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BioMed Central
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BMC Cancer
Open Access
Research article
In vitro and in vivo MMP gene expression localisation by In
Situ-RT-PCR in cell culture and paraffin embedded human breast
cancer cell line xenografts
Larisa M Haupt*1,2, Erik W Thompson3, Ann EO Trezise4, Rachel E Irving1,
Michael G Irving5 and Lyn R Griffiths*1
Address: 1Genomics Research Centre, Griffith University Gold Coast, School of Medical Science, Griffith University, Queensland, 4217,Australia ,
2IMCB, Biopolis, Singapore, 3VBCRC Invasion and Metastasis Unit, St. Vincent's Institute of Medical Research and University of Melbourne,
Department of Surgery, Melbourne, Victoria, 3065, Australia , 4School of Biomedical Sciences, University of Queensland, St Lucia, Queensland
4072, Australia and 5Institute of Health Sciences, Bond University, Queensland, 4229, Australia
Email: Larisa M Haupt* - lhaupt@imcb.a-star.edu.sg; Erik W Thompson - rik@foo.medstv.unimelb.edu.au;
Ann EO Trezise - ann.Trezise@uq.edu.au; Rachel E Irving - rairving@staff.bond.edu.au; Michael G Irving - michael_irving@bond.edu.au;
Lyn R Griffiths* - l.griffiths@griffith.edu.au
* Corresponding authors
Abstract
Background: Members of the matrix metalloproteinase (MMP) family of proteases are required for the degradation of the
basement membrane and extracellular matrix in both normal and pathological conditions. In vitro, MT1-MMP (MMP-14,
membrane type-1-MMP) expression is higher in more invasive human breast cancer (HBC) cell lines, whilst in vivo its expression
has been associated with the stroma surrounding breast tumours. MMP-1 (interstitial collagenase) has been associated with
MDA-MB-231 invasion in vitro, while MMP-3 (stromelysin-1) has been localised around invasive cells of breast tumours in vivo.
As MMPs are not stored intracellularly, the ability to localise their expression to their cells of origin is difficult.
Methods: We utilised the unique in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) methodology to localise
the in vitro and in vivo gene expression of MT1-MMP, MMP-1 and MMP-3 in human breast cancer. In vitro, MMP induction was
examined in the MDA-MB-231 and MCF-7 HBC cell lines following exposure to Concanavalin A (Con A). In vivo, we examined
their expression in archival paraffin embedded xenografts derived from a range of HBC cell lines of varied invasive and metastatic
potential. Mouse xenografts are heterogenous, containing neoplastic human parenchyma with mouse stroma and vasculature
and provide a reproducible in vivo model system correlated to the human disease state.
Results: In vitro, exposure to Con A increased MT1-MMP gene expression in MDA-MB-231 cells and decreased MT1-MMP gene
expression in MCF-7 cells. MMP-1 and MMP-3 gene expression remained unchanged in both cell lines. In vivo, stromal cells
recruited into each xenograft demonstrated differences in localised levels of MMP gene expression. Specifically, MDA-MB-231,
MDA-MB-435 and Hs578T HBC cell lines are able to influence MMP gene expression in the surrounding stroma.
Conclusion: We have demonstrated the applicability and sensitivity of IS-RT-PCR for the examination of MMP gene expression
both in vitro and in vivo. Induction of MMP gene expression in both the epithelial tumour cells and surrounding stromal cells is
associated with increased metastatic potential. Our data demonstrate the contribution of the stroma to epithelial MMP gene
expression, and highlight the complexity of the role of MMPs in the stromal-epithelial interactions within breast carcinoma.
Published: 24 January 2006
BMC Cancer 2006, 6:18 doi:10.1186/1471-2407-6-18
Received: 09 August 2005
Accepted: 24 January 2006
This article is available from: http://www.biomedcentral.com/1471-2407/6/18
© 2006 Haupt et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
BMC Cancer 2006, 6:18 http://www.biomedcentral.com/1471-2407/6/18
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Background
Human breast carcinoma (HBC) is the most predominant
cancer in females worldwide. Breast cancers can be classi-
fied histologically based upon the types and patterns of
cells of which they are composed. Carcinomas can be
invasive, extending into the surrounding stroma or non-
invasive, confined to the epithelial cells of ducts or lob-
ules [1]. In conjunction with other protease systems such
as the serine, cysteine and aspartyl proteases, members of
the matrix metalloprotease (MMP) family of proteolytic
enzymes degrade constituents of the extracellular matrix
surrounding invasive breast carcinomas [2]. Currently, 28
human MMPs have been identified and classified accord-
ing to both their substrate specificities and structural sim-
ilarities. There are four major subgroups: i) interstitial
collagenases; ii) gelatinases; iii) stromelysins; and iv) the
membrane-type (MT) -MMPs [3,4]. Collectively, MMPs
degrade all extracellular matrix proteins as well as a grow-
ing number of key regulatory genes such as cytokines,
growth factors, cell surface receptors and adhesion mole-
cules [5,6]. Although MMPs are expressed by tissues at var-
ious stages of development, they are typically absent in
normal cells of the adult organism [2], and the high fre-
quency at which MMP transcripts or proteins are detected
in invasive tumours has implicated these enzymes in the
establishment, growth, invasion and/or metastasis of
tumours [6]. The expression of MMPs is usually tightly
regulated, and a number of studies provide evidence of
carefully controlled MMP involvement in developmen-
tally regulated processes such as ovulation, embryogenic
growth and differentiation, and organ development [6-8].
