Lung Cancer 45 Suppl. 2 (2004) S163–S175

www.elsevier.com/locate/lungcan

Tumour–stroma interaction:

cancer-associated fibroblasts as novel targets

in anti-cancer therapy?

Patrick Micke*, Arne Ostman

Ludwig Institute for Cancer Research, Uppsala, Sweden

KEYWORDS

Lung cancer;

Prostate cancer;

Stroma;

Cancer-associated

fibroblasts;

Tumour–stroma

interaction;

Reactive stroma;

Myofibroblasts;

Targeted therapy;

PDGF;

TGFb-1;

Laser microdissection

Summary Stroma cells, together with extracellular matrix components,

provide the microenvironment that is pivotal for cancer cell growth, invasion

and metastatic progression. Characteristic stroma alterations accompany or

even precede the malignant conversion of epithelial cells. Crucial in this

process are fibroblasts, also termed myofibroblasts or cancer-associated

fibroblasts (CAFs) that are located in the vicinity of the neoplastic epithelial

cells. They are able to modify the phenotype of the epithelial cells by

direct cell-to-cell contacts, through soluble factors or by modification

of extracellular matrix components. Seminal functional studies in various

cancer types, including breast, colon, prostate and lung cancer, have

confirmed the concept that fibroblasts can determine the fate of the

epithelial cell, since they are able to promote malignant conversion

as well as to revert tumour cells to a normal phenotype. This review

focuses on characteristic changes of fibroblasts in cancer and provides

the experimental background elucidating functional properties of CAFs in

the carcinogenic process. A possible implication in lung carcinogenesis

is emphasised. Finally, a laser-capture- and microarray-based approach

is presented, which comprehensively characterises carcinoma-associated

fibroblasts in their in vivo environment for the identification of potential

targets for anti-cancer therapy.

© 2004 Elsevier Science Ltd.

1. Introduction

The majority of human malignancies are carcinomas

that arise from the epithelium lining glands, ducts

and surfaces of organs. Consequently the focus

* Correspondence to: Patrick Micke MD. Ludwig Institute

for Cancer Research, Husargatan 3, Box 595, S-75124 Upp-

sala, Sweden.

Tel: +46-(18)-160-415; fax: +46-(18)-160-420.

E-mail: patrick.micke@licr.uu.se [website: www.licr.uu.se]

of research to date has been on the epithelial

cell type. Indeed, the knowledge of the patho-

genesis of cancer development and progression

has advanced tremendously over the last decades.

Through the process of identification and character-

isation of oncogenes and tumour-suppressor genes,

it has become accepted that cancer is a disease due

to genetic alterations. However, there is growing

evidence that carcinogenesis is influenced and

0169-5002/$ – see front matter © 2004 Published by Elsevier Ireland Ltd.doi:10.1016/j.lungcan.2004.07.000

S164 P. Micke, A. Ostman

controlled by cellular interactions derived from a

complex relationship between stromal, epithelial,

and extracellular matrix (ECM) components. In

many human tumours the stromal microenviron-

ment is fundamentally different from the stroma of

the corresponding normal tissue. Studies in human

breast, lung, colon and prostate cancer have iden-

tified “reactive stroma” that is characterised by

modified ECM composition, increased microvessel

density, inflammatory cells and fibroblasts with

an “activated” phenotype [1]. These modified

fibroblasts, often termed myofibroblasts, reactive

stroma or cancer-associated fibroblasts (CAFs),

are considered to play a central role in the

complex process of tumour–stroma interaction and

consequently tumorigenesis. This review will focus

on this cell type and will (1) provide a description

of biological changes of fibroblasts during cancer

formation, (2) give examples for factors responsible

for these changes, (3) summarise seminal studies

evaluating the functional capacity of CAFs to

promote tumour development and progression

and its therapeutic potential, (4) discuss possible

implications in lung cancer, and (5) present a

laser-capture- and microarray-based approach for

characterisation of cancer-associated fibroblasts

and tumour cells in order to ultimately target CAFs

as drug targets.

2. Fibroblasts in tumour stroma

2.1. Myofibroblasts in inflammation and cancer

The concept that stroma may play a profound

role was first noted by pathologists who recog-

nised stromal changes during cancer formation.

