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Molecular and Cellular Probes 22 (2008) 83–89

www.elsevier.com/locate/ymcpr

Matrix metalloproteinase localisation by in situ-RT-PCR in archivalhuman breast biopsy material

Larisa M. Haupta,�, Rachel E. Irvinga, Stephen R. Weinsteinb,Michael G. Irvingc, Lyn R. Griffithsa,��

aGenomics Research Centre, School of Medical Science, Griffith University Gold Coast, PMB 50 Gold Coast Mail Centre, Qld. 9726, AustraliabDepartment of Pathology, Gold Coast Hospital, Southport, Qld., Australia

cInstitute of Health Sciences, Bond University, Qld. 4229, Australia

Received 10 April 2007; accepted 19 June 2007

Available online 4 July 2007

Abstract

Utilising archival human breast cancer biopsy material we examined the stromal/epithelial interactions of several matrix

metalloproteinases (MMPs) using in situ-RT-PCR (IS-RT-PCR). In breast cancer, the stromal/epithelial interactions that occur, and

the site of production of these proteases, are central to understanding their role in invasive and metastatic processes. We examined MT1-

MMP (MMP-14, membrane type-1-MMP), MMP-1 (interstitial collagenase) and MMP-3 (stromelysin-1) for their localisation profile in

progressive breast cancer biopsy material (poorly differentiated invasive breast carcinoma (PDIBC), invasive breast carcinomas (IBC)

and lymph node metastases (LNM)). Expression of MT1-MMP, MMP-1 and MMP-3 was observed in both the tumour epithelial and

surrounding stromal cells in most tissue sections examined. MT1-MMP expression was predominantly localised to the tumour

component in the pre-invasive lesions. MMP-1 gene expression was relatively well distributed between both tissue compartments, while

MMP-3 demonstrated highest expression levels in the stromal tissue surrounding the epithelial tumour cells. The results demonstrate the

ability to distinguish compartmental gene expression profiles using IS-RT-PCR. Further, we suggest a role for MT1-MMP in early

tumour progression, expression of MMP-1 during metastasis and focal expression pattern of MMP-3 in areas of expansion. These

expression profiles may provide markers for early breast cancer diagnoses and present potential therapeutic targets.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Matrix metalloproteinases; Breast; Cancer; Paraffin embedded; IS-RT-PCR; Human

1. Introduction

Histologically, breast cancers are classified according totheir estrogen receptor (ER) status and as invasive or non-invasive, dependent upon the types and patterns of cells thatcomprise them. Invasive carcinomas extend into the sur-rounding stroma, whilst in non-invasive carcinoma, tumourcells are confined to the epithelial cells of the ducts or lobules[1]. Invasive carcinomas can potentially spread duringthe process of metastasis. This process of intravastation from

e front matter r 2007 Elsevier Ltd. All rights reserved.

cp.2007.06.009

ing author. Present address: Institute of Molecular and

iopolis, Singapore, 138673, Singapore.

9709; fax: +65 6779 1117.

corresponded to.

ess: lhaupt@imcb.a-star.edu.sg (L.M. Haupt).

the primary lesion, avoidance of immune surveillance andextravastation at the secondary site defines metastasis as anactive process and not a consequence of tumour growth [2,3].Malignant tumour cells degrade ECM constituents essentiallyinvolving proteolytic enzymes produced either by the tumourcells themselves or by the surrounding stromal cells [4]. Theidentification of genes involved in malignant conversion andthe understanding of processes occurring during the establish-ment of secondary tumour sites, along with strategies to limitthe cancer to the primary site are of medical and socialinterest. Markers currently available for breast cancer aid inselecting subjects at high risk of developing the inherited formof the disease, assessing prognosis, guiding therapy andmonitoring patients with diagnosed disease. Currently,however, no biochemical or genetic marker/s exist that canbe used to screen and diagnose for disease onset [5]. With

ARTICLE IN PRESSL.M. Haupt et al. / Molecular and Cellular Probes 22 (2008) 83–8984

survival declining after 5 years and survival after 10 yearsdependent on the stage at diagnosis, if breast cancer isdetected early, while still localised in the breast, chances of5-year survival are 90% [6].

