Mammary carcinoma provides highly tumourigenic and invasive reactive stromal cells

Mirco Galiè *, Carlo Sorrentino 1, Maura Montani 2, Luigi Micossi 2, Emma Di Carlo 1, Tommaso D'Antuono 1, Laura Calderan, Pasquina Marzola, Donatella Benati, Flavia Merigo, Fiorenza Orlando 3, Arianna Smorlesi 3, Cristina Marchini 2, Augusto Amici 2 and Andrea Sbarbati

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

* To whom correspondence should be addressed. Tel: +39 045 8027265; Fax: +39 045 8027163; Email: mirco{at}anatomy.univr.it

Correspondence may also be addressed to A.Amici. Tel: +39 0737 403275; Fax: +39 0737 636216; Email: augusto.amici{at}unicam.it


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The progression of a lesion to a carcinoma is dependent on the engagement of ‘reactive stroma’ that provides structural and vascular support for tumour growth and also leads to tissue reorganization and invasiveness. The composition of reactive stroma closely resembles that of granulation tissue, and myofibroblasts are thought to play a critical role in driving the stromal reaction of invasive tumours as well as of physiological wound repair. In the present work, we established a myofibroblast-like cell line, named A17, from a mouse mammary carcinoma model in which tumourigenesis is triggered in a single step by the overexpression of HER-2/neu transgene in the epithelial compartment of mammary glands. We showed that although they derived from a tumour of epithelial origin and did not express HER-2/neu transgene, their subcutaneous injection into the backs of syngeneic mice gave rise to sarcomatoid tumours which expressed alpha-smooth muscle actin at the invasive edge. The expression of cytokeratin 14 suggested a myoepithelial origin but immunophenotypical profile, invasive and neoangiogenic potential of A17 cells and tumours showed many similarities with the reactive stroma that occurs in wound repair and in cancerogenesis. Our results suggest that epithelial tumours have the potential to develop highly tumourigenic and invasive reactive stromal cells and our cell line represents a novel, effective model for studying epithelial-stromal interaction and the role of myofibroblasts in tumour development.

Abbreviations: alpha-SMA, alpha-smooth muscle actin; bFGF, basic fibroblast growth factor; DCE-MRI, dynamic contrast enhanced magnetic resonance imaging; EMT, epithelial–mesenchymal transition; fPV, fractional plasma volume; IL-6, interleukin-6; Kps, endothelial transfer coefficient; PCNA, proliferating cell nuclear antigen; PDGF-A, platelet derived growth factor-A; TGF-beta, transforming growth factor beta; VEGF, vascular endothelial growth factor


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is believed to be driven by the transformation of the epithelial lineage of mammary tissue that carries out a program aimed at aberrant remodelling of the gland by cell proliferation and fibroblast recruitment. In recent years the characterization of stromal populations capable of promoting carcinogenesis in in vivo or in vitro experimental systems (13) has changed ideas about the interaction between epithelium and stroma in tumour growth and invasion. The progression of a lesion to carcinoma is dependent on the engagement of a ‘reactive stroma’ that provides structural and vascular support for tumour growth, and also leads to tissue reorganization and invasiveness (46).

As the composition and ‘invasive’ behaviour of reactive stroma closely resemble that of granulation tissue, Dvorak even defined a tumour as ‘a wound that never heals’ (7). The fact that the microenvironment of experimentally induced granulation tissue stimulates the invasive progression of trasplanted adenocarcinoma cells (8) demonstrates that similarities between tumours and granulation tissues are not only morphological. Like wound healing, tumour growth is dependent on the recruitment of stromal subpopulations that actively break up the anatomical constraints of a lesion and lead its invasive edge through surrounding tissues.

The main candidates for this role are myofibroblasts. They were initially described within granulation tissues as fibroblast-like cells endowed with a microfilamentous apparatus (9,10) which they use to generate contracting forces during wound healing processes (1113). A similar apparatus had previously been observed in cultured fibroblasts (14) and described as including alpha-smooth muscle actin as an important functional component (12,15). Along with alpha-smooth muscle actin (alpha-SMA), myofibroblasts express mesenchymal markers such as fibronectin and vimentin, and frequently appear rimmed by traces of collagen fibres (16). Desmin and cytokeratins are occasionally observed.

