The processes by which solid primary tumours, such as those of the breast, are able
to disseminate and establish growth at a secondary site are still poorly understood.
Tumour metastasis is complex, probably requiring both gain and loss of functions,
enabling escape from the primary tumour, and growth at a secondary site. This has
led to the search for metastasis-associated genes, which, unlike oncogenes, are unable
to initiate tumour formation but are able to induce a metastatic phenotype in previously
tumorigenic cells (Davies et al, 1993,1996; Oates et al, 1996). One such metastasis-associated
gene is that for the calcium-binding protein S100A4 (Barraclough et al, 1987). Increased
levels of S100A4 have been shown to induce a metastatic phenotype in several rodent
models of mammary carcinogenesis (Davies et al, 1993; Grigorian et al, 1996). Moreover,
elevated expression of S100A4 has been shown to correlate with early patient demise
of one group of breast cancer patients (Rudland et al, 2000), presumably due to metastatic
spread of the primary tumour.
A transgenic murine model that represents human metastatic breast disease in both
its pathology and development of metastasis has previously been established by mating
MMTV-neu transgenic mice with S100A4 transgenic mice (Davies et al, 1996). The resultant
neu/S100A4 transgenic offspring develop both mammary gland tumours and lung metastases,
in contrast to the parental transgenic MMTV-neu strain, which develops only mammary
gland tumours (Bouchard et al, 1989; Davies et al, 1996), and to the S100A4 transgenic
strain, which shows no pathology (Davies et al, 1995). Other transgenic murine models
showing that overexpression of S100A4 causes metastasis have also been developed (Ambartsumian
et al, 1996). S100A4 has been shown to be capable of binding to the actomyosin elements
of the cellular cytoskeleton (Davies et al, 1993; Kriajevska et al, 1994; Takenaga
et al, 1994), raising the possibility that the metastatic effect exerted by S100A4
might occur via its influence on cytoskeletal function, which in turn may regulate
cell movement. In this study, new mammary cell lines have been derived from mice carrying
transgenes for either the neu oncogene alone, or the neu oncogene in combination with
that for S100A4. The behaviour of these cell lines, and that of the neu oncogene containing
cell lines transfected with the cDNA for S100A4 in vitro have been compared using
in vitro tests to study individual aspects of S100A4-mediated effects for correlation
with in vivo phenotypes. In particular, the effect of S100A4 on cytoskeletal-dependent
processes, including motility and invasion, as well as on other potential elements
of the metastatic phenotype, including metalloproteinase-mediated matrix disruption
of an artificial matrix, gelatin, have been examined.
MATERIALS AND METHODS
Isolation and characterisation of cell lines
Mixed cell populations were isolated from mammary gland tumour extracts of either
neu or neu/S100A4 transgenic mice (Davies et al, 1996), as described previously (Davies
et al, 1993). Cells isolated from tumour extracts were cloned and subsequently grown
in Routine Medium (Dulbecco's modified Eagle's medium (DMEM), 10% (v v−1) foetal calf
serum (FCS), 100 ng ml−1 insulin, 50 ng ml−1 hydrocortisone). The cells were tested
for the expression of peanut lectin-binding protein and cytokeratin 18 (Jenkinson,
2001), as described previously for rat and human mammary cell lines (Rudland et al,
1998). All the cell lines could be grown indefinitely in routine medium and stocks
were frozen in liquid nitrogen for preservation. The characterisation and growth of
the low S100A4-expressing control cell line Rat mammary (Rama) 25 isolated from a
chemically induced rat mammary tumour has been described previously (Bennett et al,
1978).
Transfection of S100A4 DNA into cultured cells isolated from neu transgenic mice
For transfection, cell lines grown to approximately 60% confluence in 3 cm diameter
dishes were transfected with 0.5 μg of the expression vector for S100A4, pSV2neo-p9Ka
(S100A4) (Davies et al, 1993) using the Lipofectamine system (Invitrogen Ltd, Paisley,
Scotland), following the manufacturer's instructions. At 72 h after transfection,
the cells were passaged at a split ratio of 1 : 10 into Selective Medium, Routine
Medium containing 0.5 mg ml−1 Geneticin (G418). Cells were cultured in this Selective
Medium for 2 weeks, the medium being replaced every 2–3 days and the resulting colonies
for any one cell line pooled and expanded. Incubation of control nontransfected cells
with the Selective Medium caused death of all the cells within 5 days.