In general terms, MMP activity is regulated at least at three
levels: transcription/translation, proteolytic activation of
the zymogen, and inhibition of the active enzyme [2] with
upregulation of each of these associated with pathological
events [6]. Importantly in all mammalian cells except pol-
ymorphonuclear (PMN) leucocytes, most MMPs are not
stored intracellularly but are rapidly secreted after biosyn-
thesis and post-translational processing [9]. As a result it
is difficult to identify their cellular origin using standard
immunohistochemical techniques [10].
MT1-MMP is one of the membrane bound MMPs. In vivo,
MT1-MMP expression has been localised to the stroma
surrounding breast tumours [11,12], whilst in vitro, our
recent data confirms previous studies where basal levels of
MT1-MMP have been shown to be higher in the more
invasive MDA-MB-231 cells as compared to the less inva-
sive MCF-7 cells [13,14]. MT1-MMP has been shown to
activate pro-gelatinase-A (proMMP-2) in human breast
carcinoma cells [15] and can also activate proMMP-13
[16]. MT1-MMP degrades native type I collagen, fibronec-
tin, laminin, fibrin, gelatin and cartilage proteoglycan
core protein [2] and has also been classified as an intersti-
tial collagenase [3]. MMP-1 is the most ubiquitously
expressed interstitial collagenase [3]. MMP-1 cleaves
fibrillar collagens including collagens types I, II and III,
resulting in cleavage products that are rapidly denatured
at body temperature to gelatin, the substrate preferred by
the gelatinases (MMP-2 and MMP-9) [2]. MMP-1 is pro-
duced by a wide variety of normal cells (eg stromal fibrob-
lasts, macrophages and endothelial cells) [3], and is
involved in tissue remodelling and repair [3,17]. It has
been implicated in matrix invasion by the MDA-MB-231
HBC cells in vitro [18,19], but has a low incidence of
expression in the tumour cells of breast carcinomas
[2,11]. MMP-3 is a member of the stromelysin sub-family,
which show broad substrate specificity, degrading type IV
collagen, laminin, fibronectin and proteoglycans. MMP-3
is expressed in areas of tissue growth [20], focally
expressed around invasive cells in the stromal component
of breast tumours, and expressed in both benign and
malignant breast phenotypes [19].
As MT1-MMP, MMP-1 and MMP-3 have all been impli-
cated in the processes involved in human breast cancer,
this study utilised HBC cell lines both in culture and
grown as xenografts in nude mice, to investigate gene
expression changes of MT1-MMP, MMP-1 and MMP-3 in
vitro and in vivo using IS-RT-PCR. In order to demonstrate
IS-RT-PCR detection to examine MMP expression level
changes, we utilised Concanavalin A (Con A), an agent
known to modulate MMP expression, and to upregulate at
least one MMP [15,21,22], in our in vitro studies. Nude
mouse xenografts are heterogenous, containing neoplastic
human parenchyma with mouse stroma and vasculature
with many histologic features of the original tumour
maintained [23]. As xenograft growth varies with tumour
type, the ability to mimic human tumours in vivo provides
a reproducible model system that can be correlated to the
human disease state. We have previously applied the IS-
RT-PCR technique to examine in vitro gene expression of
MT1-MMP in MDA-MB-231 HBC cells [21], while others
have examined expression of MMP-2, MMP-9 and their
inhibitors TIMP-1 and TIMP-2 in cervical cancer [24].
Thus the objective of this study was to utilise the unique
IS-RT-PCR methodology to examine the localised gene
expression of members of the MMP family of proteases
implicated in breast cancer. Our findings here with IS-RT-
PCR demonstrate the detection of in vitro and in vivo gene
expression patterns of MMPs in HBC cell lines, and sug-
gest a contribution from the stroma to MMP expression in
breast carcinomas.
Methods
Cell culture
HBC cell lines MDA-MB-231, MCF-7, ZR-75-1 and MDA-
MB-453, were originally obtained from the ATCC (Vir-
ginia, USA) and maintained by the Lombardi Cancer
Center Shared Cell Culture Resource. They were grown as
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a monolayer culture in RPMI 1640 media (Invitrogen,
USA) supplemented with 10% foetal calf serum (Biowhit-
taker, USA) in the presence of 5% CO2. Cells (approx. 1 ×
105 cells/slide) were passaged onto two eight-chambered
chamber slides (Lab-Tek, Nunc, USA) and allowed to
attach for 8–12 hrs. Cells were then grown for a further
16–24 hrs in RPMI 1640 with 10% foetal calf serum in the
presence or absence of Con A (25 µg/ml, Sigma, USA).