Along with other protease families, members of thematrix metalloprotease (MMP) family of proteases areintegral to maintaining the ECM milieu during devel-opmentally regulated processes such as ovulation, embryo-genic growth and differentiation and organ development[2,3,5,7–10]. Membrane type-1-matrix metalloproteinase(MT1-MMP, MMP-14) is one of the membrane-boundMMPs localised to the cell surface, has been shown toactivate pro-gelatinase-A (proMMP-2) in human breastcarcinoma cells [11] and can also activate proMMP-13[12,13]. Interstitial collagenase (MMP-1) is secreted in vitro

by fibroblasts in a proenzyme form. MMP-1 is produced bya wide variety of normal cells (e.g. stromal fibroblasts,macrophages and endothelial cells) [14], and is involved intissue remodelling and repair [14,15]. Stromelysin-1(MMP-3) is structurally the least homologous member ofthe MMP family. MMP-3 is secreted constitutively byhuman skin fibroblasts in vitro [16,17], activates MMP-1,both in vitro and in vivo, with degradation of the ECM byMMP-3 resulting in apoptosis [18,19].

There is a tight and complex regulation in the expression ofMMP family members in breast cancer. Previous idiom wouldsuggest the expression patterns represent a host response to thetumour; however, the question of whether tumour metastasis isinstigated by stromal or epithelial cells remains unanswered[20]. The ability to localise these genes in situ to determine theirrole both collectively and individually, and importantlydetermine their cellular origin, is offered by use of IS-RT-PCR. This technique uses the mRNA as a template forin situ amplification of a localised intracellular product. Onceamplified, the labelled gene expression product can beidentified to allow spatial determinations to be made bothwith regard to cell type and pathological diagnosis.

2. Materials and methods

2.1. Source of biopsy material and tissue fixation

Surgical biopsy material was obtained from consentingpatients undergoing surgery for removal of breast tumoursat the Gold Coast Hospital between January 1995 and July1998. Biopsy tissue was excised and immediately placed in10% neutral buffered formalin, fixed (10% formalin saline,1 h, 37 1C), dehydrated through an ethanol series (75%,95% and 100% � 40min each, 100% 3 � 55min at 37 1C),washed in xylene (1 � 55min, 2 � 25min, 37 1C) andparaffin wax embedded (4 � 1 h, 60 1C) using a Tissue TekVIP Processor (Bayer, Germany). Biopsies were thensectioned by a microtome (Leica, USA) onto SuperfrostPlus positively charged microscope slides (Menzel-Glaser,USA). Sections required for morphological diagnosis werestained with H&E while those required for IS-RT-PCRwere stored at 4 1C prior to use.

2.2. IS-RT-PCR

The technique used has been described in detail else-where [21,22]. Reverse transcription was performed usinggene-specific primers for MT1-MMP, MMP-1, MMP-3and b-actin, as previously reported with the followingmodifications: dCTP concentration was at 100 mM, biotin-dCTP at 80 mM, MnCl2 at 10mM and 7.5U of rTthDNA polymerase (Perkin-Elmer Corporation, AppliedBiosystems Division, USA) was used in the 30 ml mixadded to each slide. For the amplification step, the initialdenaturation was 95 1C for 4min, MgCl2 concentration15mM with 30 ml added to each slide. Amplification wasperformed for 25 cycles. Following IS-RT-PCR detectionwas performed as reported previously with the followingmodifications: incubation in the NBT/BCIP/ASB solutionwas for 15min, slides were counterstained with eosin(Australian Biostain Pty Ltd., Australia) and mounted inUltramount (Fronine, Australia).