Detection of Neu and S100A4 protein
For Western blotting, total cellular protein was isolated from cell lines; washed
twice with cold phosphate-buffered saline (PBS), lysed in 100 μl of 0.125 M Tris-HCl
(pH 6.8), 4% (w v−1) sodium dodecyl sulphate (SDS), 20% (v v−1) glycerol; scraped
with a rubber policeman, collected in 1.5 ml tubes and protein quantitated using Bradford's
reagent (Pierce and Warriner, Chester, UK) following the manufacturer's instructions.
Samples (10 μg) of protein from each cell line in gel loading buffer (0.125 M Tris-HCl
(pH 6.8), 4% (w v−1) SDS, 20% (v v−1) glycerol, 0.04% (w v−1) bromophenol blue, 10%
(v v−1) 2-mercaptoethanol), heated to 100°C, were separated by electrophoresis on
10 or 15% (w v−1) polyacrylamide gels in the presence of SDS. The separated polypeptides
were transferred to nitrocellulose membranes and incubated with a primary antibody
to either c-erbB-2 (Dako Ltd, Ely, Cambs, UK) or S100A4 (Gibbs et al, 1995), which
were detected, in turn, by means of a peroxidase-conjugated secondary polyclonal antibody
and visualised with the Supersignal Chemi-luminescent System (Pierce and Warriner,
Chester, UK). Western blotting was performed with protein isolated from three independent
experiments. Bands were detected by autoradiography using Fuji X-Omat autoradiography
film and quantified by scanning the images using a Shimadzu CS9000 scanning densitometer.
Differences in loading were calculated by scanning and quantifying the staining of
each of the cell lines on the Coomassie blue stained protein gel.
Cell substrate-adhesion assay
Cells were trypsinised and counted using a Coulter counter, and resuspended at 2 ×
105 cells ml−1. Cell suspension (1 ml) was added to each well of a 24-well plate and
at time periods of 0.5, 1, 1.5, 2.0, 2.5 h, the number of adherent cells was found
by washing the wells three times with PBS to remove any cells in suspension. Any cells
adhering to the well were trypsinised and counted using the Coulter counter. Each
experiment was carried out in triplicate.
Cell proliferation assay
Cells were trypsinised and counted as described previously, and resuspended at 1 ×
104 cells ml−1 and 1 ml was added to each well of a 24-well plate. The cells were
incubated for 24, 48, 72 h periods, and at the end of each time period the number
of cells was found by trypsinising the cells and counting them in a Coulter counter.
Each experiment was carried out in triplicate.
Motility and invasion assays
The motile and invasive abilities of the cell lines through a filter containing 8 μm
pores were measured using a Boyden chamber in a 24-well plate assay system (Corning
Costar, High Wycombe, Bucks, UK). Chemotactic-induced motility in response to a FCS
concentration gradient was measured by adding 400 μl of Routine Medium containing
10% (v v−1) FCS to the lower compartment, and 2 × 105 cells in 200 μl of Routine Medium,
but with 2% (v v−1) FCS to the upper compartment of the Boyden chamber. The cells
were incubated for 24 h, the upper side of the filter was wiped with a cotton swab
to remove any nonmotile cells, and the motile cells on the lower side of the filter
were fixed and stained using the Diffquik histochemical stain (Dade Behring, Düdingen,
Switzerland) according to the manufacturer's instructions. The lower compartment of
the Boyden chamber was checked for cells and the number of stained cells/field of
0.50 mm2 on the lower side of the filter was counted using a Dynascope with a × 20
objective (Vision Engineering, Woking, Surrey, UK). For each cell line, four experiments
were carried out, each experiment consisting of three filters and 10 fields per filter
were counted. In controls, fixed numbers of cells incubated for different times up
to 24 h showed a linear increase in the number of motile cells on the lower side of
the filter. Thereafter, there was an appreciable number of cells present in the lower
chamber.