Xenograft tissue
Tumour xenografts of the MDA-MB-231, MDA-MB-435,
MCF-7 and Hs578T HBC cell lines were xenografted into
intact female mice as described previously [25]. Animal
studies were conducted in accordance with the National
Institutes of Health Guide for the Care and Use of Labora-
tory Animals with approval of the Georgetown University
Medical Center Animal Ethics Committee. The tissue was
excised, and material from the xenografted tumours fixed
in 10% neutral buffered formalin (Fisher Scientific, USA)
and embedded in paraffin for routine histological analy-
sis. The Lombardi Cancer Center Tissue Resource per-
formed sectioning of the paraffin blocks, and tissue
sections of 5 µm were mounted onto Pro-Bond +/- slides
(Fisher Scientific, USA).
Pre-treatment of sections
Following sectioning, slides were placed under vacuum
overnight with desiccant. Our previously described RNA
ISH procedure [26] was modified as follows. All slides
were de-waxed in xylene (4 × 10 min), washed in ethanol
(100%, 3 × 3 mins), and rehydrated through a graded eth-
anol series (85%, 70% and 50% in 0.9% NaCl × 2 min
each). Sections were then washed in saline (0.9% NaCl,
1X × 2 min) and PBS (1X × 2 min) prior to fixation with
4% paraformaldehyde (PFA; 250 ml H2O, 1 g NaOH, 10
g PFA, 1.7 g sodium acetate pH 6.5) for 20 minutes. Fol-
lowing fixation, slides were rinsed in 2X PBS (2 mins), fol-
lowed by Proteinase K digestion (10 mg/ml; Roche
Diagnostics, USA; 25 ml 1 M Tris pH 8.0, 250 µl 10 mg/
ml Proteinase K, 25 ml 0.5 M EDTA pH 8.0, 200 ml H2O).
Slides were then rinsed in 2% glycine (2 mins) and re-
fixed in 4% PFA (10 mins) to ensure complete deactiva-
tion of Proteinase K. Slides were rinsed in triethanolamine
(TEA, Sigma, USA; 0.1 M pH 8.0 × 3 min) in preparation
for further digests.
RNase digestion
Following pre-treatment all slides were rinsed in RNase
buffer (10 mM HEPES, 20 mM NaCl, 1 mM EDTA) with-
out enzyme (2 × 2 mins). Negative control sections under-
going RNase digestion were then overlayed with RNase
Digest mixture containing RNase cocktail (8 mg/ml RNase
A and 160 U/ml RNase T1 final volume, Bresatec, Aus-
tralia) in RNase buffer, placed in a humid chamber and
incubated at 37°C for 8 hrs. Untreated slides were over-
layed in RNase buffer and also incubated at 37°C for 8
hrs.
DNase pre-treatment
Prior to DNase digest, all slides were rinsed in DNase
buffer (0.1 M C2H3O2Na, 5 mM MgSO4 pH 5.0, 2 × 2
min). DNase buffer (200 µl) containing 1.5 U/ml final
volume of RNase-free DNase (Roche Diagnostics, USA)
was added to each slide and these were incubated over-
night at 37°C in a humid chamber. After incubation the
slides were rinsed thoroughly in DNase buffer without
enzyme, followed by washes in sterile water and dehydra-
tion through a graded ethanol series (50%, 70%, and 90%
in 0.9% saline, 100% × 3 mins each).
Primers
Primers encoding MT1-MMP, MMP-1 and MMP-3
(Bresatec, Australia) were designed using the Amplimer
program, product design checked by the Amplify v1.2 pro-
gram, and BLASTed (NCBI, USA) for homology to the
mRNA of these genes, with significant sequence homol-
ogy demonstrated between the human and mouse MMPs
examined. Primers encoding human β-actin were used as
a positive control (Research Genetics, USA). Primer
sequences are listed in Table 1. All primers were tested
using traditional RT-PCR on mRNA extracted from MDA-
MB-231 and MCF-7 HBC cells using the High Pure RNA
Isolation Kit (Roche Diagnostics, USA). Reverse transcrip-
tion was performed using 20 ng of isolated mRNA, by
AMV reverse transcriptase (Promega Corporation, USA) at
50°C for 30 minutes, followed by PCR amplification
using Taq DNA Polymerase (Perkin-Elmer Corporation,
Applied Biosystems Division, USA). The linear phase of
amplification was determined to be between cycles 20–25
using a standard two-step PCR cycle.
