Mammary carcinoma provides highly tumourigenic and invasive reactive stromal cells

Mammary carcinoma provides highly tumourigenic andinvasive reactive stromal cells

Mirco Galie�, Carlo Sorrentino1, Maura Montani2,Luigi Micossi2, Emma Di Carlo1, Tommaso D’Antuono1,Laura Calderan, Pasquina Marzola, Donatella Benati,Flavia Merigo, Fiorenza Orlando

3, Arianna Smorlesi

3,

Cristina Marchini2, Augusto Amici2 andAndrea Sbarbati

Department of Morphological and Biomedical Sciences, Section Anatomyand Histology, University of Verona, Verona, Italy, 1Department of Oncologyand Neuroscience, Section Surgical Pathology, University of Chieti,Chieti, Italy, 2Department of Molecular, Cellular and Animal Biology,Genetic Immunization laboratory, University of Camerino, Camerino, Italyand 3Department of INRCA Gerontology Research, Immunology Center,Ancona, Italy

�To whom correspondence should be addressed. Tel: þ39 045 8027265;Fax: þ39 045 8027163;Email: [email protected] may also be addressed to A.Amici. Tel: þ39 0737 403275;Fax: þ39 0737 636216;Email: [email protected]

The progression of a lesion to a carcinoma is dependent onthe engagement of ‘reactive stroma’ that provides struc-tural and vascular support for tumour growth and alsoleads to tissue reorganization and invasiveness. The com-position of reactive stroma closely resembles that of granu-lation tissue, and myofibroblasts are thought to play acritical role in driving the stromal reaction of invasivetumours as well as of physiological wound repair. In thepresent work, we established a myofibroblast-like cell line,named A17, from a mouse mammary carcinoma modelin which tumourigenesis is triggered in a single step bythe overexpression of HER-2/neu transgene in the epithe-lial compartment of mammary glands. We showed thatalthough they derived from a tumour of epithelial originand did not express HER-2/neu transgene, their subcuta-neous injection into the backs of syngeneic mice gave rise tosarcomatoid tumours which expressed alpha-smooth mus-cle actin at the invasive edge. The expression of cytokeratin14 suggested a myoepithelial origin but immunopheno-typical profile, invasive and neoangiogenic potential ofA17 cells and tumours showed many similarities with thereactive stroma that occurs in wound repair and in can-cerogenesis. Our results suggest that epithelial tumourshave the potential to develop highly tumourigenic andinvasive reactive stromal cells and our cell line representsa novel, effective model for studying epithelial-stromalinteraction and the role of myofibroblasts in tumourdevelopment.

Introduction

Breast cancer is believed to be driven by the transformation ofthe epithelial lineage of mammary tissue that carries out aprogram aimed at aberrant remodelling of the gland by cellproliferation and fibroblast recruitment. In recent years thecharacterization of stromal populations capable of promotingcarcinogenesis in in vivo or in vitro experimental systems(1–3) has changed ideas about the interaction between epithe-lium and stroma in tumour growth and invasion. The progres-sion of a lesion to carcinoma is dependent on the engagementof a ‘reactive stroma’ that provides structural and vascularsupport for tumour growth, and also leads to tissue reorgan-ization and invasiveness (4–6).

As the composition and ‘invasive’ behaviour of reactivestroma closely resemble that of granulation tissue, Dvorakeven defined a tumour as ‘a wound that never heals’ (7). Thefact that the microenvironment of experimentally inducedgranulation tissue stimulates the invasive progression oftrasplanted adenocarcinoma cells (8) demonstrates that simil-arities between tumours and granulation tissues are not onlymorphological. Like wound healing, tumour growth is depend-ent on the recruitment of stromal subpopulations that activelybreak up the anatomical constraints of a lesion and lead itsinvasive edge through surrounding tissues.

The main candidates for this role are myofibroblasts.They were initially described within granulation tissues asfibroblast-like cells endowed with a microfilamentous appar-atus (9,10) which they use to generate contracting forcesduring wound healing processes (11–13). A similar apparatushad previously been observed in cultured fibroblasts (14) anddescribed as including alpha-smooth muscle actin as an impor-tant functional component (12,15). Along with alpha-smoothmuscle actin (alpha-SMA), myofibroblasts express mesenchy-mal markers such as fibronectin and vimentin, and frequentlyappear rimmed by traces of collagen fibres (16). Desmin andcytokeratins are occasionally observed.

Myofibroblasts have also been described in fibrocontractivedisease (11) and tumours (4,17,18) such as cancer of thecolon (19), liver (20), lung (21), prostate (22), pancreas (23)and particularly in the stromal compartment of breastneoplasias (24). It was proposed that the appearance ofmyofibroblasts precedes the onset of invasion and contributesto tumour growth and progression (6). Their pro-invasiveactivity is dependent on the capacity to secrete extracellularmatrix degrading proteases (13,25) and to participate in thesynthesis of extracellular matrix components, which alterthe adhesive and migratory properties of epithelial cancercells (18).

In the present work we established a highly tumourigenicstromal cell line, named A17, from a model of mammarycarcinoma in which tumourigenesis is triggered in a single stepby the overexpression of HER-2/neu transgene in the epithelialcompartment of mammary glands (26). The expression of

Abbreviations: alpha-SMA, alpha-smooth muscle actin; bFGF, basicfibroblast growth factor; DCE-MRI, dynamic contrast enhanced magneticresonance imaging; EMT, epithelial–mesenchymal transition; fPV, fractionalplasma volume; IL-6, interleukin-6; Kps, endothelial transfer coefficient;PCNA, proliferating cell nuclear antigen; PDGF-A, platelet derived growthfactor-A; TGF-beta, transforming growth factor beta; VEGF, vascularendothelial growth factor.