Myofibroblasts have also been described in fibrocontractive disease (11) and tumours (4,17,18) such as cancer of the colon (19), liver (20), lung (21), prostate (22), pancreas (23) and particularly in the stromal compartment of breast neoplasias (24). It was proposed that the appearance of myofibroblasts precedes the onset of invasion and contributes to tumour growth and progression (6). Their pro-invasive activity is dependent on the capacity to secrete extracellular matrix degrading proteases (13,25) and to participate in the synthesis of extracellular matrix components, which alter the adhesive and migratory properties of epithelial cancer cells (18).

In the present work we established a highly tumourigenic stromal cell line, named A17, from a model of mammary carcinoma in which tumourigenesis is triggered in a single step by the overexpression of HER-2/neu transgene in the epithelial compartment of mammary glands (26). The expression of cytokeratin 14 suggests a myoepithelial origin, but the neoangiogenic potential and the capability of A17 cells to generate myofibroblasts at the invasive edge of the tumours arising from their subcutaneous injection shared many similarities with the reactive stroma that occurs in wound repair and in cancerogenesis.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
This study was carried out on FVB mice, line 233, which were transgenic for the activated isoform of rat HER-2/neu oncogene (FVB/neuT233), purchased from Charles River Laboratories (Calco, Italy). The mice had been bred under ‘pathogen-free’ conditions inside galvanized cages (4–6 mice per cage) at 20 ± 1°C temperature and 50 ± 1% humidity. The animals were exposed to cycles 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 routinely checked by the Polymerase Chain Reaction on tail DNA using primers specific for vector (sense: 5'-ATCGGTGATGTCGGCGATAT-3') and MMTV promoter (anti-sense: 5'-GTAACACAGGCAGATGTAGG-3') sequences.

Establishment of transgenic mammary carcinoma cell lines
Tumours surgically excised from 6-month-old transgenic mice were minced with scissors and lancet, seeded in tissue culture flasks in Dulbecco's modified minimal essential medium (DMEM) + 20% fetal bovine serum (FBS) (GIBCO) and incubated at 37°C in a humidified 5% CO2 atmosphere. After the sprouting of cells from tissue fragments, the cultures were periodically washed briefly (1–3 min) with trypsin–EDTA to detach contaminating fibroblasts without damage to epithelial areas. After several washings, 3–5 months after plating, the resulting confluent epithelial monolayer was diluted to as few as 5 cells/ml and split in a 96-well plate in order to obtain clones derived from single cells. The colonies were subsequently subcloned and the cell lines obtained were established by several subculturing steps.

Experimental tumours
5 x 105 cells suspended in 0.1 ml of phosphate-buffered saline were subcutaneously (s.c.) injected into the backs of 5–7 week-old female FVB/neuNT233 mice. Tumour growth was followed and tumour size measured weekly using calipers. The animals underwent DCE-MRI and CEUS when the longest diameter of their tumours reached 10–15 mm.

PCR
DNA extracted from BB1 and A17 cells was subjected to the polymerase chain reaction using primers specific for vector (sense: 5'-ATCGGTGATGTCGGCGATAT-3') and MMTV promoter (anti-sense: 5'-GTAACACAGGCAGATGTAGG-3') sequences.