The proliferation of fibroblasts, accompanied by

an accumulation of connective tissue, termed

“desmoplasia” [2], was regarded as a typical

feature in many solid tumours (Figure 1). A similar

morphological pattern of “reactive stroma” can

be found in wound healing. Indeed, proliferating

“activated” fibroblasts appeared not only to be

one of the key stroma features in a variety of

inflammatory conditions but also in cancer [3]. The

assumption of chronic inflammation being involved

in tumorigenesis was already postulated in 1863

by Virchow, who observed that irritants together

with ensuing inflammation enhance cell prolifera-

tion [4]. This hypothesis was later supported by the

striking similarities between the tissue reaction in

wound healing and cancer, concerning inflammatory

cells, blood vessels, connective tissue and the

“activated” fibroblasts [5]. The obvious difference,

that tissue repair in contrast to tumour growth

is usually self-limiting, leads to the concept of

Hematoxylineosin

α-smoothmuscle actin

PDGF receptor

a b

c d

e f

Fig. 1. Paraffin sections of human lung adenocarcinomas:

(a,b) Hematoxylin-eosin staining of two adenocarcinomas of the

lung. (c,d) Immunohistochemical staining of adenocarcinomas

using a monoclonal antibody against a-smooth muscle actin.

(e,f) Staining of the same specimens using a polyclonal antibody

to the PDGFb receptor.

tumours as “wounds that do not heal” [6]. In

effect, the diligent comparison of wound healing

with cancer helped to reconnoiter some of the

complex mechanisms of tumour–stroma interac-

tion. The proliferating, “activated” fibroblasts

in cancer were later identified as mesenchymal

cells with typical signs of smooth muscle dif-

ferentiation, an important cell type in wound

healing termed “myofibroblasts”. The designation

reflects the intermediate state between smooth

muscle cells and fibroblasts [7,8]. Myofibroblasts

and in particular their environmental interaction

were extensively analyzed in inflammatory models.

For instance, they participate in wound healing

through migration, proliferation and contraction

and provide critical factors regulating the patho-

logical processes [3]. Although the presence of

this cell type in cancer has been known for

decades, the interest in research has been weak

and consequently the knowledge of cancer-related

myofibroblasts is still fragmented. It should be

mentioned that myofibroblasts are not per se a

pathological cell type but are present in various

tissues under normal conditions (e.g. lung, brain,

prostate, breast, heart [3]).

2.2. Definition of tumour myofibroblasts

Although myofibroblasts possess many similarities

in their morphology and function independent

of their location, the difficulties to define

them exactly already reflects the heterogeneous

Tumour–stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy? S165

character regarding their biochemical repertoires

and properties. Myofibroblasts are morphologically

characterised by large spindle-shaped cells with

indented nuclei [3,7,9]. They possess contractile

filaments (stress fibers), a prominent rough endo-

plasmatic reticulum, intercellular gap junctions and

well-developed fibronexi (transmembrane complex

with intracellular actin, integrins and extracellular

fibronectin). Later the identification was facilitated

with the introduction of antibodies to components

of the cellular filament system [10]. In cancer,

myofibroblasts are normally defined by the concur-

rent expression of a-smooth muscle actin (a-SMA,

smooth muscle marker) and vimentin (mesenchymal

marker) [8,11,12]. The presence or absence of

other cellular filaments (e.g. lamin, desmin,

calponin, smooth muscle myosin, caldesmon) was

also used to characterise myofibroblasts more

clearly, but depending on the location and

pathological condition the expression pattern can

differ markedly [3,13,14].

2.3. Localisation of myofibroblasts in human

malignancies

Several studies have quantified myofibroblasts in

the reactive stroma of different cancer types.

In invasive breast cancer, myofibroblasts were

found in a much higher proportion than in in situ

carcinomas, and predominantly at the invasive

front [15,16]. Colon cancer myofibroblasts were

also preferentially located at the tumour–stroma

border [17]. In prostate cancer, tumour stroma is

typified by a myofibroblast and fibroblast pheno-

type with a striking reduction of differentiated

smooth muscle cells, the predominant cell type

in normal prostate stroma. Interestingly, reactive

changes were already seen in fibroblasts adjacent

to prostatic intraepithelial neoplasia [18]. A study

of endometrial cancer samples showed that nearly

all interstitial cells were myofibroblasts [19].

2.4. Origin of tumour myofibroblasts

Another topic of extensive investigation and

discussion is the origin of myofibroblasts. Different

lineages are suggested in tumour stroma. (1) Al-

ready existing fibroblasts in the tissue stroma

obviously appear to be a candidate for such

conversion, and several studies including “tumour

environment” assays favour this concept [20,21].

(2) On the other hand, vascular smooth muscle

cells and pericytes with striking similarity to

myofibroblasts brought up the hypothesis that

myofibroblasts may originate from the vascular

bed [22,23]. This is supported by the observation

that mural cells are carried along with endothelial

sprouting during angiogenesis [24,25]. (3) More re-

cent findings also suggest the possibility that the

epithelial tumour cells themselves can provide the

surrounding stroma after epithelial–mesenchymal

transition, a process that converts epithelial

cells into cells with mesenchymal features [26].

(4) Another study indicates a heterogeneous origin.