Carcinogenesis vol.26 no.11 # Oxford University Press 2005; all rights reserved. 1868

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cytokeratin 14 suggests a myoepithelial origin, but the neoan-giogenic potential and the capability of A17 cells to generatemyofibroblasts at the invasive edge of the tumours arisingfrom their subcutaneous injection shared many similaritieswith the reactive stroma that occurs in wound repair and incancerogenesis.

Materials and methods

Animals

This study was carried out on FVB mice, line 233, which were transgenicfor the activated isoform of rat HER-2/neu oncogene (FVB/neuT233), pur-chased from Charles River Laboratories (Calco, Italy). The mice had been bredunder ‘pathogen-free’ conditions inside galvanized cages (4–6 mice per cage)at 20 � 1�C temperature and 50 � 1% humidity. The animals were exposed tocycles of 12 h light and 12 h dark and were fed with standard foodstuff(Nossen, Italy) and water ad libitum. The presence of transgene was routinelychecked by the Polymerase Chain Reaction on tail DNA using primers specificfor vector (sense: 50-ATCGGTGATGTCGGCGATAT-30) and MMTV pro-moter (anti-sense: 50-GTAACACAGGCAGATGTAGG-30) sequences.

Establishment of transgenic mammary carcinoma cell lines

Tumours surgically excised from 6-month-old transgenic mice were mincedwith scissors and lancet, seeded in tissue culture flasks in Dulbecco’s modifiedminimal essential medium (DMEM) þ 20% fetal bovine serum (FBS)(GIBCO) and incubated at 37�C in a humidified 5% CO2 atmosphere. Afterthe sprouting of cells from tissue fragments, the cultures were periodicallywashed briefly (1–3 min) with trypsin–EDTA to detach contaminating fibro-blasts without damage to epithelial areas. After several washings, 3–5 monthsafter plating, the resulting confluent epithelial monolayer was diluted to as fewas 5 cells/ml and split in a 96-well plate in order to obtain clones derived fromsingle cells. The colonies were subsequently subcloned and the cell linesobtained were established by several subculturing steps.

Experimental tumours

5 � 105 cells suspended in 0.1 ml of phosphate-buffered saline were subcuta-neously (s.c.) injected into the backs of 5–7 week-old female FVB/neuNT233mice. Tumour growth was followed and tumour size measured weekly usingcalipers. The animals underwent DCE-MRI and CEUS when the longestdiameter of their tumours reached 10–15 mm.

PCR

DNA extracted from BB1 and A17 cells was subjected to the polymerase chainreaction using primers specific for vector (sense: 50-ATCGGTGATGTCGGC-GATAT-30) and MMTV promoter (anti-sense: 50-GTAACACAGGCAGATG-TAGG-30) sequences.

Western immunoblotting

The semi-confluent cell layers were lysated directly into the flask usingboiling Sample Loading Buffer (50 mM Tris (pH 6.7), 2% SDS and 10%Glycerol); the mouse mammary gland was lysated using RIPA buffer [1%NP40, 0.5% Na-deoxycolic acid and 0.1% SDS in phosphate buffered saline(PBS)] with fresh added protease inhibitors. The lysates were passed severaltimes through a 22-gauge needle in order to shatter the DNA molecules.Lysates were quantified using the Lowry method and equal amounts (20 mg)of protein from each cell lysate were separated in an SDS–PAGE and electro-phoretically blotted in a nitrocellulose support (Hybond C, AmershamBioscience, Little Chalfont, UK). For the immunoblotting of the differentproteins the following acrylamide percentages and the following antibodieswere used:

BALB/c 3T3 fibroblasts were used as positive control for vimentinand alpha-SMA expression, as negative control for HER-2/neu, and c-metexpression.

The protein lysate of a mouse mammary gland was used as positive controlfor alpha-SMA and ck14 expression.

Immunofluorescent analysis

Cells grown to confluence on coverslips were fixed in ice-cold 100% methanolat �20�C for 5 min, and subsequently permeabilized in PBS containing0.1% Triton X-100 for 10 min. After incubation in blocking buffer (1% bovineserum albumin (BSA) in PBS) for 20 min, cells were incubated for 1 h at 37�Cwith primary antibodies against E-cadherin (Transduction LaboratoriesNewington, NH, USA), N-cadherin (Transduction Laboratories) and Vimentin(Chemicon International), diluted (1:500) in blocking buffer. After washing,the coverslips were incubated with fluorescein isothiocyanate-conjugated ratanti-mouse IgG2a monoclonal antibody (Pharmingen, San Diego, CA, USA) ata dilution of 1:100 for 1 h at 37�C and washed (three times) in excess PBS.Slides were mounted on Mowiol and the preparations were viewed using aLeica confocal TCS SP2 microscope.

Flow cytometry

The overexpression of p185HER2 on the cell surface was detected usingmonoclonal antibody c-neu Ab-4 (Oncogene Research Products, Boston,MA) and a secondary antibody, fluorescein-conjugated anti-mouse IgG(Calbiochem, Darmstadt, Germany) for indirect immunofluorescence staining.

Anti-b-galattosidase Ab-1 (Oncogene Research Products, Boston, MA) wasemployed as an isotypic control.

500 000 cells per sample were harvested by trypsin–EDTA solution, washedin PBS, and incubated with the primary antibody and then, after washing inPBSþ0.1% NaN3, with secondary antibodies for 1 h at 4�C.