In situ-Reverse Transcription-Polymerase Chain Reaction
(IS-RT-PCR)
Prior to the IS-RT-PCR procedure Gene-Frame gaskets
(Advanced Biotechnologies, UK) were placed around the
sections. IS-RT-PCR was performed on consecutive sec-
tions for each xenograft: section A) RNase/DNase negative
Table 1: Primer sequences encoding the human β-actin, MT1-
MMP, MMP-1 and MMP-3 genes.
Gene Primer Sequence
β-actin sense 5'- ACC CAC ACT GTG CCC ATC TA
β-actin antisense 5'- CGG AAC CGC TCA TTG CC
MT1-MMP sense 5'- AGT GGA TGG ACA CGG AGA AT
MT1-MMP antisense 5'- TCC ATC CAT CAC TTG GTT AT
MMP-1 sense 5'- AGC GTG TGA CAG TAA GCT AA
MMP-1 antisense 5'- GTT TTC CTC AGA AAG AGC AGC AT
MMP-3 sense 5'- GTC TCA AGA TGA TAT AAA TG
MMP-3 antisense 5'- AAT TGA TTT CCT TTA AAA ATG A
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control; B) β-actin positive control; C) MT1-MMP assay
slide; D) MMP-1 assay slide; and E) MMP-3 assay slide.
Reverse transcription was performed as previously
reported [18] with the following modifications: dCTP
concentration was 100 µM, biotin-dCTP 80 µM, MnCl2 10
mM and 7.5 U of rTth DNA Polymerase (Perkin-Elmer
Corporation, Applied Biosystems Division, USA) was
used in the 30 µl mix added to each slide. Linear amplifi-
cation efficiency was determined using traditional RT-
PCR as outlined above, to be between cycles 20 and 25.
For the amplification step the initial denaturation was at
95°C for 4 minutes, the MgCl2 concentration was 15 mM,
and 30 µl were added to each slide. Amplification was per-
formed for 20 cycles.
Detection
Post IS-RT-PCR detection was performed as reported pre-
viously [21] with the following modifications: Incubation
in the NBT/BCIP/ASB solution was for 15 minutes, slides
were counterstained with eosin (Australian Biostain Pty
Ltd, Australia) and mounted in Ultramount (Fronine,
Australia). Consistency of observed gene expression in
consecutive tumour sections demonstrated the reliability
of the IS-RT-PCR technique and as expected, all cells were
found to express the ubiquitous β-actin gene.
Signal quantitation
The level of signal intensity was assigned a value from zero
to four (0+ to 4+). Slides showing no purple precipitation
and only eosin counterstain were assigned a value of zero
(0+), whilst cells stained with precipitate were assigned a
Representative photomicrographs of IS-RT-PCR analysis of Con A effects in MDA-MB-231 cells counterstained with EosinFigure 1
Representative photomicrographs of IS-RT-PCR analysis of
Con A effects in MDA-MB-231 cells counterstained with
Eosin. Negative controls include, RNase/DNase treated sec-
tions (A) and No RT (B). β-actin staining provided a positive
Control without (C) or with (D) Con A treatment. MT1-
MMP levels before (E) and after (F) Con A treatment are also
shown. Photographs were on an Nikon Eclipse TE300 Micro-
scope with a Nikon F-600 camera attachment and are × 40
(except for A, which is × 20).
Representative photomicrographs of IS-RT-PCR results of MCF-7 xenograft showing the RNase/DNase negative con-trol (A), positive control β-actin (B), MT1-MMP gene expres-sion (C), MMP-1 gene expression (D) and MMP-3 gene expression (E)Figure 2
Representative photomicrographs of IS-RT-PCR results of
MCF-7 xenograft showing the RNase/DNase negative con-
trol (A), positive control β-actin (B), MT1-MMP gene expres-
sion (C), MMP-1 gene expression (D) and MMP-3 gene
expression (E). All photographs ×40 taken on an Olympus
Microscope with a Nikon F-600 camera attachment.
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value between one and four (1+ to 4+). The observed sig-
nal intensity was assessed based on the observed level of
signal intensity and tissue morphology using light micro-
scopy. Visual characteristics including the overall level of
precipitation, direct comparison of the entire tissue area
to compensate for background, and the number and
intensity of cells exhibiting purple precipitation were also
considered. In addition, a coded labelling system was
used to ensure that slide examination was unbiased. In
vitro signal intensity following exposure to Con A and
cytokines was compared to the untreated or basal level
allowing an estimate of up/down-regulation to be deter-
mined. The in vivo xenograft material was histologically
assessed and scored from 0+ to 4+ (with 0 = no expres-
sion; 1+ = low expression; 2+ = moderate expression; and
3+ = strong expression). We compared the relative levels
of gene expression of each MMP within the xenografts
analysed to controls. Following independent examination
of slides and sections by three individuals the signal level
intensity was averaged. The photographs presented in Fig-
ures 1, 2 and 3 are of the same area within consecutive sec-
tions and are therefore a representation of the overall
observed gene expression levels.
