
human fibroblasts when co-cultured with MCF-7 cells.54 In
addition, MCF-7 cell conditioned media induced elevated ex-
pression of tenascin55 and glycosaminoglycans56 in fibro-
blasts. Similarly myofibroblasts isolated from granulation
tissue exhibited increased collagen types I and III, and ver-
sican mRNA expression.57 Therefore, it appears that cancer
cells are capable of inducing stromal cells to create an extra-
cellular matrix similar to that of granulation tissue. Consid-
ering how the extracellular matrix influences cell behavior it
follows that the reactive stromal microenvironment would be
conducive to cancer cell survival, proliferation and migration.
Production of growth factors by reactive stroma. As carci-
noma derived growth factors regulate stroma, increased ex-
pression of growth factors by reactive stromal cells recipro-
cally regulate carcinoma cells directly or indirectly. Foremost
in this list are members of the fibroblast growth factor fam-
ily. In human prostate cancer specimens fibroblast growth
factor-2 expression has been shown to be significantly ele-
vated in stromal fibroblasts and in endothelial cells com-
pared with normal tissue.58 Fibroblast growth factor-2 stim-
ulates fibroblast proliferation, extracellular matrix
production and protease secretion.11 It also functions as an
angiogenic factor that induces endothelial cell migration,
proliferation and differentiation into new blood vessels.59
Thus, fibroblast growth factor-2 may promote cancer progres-
sion by inducing angiogenesis and reactive stroma formation.
This growth factor may also directly regulate tumor cell
proliferation and invasion. During cancer progression pros-
tate carcinoma cells have been shown to up-regulate fibro-
blast growth factor receptor isoforms with a high affinity for
fibroblast growth factor-2.58, 60 Accordingly, elevated sensi-
tivity to fibroblast growth factor-2 may stimulate cancer cell
proliferation and protease expression, thereby, supporting
tumor growth and invasion. Intense efforts are also under
way to examine the role of other fibroblast growth factor
family members in prostate cancer.
Another stromal derived growth factor that is important in
prostate cancer progression is hepatocyte growth factor. It is
produced by stromal cells and has been shown to stimulate
proliferation and migration of epithelial and endothelial cells
during development and tissue remodeling.61 Increased ex-
pression of the hepatocyte growth factor receptor c-MET
proto-oncogene has been associated with progression of sev-
eral types of carcinoma, including that of the prostate.62
Moreover, hepatocyte growth factor acts to stimulate cell
growth, scattering and invasion of DU145 human prostate
cancer cells.62, 63 In addition, co-culture with DU145 or PC-3
cells significantly increased hepatocyte growth factor expres-
sion in human prostate stromal cells.63 These data provide
direct evidence that human prostate cancer cells can induce
surrounding stromal cells to secrete factors that promote
tumor growth and invasion. In addition, hepatocyte growth
factor may contribute to cancer progression by stimulating
angiogenesis.61
Protease/protease inhibitor balance. Serine proteases and
matrix metalloproteinases (MMPs) have significant roles in
physiological and pathological tissue remodeling, including
wound repair and tumorigenesis.64, 65 Furthermore, it is well
established that elevated protease activity strongly corre-
lates with metastatic potential in advanced cancer.66, 67 Al-
though many cancer cell lines have been shown to express
serine proteases and MMPs in vitro,68, 69 in vivo analysis of
various human tumors demonstrated that these proteases
were predominantly expressed by stromal cells.66 For exam-
ple, myofibroblasts in human breast cancer reactive stroma
expressed the serine protease urokinase plasminogen activa-
tor,70 and MMP-2 and MMP-9 expression was localized to
stromal cells in human prostate cancer.71 Thus, reactive stro-
mal cells may contribute to cancer progression by production
of proteases.