In vitro TGF-b stimulation

BB1 and A17 cells were starved in serum-free medium for 24 h and then5 ng/ml of recombinant TGF-b 1 (R&D, Minneapolis, MN, USA) were added.At 3 and 7 days time points, cell cultures were analysed for morphologicaltransition and lysated for electrophoresis.

Wound healing assay

The confluent A17 and BB1 cell monolayers were wounded by scraping witha pipette tip and were monitored for wound closure after 4 and 24 h.

Histologic analysis

For histologic evaluation, tissue samples were fixed in 4% neutral bufferedformalin, embedded in paraffin, sectioned at 4 mm, and stained with H&E.

Immunohistochemistry

Paraffin-embedded sections were immunostained with anti-p185neu (SantaCruz Biotechnology), anti-cytokeratins 8/18 (Clone GP11; Progen Biotechnik,Heidelberg, Germany), anti-PCNA (clone PC10), anti-desmin (clone D33),anti-myoglobin and anti-alpha-SMA (clone 1A4) antibodies (all from DAKO).After washing, sections were overlaid with biotinylated goat anti-guinea pig,goat anti-rabbit and horse anti-mouse Ig (Vector Laboratories, Burlingame,CA) for 30 min. Unbound Ig was removed by washing and slides wereincubated with ABCcomplex/HRP (DAKO).

Acetone-fixed cryostat sections were immunostained with anti-bFGF, anti-VEGF and anti-PDGF-A (all from Santa Cruz Biotechnology), anti-IL6 (cloneMP5-20F3; BD Pharmingen), anti-endothelial cells (clone: mEC-13.324) (pro-vided by Dr A.Vecchi, Istituto Mario Negri, Milan, Italy) and anti-tenascin-C(clone: Mtn-12; Abcam, Cambridge, UK) antibodies. After washing, sectionswere overlaid with biotinylated goat anti-rat and goat anti-rabbit Ig (VectorLaboratories) for 30 min. Unbound Ig was removed by washing and slideswere incubated with ABComplex/AP.

Proteins assessed Acrylamide(%)

Primary ab Workingconcentration

Secondary ab

HER-2/neu 8 C-18 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1:1000 Anti-rabbit–HRPVimentin 10 Polyclonal ab AB1620 (Chemicon International,Temecula, CA, USA) 1:100 Horse anti-goat–HRPCytokeratincocktail (5/6/8/17/19)

10 Monoclonal ab clone MNF116 (Dako Cytomation, Glostrup, Denmark) 1:1000 Anti-mouse–HRP

Cytokeratin 14 10 Polyclonal antibody AF 64 (Covance, Berkley, CA, USA) 1:1000 Anti-mouse–HRPAlpha-SMA 10 Monoclonal antibody 1A4 (Dako Cytomation, Glostrup, Denmark)C-MET 6 Polyclonal ab SP260 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1:500 Anti-rabbit–HRPBeta-actin 10 Monoclonal MAB1501R (Chemicon International, Temecula, CA, USA) 1:1000 Anti-mouse–HRP

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For CD-31 immunostaining, tumours were fixed overnight in zincfixative. Endothelial cells were identified by immunohistochemical stainingfor platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD-31), usinga rat monoclonal antibody directed against mouse CD-31 (Pharmingen). Forimmunoperoxidase staining, sections were incubated in 3% hydrogen peroxideto block endogenous peroxidase activity. To prevent aspecific antibody bind-ing, sections were preincubated for 20 min in PBS (pH 7.4) containing 10%normal rabbit serum. Next, sections were incubated overnight at 4�C with theprimary antibody directed against CD-31 (dilution of 1:100). Sections wererinsed for 15 min with PBS, followed by incubation with biotinylated second-ary antibody for 45 min at room temperature. After washing with PBS, thebound antibody was visualized by the peroxidase ABC method usingdiaminobenzidine (Sigma Chemical, St Louis, MO) as the developer. Thesections were rinsed with distilled water and mounted in aqueous solutions.For the above immunohistochemical procedures, controls were performedby replacing the primary antibody with 10% non-immune serum. Furthercontrols were made by omitting the secondary antibody. Controls were alwaysnegative.

Analysis of immunostained tissue sections

The immunoreactivity of proliferating cell nuclear antigen (PCNA) was ratedby counting the number of positive cells/number of total cells under a micro-scope � 600 field (0.120 mm2 per field).

Vessel counts were performed at 400� in a 0.180 mm2/field. At leastthree samples (one sample per tumour growth area) and 10 randomly chosenfields per sample were evaluated. Results are expressed as mean � SD ofpositive vessels per field evaluated on cryostat sections by immunohisto-chemistry.

The expression of p185neu, cytokeratins 8/18, desmin, myoglobin and alpha-SMA and the expression of angiogenic factors were defined as absent (�),scarce (�), moderate (þ), frequent (þþ) or very frequent (þþþ) on formalin-fixed paraffin-embedded sections or on cryostat sections stained with thecorresponding antibody.