Results
Primer specificity
Primers were tested using traditional RT-PCR on mRNA
extracted from cultured MDA-MB-231 cells. Bands of the
appropriate size for β-actin (289 bp), MT1-MMP (274
bp), MMP-1 (330 bp) and the MMP-3 (330 bp) tran-
scripts were visualised by ethidium bromide agarose gel
electrophoresis (not shown). For IS-RT-PCR two negative
controls were used to ensure assay validity. The "No RT"
control was used as a measure of the detection system spe-
cificity. Lack of precipitation in this control ensures that a
positive signal is due to expression of the gene of interest.
The RNase/DNase control was used for two reasons, to
control for false positive results due to insufficient
genomic DNA digestion, and to ensure that there was no
signal due to any residual biotin-dCTP in the tissue sec-
tion. Examples of these controls are given for the MDA-
MB-231 cell line (Figure 1A and 1B), where the only col-
ouration in the slides is due to the eosin counterstain, and
purple precipitation which would be indicative of positive
gene expression signal, is not observed. Primers amplify-
ing β-actin mRNA were used as a positive control for dem-
onstration of mRNA preservation in the tissue, and the
ability to detect gene expression. We found a clear positive
signal, the purple precipitate localised to all cells exam-
ined, demonstrating β-actin expression. An example of
this is given in Figure 1C for the MDA-MB-231 cell line.
Induction of in vitro MMP gene expression
IS-RT-PCR results following Con A treatment
Expression levels of the three MMPs were examined with
respect to β-actin in the MDA-MB-231, and MCF-7 cell
lines, with or without exposure to Con A. In the MDA-MB-
231 cell line, MT1-MMP expression was increased from 2+
to 4+ by Con A treatment, while MMP-1 and MMP-3
remained unchanged at 2+ following exposure to Con A.
MT1-MMP induction by Con A in the MDA-MB-231 cell
line is shown in Figure 1E and 1F. In the MCF-7 cell line,
the MMP-1, MMP-3 and β-actin expression levels also
remained unaffected by Con A at 2+, however, the MT1-
MMP expression was decreased by Con A from 3+ in the
absence of Con A to 1+ in the presence of Con A (not
shown).
Representative photomicrographs of IS-RT-PCR results of MDA-MB-435 xenograft showing the No RT control (A), RNase/DNase negative control (B), positive control β-actin (C), MT1-MMP gene expression (D), MMP-1 gene expression (E) and MMP-3 gene expression (F)Figure 3
Representative photomicrographs of IS-RT-PCR results of
MDA-MB-435 xenograft showing the No RT control (A),
RNase/DNase negative control (B), positive control β-actin
(C), MT1-MMP gene expression (D), MMP-1 gene expression
(E) and MMP-3 gene expression (F). All photographs ×40
taken on an Olympus Microscope with a Nikon F-600 cam-
era attachment.
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Adaptation of IS-RT-PCR to paraffin tissue
We adapted the IS-RT-PCR protocol for analyses of these
MMPs in paraffin embedded xenografts of MCF-7, MDA-
MB-231, MDA-MB-435, and Hs578T HBC cell lines
grown subcutaneously in the mammary region of nude
mice. Primary xenograft material from each cell line
(except MDA-MB-231) were examined, as were metastatic
deposits of MDA-MB-231 cells in the spleen and mesen-
tery. The primary MDA-MB-231 tumour was unavailable
for examination. A summary of the results obtained from
the xenograft tissue is given in Table 2.
In the MCF-7 xenograft, low expression of MT1-MMP,
MMP-1 and MMP-3 was observed in the tumour cells (1+)
as indicated by the faint purple precipitation (Figure 2C,
2D, 2E). A low level of gene expression for MT1-MMP,
MMP-1 and MMP-3 (1+) was also seen in the stromal
compartment surrounding the MCF-7 cells (Figure 2C,
2D, 2E). However, β-actin expression was low in the stro-
mal component (1+) and at a moderate level in the
tumour component (2+) suggesting that the relative stro-
mal levels are even higher (Figure 2B). A low level of
expression of β-actin was consistent between the two
compartments (1+). The MDA-MB-435 No RT control
(Figure 3B) served as the No RT control section for the
MCF-7 experiment, with the experiments run concur-
rently. In the MDA-MB-435 cell line xenograft, we
observed moderate expression of MT1-MMP and MMP-1
(2+) (Figure 3C and 3D), and high levels of MMP-3 (3+)
(Figure 3E) in the tumour cells. No expression of MT1-
MMP (0+), MMP-1 (0+) or MMP-3 (0+) was observed in
the stromal tissue surrounding the MDA-MB-435 cells
(Figure 3D, 3E and 3F). β-actin expression was consist-
ently moderate in both compartments (2+) (Figure 3C).