Using breast-cancer cell lines and corresponding

fibroblasts in in vitro and in vivo models,

it was demonstrated that tumour “fibroblasts”

are recruited from residential fibroblasts, to a

minor extent from vascular smooth muscle cells,

and very rarely from pericytes [9]. (5) Some

other authors propose that myofibroblasts are

recruited from circulating CD34-positive (marker

for haematopoietic progenitors) stem cells derived

from the bone marrow [27,28].

However, because of the fact that not all fibro-

blasts in the tumour stroma show smooth muscle

differentiation, and the uncertainty of their origin,

many authors prefer to comprise the heterogeneous

tumour fibroblast populations in the term “cancer-

associated fibroblasts” (CAFs [29]). It should be

emphasised that this cell population includes all

fibroblasts in the tumour stroma, regardless of

whether they exhibit the myofibroblastic phenotype

or not.

3. Signals involved in the recruitment

and differentiation of CAFs

One obvious question arises from the observations

made on the tumour stroma: Which are the

underlying mechanisms that lead to the activation

of stroma and, more precisely, to the conversion

and/or recruitment of fibroblasts that exhibit

myofibroblastic phenotype?

Again, corresponding to stromal reaction in

inflammation, molecular mechanisms involved in

the recruitment of CAFs include microvessel

injuries, direct cell–cell contacts and soluble fac-

tors, comprising hormones, cytokines, chemokines,

ECM proteins, proteinases, protease inhibitors and

growth factors. Various growth factors, such as

transforming growth factor b (TGFb), platelet-

derived growth factor (PDGF), insulin-like growth

factor I and II and granulocyte macrophage-colony

stimulating factor (GM-CSF) were suggested to take

part in the stroma conversion [1,3,30]. TGFb and

PDGF are often considered to be most important,

because they are key molecules in the wound-

healing process [12,31–34]. Since their effects are

best investigated in a variety of tumour–stroma

models, both are described in more detail.

S166 P. Micke, A. Ostman

3.1. TGFb and CAF recruitment

The cytokine TGFb exists in mammals in three

isoforms (TGFb-1, TGFb-2, TGFb-3) that generally

have different potencies, but similar properties

in vitro and in vivo. TGFb is secreted to the

ECM as a latent complex consisting of bioactive

TGFb, the latency-associated peptide and the

latent TGFb-binding protein-1. Activation of TGFbis regulated by proteases and other extracellular-

matrix proteins (e.g. plasmin, cathepsin, human

mast cell chymase, MMP2, MMP9, integrin avb6,

integrin avb8 and thrombospondin [35]). TGFbsignals through a heteromeric cell surface complex

of two transmembrane serine/threonine kinase

receptors (I and II) leading to activation of

downstream molecules, mainly SMADs and small

G-proteins (e.g. Rho, RhoA, Ras). Well-described

target genes are fibronectin, PAI-I, and cyclin-

dependent kinase inhibitors [36,37]. The responses

to TGFb are diverse and frequently depend on

the cellular context. TGFb effects can therefore

only be described here in a simplified and reduced

manner. TGFb is a potent inhibitor of epithelial

cell growth and migration [38,39]. It can stimulate

migration, proliferation and contractility of mes-

enchymal cells and it increases the production of

typical extracellular-matrix components, like col-

lagens, tenascin-C, fibronectin, thrombospondin,

osteopontin, osteonectin, elastin and protease

inhibitors [12,38,40–42]. Further, it is the only

growth factor that is able to directly transdifferen-

tiate fibroblasts to myofibroblasts, i.e. stimulation

by TGFb leads in vitro and in vivo to charac-

teristic morphological changes and up-regulation

of a-SMA [12,43,44]. However, TGFb reveals

antiproliferative properties after fibroblasts are

transformed to myofibroblasts [45,46]. Thus, under

these circumstances, TGFb is considered as a

predominantly cytodifferentiating rather than a

proliferating growth factor on fibroblasts [3].

The role of TGFb in cancer is complex and

often ambiguous. It is well known that TGFb, as

a negative regulator of epithelial cell growth, can

effectively inhibit migration and proliferation of

malignant cells in vivo and in vitro [47,48]. The

action as tumour suppressor is best illustrated by

the occurrence of inactivating mutations in genes

encoding the TGFb receptors and SMADs in colon,

gastric and pancreatic cancers [49–51].

With this background it is paradoxical that on

the other hand TGFb is positively implicated in

the development of many cancers with epithelial

origin [52,53]. This might be due to the fact

that cancer cells become resistant to the growth-

inhibitory effects of TGFb, either by defective

receptors or signaling pathways or by other mech-

anisms, although they generate abundant active

TGFb [54]. Relieved from TGFb growth constraints,

tumour cells can utilise the cytokine as a tumour-

promoting factor. The obviously dualistic effects

of TGFb can be explained at least partially by its

paracrine influence on the tumour environment.