Double immunofluorescent staining

Acetone-fixed cryostat sections were washed for 5 min in PBS and incubatedfor 30 min with anti-p185neu (Santa Cruz Biotechnology). The slides were thenwashed in PBS for 5 min. Next, sections were incubated for 30 min withbiotinylated goat anti-rabbit Ig (Vector Laboratories), washed and incubatedwith Alexa Fluor 488 conjugated StreptAvidin (Molecular Probes, Eugene,OR) (1/800) for 20–30 min. After washing, sections were incubated for 30 minwith anti-endothelial cells (clone: mEC-13.324) (provided by Dr A.Vecchi),washed again and incubated for 30 min with biotinylated goat anti-rat Ig(Vector Laboratories). After washing, sections were incubated with AlexaFluor 594 conjugated StreptAvidin (Molecular Probes) (1/800) for 20–30 minand then washed. Cross-reaction between the first secondary antibody andAlexa Fluor 594 was prevented by saturation of all its binding sites with AlexaFluor 488. Slides were mounted with vectashield medium (Vector Laborator-ies) and examined with a Zeiss LSM 510 Meta laser scanning confocal micro-scope (Zeiss, Oberkochen, Germany).

DCE-MRI with Gd-DTPA-albumin

Six animals which had been s.c. injected with the A17 cell line and six animalsinjected with the BB1 cell line underwent Dynamic Contrast EnhancedMagnetic Resonance Imaging (DCE-MRI) exams using Gd-DTPA-albuminas the contrast agent.

The animals were anesthetized by the inhalation of a mixture of air and O2

which contained 0.5–1% halotane, and were placed in a prone position insidea 3.5 cm i.d. transmitter–receiver birdcage coil. Images were acquired usinga Biospec tomograph (Burker, Karlsruhe, Germany) equipped with a 4.7 T,33-cm bore horizontal magnet (Oxford, Oxford, UK). Coronal Spin Echo (SE)and transversal multislice, fast spin-echo T2w (RARE, TE ¼ 70 ms) imageswere acquired for tumour localization. Afterwards a dynamic series of 3D,transversal spoiled-gradient echo (SPGR) images were acquired with the fol-lowing parameters: repetition time (TR)/echo time (TE) ¼ 50/3.5, flip angle(a) ¼ 90�, matrix size 128 � 64 � 32, field-of-view (FOV) 5 � 2.5 � 3 cm3

(corresponding to 0.39 � 0.39 mm2 in-plane resolution and 0.94 mm slicethickness), number of acquisitions (NEX)¼ 1. The acquisition time for a single3D image was 104 s; a dynamic scan of 24 images was acquired with 30-sintervals between each image (total acquisition time 53 min). The contrastagent was injected in bolus during the interval between the first and the secondscan. A phantom containing 1 mM Gd-DTPA in saline was inserted in the fieldof view and used as an external reference standard in order to normalizepossible spectrometer drifts during the acquisition (27). The experimentalprotocol closely followed that previously described by Daldrup et al. (27),except that pre-contrast T1 values were measured using the IR-SnapShot Flashtechnique (28).

DCE-MRI with Gd-DTPA

With the aim of confirming the vascular differences between tumour histotypesobserved by means of DCE-MRI with macromolecular contrast agent(Gd-DTPA-albumin), two animals per histotype group underwent DCE-MRIwith Gd-DTPA as contrast agent, within the 24 h prior to contrast enhancedultrasound (CEUS) examination. After SE and transversal multislice, fastspin echo T2-weighted (RARE, TEeff ¼ 70 ms) images were acquiredfor tumour localization, a dynamic series of T1-weighted, spoiled GREimages was acquired with the following parameters: TR ¼ 65 ms, TE ¼ 3.8ms, a ¼ 90�, matrix size 128 � 64 zero filled at 256 � 256, FOV ¼ 5 �2.5 cm2, space resolution ¼ 391 � 391 m2. A single slice (slice thickness ¼1.5 mm and slice separation ¼ 1.5 mm) was acquired at the tumour centre. Theacquisition time for a single image was 4.1 s and there was an interval of 1 sbetween successive images (time resolution ¼ 5.1). A total of 60 images wasacquired, 3 before and 57 after the contrast medium bolus injection; Gd-DTPAat 100 mmol/kg dosage was used. The dynamic evolution of the signal wasobserved for �5 min.

Statistics

The Student t-test was used to compare differences between BB1 and A17microvessel density. The statistical significance of differences in fractionalplasma volume (fPV) and endothelial transfer coefficient (Kps) was evaluatedby univariate ANOVA analysis. P 5 0.005 was adopted as the significancecut-off.

Results

Immunocytochemical characterization of FVB/neuT233 cell lines

From FVB/neuT233 spontaneous mammary tumours we estab-lished several epithelial-shaped cell lines, expressing the trans-gene to a different extent, and one spindle-shaped cell line,named A17, not expressing the transgene product (p185neu)(Figure 1). In order to investigate the histogenesis of thisphenotypical divergence, we comparatively characterized theimmunophenotypical and tumourigenic features of A17 cellswith those of one epithelial cell line, named BB1, and withthose of the parental spontaneous tumour.

The PCR analysis performed using primers specific for thevector and MMTV promoter sequences demonstrated thepresence of transgene in both clones. A17, but not BB1 cells,exhibited adhesion-independent growth (data not shown). BB1cells were negative for vimentin, N-cadherin, cytokeratin 14and alpha-SMA, but positive for E-cadherin, and C-MET.A17 cells were positive for vimentin, N-cadherin, cytokeratin14 and c-met, but negative for E-cadherin and alpha-SMA(Figure 1d and e).

The stimulation with TGF-beta did not induce epithelialmesenchymal transition of BB1 cells and failed to upregulatethe expression of alpha-SMA by A17 cells in culture (data notshown).

Tumourigenicity

When s.c. injected into the backs of animals, the BB1 and A17cells gave rise to tumours growing with a short latency time asunifocal masses composed of one or more lobes. The histo-logical and immunohistochemical profiles of BB1 and A17tumours were analysed in comparison with that of the parentalFVB/neuT233 tumour.