2.3. Signal semi-quantitation

The level of signal intensity was assigned a value fromzero to four (0+ to 4+). Slides showing no purpleprecipitation and only eosin counterstain were assigned avalue of zero (0+), whilst cells stained with precipitatewere assigned a value between one and four (1+ to 4+)using light microscopy. Visual characteristics including theoverall level of precipitation, direct comparison ofthe entire tissue area to compensate for background, andthe number and intensity of cells exhibiting purpleprecipitation were also considered. The sectionswere histologically assessed and scored from 0+ to 4+(0, no expression; 1+, low expression; 2+, moderateexpression; 3+, strong expression). We compared therelative levels of gene expression of each MMP within thesamples analysed to controls. The signal intensity wasaveraged following independent examination by threeindividuals. The photographs presented in Figs. 1–6 areof the same area within consecutive sections and aretherefore a representation of the overall observed geneexpression levels.

3. Results

3.1. Localisation of signal

All IS-RT-PCR results were correlated with estrogenreceptor (ER) status and morphological controls. Themorphological controls ensured that any positive signalobserved was due to expression of the gene of interest andnot a result of contaminating genomic DNA or inadequatestringency throughout the detection protocol. As pre-viously described [22], these included the use of theubiquitous gene b-actin as a positive control for IS-RT-PCR and the histologic H&E stain. These results aresummarised in Table 1.

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Fig. 1. IS-RT-PCR results of PDIBC-1 showing (A) the no RT control,

(B) the RNAse/DNAse negative control, (C) the H&E morphology

control, (D) positive control b-actin, (E) MT1-MMP gene expression, (F)

MMP-1 gene expression, and (G) MMP-3 gene expression. All photo-

graphs 40� taken on a Minolta-RD-175 Digital Camera attached to an

Olympus microscope.

Fig. 2. IS-RT-PCR results of PDIBC-2 showing (A) the no RT control,

(B) the RNAse/DNAse negative control, (C) the H&E morphology

control, (D) positive control b-actin, (E) MT1-MMP gene expression, (F)

MMP-1 gene expression, and (G) MMP-3 gene expression. All photo-

graphs 40� taken on a Minolta-RD-175 Digital Camera attached to an

Olympus microscope.

L.M. Haupt et al. / Molecular and Cellular Probes 22 (2008) 83–89 85

3.2. Poorly differentiated invasive breast carcinoma

(PDIBC)

In PDIBC-1, expression of both MT1-MMP and MMP-3 was shown to be strongest in the tumour component ofthe biopsy section with a signal intensity level of three (3+)for both genes, compared with the stromal componentshowing a signal intensity at a level of two (2+). MMP-1gene expression was a factor lower than these two genes,with a signal intensity level of two (2+) in both the tumourcells and surrounding stromal component (Fig. 1). InPDIBC-2, a high level of signal intensity of all MMP genesexamined was observed in the stromal component ofthis tumour (3+). This level of gene expression of MT1-MMP was maintained in the tumour component (3+),increased for MMP-1 (4+) and decreased for MMP-3

(1+) (Fig. 2 and Table 1). As this section showed sometissue heterogeneity, it was also noted in this sample thatthe non-tumour associated fibroblasts showed an expres-sion level of three (3+) for b-actin and MMP-1, a level oftwo (2+) for MT1-MMP and a level of one (1+) forMMP-3. These fibroblasts were localised to a site distantfrom the tumour component, but within the same biopsysection, in a section of non-tumourous tissue.

3.3. Invasive breast carcinoma (IBC) IS-RT-PCR

In IBC-1, there was a high level of expression of MMP-1in the tumour component (3+) and an absence of observedsignal of MMP-3 (0+) in the tumour component of

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Fig. 3. IS-RT-PCR results of IBC-1 showing (A) the no RT control, (B)

the RNAse/DNAse negative control, (C) the H&E morphology control,

(D) positive control b-actin, (E) MT1-MMP gene expression, (F) MMP-1

gene expression, and (G) MMP-3 gene expression. All photographs 40�

taken on a Minolta-RD-175 Digital Camera attached to an Olympus

microscope.