The tumour-promoting effects of TGFb are

illustrated by an experiment that again highlights

the connection between wound healing and cancer,

and stresses the pivotal function of TGFb in

this process. Chicken were infected with the

Rous sarcoma virus, a retrovirus that transduces

the oncogene src. The oncogene alone, even

though active, was not sufficient to transform the

chicken cells. Surprisingly, tumours only occurred

at the site of injection and at distant sites by

simply wounding the chicken [55]. Subsequently,

the administration of TGFb was sufficient to induce

tumour formation in the infected hens without

wounding [56].

In tumour tissue, TGFb can be derived from

epithelial cells, from inflammatory cells or from

CAFs [57,58]. The in vitro findings that TGFbinduces myofibroblast transdifferentiation in cancer

were confirmed in prostate- and colon-cancer

mice models [44,45,59]. TGFb expression has

been observed in breast-, bladder-, prostate-,

pancreatic- and lung-cancer cells and was cor-

related to disease progression in several studies

[34,60–63]. Higher TGFb activity in tumours can

also be explained by the increased release of

active TGFb from its latent complex by matrix-

associated proteases that are commonly found

in the ECM in the tumour stroma [64]. In an

elegant study by Berking and coworkers [65],

22 melanoma cell lines were transfected with TGFbusing adenoviral vectors. In cell culture, most of

these cell lines were either inhibited or unaffected

by TGFb stimulation. After co-injection of TGFb-or control-infected cells together with fibroblasts

and matrix components (Matrigel), the stroma–

tumour ratio clearly increased in TGFb-transfected

tumours. After 39 days, TGFb-1 tumours were

1.7-fold larger and the number and size of lung

metastases were significantly increased. In parallel,

fibroblasts were transduced with the TGFb-1 gene

in an organotypic cell culture. The gene-expression

analysis demonstrated the induction of the a1 chain

of collagens XV and XVIII, osteonectin, tenascin,

PAI-I and -II, VEGF-A and TGFb-RII which mirrored

common expression patterns of melanoma stroma

in vivo [65].

In another study, a weak tumorigenic prostate

cancer cell line developed visible tumours in only

8% of mice, in contrast to 100% when the cells were

Tumour–stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy? S167

co-injected with prostate fibroblasts and matrigel.

These tumours were rich in stroma and showed

well-developed blood vessels [59]. In a following

study, TGFb was identified as the main factor

that promoted the striking differences between

the two tumours, including a switch of fibroblast

to myofibroblasts [44]. This study, in accordance

with previous observations [66,67], highlighted the

neoangiogenic properties of TGFb. This includes

direct and indirect (e.g. via VEGF induction) effects

on blood-vessel formation, another effect that

might explain tumour-growth promoting properties

of TGFb [68,69]. TGFb is also considered to bear pro-

metastatic functions. For instance, TGFb signaling

enhances the development of metastases of human

breast-cancer cells to the bone [70]. A very

recent study confirmed the dualistic action of

TGFb depending on the environment in vivo [71].

Transgenic mice, expressing the activated form

of the neu oncogene, spontaneously developed

mammary tumours accompanied by metastases in

the lung. When these mice were crossed with mice

bearing the activated form of TGFb receptor-I,

an increased frequency of lung metastasis, but

at the same time a prolonged latency of neu-

driven primary mammary tumour formation was

observed.

However, because of the complexity of TGFb func-

tions, all conclusions regarding its tumour-promoting

or -inhibiting effects should be interpreted with

caution. Referring to the current knowledge and

as a rough rule-of-thumb, TGFb acts tumour-

suppressive in the premalignant stages of tumori-

genesis but pro-oncogenic at later stages of cancer

leading to metastases [53].

3.2. Tumour–stroma interaction and

PDGF signaling

Another attractive candidate involved in stroma

remodeling is PDGF. Four PDGF polypeptide chains

have been identified, which generate five dimeric

PDGF isoforms: PDGF-AA, -AB, -BB, -CC and

-DD [72]. The isoforms exert their cellular effects

with different affinities through the tyrosine-kinase

a- and b-receptor, that homo- and hetero-dimerise

after ligand binding. The role of PDGF signaling

in cancer is well described since the discovery

that the transforming protein product of the v-sis

gene of the simian sarcoma virus is identical to

PDGF-B [73,74]. In general PDGF tumour-promoting

effects can be itemised in (1) autocrine, (2) stromal

and (3) angiogenic effects [75].