The Hs578T xenograft showed low expression of MT1-
MMP (1+), MMP-1 (1+) and MMP-3 (1+) in the tumour
cells. In the surrounding stromal cells, we observed low
MT1-MMP expression (1+), moderate MMP-3 expression
(2+) and no MMP-1 expression (0+). β-actin expression
levels were consistently low in both compartments (1+)
(Table 2). Although the primary MDA-MB-231 xenograft
was unavailable for examination, mesenteric deposits in
the spleen and mesentery of the same mouse were exam-
ined (Table 2). In the MDA-MB-231 spleen metastasis, the
tumour cell showed high MT1-MMP (3+), and low MMP-
3 expression (1+), but no detectable MMP-1 gene expres-
sion (0+). No detectable expression of MT1-MMP, MMP-
1 and MMP-3 was observed in the host stromal cells (0+).
β-actin gene expression was consistent between the two
compartments at a moderate level (2+) (Table 2). In the
mesenteric metastasis, the MDA-MB-231 tumour cells
showed a moderate expression of both MT1-MMP and
MMP-1 (2+), and low expression of MMP-3 (1+). In con-
trast to the splenic metastasis, the host stromal tissue sur-
rounding the mesenteric metastasis showed low
expression of MT1-MMP (1+), MMP-3 (1+) and moderate
MMP-1 expression (2+). Again, β-actin was consistent in
both compartments at a moderate level (2+) (Table 2).
Discussion
The central aim of investigations into molecular carcino-
genesis is the identification of gene products involved in
cancer progression during which tumour cells acquire the
capacity for invasion with resulting metastasis [27].
Metastasis is an active process involving the altered attach-
ment of the tumour cell to the basement membrane,
localised degradation of connective tissue, and migration
through stromal tissue [28,29]. Such matrix degradation
is mediated by members of several proteinase families
(the serine, cysteine, aspartate proteinases and MMPs),
with substantial tissue destruction being carried out by
members of the MMP family [3]. The expression of mem-
bers of the MMP family and their inhibitors, the TIMPs,
have been examined in both physiological and patholog-
ical conditions by many methods both in vitro and in vivo,
including the use of total RNA from tissues and cell lines,
Northern analysis, RT-PCR in situ and standard in situ
Table 2: Summary of IS-RT-PCR results obtained for expression of β-actin, MT1-MMP, MMP-1 and MMP-3 in HBC cell line derived
xenograft tissues. The intensity is a value from zero to four (0+ to 4+) based on the overall level of signal intensity observed throughout
the entire section.
Cell Line β-actin MT1-MMP MMP-1 MMP-3
MCF-7 Tumour 2+ Tumour 1+ Tumour 1+ Tumour 1+
Stroma 1+ Stroma 1+ Stroma 1+ Stroma 1+
MDA-MB-231 Tumour 2+ Tumour 3+ Tumour 0+ Tumour 1+
Spleen Stroma 2+ Stroma 0+ Stroma 0+ Stroma 0+
MDA-MB-231 Tumour 2+ Tumour 2+ Tumour 2+ Tumour 1+
Mesenteric Stroma 2+ Stroma 1+ Stroma 2+ Stroma 1+
MDA-MB-435 Tumour 2+ Tumour 2+ Tumour 2+ Tumour 3+
Stroma 2+ Stroma 0+ Stroma 0+ Stroma 0+
Hs578T Tumour 1+ Tumour 1+ Tumour 1+ Tumour 1+
Stroma 1+ Stroma 1+ Stroma 0+ Stroma 2+
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hybridisation (ISH) [13,21,24,30-33]. Xenografts of cell
lines representing increased progression of breast cancer
were examined in the present study using the IS-RT-PCR
technique. The samples ranged from the poorly invasive,
estrogen-receptor positive cells (MCF-7), through to
examples of more aggressive, estrogen-receptor negative
human breast carcinoma (MDA-MB-231, MDA-MB-435,
Hs578T) [25]. We analysed and compared the localised
expression of MT1-MMP, MMP-1, MMP-3 and β-actin, in
both the tumour parenchyma and the surrounding
stroma. Both the MDA-MB-231 splenic and MDA-MB-435
xenografts demonstrate no detectable MMP gene expres-
sion in the stromal compartment. This highlights that
although significant sequence homology exists between
the human and mouse MMPs examined, minor differ-
ences may influence the detection of gene expression.
Localised gene expression in both the epithelial tumour
cells and the stroma compartments in the remaining
xenografts however, demonstrate the detectable homol-
ogy between human and mouse for the IS-RT-PCR primer
sequences used.
MT1-MMP analysis of the HBC xenografts demonstrated
mRNA in the tumour cells of all of the samples examined.