Notably overall proteolytic activity is regulated by the bal-
ance of activated enzymes and their specific inhibitors.65
Examination of MMP and tissue inhibitor of metalloprotein-
ase (TIMP) expression in human prostate cancer by in situ
hybridization revealed that low grade (Gleason score 5) spec-
imens showed elevated TIMP-1 and TIMP-2 expression in
the stroma compared with MMP expression. However, high
grade (Gleason scores 8 to 10) specimens showed increased
levels of MMP-2 and MMP-9, while TIMPs were not ex-
pressed.71 These observations indicate that the balance be-
tween proteases and their inhibitors shifts during prostate
cancer progression. Similarly a study that measured total
MMP enzyme activity and TIMP-1 expression in tissue ex-
tracts showed that the MMP-to-TIMP-1 ratio is higher in
 REACTIVE STROMA IN PROSTATE CANCER PROGRESSION
prostate cancer than in normal prostate tissues.72 Together
these data suggest that the altered protease/protease inhib-
itor balance in prostate tumors may promote cancer progres-
sion.
Studies which shift the protease/protease inhibitor bal-
ance, indicate that proteases regulate prostate cancer pro-
gression. For example, PC-3 human prostate carcinoma cells
transfected to over express the plasminogen activator
inhibitor-1 formed significantly smaller tumors in nude mice
than in controls, and plasminogen activator inhibitor-1 ex-
pressing tumors showed reduced angiogenesis and metasta-
sis.73 Similar results were obtained using the synthetic MMP
inhibitor A-177430. Mat Ly Lu rat prostate cancer cells
formed large primary tumors and distant metastases when
injected into rats. However, treating the animals with
A-177430 resulted in decreased tumor growth, reduced an-
giogenesis and prevention of metastasis.74
Since it has been shown that carcinoma cells affect the
expression of extracellular matrix components and growth
factors in adjacent stromal cells, it would be predicted that
cancer cells also influence stromal cell expression of pro-
teases. Co-culture of MCF-7 human breast cancer cells with
human fibroblasts induced expression of MMP-1, MMP-2,
MMP-3 and TIMP-1 in the fibroblasts.75 In contrast, co-
culture of normal mammary epithelial cells with fibroblasts
only induced TIMP-1 expression. This study provides an-
other example of how stromal cell activity may be modulated
by tumor cells to promote cancer progression.
Interestingly it is becoming clear that proteolytic degradation
of the extracellular matrix is more than a physical mechanism
for tumor cell invasion through adjacent tissue.76 Extracellular
matrix remodeling by proteases alters extracellular matrix
composition by releasing growth factors, generating bioactive
extracellular matrix fragments and exposing cryptic or conceal-
ing existing cell adhesion sites.7, 77 These changes affect cell
growth, survival and migration.76 Thus, the expression and
activation of proteases further contribute to creating the reac-
tive stroma microenvironment that promotes tumor progres-
sion.
Angiogenesis. Substantial evidence shows that angiogene-
sis is required for tumor growth and metastasis.78 Recruit-
ment of new blood vessel growth clearly illustrates the im-
portance of carcinoma-stroma interactions during cancer
progression. Endothelial cells from microvessels in the sur-
rounding stroma must be induced to migrate into the tumor,
where they proliferate and form new blood vessels. This
complex process is regulated by the relative balance of an-
giogenesis inducers and angiogenesis inhibitors in the extra-
cellular milieu. These factors may be secreted by cancer or
stromal cells, or mobilized from the extracellular matrix.
Increased concentration of an activator or decreased levels of
an inhibitor alter the balance and lead to the growth of new
blood vessels.79 Increased expression of the angiogenesis ac-
tivator, VEGF is observed in many types of cancer, including
that of the prostate.
In normal human prostate tissue it has been reported that
VEGF is expressed at low levels and expression is restricted
to stromal cells.80, 81 However in high grade prostatic intra-
epithelial neoplasia and prostate cancer elevated expression
was observed in cancer, stroma and vascular endothelial
cells.80 – 83 Also, increased expression by human prostate can-
cer cell lines correlated with tumor microvessel density and
metastatic potential when grown in nude mice.84 Additional
studies have shown that VEGF expression in the prostate is
androgen regulated. Dihydrotestosterone stimulated expres-
sion in human fetal prostate fibroblasts,83 and testosterone
replacement in castrated rats induced VEGF synthesis in the
prostate epithelium.85 In addition, castrating mice bearing
androgen responsive human prostate cancer xenografts re-
sulted in a significant decrease in tumor VEGF expression
and tumor volume.86 To determine the role of VEGF in pros-
2477
tate cancer progression others have examined the effects of
anti-vascular endothelial growth factor neutralizing antibod-
ies on tumor growth in mice. In all cases treatment with the
antibody suppressed angiogenesis, tumor growth and metas-
tasis even in well established tumors.87– 89
Although prostate cancer cells produce significant levels of
VEGF and other angiogenic factors, it is likely that surround-
ing stromal cells are also involved in angiogenesis. A recent
study used species specific anti-vascular endothelial growth
factor antibodies to treat a human rhabdomyosarcoma xeno-
graft in nude mice. Inhibition of VEGF produced by the host
stroma further reduced tumor growth compared with only
blocking VEGF produced by the cancer cells.90 Stromal cells
also produce several other growth factors that promote an-
giogenesis, such as PDGF, TGF-␤ and connective tissue
growth factor.3 A 3-dimensional co-culture model using fibrin
and collagen matrix showed that PC-3 human prostate can-
cer cells did not induce endothelial cell formation of capillary-
like structures unless fibroblasts were present.91 It is becom-
ing more clear that angiogenesis requires the cooperation of
cancer cells and multiple cell types in reactive stroma.