PDGF production and autocrine stimulation of

cancer growth is described for only a subset

of cancer types, e.g. glioblastomas and sarco-

mas [76–78]. Most solid tumours, while secreting

PDGF ligands, lack the expression of the corre-

sponding PDGF receptors, suggesting that PDGF

may act as a paracrine growth factor. Indeed,

PDGF-receptor expression is frequently (20–90%)

found in stroma fibroblasts (Figure 1) of most

solid tumours [77,79,80]. Since the PDGF ligands

are potent mitogens and chemoattractants for

mesenchymal cells [72] and are considered to

be involved in nearly all proliferative disorders

of fibroblastic origin [78], the implication in tu-

mour stroma formation becomes obvious. Although

PDGF is not capable of converting fibroblasts

into myofibroblasts directly, it indirectly, e.g.

through stimulation of TGFb release from stromal

macrophages or other stromal cells, promotes myo-

fibroblastic differentiation [12,43]. Taken together

with the observation that TGFb stimulation can lead

to PDGF-receptor expression in fibroblasts [81–84]

it can be hypothesised that PDGF mainly serves to

mobilise the target cells, rather than to transform

them [3].

Proof of the important role of PDGF in tumour–

stroma interaction has come from studies in which

PDGF-B was overexpressed in melanoma cells

that lack PDGF-receptor expression. These weakly

tumorigenic cells formed robust tumours after

subcutaneous injection into nude mice, with

distinct connective tissue and abundant blood

vessels [85]. In another study, a non-tumorigenic

PDGF-receptor-negative keratinocyte cell line was

stably transfected with PDGF-B [86]. Although the

in vitro growth remained unaltered, these cells

formed tumours after injection in nude mice.

The tumours were slowly growing and non-

invasive. In a recent study PDGF has been shown

to be essential for desmoplastic response in a

human xenograft breast-cancer model [87]. Human

oestrogen-dependent breast-cancer cells (MCF-7)

were usually weakly tumorigenic in ovariectomised

mice. After H-ras transfection the cells were

injected in nude mice and compared with wild-type

controls. Transfected tumours were desmoplastic

and comprised of 30% myofibroblasts. In order

to test whether PDGF is responsible for the

desmoplastic effects, H-ras-transfected cells with a

PDGF-A dominant negative mutant were generated.

The injected cells formed tumours, but without

clear desmoplastic response [87].

Another interesting impact of PDGF on stroma

function in tumours was discovered recently [88].

Solid tumours frequently demonstrate increased

interstitial fluid pressure (IFP, the pressure within

the connective tissue), which is one hindrance

for effective drug delivery [89]. The IFP is

S168 P. Micke, A. Ostman

considered to be controlled by fibroblasts through

the action of PDGF. Treatment of animals with

PDGF-receptor antagonists decreased the IFP in

tumours and increased the uptake of chemotoxic

agents. Consequently the anti-tumour efficiency

of the chemotherapy was much higher with the

addition of the PDGF-receptor inhibitor [88,90,91].

PDGF itself can induce the release of matrix

components and a pool of growth factors from

fibroblast, like IGF-I, HGF, bFGF and ET3 that

subsequently can also modulate the tumour growth

and/or the tumour environment [66,67,92,93,94].

In addition, PDGF is important for the recruitment

and differentiation of pericytes and is considered to

stabilise immature blood vessels and contribute to

their functional integrity [95,96], hence it may also

be important for tumour vascularisation [97,98].

4. Tumour-promoting effects of CAFs

Virchow’s hypothesis that chronic inflammation or

wounding can predispose to tumour development

has been confirmed clinically for a multitude of

tissues [99–101]. This concept is complemented by

the “seed and soil”-observation of Paget (1889),

who examined 735 breast-cancer patients and found

that the distribution of metastases is not random

but that there are clear organ preferences [102].

Paget proposed that tumour cells (“seeds”) were

randomly distributed by the blood stream, but

can only form metastatic deposits if they land in

appropriate territories (“soil”).

The experiment already mentioned in the context

of TGFb [55,56], demonstrating tumour develop-

ment only at sites of wounding in RSV-infected

chicken, fortifies such concept. The “activation”

of the stroma can also be induced by other

mechanisms than infection. Barcellos-Hoff and

colleagues [103] have shown that irradiation of

the stromal tissue promotes the tumorigenic

potential of non-irradiated mammary epithelial in

fat pads transplanted into mice [103]. Interestingly,

an examination of several independent human

cases with breast cancer identified chromosomal

rearrangements that were present in the stromal

cells but not present in the malignant carcinoma

cells [104]. Thus, aberrations in the stroma may

both precede and stimulate the development of

tumours.

4.1. Co-culture models of tumour–stroma

interaction

Focusing on the fibroblasts again, early ex-

periments, typically co-culture experiments with

embryonic or differentiated fibroblasts, illustrated

rather an inhibiting effect on tumour growth.

Co-cultivation of mesenchymal cells with mammary

carcinoma cells exhibits a more orderly histodif-

ferentiation [105,106]. Basal-cell carcinoma cells

grown in association with stromal cells demon-

strated an apparent loss of their malignant proper-

ties [107]. On the contrary, a variety of experiments

later showed a growth-stimulating effect [12], in

particular when tumour-derived fibroblasts were

used in co-culture experiments [108,109]. Although

co-culture studies are very useful tools for studying

effects under controlled conditions, they definitely

cannot simulate the complex in vivo situation.