In vitro, we have previously demonstrated MT1-MMP
expression in MCF-7 cells by IS-RT-PCR [21], however
Northern analysis of MCF-7 cells failed to find MT1-MMP
in MCF-7 cells, whereas more invasive HBC cell lines
showed MT1-MMP expression in concordance with their
ability to be induced by Con A to activate MMP-2
[12,34,35]. MT1-MMP has also been shown to correlate
with increased invasion and to enhance migration of
MCF-10A epithelial cells [6,12,36], and transfection of
MT1-MMP into MCF-7 cells stimulates higher migration
and invasiveness [37,38], and also stimulates VEGF pro-
duction, angiogenic stimulation, and xenograft take rate
in nude mice [39,40]. Presumably the IS-RT-PCR method
is more sensitive than Northern analysis, as may be
expected. Indeed, we detected lower levels of MT1-MMP
in the MCF-7 xenografts than in the majority of other
xenografts derived from more invasive HBC cells. More
recently, using isolated RNA for quantitative analyses, we
were unable to detect MT1-MMP in MCF-7 derived
xenografts, but consistent with our observations here,
increasing levels of MT1-MMP was detected in MDA-MB-
231 derived xenografts as compared to the parental cells
[13]. The reasons for these differences are not clear and
require further experimentation, but it is important to
note that the cultured MCF-7 cells would have received
estrogenic stimuli from both the phenol red and foetal
calf serum in the medium [41].
MT1-MMP overexpression in the mammary gland results
in abnormalities including hyperplasia, fibrosis, lym-
phocytic infiltration and adenocarcinoma [42], suggest-
ing a pivotal role for MT1-MMP in carcinogenesis.
However, ISH analyses of human breast carcinomas have
primarily localised MT1-MMP mRNA expression to the
stromal cells [11,29,43-45], although one study found
localised MT1-MMP in the tumour [46]. An immunocyto-
chemical analysis showed expression of MT1-MMP in
both tumour and stromal cells [47]. More recently, one
study by Bisson et al, combining ISH and IHC data has co-
localised MT1-MMP to the α-smooth muscle positive
myofibroblast cells in close contact with tumour cells
[48]. Our studies certainly show the potential for the cell
lines examined to make MT1-MMP, even the relatively
well-conserved cell lines such as MCF-7. Although it is
acknowledged that in vitro propagation of cell lines may
alter some genetic pathways, it is also apparent that cell
lines and tumour samples have distinctive gene expres-
sion patterns in common [49]. Being ER-positive, and pre-
dominantly epithelial, MCF-7 cells far better represent
what one may find clinically in breast cancers than the
other cell lines examined here [50]. The invasive HBC cell
lines show a mesenchymal-like phenotype that may have
resulted from epithelial-mesenchymal transition (EMT)
in breast carcinoma. EMT is thought to have occurred in
the MDA-MB-231, MDA-MB-435 and Hs578T cell lines
[51]. The demonstration of MT1-MMP expression
observed in the stromal component is not totally unex-
pected. MT1-MMPs ability to stimulate invasion and
metastasis in in vitro systems along with the estrogen
receptor status of the cells is well documented as
described above. Also, the basal levels observed between
the MCF-7 and more invasive HBC cell lines may also be
of impact in respect to the higher invasive potential of the
cells.
MMP-1 mRNA was detected in the tumour cells of five of
the six xenografts examined, the exception, surprisingly,
being the MDA-MB-231 splenic metastasis. This is unex-
pected since MDA-MB-231 cell overexpress MMP-1 in cul-
ture [52], and these cells have a mutation/polymorphism
in the promotor region which drives strong MMP-1
expression [18]. This transcriptional repression has not
been found to occur extensively in breast cancer [18]. The
reasons for suppression of the MMP-1 expression in
splenic metastasis are not clear, but could include a strong
repression, possibly due to altered signalling from the
stroma. The splenic metastasis stroma was notably also
lacking all three MMPs examined, but showed a strong β-
actin signal in both compartments. One cannot rule out
the possibility of selective aberrant loss of MMP mRNA
rather than the β-actin, however, this is unprecedented.
Further analysis of splenic metastases of the MDA-MB-
231 cells, and primary human tumours, would be war-
ranted. MMP-1 has been previously demonstrated to be
equally expressed in vitro in both MCF-7 and MDA-MB-
231 cells [53], although in a similar study using Northern
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analysis we found selective expression in MDA-MB-231
cells but not MCF-7 [51]. Increased production and secre-
tion of MMP-1 has been correlated with increased meta-
static potential [52,54]. More recently, Bachmeir et al have
demonstrated a cell density-dependent regulation of gene
expression of several MMPs in HBC cell lines [31].
Increased cell density resulted in decreasing levels of both
MMP-1 and MMP-3 expression levels, with a resultant
decreased invasion concurrent with invasive potential
[31-33]. Increased expression of MMP-1 has been
reported on contact with Matrigel [55], and also in
response to fibronectin fragments [56], attesting to the
potential regulation of this MMP by the microenviron-
ment. In vivo, MMP-1 expression has been demonstrated
in the stroma of nine of thirty-four breast carcinomas [11]
when examined by ISH, localised to stromal constituents
during tumour formation [57], and to be upregulated in
the stromal component of ductal carcinomas when exam-
ined by ISH and IHC [57-59]. Thus, again, our observa-
tion that MMP-1 expression was absent in the stroma of
three out of six HBC xenografts appears to contrast the
clinical situation. This may reflect the increased sensitivity
of IS-RT-PCR and may also reflect the stage in tumour for-
mation of the MCF-7 and MDA-MB-231 mesenteric
xenografts that we examined.