In addition to providing the necessary blood supply, angio-
genesis may contribute to cancer progression in other ways.
For example, vascular endothelial cells secrete growth fac-
tors and proteases that likely contribute to prostate cancer
growth and invasion.66, 92 It has also been proposed that
tumor associated vasculature stimulates the generation of a
granulation tissue-like environment. Blood vessels in tumors
have enhanced permeability to circulating macromolecules,
which is likely due to high levels of VEGF.93 Increased vas-
cular permeability leads to the extravasation of plasma pro-
teins, including fibrinogen, fibronectin and clotting factors.
Procoagulants produced by cancer cells, such as tissue factor,
rapidly clot fibrinogen to form a fibrin matrix.10, 93 As in
wound repair, the fibrin-fibronectin matrix recruits macro-
phages, stimulates fibroblast proliferation and induces an-
giogenesis. In addition, it has been shown that the provi-
sional matrix is remodeled into mature tumor stroma that
resembles granulation tissue.10, 93 Fibrin deposits have been
observed in many types of human cancer, including breast
carcinoma.10 To our knowledge fibrin deposition has not been
examined in prostate cancer, although elevated levels of tis-
sue factor have been reported.94 Therefore, this process may
significantly contribute to the formation of reactive stroma in
prostate cancer.
Tumor-associated macrophages. Considering the similari-
ties of the cancer reactive stroma microenvironment and
granulation tissue, the recruitment of macrophages to the
tumor would be expected. In fact, it has been reported that
the macrophage density is significantly higher in malignant
prostate tissue than in adjacent benign areas.95 The che-
moattraction of macrophages is likely due to fibrin deposits
and fragments of collagen and elastin in the extracellular
matrix.96 In addition, expression of macrophage recruitment
factors, such as monocyte chemotactic protein-1, VEGF and
TGF-␤, have been observed in cancer and stromal cells.97, 98
Tumor associated macrophages have multiple functions
that may inhibit or promote tumor growth. For example, they
can release cytotoxic molecules and present tumor associated
antigens to other components of the immune system to elim-
inate tumor cells.97 However, macrophage functions associ-
ated with wound repair may aid cancer progression. Tumor
associated macrophages have been shown to secrete numer-
ous growth factors that induce angiogenesis and may pro-
mote tumor growth.99 Also, it has been reported that tumor
associated macrophages release urokinase plasminogen acti-
vator and MMPs, and show procoagulant activity to initiate
fibrin deposition. The balance of these activities determines
how they influence tumor growth.98 As may be predicted,
evidence suggests that cancer cells secrete factors that redi-
rect tumor associated macrophage activities to promote tu-
2478 REACTIVE STROMA IN PROSTATE CANCER PROGRESSION
mor progression.97 Thus, tumor associated macrophages are
another key component of reactive stroma in cancer.
Reactive stroma promotes cancer progression in vivo. It has
been proposed that a reactive stroma environment similar to
granulation tissue may favor tumorigenesis. To investigate
this hypothesis experimentally, fragments of rat colon carci-
noma tissue were implanted into undisturbed rat subcutane-
ous tissue or into experimentally, induced granulation tis-
sue.100 Carcinoma cells transplanted into undisturbed
subcutaneous tissue “were arranged in compact groups with
a strong tendency to form acini.”100 In contrast, carcinoma
cells transplanted into granulation tissue were widely dis-
persed and showed an invasive morphotype, including an
elongated shape with extensive tumor cell protrusions at the
ultrastructural level. Tumor growth was not measured, al-
though these results suggest that granulation tissue en-
hances cancer cell invasion.