Mammary tumours which had spontaneously arisen in trans-genic animals appeared as multifocal carcinomas with lobular,lobulo–ductal and sometimes papillary features (Figure 2a).Edematous and hemorrhagic areas were frequently observedinside the tumour parenchyma.

The epithelial compartment of spontaneous tumoursexhibited heterogeneous staining with anti-p185neu antibody(Figure 2b and Table I). Some elements of lobular and ductu-lar parenchyma proved positive, others negative. The same

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heterogeneity was observed for proliferating activity assessedby PCNA immunostaining (Figure 2c and Table I).

The BB1 tumours showed the features of a lobular carcinomavery similar to the transgenic parental tumour. The tumour par-enchyma appeared to be formed of round to polygonal cells(Figure 2d), rich in haemorrhagic areas, and positive bothfor p185neu (Figure 2e and Table I) and PCNA (Figure 2f andTable I).

The s.c. injection of A17 cells gave rise to sarcomascomposed of spindle-shaped cells organized in a vorticoidfashion (Figure 2g), which were p185neu negative (Figure 2hand Table I) and expressed PCNA (Figure 2i and Table I). A17tumours were not encapsulated; the cutis of the animalsshowed ulcers within 6 weeks of s.c. cell injection. The prim-ary tumour mass frequently disappeared 10–12 weeks aftercell injection; but the animals showed evident symptoms ofpain, ulcers appeared on their tails and legs, and post-mortemanalysis revealed the occurrence of micrometastases or sec-ondary tumours in the liver and gut, indicating intraperitonealinvasion (Figure 3a and b).

Histological characterization

We performed an immunohistochemical analysis of spontan-eous and experimental tumours for epithelial (ck 8.18) andmyofibroblast markers (desmin, myoglobin, alpha-SMA).

As expected, spontaneous and BB1 tumours proved positivefor ck 8.18, which specifically stains luminal cells of the mam-mary gland, but did not appear reactive for desmin, myoglobinand alpha-SMA (Table 1).

A17 tumours were negative for ck 8.18 and for desmin andmyoglobin, but expressed alpha-SMA at the invasive edge ofparenchyma (Table I and Figure 3c and d), indicating a plasticpropensity to myofibroblast differentiation.

Ultrastructural analysis of A17 tumours revealed the pres-ence of collagen tracks surrounding spindle-shaped to poly-gonal neoplastic cells (Figure 3e).

Wound closure

The migration of the A17 cells, but not of the BB1 cells, intothe gap was evident within 4 h after scraping of confluent cellsheet. The wound closure of the A17 cells appeared complete24 h after scraping (Figure 3f ), whereas the gap of theBB1 partially reduced in size, probably due to the contributionof cell proliferation, and no sign of actively migrating cellwas evident.

Vascular analysis

Analysis of the vascularization of A17 and BB1 tumours wasperformed by integrating histological information, obtained byanti-CD-31 immunohistochemistry, and dynamic information,

Fig. 1. Immunocytochemical characterization of FVB/NeuT cell lines. From mammary tumours of FVB/NeuT233 transgenic mice we established a fibroblast-likecell line, named A17 (a), and an epithelial cell line, named BB1(b). Both of them imported HER-2/neu transgene into their genome, but the p185neu proteinwas expressed only by the BB1 cell line (c). BB1 cells proved positive for pan-cytokeratins and C-MET, negative for cytokeratin 14, vimentin and alpha-SMA;A17 cell expressed vimentin, pan-cytokeratins, cytokeratin 14 and C-MET, but not alpha-SMA (d). At immunofluorescent analysis (e) BB1 cells proved positivefor E-cadherin (I), and negative for N-cadherin (II) and vimentin (III); A17 cells proved negative for E-cadherin (IV), moderately positive for N-cadherin(V) and strongly reactive for vimentin (VI).


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obtained by dynamic contrast-enhanced magnetic resonanceimaging (DCE-MRI) with Gd-DTPA-albumin or Gd-DTPAcontrast agents (Figure 4).

CD-31

At CD-31 immunostaining analysis, A17 vasculature appearedto be composed of extremely numerous and homogeneously

distributed vessels. Vessels of BB1 tumours proved signifi-cantly fewer in number and were confined to the stromalcompartment of tumour parenchyma (Figure 4a–e and TableII). Spontaneous tumours proved significantly less vascular-ized than BB1 tumours (Table II).

A17 vessels frequently appeared very small (Figure 4c) andmorphologically immature (Figure 4d), whereas BB1 vesselsappeared larger and well developed (Figure 4e).

DCE-MRI

Contrast-enhanced MRI performed by injecting Gd-DTPA-albumin was used to evaluate the fPV and Kps of the tumourvascular network (Figure 4f).

The fPV is measured as the signal intensity increase imme-diately after contrast agent injection. The tumour parenchymaof A17 tumours showed significantly higher fPV than the BB1tumours. The Kps parameter is calculated on the basis of thekinetic increase of signal intensity after contrast agent injec-tion (Figure 4f ), and is a measure of vessel permeability.It proved significantly higher in A17 tumours than in BB1tumours.