Consequently in vivo models have been developed.

4.2. In vivo models of tumour–stroma interaction

The most commonly used model for characterising

the stromal effects is to simply mix stromal

cells (and/or stromal components) with epithelial

cells and co-inject them into animals. Camps and

coworkers [110] studied five weak tumorigenic

or non-tumour-forming human cancer cell lines

(prostate PC3, breast MDA436; bladder, cell strains

derived from ascites) inoculated into athymic mice.

The addition of fibroblasts (tumorigenic prostate

fibroblasts or neu transformed NIH-3T3) shortened

the latency and increased the frequency of

tumour formation [110]. Beside the tumour-growth

enhancing effects of fibroblasts, another study

revealed that the addition of fibroblasts to different

cancer cell lines (animal and human origin) can

impressively reduce the dose of cells needed

to form tumours [111]. Later the same group

co-inoculated weakly tumorigenic human prostate

cancer cells (LNCaP) and various non-tumorigenic

fibroblasts into mice. Apparently different fibro-

blasts induced tumour formation differentially

(with human bone fibroblasts (62%), embryonic

rat urogenital sinus mesenchymal cells (31%), rat

prostate fibroblasts (17%), NIH-3T3 (0%), normal rat

kidney (0%), human lung fibroblasts (0%) [112]).

Tumour growth was significantly reduced if normal

ovarian stroma cells were co-injected. Interestingly

the stromal cells were not able to long-term survive

in the xenograft, rather the cancer cells recruited

new stroma of murine origin [113].

Olumi and coworkers [108] used 4 different

combinations of normal fibroblasts and cancer-

associated fibroblasts with normal prostate epi-

thelium and initiated epithelium (SV-40T immor-

talised, non-tumorigenic) in collagen grafts. Only

the combination of CAFs with initiated prostate

epithelial cells revealed a dramatic increase

of the epithelial cell population when grafted

into mice [108]. Combining human transformed

Tumour–stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy? S169

mammary epithelium (SV40T-Antigen, telomerase

catalytic subunit or H-ras) with normal fibroblast

doubled the incidence of tumours and reduced

the tumour latency period in mice [114]. Using

a weak tumorigenic prostate cancer cell line in

mouse xenografts, the frequency of visible tumours

could be increased from 8% up to 100% when

Matrigel and primary prostate stromal cells were

added. Remarkably, only three of five stromal cells

isolated from human specimens could promote

tumour growth, whereas two of them had no

effects [59]. Taken together these studies support

the concept that fibroblasts and their protein

repertoire are involved in all stages of tumour

development including initiation, tumour growth,

local invasion and metastasis, but also can inhibit

tumour growth.

4.3. Ectopic expression of proteins derived

from CAFs

Another set-up to evaluate different effects in vitro

and in vivo is to ectopically express the desired

stromal factor in cancer cells that usually do

not express this molecule. Recently it could

be shown that only one single stromal factor

can be sufficient to initiate tumorigenesis. The

ECM protease stromelysin 1 (MMP3) was expressed

under the control of a tetracycline-regulated

promoter in a murine immortal mammary cancer

cell line epithelium. Activation of MMP3 after

injection in mammary fat pads of mice resulted

in the degradation of the basement membrane

and morphological changes in the epithelium.

Concomitantly with the alteration in the epi-

thelium, the underlying stroma transformed into

“reactive stroma” with typical ECM accumulation

and neovascularisation. Subsequently many of the

mice developed mammary hyperplasia, dysplasia or

cancer [115–117].

4.4. Transgenic models used to study tumour

stroma interaction

With the implementation of genetically modified

mice strains, effects of modifications or the

absence of single genes in the whole organism,

i.e. in the complete stromal tumour environment,

could be analyzed. Mice deficient for stromelysin 3

were found to have reduced tumorigenesis when

challenged with chemical carcinogens [118]. Fuku-

mura et al. [119] used transgenic mice that express

the green fluorescent protein (GFP) under the

control of the promoter for VEGF. Either after

implantation of solid tumours (syngenic mammary

and hepatocellular carcinoma) into GFP mice or

Fig. 2. Schematic drawing of CAF–cancer cell interaction.

Cancer cells activate surrounding fibroblasts and blood ves-

sels. Responding fibroblasts secrete tumour-promoting factors,

angiogenic factors and modulate the extracellular matrix.

Leaking blood vessels release components modulating the

tumour environment. Not included in this scenario are

immune cells that are also integral part of the complex

interaction. Abbreviations: TGFb, transforming growth factor b;PDGF, platelet-derived growth factor; bFGF, basic fibroblast

growth factor; HGF, hepatocyte growth factor; VEGF, vascular

endothelial growth factor, MMP, matrix metalloproteinase;

ET, endothelin; FN, fibronectin; IGF, insulin-like growth factor;

EGF, epidermal growth factor.

after crossing with a transgenic mouse strain

(transgene for the polyoma virus middle T onco-

gene) developing spontaneous mammary tumours,

fluorescent fibroblasts were observed surrounding

or infiltrating the tumour mass. Thus predominantly

the CAFs were the source of VEGF, the main inducer

of tumour angiogenesis in these mice [119].