The synthesis of extracellular matrix components, growth
factors and proteases by tumor associated stromal cells im-
plies that these cells are important for creating the reactive
stroma environment. However, it does not directly demon-
strate their role in promoting tumor growth. Recent studies
have examined this issue by combining stromal cells with trans-
formed epithelium in models of tumorigenesis. Elenbaas et al
reported that co-injection of primary human fibroblasts with
primary transformed human mammary epithelial cells en-
hances tumor formation and accelerates tumor growth in nude
mice.101 Although primary human mammary epithelial cells
transformed with the 3 oncogenes formed tumors when injected
subcutaneously, the incidence was only 52%. When these cells
were mixed with normal human mammary fibroblasts, tumors
formed at 100% of the injection sites, and average time to the
development of palpable tumors was reduced from 52 to 20
days. These data suggest that while oncogene expression is
critical for tumor formation, the stromal microenvironment can
further promote tumorigenesis.
Another study used carcinoma associated fibroblasts iso-
lated from human prostate cancer tissue and human prostate
epithelial cells immortalized with SV40 large T antigen.102 In
vivo recombination of SV40 large T antigen with human
prostate carcinoma associated fibroblasts in athymic rodent
hosts resulted in the rapid growth of tumors that resembled
poorly differentiated adenocarcinoma. Interestingly normal
human prostate fibroblasts did not support SV40 large T
antigen tumor growth. This study indicates that normal
prostate stromal cells and stromal cells adjacent to prostate
carcinoma show fundamental differences in the cell pheno-
type. Notably carcinoma associated fibroblasts showed no
effect on normal epithelium, suggesting that epithelial cells
must sustain genetic alterations to initiate tumorigenicity.
It seems likely that the ability of stromal cells to support
tumor growth in vivo is related to their initial phenotype and
to the cancer cell ability to convert them to the activated
myofibroblast phenotype. For example, the study of Olumi et
al implies that carcinoma associated fibroblasts show a reac-
tive phenotype and, therefore, support the growth of SV40
large T antigen tumors.102 However, SV40 large T antigen
cells do not appear to induce normal human prostate fibro-
blasts to switch to the reactive phenotype since tumors did
not form when these cells were recombined in vivo. In con-
trast, transformed primary human mammary epithelial cells
in the study of Elenbaas et al appear to have induced normal
stromal cells to support tumor growth.101 Histological anal-
ysis of tumors generated by injecting transformed epithelium
revealed that mouse host stromal cells were recruited into
the tumors. Furthermore, co-injection of transformed epithe-
lium with normal human mammary fibroblasts significantly
promoted tumorigenesis.
This theory may also explain the differential support of
LNCaP tumor growth that has been observed in different
fibroblast lines. LNCaP cells are considered stromal depen-
dent since co-injection of Matrigel extracellular matrix or
stromal cells is required to form subcutaneous tumors in
nude mice. However, not all stromal cell lines support LN-
CaP tumor growth. LNCaP cells formed tumors when co-
injected with fibroblasts derived from a human osteogenic
sarcoma or with fibroblasts derived from rat fetal urogenital
sinus mesenchyme.103 Conversely co-injection of normal rat
prostate stromal cells with LNCaP cells rarely supported
tumor growth, and normal adult human lung fibroblasts did
not support tumor growth at all.103 It may be that stromal
cells that supported tumor growth had an activated pheno-
type independent of interaction with LNCaP cells. However,
it is also possible that stromal cells that failed to support
LNCaP tumor growth were unable to create the reactive
stromal environment. Accordingly we predict that successful
tumorigenesis requires the ability of carcinoma to induce
phenotypic switching in adjacent stromal cells and the capa-
bility of activated stromal cells to create the reactive stromal
environment.