Western immunoblotting
The semi-confluent cell layers were lysated directly into the flask using boiling 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 several times through a 22-gauge needle in order to shatter the DNA molecules. Lysates were quantified using the Lowry method and equal amounts (20 µg) of protein from each cell lysate were separated in an SDS–PAGE and electrophoretically blotted in a nitrocellulose support (Hybond C, Amersham Bioscience, Little Chalfont, UK). For the immunoblotting of the different proteins the following acrylamide percentages and the following antibodies were used:
Proteins assessed

Acrylamide (%)

Primary ab

Working concentration

Secondary ab

HER-2/neu 8 C-18 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) 1:1000 Anti-rabbit–HRP
Vimentin 10 Polyclonal ab AB1620 (Chemicon International,Temecula, CA, USA) 1:100 Horse anti-goat–HRP
Cytokeratin cocktail (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–HRP
Alpha-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–HRP
Beta-actin 10 Monoclonal MAB1501R (Chemicon International, Temecula, CA, USA) 1:1000 Anti-mouse–HRP

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

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

Immunofluorescent analysis
Cells grown to confluence on coverslips were fixed in ice-cold 100% methanol at –20°C for 5 min, and subsequently permeabilized in PBS containing 0.1% Triton X-100 for 10 min. After incubation in blocking buffer (1% bovine serum albumin (BSA) in PBS) for 20 min, cells were incubated for 1 h at 37°C with primary antibodies against E-cadherin (Transduction Laboratories Newington, 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 rat anti-mouse IgG2a monoclonal antibody (Pharmingen, San Diego, CA, USA) at a 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 a Leica confocal TCS SP2 microscope.

Flow cytometry
The overexpression of p185HER2 on the cell surface was detected using monoclonal 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-ß-galattosidase Ab-1 (Oncogene Research Products, Boston, MA) was employed as an isotypic control.

500 000 cells per sample were harvested by trypsin–EDTA solution, washed in PBS, and incubated with the primary antibody and then, after washing in PBS+0.1% NaN3, with secondary antibodies for 1 h at 4°C.

In vitro TGF-ß stimulation
BB1 and A17 cells were starved in serum-free medium for 24 h and then 5 ng/ml of recombinant TGF-ß 1 (R&D, Minneapolis, MN, USA) were added. At 3 and 7 days time points, cell cultures were analysed for morphological transition and lysated for electrophoresis.

Wound healing assay
The confluent A17 and BB1 cell monolayers were wounded by scraping with a 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 buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with H&E.

Immunohistochemistry
Paraffin-embedded sections were immunostained with anti-p185neu (Santa Cruz 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 were incubated 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 (clone MP5-20F3; BD Pharmingen), anti-endothelial cells (clone: mEC-13.324) (provided by Dr A.Vecchi, Istituto Mario Negri, Milan, Italy) and anti-tenascin-C (clone: Mtn-12; Abcam, Cambridge, UK) antibodies. After washing, sections were overlaid with biotinylated goat anti-rat and goat anti-rabbit Ig (Vector Laboratories) for 30 min. Unbound Ig was removed by washing and slides were incubated with ABComplex/AP.

For CD-31 immunostaining, tumours were fixed overnight in zinc fixative. Endothelial cells were identified by immunohistochemical staining for platelet endothelial cell adhesion molecule-1 (PECAM-1 or CD-31), using a rat monoclonal antibody directed against mouse CD-31 (Pharmingen). For immunoperoxidase staining, sections were incubated in 3% hydrogen peroxide to block endogenous peroxidase activity. To prevent aspecific antibody binding, 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 the primary antibody directed against CD-31 (dilution of 1:100). Sections were rinsed for 15 min with PBS, followed by incubation with biotinylated secondary antibody for 45 min at room temperature. After washing with PBS, the bound antibody was visualized by the peroxidase ABC method using diaminobenzidine (Sigma Chemical, St Louis, MO) as the developer. The sections were rinsed with distilled water and mounted in aqueous solutions. For the above immunohistochemical procedures, controls were performed by replacing the primary antibody with 10% non-immune serum. Further controls were made by omitting the secondary antibody. Controls were always negative.

Analysis of immunostained tissue sections
The immunoreactivity of proliferating cell nuclear antigen (PCNA) was rated by counting the number of positive cells/number of total cells under a microscope x 600 field (0.120 mm2 per field).

Vessel counts were performed at 400x in a 0.180 mm2/field. At least three samples (one sample per tumour growth area) and 10 randomly chosen fields per sample were evaluated. Results are expressed as mean ± SD of positive vessels per field evaluated on cryostat sections by immunohistochemistry.