Taking only the peripheral rim of tumour parenchyma intoconsideration, no significant differences were observedbetween the two cell clones either in the fPV or Kps parameter

Fig. 2. Histologic and immunohistochemical features of spontaneous and experimentally induced tumours in FVB/neuNT233 mice. Mammary carcinomas whichspontaneously developed in 6-month-old female FVB/neuNT233 mice (a) were constituted by r-p185neu expressing (b) and highly proliferating cell nuclearantigen (PCNA) positive (c) neoplastic cells. Experimental carcinomas which developed 15 days after s.c. injection of BB1 cells into syngeneic mice weremorphologically (d) and immunohistochemically similar (r-p185 and PCNA positivity) to the spontaneous tumours (c and d). On the contrary, tumours whichdeveloped 15 days after s.c. injection of A17 cells were formed mostly of highly proliferating (i), but r-p185neu– (h) spindle cells organised in vorticoid structures(g). (a: 200�; b–i: 400�)

Table I. Histological and immunohistochemical analyses of spontaneousmammary tumours and BB1 and A17 tumours experimentally inducedin FVB/neuNT233 micea

Mammary tumour BB1 A17

P185neu þþ þþ �PCNA 86 � 8%b 80 � 6%b 70 � 6%b

Ck 8.18 þ þ �Desmin � � �Myoglobin � � �c

Alpha-sma � � �c

aThe expression of the markers reported in this table was defined as absent(–), scarce (�), or moderately (þ) or frequently (þþ) expressed in formalin-fixed paraffin-embedded sections stained with the corresponding ab.bThe immunoreactivity of PCNA was rated by counting the number of positivecells/number of total cells under a microscope 600� field (0.120 mm2 per field).cPositive immunostaining was observed in some tumour cell subpopulations.

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(data not shown). It is reasonable to suppose that the analysisof the BB1 tumours’ periphery included the vessels of thecapsula, which are impossible to distinguish from tumourparenchyma in our experimental system.

The differences in tumour vascularization between A17 andBB1 tumours were confirmed by DCE-MRI using Gd-DTPAas a contrast agent (Figure 4g). The signal increase after con-trast agent injection was conspicuously and markedly higher inA17 than in BB1 tumours.

Expression of angiogenic factors

In order to investigate their neoangiogenic potential, theimmunohistochemical profile for proangiogenic factors wastraced for BB1 and A17 tumours and compared with that ofspontaneous tumours.

Basic fibroblast growth factor (bFGF) and interleukin-6(IL-6) were scarce in the stroma of spontaneous tumours, butwere moderately expressed in the stromal compartment ofBB1 tumours and very frequently expressed in A17 tumours(Figure 5a–f ). Platelet-derived growth factor-A (PDGF-A)was found in the delicate stroma surrounding spontaneousand BB1 tumours, and was frequently expressed in A17tumours (Figure 5g–i). Frequent expression of Tenascin-Cwas observed in the stromal compartment of spontaneous andBB1 tumours, while it was homogeneously distributed withinthe parenchyma of A17 tumours (Figure 5j–l).

Vascular endothelial growth factor (VEGF) proved to bemoderately expressed in spontaneous and in BB1 and A17tumours (Table II).

Discussion

Our results show that mammary tumours of epithelial originmay generate reactive stromal subpopulations exhibiting astrong invasive potential and the capacity to organize a com-plex neovascular network. The cytokeratin 14 expressionsuggested a myoepithelial origin of A17 cells, but the loss ofE-cadherin indicated a partial transdifferentiation towards amesenchymal phenotype. On the basis of their functional prop-erties, revealed by in vitro and in vivo experiments, these cells

Fig. 3. Metastatic and invasive behaviour of A17 cells. Sarcomatoid tumours which arose from s.c. injection of A17 cells into the backs of syngeneic mice werehighly invasive. Within 12–15 weeks of cell challenge, post-mortem analysis revealed intraperitoneal invasion and the presence of hepatic micrometastases(a and b).The invasive edge (c), but not the inner (d) of A17 tumours exhibited high expression of alpha-SMA. Ultrastructural analysis of A17 tumours frequentlyrevealed collagen fibres (e). At wound healing assay the migration of A17 cells into the gap (f, I) resulted within 4 h of scraping (f, II). The gap completelydisappeared within 24 h (f, III). The BB1 wound (f, IV) appeared to be reduced in size only after 24 h (f, V and VI), probably due to the contribution of cellproliferation, and no sign of active cell migration appeared evident.

Table II. Production of angiogenic factors and evaluation of microvesseldensity in spontaneous mammary tumours and BB1 and A17 tumoursexperimentally induced in FVB/neuNT233 mice

Mammary tumour BB1 A17

Angiogenic factorsa

bFGF þ/� þþ þþþIL-6 � þ þþþPDGF-A �� þþþVEGF þ/� þ/� þ

Microvessel densityb 9 � 3 14 � 3 32 � 6c

aThe expression of angiogenic factors was defined as absent (–), scarce (�), ormoderately (þ), frequently (þþ) or very frequently (þþþ) expressed incryostat sections stained with the corresponding ab.bVessel counts performed at 400� in a 0.180 mm2 field. At least 3 samples(1 sample/tumour growth area) and 10 randomly chosen fields/sample wereevaluated. Results are expressed as mean � SD of positive vessels/fieldevaluated on cryostat sections by immunohistochemistry.cValues significantly different (P � 0.005) from corresponding values ofmammary tumours which spontaneously developed in FVB/neuNT233 micefrom which the BB1 and A17 cell lines were derived.�Expression was detected only in the stroma surrounding tumour nests(see Figure 2).