Together these studies suggest that human prostate cancer
is accompanied by the induction of reactive stroma, charac-
terized by stromal cell phenotypic switching, extracellular
matrix remodeling, altered growth factor and protease bio-
availability, increased angiogenesis and an influx of tumor
associated macrophages. As in the stromal response de-
scribed in breast and colon cancer, the reactive stroma envi-
ronment mimics that of granulation tissue in wound repair.
These conditions likely support cancer cell proliferation, can-
cer cell migration/invasion and stimulate angiogenesis to
promote tumorigenesis. Furthermore, these studies indicate
that cancer cells secrete factors that induce and regulate
reactive stroma.
POTENTIAL REGULATORS OF REACTIVE STROMA
Growth factors that regulate wound repair, namely TGF-␤,
fibroblast growth factor-2, PDGF and VEGF,9 are also likely
to modulate the reactive stroma environment in cancer. Ex-
pression of these growth factors is typically restricted to
stromal components but many cancer cells acquire the ability
to express them with time. It follows that over expression of
TGF-␤, PDGF, fibroblast growth factor-2 and VEGF by pros-
tate carcinoma cells may function to induce reactive stroma
and angiogenesis.
Transforming growth factor-␤. Transforming growth
factor-␤ is a multifunctional growth factor that regulates
several biological processes, including development, wound
healing and tumorigenesis.104, 105 Its release from degranu-
lating platelets initiates the stromal response during wound
repair. In fact, it has been shown that TGF-␤1 is sufficient to
induce the generation of granulation tissue and angiogenesis
in vivo.106, 107 Moreover, TGF-␤1 is the only growth factor
known to induce differentiation of fibroblasts to the myofi-
broblast phenotype.107, 108 Transforming growth factor-␤ has
also been shown to attract immune cells by chemotaxis and
stimulate extracellular matrix synthesis and remodeling.105
In many types of cancers TGF-␤ is over expressed by carci-
noma cells.22, 23, 109 Although it inhibits epithelial cell growth in
vitro, studies show that TGF-␤ over expression by prostate
cancer cells promotes tumor growth in vivo.110 It has been
shown that prostate cancer cells avoid TGF-␤ growth inhibition
by down-regulating TGF-␤ receptor expression.111–114 However,
the stroma maintains expression of these receptors and, there-
fore, it is still responsive to this growth factor.111 These obser-
vations suggest that TGF-␤ acts primarily on the stromal com-
partment during prostate cancer progression.
Considering the role of TGF-␤ in wound repair and the sim-
ilarities of granulation tissue and reactive stroma, it has been
proposed that TGF-␤ expression by carcinoma cells induces
reactive stroma in cancer.22–24, 47 Transforming growth
factor-␤1 has been shown to modulate the myodifferentiation of
 REACTIVE STROMA IN PROSTATE CANCER PROGRESSION
prostate fibroblasts29, 30 and mammary gland fibroblasts108 to a
myofibroblast phenotype. It has also been shown to induce the
synthesis of many extracellular matrix components in reactive
stroma, such as collagen types I and III, fibronectin, ED-A⫹ and
ED-B⫹ fibronectin, tenascin, thrombospondin and versi-
can.35, 55, 57, 106, 115 In addition, TGF-␤ up-regulated stromal cell
expression of other growth factors involved in tissue repair and
angiogenesis, including fibroblast growth factor-2, VEGF and
connective tissue growth factor.116 –119 Moreover, TGF-␤ may
function to suppress the immune system, possibly by inhibiting
the production of cytotoxic molecules by tumor associated mac-
rophages.97, 109
Studies of in vivo tumor models further suggest that
TGF-␤ over expression may support cancer progression by
acting through the stroma. For example, tumors formed by
human prostate cancer cells transfected to over express
TGF-␤1 showed elevated angiogenesis and an increased rate
of metastasis.120 In another study CHO cells transfected to
over express this growth factor formed significantly larger
tumors at a more rapid rate in nude mice compared with
control cells.121 Over production did not affect the prolifera-
tion rate of CHO cells in vitro, although it significantly in-
creased angiogenesis in vivo. The specificity of TGF-␤1 was
confirmed by co-injection of a neutralizing antibody that re-
duced tumor growth and angiogenesis to control levels. Since
angiogenesis is a key component of reactive stroma, these
studies imply that TGF-␤1 promotes tumor progression by
inducing reactive stroma.