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

Double immunofluorescent staining
Acetone-fixed cryostat sections were washed for 5 min in PBS and incubated for 30 min with anti-p185neu (Santa Cruz Biotechnology). The slides were then washed in PBS for 5 min. Next, sections were incubated for 30 min with biotinylated goat anti-rabbit Ig (Vector Laboratories), washed and incubated with Alexa Fluor 488 conjugated StreptAvidin (Molecular Probes, Eugene, OR) (1/800) for 20–30 min. After washing, sections were incubated for 30 min with 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 Alexa Fluor 594 conjugated StreptAvidin (Molecular Probes) (1/800) for 20–30 min and then washed. Cross-reaction between the first secondary antibody and Alexa Fluor 594 was prevented by saturation of all its binding sites with Alexa Fluor 488. Slides were mounted with vectashield medium (Vector Laboratories) and examined with a Zeiss LSM 510 Meta laser scanning confocal microscope (Zeiss, Oberkochen, Germany).

DCE-MRI with Gd-DTPA-albumin
Six animals which had been s.c. injected with the A17 cell line and six animals injected with the BB1 cell line underwent Dynamic Contrast Enhanced Magnetic Resonance Imaging (DCE-MRI) exams using Gd-DTPA-albumin as 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 inside a 3.5 cm i.d. transmitter–receiver birdcage coil. Images were acquired using a 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 = 70 ms) images were acquired for tumour localization. Afterwards a dynamic series of 3D, transversal spoiled-gradient echo (SPGR) images were acquired with the following parameters: repetition time (TR)/echo time (TE) = 50/3.5, flip angle ({alpha}) = 90°, matrix size 128 x 64 x 32, field-of-view (FOV) 5 x 2.5 x 3 cm3 (corresponding to 0.39 x 0.39 mm2 in-plane resolution and 0.94 mm slice thickness), number of acquisitions (NEX) = 1. The acquisition time for a single 3D image was 104 s; a dynamic scan of 24 images was acquired with 30-s intervals between each image (total acquisition time 53 min). The contrast agent was injected in bolus during the interval between the first and the second scan. A phantom containing 1 mM Gd-DTPA in saline was inserted in the field of view and used as an external reference standard in order to normalize possible spectrometer drifts during the acquisition (27). The experimental protocol closely followed that previously described by Daldrup et al. (27), except that pre-contrast T1 values were measured using the IR-SnapShot Flash technique (28).

DCE-MRI with Gd-DTPA
With the aim of confirming the vascular differences between tumour histotypes observed by means of DCE-MRI with macromolecular contrast agent (Gd-DTPA-albumin), two animals per histotype group underwent DCE-MRI with Gd-DTPA as contrast agent, within the 24 h prior to contrast enhanced ultrasound (CEUS) examination. After SE and transversal multislice, fast spin echo T2-weighted (RARE, TEeff = 70 ms) images were acquired for tumour localization, a dynamic series of T1-weighted, spoiled GRE images was acquired with the following parameters: TR = 65 ms, TE = 3.8 ms, {alpha} = 90°, matrix size 128 x 64 zero filled at 256 x 256, FOV = 5 x 2.5 cm2, space resolution = 391 x 391 µ2. A single slice (slice thickness = 1.5 mm and slice separation = 1.5 mm) was acquired at the tumour centre. The acquisition time for a single image was 4.1 s and there was an interval of 1 s between successive images (time resolution = 5.1). A total of 60 images was acquired, 3 before and 57 after the contrast medium bolus injection; Gd-DTPA at 100 µmol/kg dosage was used. The dynamic evolution of the signal was observed for ~5 min.

Statistics
The Student t-test was used to compare differences between BB1 and A17 microvessel density. The statistical significance of differences in fractional plasma volume (fPV) and endothelial transfer coefficient (Kps) was evaluated by univariate ANOVA analysis. P < 0.005 was adopted as the significance cut-off.