Fig. 4. IS-RT-PCR results of IBC-2 showing (A) the no RT control, (B)

the RNAse/DNAse negative control, (C) the H&E morphology control,

(D) positive control b-actin, (E) MT1-MMP gene expression, (F) MMP-1

gene expression, and (G) MMP-3 gene expression. All photographs 40�

taken on a Minolta-RD-175 Digital Camera attached to an Olympus

microscope.

L.M. Haupt et al. / Molecular and Cellular Probes 22 (2008) 83–8986

this tissue sample. MT1-MMP appeared to be evenlydistributed between the two tissue compartments (1+)with MMP-1 and MMP-3 observed at a similar level (1+)in the stroma (Fig. 3). In the IBC-2 tissue sample,expression levels of both MT1-MMP and MMP-1appeared to be evenly distributed between the two tissuecompartments (2+ and 1+, respectively). In contrast, geneexpression levels of MMP-3 were shown to be higher in thetumour component (2+) when compared to the stromalcomponent (1+) (Fig. 4 and Table 1).

3.4. Lymph node metastasis (LNM) IS-RT-PCR

The LNM-1 patient sample demonstrated higher geneexpression levels of MT1-MMP and MMP-1 (3+) in thetumour component of the sample when compared with

MMP-1 (2+). The expression levels of MT1-MMP andMMP-3 were comparable (2+) in the stromal component,with MMP-1 absent (0+) (Fig. 5). In LNM-2, MT1-MMPwas determined to be absent in the tissue samplecompletely, while both MMP-1 (1+) and MMP-3 (2+)showed moderate gene expression in the tumour compo-nent only (Fig. 6 and Table 1).

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Fig. 5. IS-RT-PCR results of LNM-1 showing (A) the no RT control, (B)

the RNAse/DNAse negative control, (C) the H&E morphology control,

(D) positive control b-actin, (E) MT1-MMP gene expression, (F) MMP-1

gene expression, and (G) MMP-3 gene expression. All photographs 40�

taken on a Minolta-RD-175 Digital Camera attached to an Olympus

microscope.

Fig. 6. IS-RT-PCR results of LNM-2 showing (A) the no RT control, (B)

the RNAse/DNAse negative control, (C) the H&E morphology control,

(D) positive control b-actin, (E) MT1-MMP gene expression, (F) MMP-1

gene expression and (G) MMP-3 gene expression. All photographs 40�

taken on a Minolta-RD-175 Digital Camera attached to an Olympus

microscope.

L.M. Haupt et al. / Molecular and Cellular Probes 22 (2008) 83–89 87

4. Discussion and conclusions

The biopsy specimens used were consecutively sectionedand included the histological H&E stain to confirmmorphological integrity of the tissue and pathologicaldiagnoses. The ubiquitous gene b-actin was used only as apositive control for IS-RT-PCR. It is important to notethat expression of this gene is under the same transcrip-tional and translational controls as all other genes withinthe cell, and hence its expression level varies.

Several adjustments were made to our previous in vivo

protocol to accommodate for the increased adipose contentof human tissue versus mouse xenografts. Estimates rangefrom 2% to 78% adipose contents in adult breasts, with anestablished average of 48% [23]. The high adipose tissuecontent in human breast tissue samples limits the number

of manipulations able to be performed whilst maintainingtissue integrity.In terms of clinical parameters in the six cases examined

in this study, the number of lymph nodes determined to bepositive for metastasis of tumour cells correlated with thediagnosis and the expression of MT1-MMP, MMP-1 andMMP-3. As the samples progressed from poorly invasivetumours, to invasive tumours, and finally to LNM, themore metastatic and invasive tumours had increased lymphnode involvement. The correlation of lymph node involve-ment and ER status therefore serve as excellent prognosticindicators in the six biopsy sections examined. Theexpression profiles of the three MMPs examined by IS-RT-PCR have demonstrated a clear interaction in terms oftheir gene expression, between the tumour epithelial cellsand the surrounding stromal cells. These stromal/epithelial