Recent evidence suggests TGF-␤1 induction of reactive
stroma may be mediated by the expression of connective
tissue growth factor. Connective tissue growth factor expres-
sion was shown to be induced in fibroblasts after activation
with TGF-␤1117, and stimulated fibroblast proliferation, ex-
tracellular matrix synthesis and angiogenesis.122 In addition,
connective tissue growth factor antibodies or antisense oligo-
nucleotides blocked TGF-␤ induced collagen type I synthesis
in fibroblasts. Moreover, connective tissue growth factor and
TGF-␤ were found to be co-expressed at sites of wound repair,
fibrosis and in the reactive stroma of cancer.122, 123 In situ
hybridization for connective tissue growth factor and TGF-␤1
in mammary carcinomas showed that the former was ex-
pressed in stromal cells, while the latter was expressed in
cancer cells.123 These data further indicate that TGF-␤1 pro-
duced by carcinoma cells acts on the surrounding stromal
cells to induce reactive stroma.
Together these studies provide evidence that TGF-␤ affects
several aspects of reactive stroma that are likely to promote
cancer progression. As in its role in wound repair, TGF-␤
regulates stromal cell differentiation, induces stromal cell
expression of extracellular matrix components and growth
factors, and promotes angiogenesis to create a granulation
tissue-like environment. Accordingly it is predicted to be a
primary regulator of reactive stroma in cancer. However, it is
known that the effects of TGF-␤ can be modulated by the
context of other growth factors, such as PDGF, epidermal
growth factor and fibroblast growth factor-2.108, 124 Therefore,
the specific mechanisms by which TGF-␤ stimulates reactive
stroma may be complex and likely result through the inte-
grated actions of TGF-␤ and many other regulatory factors.
Other growth factors. PDGF has also been suggested as a
mediator of reactive stroma in cancer.22, 24 It is another com-
ponent of the wound repair pathway that stimulates fibro-
blast and smooth muscle cell proliferation and migration. It
also induces the production of extracellular matrix compo-
nents, growth factors and proteases.125 Human melanoma
cells transfected to over express platelet derived growth
factor-BB produced tumors in nude mice that contained an
abundance of connective tissue and blood vessels.126 In con-
trast, melanoma cells lacking PDGF expression formed tu-
mors that were poorly vascularized and necrotic with no
connective tissue. The role of PDGF is not well characterized
2479
in prostate cancer, but PDGF-A and PDGF receptor ␣ were
expressed by epithelial and stromal cells in prostatic intra-
epithelial neoplasia.127 In addition, the human prostate can-
cer cell lines DU145 and PC-3 express PDGF,128 and PDGF
stimulated human prostate stromal cell proliferation in
vitro.129 Thus, PDGF may be predicted to promote reactive
stroma in the prostate.
As discussed, the recent study of Giri et al indicated that
the elevated level of fibroblast growth factor-2 in human
prostate cancer was “due to induction of stromal FGF-2 pro-
duction.”58 However, it was also suggested that carcinoma
cells in more advanced prostate cancers over express fibro-
blast growth factor-2. For example, the human prostate can-
cer cell lines DU145 and PC-3 have been shown to express
this growth factor, and it has been reported that prostate
cancer cells that metastasize to bone also express it.59 Fur-
thermore, studies of the Dunning rat model showed that
activation of fibroblast growth factor-2 expression accompa-
nied “progression of the epithelial cell to malignancy.”60 The
ability of carcinoma cells to express fibroblast growth factor-2
would be expected to stimulate angiogenesis and reactive
stroma, and may further promote cancer cell proliferation
and invasion via an autocrine loop.
As described, over expression of VEGF by carcinoma cells
has been reported in many tumor types, including prostate82
and breast.32 It has been suggested that VEGF promotes
cancer progression in several ways.93 It has been shown to
increase microvascular permeability, which allows circulat-
ing plasma proteins to leak into the surrounding extracellu-
lar matrix. This leakage results in generation of a fibrin
matrix that attracts fibroblasts, macrophages and endothe-
lial cells to initiate the wound repair response and stimulate
angiogenesis. Also, VEGF stimulated endothelial cell prolif-
eration and migration to drive angiogenesis. These activities
suggest that VEGF has a significant role in the regulation of
reactive stroma in cancer.