The performed experiments revealed that dis-

tinct fibroblasts are able to either inhibit or

promote carcinogenesis. The effects were strongly

dependent on the type of fibroblasts, epithelial

cells or the combinations used. In this respect, CAFs

are considered to rather support tumour formation.

Possible tumour–stroma interactions are illustrated

schematically in Figure 2.

5. Role of CAFs in lung cancer

In accordance with the observation in other solid

tumours, there is evidence that CAFs have similar

tumorigenic potential in lung cancer. First of all, the

protein expression pattern of PDGF-A,B and TGFbindicates a paracrine action of these growth factors

on the tumour environment [120]. Indeed, the

PDGFb receptor is frequently expressed by CAFs in

non-small-cell lung cancer (NSCLC, Figure 1) but

not on tumour cells that express instead the ligand

S170 P. Micke, A. Ostman

PDGF-B [77,121,122]. Overexpression of PDGF-B

was associated with decreased survival [122]. In

53 human NSCLC samples TGFb-1 levels correlated

with angiogenesis, tumour progression and prog-

nosis [123]. Already the stroma proportion alone

was demonstrated to be an independent prognostic

factor in NSCLC [124]. In the connective tissue of

NSCLC the amount of hyaluronan, a polysaccharide

synthesised by lung fibroblasts and an important

ECM component, was negatively correlated with

patient prognosis [125]. The growth-factor receptor

c-Met and its ligand HGF are overexpressed in

myofibroblasts of small-sized adenocarcinomas,

indicating autocrine stromal growth stimulation

in the stromal compartment. Interestingly, sta-

tistical analysis revealed an independent impact

of c-Met expression in the stroma on survival

of these patients [126]. Co-culture models of

lung fibroblasts and NSCLC cells resembled the

observation obtained in other cancer types. For

instance, NSCLC cells induce the secretion of

angiogenic factors (e.g. bFGF, IL-8 [127]) as well

as matrix proteases (e.g. MMP11 [128]) in lung

fibroblasts. Comparable patterns were confirmed

with a more comprehensive method. Using cDNA

microarrays, the gene-expression pattern of fibro-

blasts co-cultured with lung cancer was analyzed.

Among up- and down-regulated genes classical

candidates for stroma tumour interaction were

identified (bFGF, IL-8; IGFBP, TIMP3, PAI1; IGFBP1,

IGFBP 3, IGFBP 5, u-PA [129]).

In small-cell lung cancer (SCLC), the ad-

dition of laminin or matrigel to SCLC cells

injected into nude mice dramatically increased

tumour growth [130]. Extracellular-matrix pro-

teins (laminin, tenascin, fibronectin) protect from

chemotherapy-induced apoptosis in SCLC lines.

In addition, the retrospective analysis of human

SCLC specimens revealed a significantly shorter

survival for patients having tumours with extensive

matrix formation [131]. The role of MMPs in lung

cancer and their ability to degrade basement

membrane and ECM have been investigated ex-

tensively [132–134]. These proteases are found to

be secreted by both tumour and stromal cells in

lung cancer. Elevated MMP expression has also been

identified as an independent negative predictor of

survival in SCLC [135].

6. Cancer-associated fibroblasts as new

targets of cancer therapy

With regard to accumulating evidence that the

tumour–host cross-talk affects the behaviour of

malignant cells, stromal therapy could emerge

Fig. 3. Strategies for targeting tumour–stroma interaction.

(a) Signals from CAFs that initiate or promote tumour growth,

invasion and metastases can be inhibited. (b) Signals from the

cancer cells that are responsible for the recruitment of CAFs

can be blocked and inhibit myofibroblastic differentiation or

angiogenesis. (c) CAF eradication leads to elimination of signals

in both directions and additionally abolishes CAF effects on other

stromal cells.

as a viable approach to cancer prevention and

intervention. It was shown that cancer-associated

fibroblasts – the signals influencing the stromal

cells and the opposite signals effecting the

fibroblasts – play a central role in this interaction.

Therefore targeting these mechanisms offers a

fundamental new direction and potential for future

cancer therapies. In principle, three main points of

attack are conceivable (Figure 3):

(1) Targeting the tumour signals that are responsi-

ble for the development of “adequate” tumour

fibroblasts.

(2) Targeting CAF signals that initiate and promote

tumour growth and facilitate invasion and

metastases.

(3) Eliminating the CAFs themselves to abolish

all interaction between the heterotypic cell

types.