    Results
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
Immunocytochemical characterization of FVB/neuT233 cell lines
From FVB/neuT233 spontaneous mammary tumours we established several epithelial-shaped cell lines, expressing the transgene 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 this phenotypical divergence, we comparatively characterized the immunophenotypical and tumourigenic features of A17 cells with those of one epithelial cell line, named BB1, and with those of the parental spontaneous tumour.



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Fig. 1. Immunocytochemical characterization of FVB/NeuT cell lines. From mammary tumours of FVB/NeuT233 transgenic mice we established a fibroblast-like cell 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 protein was 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 positive for 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).

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

The stimulation with TGF-beta did not induce epithelial mesenchymal transition of BB1 cells and failed to upregulate the expression of alpha-SMA by A17 cells in culture (data not shown).

Tumourigenicity
When s.c. injected into the backs of animals, the BB1 and A17 cells gave rise to tumours growing with a short latency time as unifocal masses composed of one or more lobes. The histological and immunohistochemical profiles of BB1 and A17 tumours were analysed in comparison with that of the parental FVB/neuT233 tumour.

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



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Fig. 2. Histologic and immunohistochemical features of spontaneous and experimentally induced tumours in FVB/neuNT233 mice. Mammary carcinomas which spontaneously developed in 6-month-old female FVB/neuNT233 mice (a) were constituted by r-p185neu expressing (b) and highly proliferating cell nuclear antigen (PCNA) positive (c) neoplastic cells. Experimental carcinomas which developed 15 days after s.c. injection of BB1 cells into syngeneic mice were morphologically (d) and immunohistochemically similar (r-p185 and PCNA positivity) to the spontaneous tumours (c and d). On the contrary, tumours which developed 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: 200x; b–i: 400x)

 
The epithelial compartment of spontaneous tumours exhibited heterogeneous staining with anti-p185neu antibody (Figure 2b and Table I). Some elements of lobular and ductular parenchyma proved positive, others negative. The same heterogeneity was observed for proliferating activity assessed by PCNA immunostaining (Figure 2c and Table I).


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Table I. Histological and immunohistochemical analyses of spontaneous mammary tumours and BB1 and A17 tumours experimentally induced in FVB/neuNT233 micea

 
The BB1 tumours showed the features of a lobular carcinoma very similar to the transgenic parental tumour. The tumour parenchyma appeared to be formed of round to polygonal cells (Figure 2d), rich in haemorrhagic areas, and positive both for p185neu (Figure 2e and Table I) and PCNA (Figure 2f and Table I).

The s.c. injection of A17 cells gave rise to sarcomas composed of spindle-shaped cells organized in a vorticoid fashion (Figure 2g), which were p185neu negative (Figure 2h and Table I) and expressed PCNA (Figure 2i and Table I). A17 tumours were not encapsulated; the cutis of the animals showed ulcers within 6 weeks of s.c. cell injection. The primary tumour mass frequently disappeared 10–12 weeks after cell injection; but the animals showed evident symptoms of pain, ulcers appeared on their tails and legs, and post-mortem analysis revealed the occurrence of micrometastases or secondary tumours in the liver and gut, indicating intraperitoneal invasion (Figure 3a and b).



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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 were highly 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 frequently revealed 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 completely disappeared 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 cell proliferation, and no sign of active cell migration appeared evident.

 
Histological characterization
We performed an immunohistochemical analysis of spontaneous and experimental tumours for epithelial (ck 8.18) and myofibroblast markers (desmin, myoglobin, alpha-SMA).

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

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

Ultrastructural analysis of A17 tumours revealed the presence of collagen tracks surrounding spindle-shaped to polygonal neoplastic cells (Figure 3e).

Wound closure
The migration of the A17 cells, but not of the BB1 cells, into the gap was evident within 4 h after scraping of confluent cell sheet. The wound closure of the A17 cells appeared complete 24 h after scraping (Figure 3f), whereas the gap of the BB1 partially reduced in size, probably due to the contribution of cell proliferation, and no sign of actively migrating cell was evident.