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resembled stromal reactive cells of granulation tissue, whichexploit invasiveness and neoangiogenesis to repair woundsand reconstruct missing tissue. The ‘driving force’ of granula-tion is dependent on a cytoskeletal machinery composedmainly of alpha-SMA (12,15). A17 cells did not expressalpha-SMA when grown in culture, even upon stimulationwith TGF-beta, but frequently induced its production at theinvasive edge of tumours arising from their s.c. injection intoanimals. Thus, the in vivo microenvironment induces A17 cellsto commit the myofibroblast phenotype when it is needed forthe accomplishment of an ‘invasion program’. The generationof myofibroblasts at the invasive edge of A17 tumours couldbe due to a direct transdifferentiation of A17 cells or to theirlocal release of growth factors, such as TGF-beta, bFGF orPDGF, that induce myofibroblasts recruitment from the hoststroma.

The invasive phenotype of A17 cells underlay the expressionof proteins that have been described as produced by reactive

stromal cells both in physiological wound repair mechan-isms and in cancer cell invasion. Vimentin is essential for cellmotility and contraction mechanisms. Embryonic and adultvimentin-knockout mice exhibit impaired wound healingand their fibroblasts prove defective in mechanical stability,migratory and contractile capacity (30). Vimentin immunore-activity is occasionally observed in breast cancer and correl-ates with malignancy in node-negative ductal carcinomas (31).N-cadherin is involved in cell–cell adhesion and its expression,usually complementary to E-cadherin loss, is responsible forweakness of intracellular junctions. It proved to be critical forthe invasive phenotype of colon cancer-derived myofibroblastsin an in vitro wound healing assay (32). Exogenous expressionof N-cadherin and loss of E-cadherin induce epithelial cancercells to break down cell–cell and cell–matrix adhesiveness andto acquire motile phenotype (33, 34). C-MET has been foundin epithelial cells of a variety of organs and HGF/C-METsystem may mediate a signal exchange between mesenchyma

Fig. 4. Vascular analysis. At laser scanning confocal microscopic analysis, BB1 tumours appeared constituted by nests and lobules of r-p185neu positive tumourcells (green stained) which were surrounded by a delicate microvessel network (red stained) (a). In contrast microvessels (red stained) were numerous in A17tumours, which were formed by r-p185neu negative (green unstained) tumour cells (b). (a and b: 400�). The A17 tumour vasculature was frequently composed ofsmall and flattened vessels (c). Although the lumen of these vessels appeared compressed, the presence of pinocytotic vesicles proved the effectiveness of fluidflow (d, white arrow). Endothelial cells showed features of immaturity such as hypertrophic organelles, particularly polyribosomes (d, arrowhead). In contrast,BB1 vessels were large and well-developed (e). MRI with Gd-DTPA albumin contrast agent revealed that A17 tumours had both fPV and Kps higher than BB1tumours (f). The upper images represent time-course signal enhancement after contrast agent injection. The lower graphs show a quantification of fPV and Kpsparameters. The vascular differences between BB1 and A17 tumours were confirmed by MRI with Gd-DTPA contrast agent (g). The upper images in the ‘g’ panelshow maps of enhancement obtained by pixel by pixel subtraction between post- and pre-contrast agent injection images. The lower images show time-coursesignal intensity before (first three acquisitions) and after (from the 4th up to the 60th acquisition) contrast agent injection.

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and epithelia during mouse development (35). C-MET expres-sion has been described in myofibroblasts of the invasive areaof lung adenocarcinomas and is significantly associated withshortened survival, suggesting that the HGF/C-met systemconstitutes an autocrine activation loop in cancer–stromalmyofibroblasts (36). Signaling pathways triggered by auto-crine activation of C-MET receptors has recently beendescribed as inducing myofibroblasts to secrete pro-invasiveextracellular matrix components such as Tenascin-C (37).Tenascin-C is produced by stromal cells in carcinomas (38),including myofibroblasts of reactive stroma (39). It is impli-cated in the extracellular matrix remodeling that stimulatescancer cell growth and migration (40–42), and is involved inthe recruitment of stromal cells (43). Together with tenascin,PDGF and basic FGF (bFGF) play a key role in generating

a supportive microenvironment for ‘invasive tissues’, both inphysiological and pathological conditions. It has long beensuggested that PDGF is a major player in wound healing.It is a potent chemoattractant for fibroblasts migrating intothe healing wound, it enhances the proliferation of fibroblasts,induces these cells to secrete extracellular matrix and hasbeen observed to directly or indirectly promote fibroblast–myofibroblast differentiation (44–46). In tumours PDGF is apotent initiator of desmoplasia (47), and stromal activationinduced by PDGF has been reported to promote angiogenesisand tumourigenesis in cancers (48,49). The role of bFGF inpromoting stromal reaction and angiogenesis in several neo-plasias has been largely clarified (50). The stromal compart-ment provides neoangiogenic support for physiological tissueregeneration and for tumour development. Like that at the site

Fig. 5. Expression of angiogenic factors in spontaneous and experimental tumours in FVB/neuN T233 mice. Basic FGF (a–c), IL6 (d–f ) and PDGF-A (g–I) werefound to be scarce in the delicate stroma surrounding lobules of spontaneous tumours (left panels), moderately expressed in the stroma of BB1 tumours (centralpanels) and frequently and homogeneously expressed in A17 tumours (right panels). Tenascin-C was strongly expressed in the stroma of spontaneous ( j) and BB1(k) tumours, and in A17 tumours (l). (a–i, l: 400�; j, k: 630�).


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of wound repair, the reactive stroma of many cancers showsincreased microvessel density (4,17,18,40). bFGF, PDGF,VEGF, IL-6 and tenascin are partially responsible for thevascularization observed in A17 sarcomas and in the stromalcompartment of spontaneous and BB1 carcinomas, suggestingthat these molecules exert a potent autocrine and paracrineeffect on stromal cells and that A17 cells are a representativesubpopulation of reactive stroma.