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Table 1

Summary of IS-RT-PCR results obtained for expression of b-actin, MT1-MMP, MMP-1 and MMP-3 in archival breast biopsy material

Tissue sample Grade ER status Lymph node metastasis b-Actin MT1-MMP MMP-1 MMP-3

PDIBC-1 III +ve None T 3+ T 3+ T 2+ T 3+

S 1+ S 2+ S 2+ S 2+

PDIBC-2 III �ve 3/16 T 3+ T 3+ T 4+ T 1+

S 3+ S 3+ S 3+ S 3+

IBC-1 I +ve 3/4 T 1+ T1+ T 3+ T 0+

S 1+ S 1+ S 1+ S 1+

IBC-2 III +ve 1/9 T 1+ T 2+ T 1+ T 2+

S 1+ S 2+ S 1+ S 1+

LNM-1 – +ve 2/4 T 4+ T 3+ T 2+ T 3+

S 4+ S 2+ S 0+ S 2+

LNM-2 – �ve 3/16 T 1+ T 0+ T 1+ T 2+

S 1+ S 0+ S 0+ S 0+

The level of signal intensity is given from zero to four (0+ to 4+). The observed signal intensity was assessed based on the observed level of signal

intensity and tissue morphology using light microscopy.

L.M. Haupt et al. / Molecular and Cellular Probes 22 (2008) 83–8988

interactions are poorly understood in terms of tumourinitiation and the active processes of invasion andmetastases not only in breast cancer, but also in othercancers.

In this study, MT1-MMP appears to have the highestlevels of gene expression in the pre-invasive tumoursexamined, and hence we postulate a role for this gene inthe instigation of invasion. Recent in vivo studies havecorrelated MT1-MMP with tumour and epithelial cells andcorrelated this expression profile with invasive potentialand metastases, increased mortality and decreased survival,further implicating MT1-MMP as a potential prognosticmarker in breast cancer and a possible target of futuretherapeutics [24–26]. Interestingly, in vitro, we and othershave previously demonstrated the use involvement ofMT1-MMP in the invasive potential of breast cancer cells[21,22,27]. MMP-1 was observed in both the tumour andstromal compartments at a similar level of gene expressionin most of the tumours examined, indicating activestromal/epithelial cell communication and implicating thisgene in the active processes of invasion and metastases.MMP-3 expression was observed predominantly in thestromal component, but interestingly was also present inthe tumour cells, potentially indicating the production ofthese matrix-degrading enzymes by the tumour cells inpreparation for intravastation. Indeed, recent data fromother groups have demonstrated MMP-1 in stromal cellsand MMP-3 in both the stromal and tissue compartmentssurrounding renal cell carcinomas, with the MMP-3expression levels correlated to protein levels [28]. Inaddition, in breast cancer, specifically, two recent studieshave indicated stromal-mediated migration and invasionregulated by MMP-1 dependent upon the interactions ofthe protease activated receptor (PAR 1) and the angiogenicfactor Cyr61 (CCN1) [29]; and the potential use of MMP-1as a prognostic indicator in pre-cancerous lesions [30].

Here, we have demonstrated the use of IS-RT-PCR forthe examination of archival paraffin-embedded biopsymaterial. In addition, we propose that MT1-MMP is

involved in the initiation of breast tumour invasion, andthat MMP-1 and MMP-3 are involved individually and ina co-ordinated manner in localised invasion, involvingbasement membrane degradation, and the active process ofmetastasis and expansion, where the epithelial and stromalcells interact to maintain the local environment.

Acknowledgements

L.M.H. was supported by the Queensland Cancer Fund,Kathleen Cunningham Foundation. We also thank JodiWiggins for her expertise along with members of thePathology Department and patients at the Gold CoastHospital.

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