PERSPECTIVES
Reactive stroma hypothesis. Based on an understanding of
the stromal wound repair response we can begin to outline a
working hypothesis describing the generation of reactive
stroma in response to prostate carcinoma. Initially carci-
nogenesis at the prostatic intraepithelial neoplasia stage
results in altered epithelial proliferation, over expression
of growth factors and disruption of the basement mem-
brane extracellular matrix. These processes produce a dis-
ruption in normal prostate tissue homeostasis, which is
considered a wound situation by the host. The stroma
reacts by activating fibroblasts to the myofibroblast phe-
notype to repair the tissue disorder. In effect, an endoge-
nous wound repair response has been initiated. The result
in prostate cancer is the generation of a reactive stroma
that accompanies proliferative carcinoma cells. Accord-
ingly this reactive stroma shows stromal cell phenotypic
switching, extracellular matrix remodeling, increased bio-
availability of growth factors and induced angiogenesis to
drive the repair process. As in any wound repair situation,
the microenvironment would be expected to be growth
promoting. Therefore, these alterations in the microenvi-
ronment during early cancer progression would be pre-
dicted to promote survival and proliferation of the carci-
noma cell, resulting in enhanced tumorigenesis.
Several potential factors may induce reactive stroma in
prostate cancer, although TGF-␤1 emerges as the most likely
candidate. It is clear that it has a fundamental role in the
wound repair response and it is over expressed by many
carcinoma cells, including those of the prostate. Further-
more, its action appears to be directed toward the stroma
since transforming growth factor-␤ receptor expression is
present in stromal cells but down-regulated in prostate car-
2480 REACTIVE STROMA IN PROSTATE CANCER PROGRESSION
cinoma cells.111 The expected outcome of TGF-␤1 action in
these conditions would be the induction of reactive stroma,
while prostate cancer cells escape TGF-␤ growth inhibitory
effects. It is likely that fibroblasts immediately adjacent to
epithelial acini are activated to switch to the reactive myofi-
broblast phenotype in response to carcinoma produced
TGF-␤1 This hypothesis is consistent with observations in
breast cancer reactive stroma27, 108 and indications that
transforming growth factor-␤1 regulates myodifferentiation
of prostate fibroblasts.29, 30 Carcinoma derived TGF-␤1 would
also be expected to regulate the reactive stromal cell secre-
tion of extracellular matrix components, growth factors and
proteases to create a tumor promoting environment that
resembles granulation tissue. Therefore, it is proposed that
TGF-␤1 over expression by prostate cancer cells activates the
host wound repair pathway to generate reactive stroma,
which functions to promote tumorigenesis. This hypothesis
would predict that the carcinoma cell does not simply invade
the normal stroma. Rather it is likely that the developing
tumor actively recruits fibroblasts and myofibroblasts to gen-
erate its own stroma, which is a reactive stroma by nature of
the generic host response to wounding.
Progression to stromal independence. Unlike early stages of
prostate cancer there is an overall reduction in the amount of
stroma associated with advanced prostate tumors. Figure 3
shows that only thin strands of stroma are evident in poorly
(Gleason 4) compared with moderately differentiated (Glea-
son 3) carcinoma and normal tissue. These data suggest that
carcinoma cells have progressed to a stromal independent
stage. However, it may be that the tumor still requires com-
ponents normally secreted by stroma for progression. In this
scenario as the tumor overgrows the surrounding stroma,
data indicate that carcinoma cells begin to express factors
typically produced by stromal cells. For example, prostate
cancer cells over express VEGF and possibly fibroblast
growth factor-2, presumably to induce angiogenesis. These
factors are usually expressed in the stroma. Carcinoma cells
also express proteases, such as urokinase plasminogen acti-
vator and MMPs, which would enhance invasion and may
activate growth factors in the extracellular matrix. Cancer
cells may even secrete extracellular matrix components nor-
mally produced by stroma. Our reactive stroma hypothesis
suggests that as carcinoma acquires the ability to express
these factors, reactive stromal cells are no longer required to
support tumor growth. Accordingly, at this stage the tumor is
considered stromal cell independent. Thus, reactive stroma
may have key significance in affecting progression in early
compared with more advanced prostate cancer.