MMP-3 mRNA was localised to the tumour component in
all six xenograft samples examined. In vitro, MMP-3
mRNA has been demonstrated in the MDA-MB-231 HBC
cell line [55], where it is stimulated by fibroblasts via the
extracellular matrix metalloproteinase inducer
(EMMPRIN) [60], and can enhance tumourigenicity and
migration [54]. In vivo, MMP-3 is centrally involved in
mammary gland development [61], and has been demon-
strated to promote tumour initiation and formation in the
tetracycline-regulated mouse mammary model [62,63].
IHC and ISH in vivo analysis has demonstrated MMP-3 in
both the tumour and stroma cellular compartments of
both invasive and non-invasive tumours, with the level of
stromal expression increasing with tumourigenicity
[59,64,65], and in the extracellular matrix adjacent to
breast tumours [66]. Our observation of MMP-3 gene
expression in the stroma of four out of six of the
xenografts examined, and in particular in the Hs578T
xenograft, further support a role for this MMP in breast
cancer.
Although HBC cell lines have been demonstrated to
express certain MMPs, this is less evident in vivo where, as
detailed above, the surrounding stromal cells contribute
to much of the MMP activity. Important differences in
MMP expression between the stromal cells recruited by
each cell line and the tumour cells themselves, were
detected in the current study. The MCF-7 xenograft stroma
demonstrated low stromal MT1-MMP, MMP-1 and MMP-
3 expression. MT1-MMP expression was observed at a
lower level in the stroma associated with the MDA-MB-
231 mesenteric metastasis. In the Hs578T xenograft
stroma, MMP-1 was absent while MMP-3 was observed at
a higher level in the stroma as compared to the tumour
cells. No stromal expression of the three MMPs examined
was observed in either the MDA-MB-231 spleen metasta-
sis or the MDA-MB-435 xenografts. The level of stromal
gene expression will depend on signals from the different
HBC lines in the primary site, and also on the different
responsivities in the different host sites for the splenic and
mesenteric metastases. In regard to the primary site, over-
all recruitment of the stroma will differ among the HBC
lines; however, β-actin mRNA detection was used to nor-
malise the data. We did detect lower levels of β-actin in
the Hs578T xenograft, and indeed higher levels of β-actin
in the tumour cell component of the MCF-7 xenograft.
However, this does not appear to have influenced the
detection of the MMPs as indicated by the moderate levels
of all MMPs examined in both compartments in the MCF-
7 xenograft, and the moderate level of MMP-3 in the
Hs578T tumour compartment.
Conclusion
These data combined indicate communication, and in
particular HBC cell-line specific communication, between
the epithelial and stromal cellular compartments for
MMP production and utilisation. Considerable work has
been performed in vitro to examine the effects of different
HBC cell lines on MMP expression by co-culture with
fibroblasts and other factors known to be produced by the
tumour cells which mediate induction of MMPs (eg Con
A, TPA, EMMPRIN) [12,15,21,31-33,53,60]. These and
other studies, implicate increasing MMP levels associated
with both epithelial tumour cells and surrounding stro-
mal cells, in association with increasing tumourigenicity
and metastatic potential [12,15,21,24,53,54,60,67]. Our
in vivo data indicate that, the MDA-MB-231, MDA-MB-
435 and Hs578T HBC cell lines are able to influence MMP
gene expression in the surrounding stroma. This is rein-
forced by a recent study demonstrating the influence of
tumour associated fibroblasts on MCF-7 tumour cells to
activate MMP-2 in vitro and increase tumour size in vivo
[68]. These data combined indicate IS-RT-PCR may serve
as a more sensitive, one-step procedure for the examina-
tion of MMP gene expression in human breast cancers.
Competing interests
The author(s) declare that they have no competing inter-
ests.
Authors' contributions
L.M.H. performed all in vitro and in vivo studies and
drafted the manuscript. E.W.T. contributed toward the
design of the study and manuscript finalisation. A.E.O.T.
BMC Cancer 2006, 6:18 http://www.biomedcentral.com/1471-2407/6/18
Page 9 of 10
(page number not for citation purposes)
contributed toward the design of the in situ sample prepa-
ration. R.E.I. contributed toward data management and
manuscript finalisation. M.G.I. participated in the con-
ception and design of the study. L.R.G. participated in the
conception and design of the study and its coordination.
All authors read and approved the final manuscript.
Acknowledgements
L.M.H. was supported by the Queensland Cancer Fund, Kathleen Cunning-
ham Foundation. We also thank the Tissue Culture Shared Resources of
Lombardi Cancer Center, which are partially supported by PHS grant NIH
1P30-CA-51008 (Cancer Center Support Grant) to Lombardi Cancer
Center.
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