Vascular analysis
Analysis of the vascularization of A17 and BB1 tumours was performed by integrating histological information, obtained by anti-CD-31 immunohistochemistry, and dynamic information, obtained by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) with Gd-DTPA-albumin or Gd-DTPA contrast agents (Figure 4).



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Fig. 4. Vascular analysis. At laser scanning confocal microscopic analysis, BB1 tumours appeared constituted by nests and lobules of r-p185neu positive tumour cells (green stained) which were surrounded by a delicate microvessel network (red stained) (a). In contrast microvessels (red stained) were numerous in A17 tumours, which were formed by r-p185neu negative (green unstained) tumour cells (b). (a and b: 400x). The A17 tumour vasculature was frequently composed of small and flattened vessels (c). Although the lumen of these vessels appeared compressed, the presence of pinocytotic vesicles proved the effectiveness of fluid flow (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 BB1 tumours (f). The upper images represent time-course signal enhancement after contrast agent injection. The lower graphs show a quantification of fPV and Kps parameters. The vascular differences between BB1 and A17 tumours were confirmed by MRI with Gd-DTPA contrast agent (g). The upper images in the ‘g’ panel show maps of enhancement obtained by pixel by pixel subtraction between post- and pre-contrast agent injection images. The lower images show time-course signal intensity before (first three acquisitions) and after (from the 4th up to the 60th acquisition) contrast agent injection.

 
CD-31
At CD-31 immunostaining analysis, A17 vasculature appeared to be composed of extremely numerous and homogeneously distributed vessels. Vessels of BB1 tumours proved significantly fewer in number and were confined to the stromal compartment of tumour parenchyma (Figure 4a–e and Table II). Spontaneous tumours proved significantly less vascularized than BB1 tumours (Table II).


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

 
A17 vessels frequently appeared very small (Figure 4c) and morphologically immature (Figure 4d), whereas BB1 vessels appeared 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 tumour vascular network (Figure 4f).

The fPV is measured as the signal intensity increase immediately after contrast agent injection. The tumour parenchyma of A17 tumours showed significantly higher fPV than the BB1 tumours. The Kps parameter is calculated on the basis of the kinetic increase of signal intensity after contrast agent injection (Figure 4f), and is a measure of vessel permeability. It proved significantly higher in A17 tumours than in BB1 tumours.

Taking only the peripheral rim of tumour parenchyma into consideration, no significant differences were observed between the two cell clones either in the fPV or Kps parameter (data not shown). It is reasonable to suppose that the analysis of the BB1 tumours' periphery included the vessels of the capsula, which are impossible to distinguish from tumour parenchyma in our experimental system.

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

Expression of angiogenic factors
In order to investigate their neoangiogenic potential, the immunohistochemical profile for proangiogenic factors was traced for BB1 and A17 tumours and compared with that of spontaneous tumours.

Basic fibroblast growth factor (bFGF) and interleukin-6 (IL-6) were scarce in the stroma of spontaneous tumours, but were moderately expressed in the stromal compartment of BB1 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 spontaneous and BB1 tumours, and was frequently expressed in A17 tumours (Figure 5g–i). Frequent expression of Tenascin-C was observed in the stromal compartment of spontaneous and BB1 tumours, while it was homogeneously distributed within the parenchyma of A17 tumours (Figure 5j–l).



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Fig. 5. Expression of angiogenic factors in spontaneous and experimental tumours in FVB/neuN T233 mice. Basic FGF (ac), IL6 (df) and PDGF-A (gI) were found to be scarce in the delicate stroma surrounding lobules of spontaneous tumours (left panels), moderately expressed in the stroma of BB1 tumours (central panels) 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: 400x; j, k: 630x).