Diagnostics and therapeutics. Several studies have shown
a correlation of cancer progression with markers of reactive
stroma. For example, expression of extracellular matrix com-
ponents, such as tenascin-C45 and versican,50 have been as-
sociated with prostate cancer progression. It has also been
reported that angiogenesis correlates with prostate cancer
metastasis.130 In addition, pronounced fibrosis in colon can-
cer is associated with a poor prognosis.20, 131 We suggest that
analysis of reactive stroma induction may be a valuable tool
in prostate cancer diagnosis. Figure 3 shows that the reactive
stroma environment may differ among specimens assigned
the same Gleason score. Reactive stroma at some Gleason
grade 3 cancer sites contains a significant smooth muscle
component, while reactive stroma at other Gleason grade 3
sites has little smooth muscle (fig. 3, B and C). Thus, the
extent of the stromal response may identify aggressive tu-
mors and may potentially be used to predict the progression
rate in patients with the same Gleason score. This technique
would have clinical benefit since it is currently difficult to
predict the outcome of moderately differentiated prostate
tumors.
If reactive stroma promotes cancer progression as our hy-
pothesis predicts, it follows that the stromal compartment
may be a potential site for prostate cancer therapy. For
example, agents that block angiogenesis are receiving con-
siderable attention as potential therapeutics against solid
tumors. Other therapies may target the reactive stromal
myofibroblasts to alter the microenvironment. By inhibiting
production of stromal derived factors that promote cancer
progression or by stimulating the synthesis of stromal de-
rived inhibitors, the microenvironment may be modulated to
inhibit tumor growth. A potential advantage of this indirect
approach is that therapy would be directed toward a normal
host reactive stroma response rather than a carcinoma cell
with genomic instability and less biological predictability.
CONCLUSIONS
It is becoming more clear that reactive stroma is a general
feature of most types of carcinoma. Considerable evidence
suggests that stromal cells are activated by the carcinoma
cells and subsequently function to promote tumorigenesis.
Any disruption of tissue homeostasis, such as physical
wounding, microorganism invasion or carcinogenesis of
epithelial cells, is predicted to induce a generic repair
response that leads to stromal cell activation. Thus, it is
not surprising that the features of reactive stroma in can-
cer closely parallel the features of stromal granulation
tissue in wound healing responses. Changes in reactive
stroma include stromal cell phenotypic switching to acti-
vated myofibroblasts, altered extracellular matrix compo-
sition, increased growth factor bioavailability, elevated
protease activity, influx of tumor associated macrophages
and induction of angiogenesis. Together these alterations
create a reactive stroma microenvironment that is permis-
sive or promotive to carcinoma cell growth or survival and,
hence, supports tumor progression.
The concept of reactive stroma in prostate cancer is just
now emerging and preliminary data suggest that this stroma is
similar to the reactive stroma characterized in breast and colon
cancer. Our current data suggest that the myofibroblast is the
key cell type in human prostate cancer reactive stroma, al-
though the specific mechanisms that generate reactive stroma
in prostate cancer are less clear. Due to its role in stromal
responses in wound healing and the fact that it is over ex-
pressed in prostate cancer TGF-␤ may be the principal mediator
of reactive stroma in prostate cancer. Over expression of other
growth factors that function in stromal wound repair, such as
fibroblast growth factor-2, VEGF, and PDGF, suggests that
they are also potential regulators of the reactive stroma envi-
ronment in prostate cancer. Although reactive stroma is pre-
dicted to have a key role in early tumorigenesis, advanced
stages of prostate cancer are likely to evolve to stromal inde-
pendence. It is possible that the expression of certain markers of
reactive stroma may have clinical diagnostic value for providing
information in addition to Gleason score for assessing the pro-
gression potential of prostate cancer. Moreover, reactive stroma
may emerge as an additional compartment of cells to target for
novel therapeutic approaches. Since the evidence suggests that
reactive stroma may have a role in early progression, a thera-
peutic approach toward altering the tumor promotive feature of
reactive stroma may be a method of treating early prostate
cancer. Currently little is understood about the biology of reac-
tive stroma in prostate cancer. However, the many similarities
of reactive stroma in different types of carcinoma and reactive
stroma at wound repair sites suggest that the reactive stromal
environment has a fundamental and important role in promot-
ing prostate cancer progression.
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