Qualified candidates, like TGFb- or PDGF-signal

pathways, have been presented in this review. In

this regard, inhibitors against these molecules have

been developed that are now at different stages of

clinical trials. The small compound Imatinib (Glivec)

inhibits effectively the Abl tyrosine kinase, the re-

ceptor tyrosine kinase cKIT and the PDGF-receptor

tyrosine kinase. Imatinib has already been approved

for the treatment of different malignancies, in

which activating mutations of the tyrosine kinases

drive the disease [136]. Comparable components

blocking the TGFb-receptor I kinase (SBI-14352;

NPC 30345, LY364947) and antibodies against the

Tumour–stroma interaction: cancer-associated fibroblasts as novel targets in anti-cancer therapy? S171

receptor or the ligand are at the preclinical

development stage [137].

Except from clinical studies based on anti-

angiogenic approaches, only few lung-cancer trials

aimed at inhibiting tumour-promoting stroma–

tumour interaction have been published up to

now. Promising preclinical studies with synthetic

inhibitors of MMPs [132] have led to the initiation

of phase-II and phase-III trials in a variety of

malignancies. However, two large randomised trials

in SCLC failed to show any benefit of MMP inhibitor

treatment [138]. One explanation for failure might

be that these proteases simply have only minor

impact on this extremely aggressive disease. On the

other hand, the control of the extracellular matrix

is obviously very complex, and the inhibition of only

a subset of MMPs among almost 30 family members

is unlikely to have a significant impact on tumour

growth and spread, particularly in the advanced

disease setting.

A phase-I dose-escalation trial published re-

cently utilised humanised monoclonal antibodies

(sibrotuzumab) against the fibroblast activation

protein (FAP), which is expressed specifically on

activated fibroblasts. 26 patients (20 colorectal

cancers, 6 NSCLC) were treated with different

dosages of sibrotuzumab. Labeled sibrotuzumab

accumulated in the tumour masses, which was

confirmed by gamma camera images. No objective

response was seen after three months of treatment,

but two patients showed stable disease, one of

them for two years at time of publication [139].

With the background of the obvious heterogeneity

of tumour–stroma interaction, it is questionable

whether one single factor is the “magic bullet”

in the concert of tumorigenesis. Therefore it is

even more important to evaluate cancer in its

complex environment and elucidate fundamental

mechanisms. The fragmentary knowledge so far ask

for even more sophisticated and comprehensive

techniques to understand the potential of CAFs.

Combining new tools for isolation of homogeneous

cell populations and the advances of microarray

gene-expression analysis may help in this process.

7. A novel approach to characterise

cancer-associated fibroblasts

The aim of our current project is to combine laser-

capture microdissection with cDNA microarray anal-

yses to obtain a comprehensive characterisation of

differences between CAFs and normal fibroblasts. In

a pilot study, human samples of basal cell carcinoma

and normal skin from the same patients were

used for laser microdissection of CAFs and normal

Fig. 4. Laser microdissection of CAFs. Cells were microdissected

with the PALM Laser-MicroBeam System and catapulted into a lid

of a PCR tube. To improve morphology, PALMLiquid Coverglass

was applied [40].

fibroblasts (Figure 4 [140]). From each sample RNA

was extracted, amplified and labeled with Cy3- or

Cy5-modified nucleotides. The labeled RNA from

CAFs and normal fibroblasts was then competitively

hybridised on microarrays. The analysis revealed

415 up-regulated and 458 down-regulated genes.

Among these were genes involved in growth

regulation (amphiregulin, SDF-1, IGF-1, TGFb-3),

angiogenesis (angiopoietin 2) and matrix remodel-

ing (kallikrein-6, -10, -11, MMP-5, -11, TIMP-4). For

6 of the genes differential expression was confirmed

by quantitative real-time PCR in the original, non-

amplified material. The results confirmed that the

strategy is feasible and reliable. The approach is

now extended to different types of solid tumours

(breast, prostate, colon, lung cancer). Significantly

regulated genes in normal fibroblasts and CAFs

will subsequently provide two catalogues with

significantly up- and down-regulated genes. The

comparison of these lists will reveal differentially

regulated genes, and thus a unique gene signature

for both cell types (normal fibroblasts versus CAFs).

These explicit candidates will further be evaluated

in in vitro and in vivo studies to additionally

validate the functional relevance in tumour growth,

with the ultimate goal to utilise CAFs as targets for

novel anti-cancer therapies.

8. Conclusion

Carcinoma-associated fibroblasts present a central

cell type in carcinogenesis. Further in vitro and

in vivo studies aimed at understanding the complex

cross-talk between CAFs and cancer cells are clearly

warranted to exploit them as valid targets for novel

therapies.

S172 P. Micke, A. Ostman

Acknowledgement

We thank Dr. Mitsuhiro Ohshima for critical reading

of the manuscript.

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