 
Vascular endothelial growth factor (VEGF) proved to be moderately expressed in spontaneous and in BB1 and A17 tumours (Table II).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Our results show that mammary tumours of epithelial origin may generate reactive stromal subpopulations exhibiting a strong invasive potential and the capacity to organize a complex neovascular network. The cytokeratin 14 expression suggested a myoepithelial origin of A17 cells, but the loss of E-cadherin indicated a partial transdifferentiation towards a mesenchymal phenotype. On the basis of their functional properties, revealed by in vitro and in vivo experiments, these cells resembled stromal reactive cells of granulation tissue, which exploit invasiveness and neoangiogenesis to repair wounds and reconstruct missing tissue. The ‘driving force’ of granulation is dependent on a cytoskeletal machinery composed mainly of alpha-SMA (12,15). A17 cells did not express alpha-SMA when grown in culture, even upon stimulation with TGF-beta, but frequently induced its production at the invasive edge of tumours arising from their s.c. injection into animals. Thus, the in vivo microenvironment induces A17 cells to commit the myofibroblast phenotype when it is needed for the accomplishment of an ‘invasion program’. The generation of myofibroblasts at the invasive edge of A17 tumours could be due to a direct transdifferentiation of A17 cells or to their local release of growth factors, such as TGF-beta, bFGF or PDGF, that induce myofibroblasts recruitment from the host stroma.

The invasive phenotype of A17 cells underlay the expression of proteins that have been described as produced by reactive stromal cells both in physiological wound repair mechanisms and in cancer cell invasion. Vimentin is essential for cell motility and contraction mechanisms. Embryonic and adult vimentin-knockout mice exhibit impaired wound healing and their fibroblasts prove defective in mechanical stability, migratory and contractile capacity (30). Vimentin immunoreactivity is occasionally observed in breast cancer and correlates 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 for weakness of intracellular junctions. It proved to be critical for the invasive phenotype of colon cancer-derived myofibroblasts in an in vitro wound healing assay (32). Exogenous expression of N-cadherin and loss of E-cadherin induce epithelial cancer cells to break down cell–cell and cell–matrix adhesiveness and to acquire motile phenotype (33.34). C-MET has been found in epithelial cells of a variety of organs and HGF/C-MET system may mediate a signal exchange between mesenchyma and epithelia during mouse development (35). C-MET expression has been described in myofibroblasts of the invasive area of lung adenocarcinomas and is significantly associated with shortened survival, suggesting that the HGF/C-met system constitutes an autocrine activation loop in cancer–stromal myofibroblasts (36). Signaling pathways triggered by autocrine activation of C-MET receptors has recently been described as inducing myofibroblasts to secrete pro-invasive extracellular 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 implicated in the extracellular matrix remodeling that stimulates cancer cell growth and migration (4042), and is involved in the 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 in physiological and pathological conditions. It has long been suggested that PDGF is a major player in wound healing. It is a potent chemoattractant for fibroblasts migrating into the healing wound, it enhances the proliferation of fibroblasts, induces these cells to secrete extracellular matrix and has been observed to directly or indirectly promote fibroblast–myofibroblast differentiation (4446). In tumours PDGF is a potent initiator of desmoplasia (47), and stromal activation induced by PDGF has been reported to promote angiogenesis and tumourigenesis in cancers (48,49). The role of bFGF in promoting stromal reaction and angiogenesis in several neoplasias has been largely clarified (50). The stromal compartment provides neoangiogenic support for physiological tissue regeneration and for tumour development. Like that at the site of wound repair, the reactive stroma of many cancers shows increased microvessel density (4,17,18,40). bFGF, PDGF, VEGF, IL-6 and tenascin are partially responsible for the vascularization observed in A17 sarcomas and in the stromal compartment of spontaneous and BB1 carcinomas, suggesting that these molecules exert a potent autocrine and paracrine effect on stromal cells and that A17 cells are a representative subpopulation of reactive stroma.