Spindle-cell morphology occasionally occurs in humancancers (51). These tumours can appear as monophasic sarco-mas or exhibit a combination of epithelial, myoepithelial andmesenchymal traits, usually malignant in either case (52,53).Tumours belonging to the latter category are named with avariety of terms, such as ‘spindle cell carcinoma’, ‘carcinosar-coma’, ‘sarcomatoid carcinoma’ or ‘metaplastic carcinoma’or, when they are alpha-SMA positive, ‘myofibrosarcoma’ or‘myoepithelioma’ (54). However, there is no consistentnomenclature for these lesions in the literature, reflectinguncertainty about their histogenesis. Pathologists have pro-posed two opposing hypotheses. The multiclonal hypothesisproposes that epithelial and sarcomatoid populations derivefrom different stem tumour cells—epithelial and mesen-chymal, respectively; the monoclonal hypothesis proposesthat both histotypes derive from the same pluripotent stemcells. Several cytogenetic and immunohistochemical studiesstrongly supported the monoclonal hypothesis, also in cases inwhich sarcomatoid subpopulations did not show any epithelialcharacteristics (55–57).

An intriguing possibility is that the spindle-shaped cells inthese tumours derive from transdifferentiation of epithelialcells by epithelial–mesenchymal transition (EMT) (58).EMT is characterized by the loss of epithelial morphology,distruption of growth constraints, rearrangement of cell–matrixinteractions, and acquisition of a motile phenotype, and hasbeen widely studied in vitro. The EMT which occurs inaggressive carcinoma is the aberrant counterpart of a physio-logical programme which was originally identified in theembryonic development, and an analogous process has alsobeen described during wound healing in epithelial sheets,where some epithelial-committed cells need to convert toindividual migratory cells.

Several immunocytochemical aspects of A17 cells, suchas loss of E-cadherin, positivity for N-cadherin and vimentin,are associated with induction of EMT in in vitro systems(33,59). In breast cancer, epithelial cells may acquire myo-epithelial characteristics (alpha-SMA, cytokeratins) and,given the right conditions, may transdifferentiate into cellswith myofibroblast characteristics (alpha-SMA, cytokeratin-negativity). The epithelial–myoepithelial–mesenchymal trans-ition sequence has been hypothesized and supported by someexperimental evidence (5).

A large body of literature decribes the roles of TGF-beta intransdifferentiation of epithelial cells toward myofibroblasts(as reviewed in ref. 6). However, the failed mesenchymalconversion of BB1 cells upon TGF-beta stimulation appearedin accordance with a recent report which demonstrates thatTGF-beta-induced EMT is a rare event in vitro (60).

HGF/C-MET system is the EMT inducer which has beenstudied the most (61). A recent work (62) suggests thathypoxia can induce the acquisition of invasive phenotype bycarcinoma cells via C-MET overexpression. We observedc-met expression in BB1, sB7 and, to a lesser extent, in A17cells. So in poorly vascularized tumours prone to necrosis

such as our model, transdifferentiation should be particularlyfavoured. The moderate expression in A17 cells may beresponsible for self-mantaining mesenchymal differentiationby autocrine loop.

The role of Her-2/neu overexpression in mediating theinduction of EMT has been widely investigated but with con-tradictory results. In some breast tumour cell lines, Her-2/neuhas been described as promoting motile phenotype and invas-iveness (63), but in others as enhancing an epithelial morpho-logical organization (64). It has also been observed that theactivated isoform of HER-2/neu receptor, but not its wild-typecounterpart, can induce EMT phenotype in MDCK celllines (65).

However, the great tumour heterogeneity that is observedin vivo makes it impossible to recognize EMT withoutambiguity. The recent finding that EMT in human breast car-cinomas can provide a nonmalignant stroma (66) supportedthe evidence of the complexity of cellular organization intumours (67).

Our results demonstrate that mammary carcinomas, even ina highly simplified in vivo model, where tumour onset andgrowth are triggered by the hyperactivity of a single oncogenein the epithelial compartment of mammary glands, can providehighly tumourigenic, vasculogenic and invasive reactive stro-mal cells, which potentially play a critical role in epithelial-stroma neoplastic reorganization. The hybrid phenotype ofA17 suggests that myoepithelial cells and myofibroblasts intumours, not inevitably, represent two distinct populations.

The fact that we obtained highly tumourigenic mesenchy-mal cells from tumours of epithelial origin suggests that thepotential to develop carcinosarcomas is inherent in epithelialtumours. This evidence should alert us to the risk that therapiesagainst p185neu in carcinomas can trigger the invasive poten-tial of tumour subpopulations that in a ‘highly epithelial’neoplasm would remain latent.

The epithelial and stromal reactive cell lines that we estab-lished from the same tumour provide a novel and intriguingexperimental model for studying epithelial–stromal interac-tions in mammary carcinogenesis and for developing therapiesaddressed against what we, and others, consider to be the‘driving force’ of tumour invasion.

Acknowledgements

We thank Dr C.Boccaccio, who kindly gave us anti-C-MET antibody. Thestudy has been sponsored by a grant from Fondazione Cassa di Risparmio diVerona (Bando 2001 ‘Ambiente e sviluppo sostenibile’).

Conflict of Interest Statement: None declared.

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Received December 17, 2004; revised June 10, 2005; accepted June 15, 2005

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