Spindle-cell morphology occasionally occurs in human cancers (51). These tumours can appear as monophasic sarcomas or exhibit a combination of epithelial, myoepithelial and mesenchymal traits, usually malignant in either case (52,53). Tumours belonging to the latter category are named with a variety of terms, such as ‘spindle cell carcinoma’, ‘carcinosarcoma’, ‘sarcomatoid carcinoma’ or ‘metaplastic carcinoma’ or, when they are alpha-SMA positive, ‘myofibrosarcoma’ or ‘myoepithelioma’ (54). However, there is no consistent nomenclature for these lesions in the literature, reflecting uncertainty about their histogenesis. Pathologists have proposed two opposing hypotheses. The multiclonal hypothesis proposes that epithelial and sarcomatoid populations derive from different stem tumour cells—epithelial and mesenchymal, respectively; the monoclonal hypothesis proposes that both histotypes derive from the same pluripotent stem cells. Several cytogenetic and immunohistochemical studies strongly supported the monoclonal hypothesis, also in cases in which sarcomatoid subpopulations did not show any epithelial characteristics (5557).

An intriguing possibility is that the spindle-shaped cells in these tumours derive from transdifferentiation of epithelial cells by epithelial–mesenchymal transition (EMT) (58). EMT is characterized by the loss of epithelial morphology, distruption of growth constraints, rearrangement of cell–matrix interactions, and acquisition of a motile phenotype, and has been widely studied in vitro. The EMT which occurs in aggressive carcinoma is the aberrant counterpart of a physiological programme which was originally identified in the embryonic development, and an analogous process has also been described during wound healing in epithelial sheets, where some epithelial-committed cells need to convert to individual migratory cells.

Several immunocytochemical aspects of A17 cells, such as 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 myoepithelial characteristics (alpha-SMA, cytokeratins) and, given the right conditions, may transdifferentiate into cells with myofibroblast characteristics (alpha-SMA, cytokeratin-negativity). The epithelial–myoepithelial–mesenchymal transition sequence has been hypothesized and supported by some experimental evidence (5).

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

HGF/C-MET system is the EMT inducer which has been studied the most (61). A recent work (62) suggests that hypoxia can induce the acquisition of invasive phenotype by carcinoma cells via C-MET overexpression. We observed c-met expression in BB1, sB7 and, to a lesser extent, in A17 cells. So in poorly vascularized tumours prone to necrosis such as our model, transdifferentiation should be particularly favoured. The moderate expression in A17 cells may be responsible for self-mantaining mesenchymal differentiation by autocrine loop.

The role of Her-2/neu overexpression in mediating the induction of EMT has been widely investigated but with contradictory results. In some breast tumour cell lines, Her-2/neu has been described as promoting motile phenotype and invasiveness (63), but in others as enhancing an epithelial morphological organization (64). It has also been observed that the activated isoform of HER-2/neu receptor, but not its wild-type counterpart, can induce EMT phenotype in MDCK cell lines (65).

However, the great tumour heterogeneity that is observed in vivo makes it impossible to recognize EMT without ambiguity. The recent finding that EMT in human breast carcinomas can provide a nonmalignant stroma (66) supported the evidence of the complexity of cellular organization in tumours (67).

Our results demonstrate that mammary carcinomas, even in a highly simplified in vivo model, where tumour onset and growth are triggered by the hyperactivity of a single oncogene in the epithelial compartment of mammary glands, can provide highly tumourigenic, vasculogenic and invasive reactive stromal cells, which potentially play a critical role in epithelial-stroma neoplastic reorganization. The hybrid phenotype of A17 suggests that myoepithelial cells and myofibroblasts in tumours, not inevitably, represent two distinct populations.

The fact that we obtained highly tumourigenic mesenchymal cells from tumours of epithelial origin suggests that the potential to develop carcinosarcomas is inherent in epithelial tumours. This evidence should alert us to the risk that therapies against p185neu in carcinomas can trigger the invasive potential of tumour subpopulations that in a ‘highly epithelial’ neoplasm would remain latent.

The epithelial and stromal reactive cell lines that we established from the same tumour provide a novel and intriguing experimental model for studying epithelial–stromal interactions in mammary carcinogenesis and for developing therapies addressed against what we, and others, consider to be the ‘driving force’ of tumour invasion.


    Acknowledgments
 
We thank Dr C.Boccaccio, who kindly gave us anti-C-MET antibody. The study has been sponsored by a grant from Fondazione Cassa di Risparmio di Verona (Bando 2001 ‘Ambiente e sviluppo sostenibile’). Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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