For cell invasion, the filters were coated with 50 μg Matrigel (Collaborative Biomedical
Products, Becton Dickinson, Oxford, UK) and the experiments were carried out as for
the chemotactic motility assays. The number of invasive cells/field on the lower side
of the filters were determined as for the motility assays for three experiments for
each cell line.
Zymography
Cell lines were washed twice with PBS and incubated in 8 ml of growth medium consisting
of phenol-red-free DMEM, 2% (v v−1) FCS, 200 ng ml−1 insulin and 50 ng ml−1 hydrocortisone
for 48 h. The media were harvested from each of the cell lines, grown to either 40–50%
confluency (sparse cultures) or 80–90% confluency (dense cultures) and any protein
concentrated into a 1 ml volume or less using an Ultrafree-15 centrifugal filter unit
(Biomax-5 K, Millipore, Watford, UK). The concentration of protein in each sample
was then determined by measuring the absorbance at 595 nm using Bradford's reagent
(Pierce and Warriner, Chester, UK) following the manufacturer's instructions and relating
it to the absorbance found for standards containing a known concentration of bovine
serum albumin.
Conditioned medium was analysed on 10% (w v−1) SDS polyacrylamide resolving gels containing
1 mg ml−1 of gelatin, with 4% (w v−1) polyacrylamide stacking gels using the Bio-Rad
Miniprotean system, as described previously (Heussen and Dowdle, 1980). Briefly, molecular
weight markers or samples consisting of 2 or 20 μg of conditioned medium in loading
buffer (0.4 M Tris-HCl (pH 6.8), 5% (w v−1) SDS, 20% (w v−1) glycerol, 0.03% (w v−1)
bromophenol blue) were loaded onto the gel, without being heated and in the absence
of 2-mercaptoethanol. Gels were run at 4°C until the dye front reached the anode end
of the gel. The resolving gel was washed for 2 × 30 min in 2.5% (v v−1) Triton X-100
to remove the SDS from the gel. Following which the gels were incubated for 18 h at
37°C in substrate buffer (50 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 75 mM NaCl). The gel
was subsequently incubated in Coomassie blue stain for 1 h and then incubated in destain
(45% (v v−1) methanol, 5% (v v−1) glacial acetic acid) for 1 h, until the digested
bands in the gel became clear. The gels were dried on gel drying frames between cellophane
sheets (Web Scientific, Crewe, Cheshire, UK).
Tumorigenicity studies and assays for metastasis
The 8Neu, 8Neup9Ka or PN2 cell lines were injected at 1 × 106 cells in 0.1 ml PBS
into three groups of 20, 6-week-old female nude, nu-nu mice, at a single subcutaneous
site in the left inguinal mammary fat pad. Mice were autopsied when tumours reached
approximately 10% of their body weight, and the tumours, mammary glands, lymph nodes,
lungs and any suspicious looking tissues were fixed in Methacarn, processed and embedded
in paraffin wax as for the rat (Dunnington et al, 1983). Samples were sectioned and
stained with haematoxylin and eosin. Further sections were immunocytochemically stained
with antibodies to either c-erbB-2 (Dako Ltd, Ely, Cambs, UK) or S100A4 (Gibbs et
al, 1995), using a peroxidase-conjugated secondary antibody and photographed using
a Reichert Polyvar microscope, as described previously (Davies et al, 1996). Sections
of both lungs and lymph nodes for each animal were examined for metastases. Animals
were maintained under UK Home Office Project Licence no. 40/1515 to Professor PS Rudland,
in accordance with the guidelines set down by the United Kingdom Coordinating Committee
for Cancer Research (Workman et al, 1998).
RESULTS
Establishment of clonal cell lines
Cells were initially isolated from frozen samples of mammary gland tumours from both
neu and neu/S100A4 transgenic mice (Materials and Methods). The tumours were selected
from mice representative of the general phenotype of the transgene status, as demonstrated
by haematoxylin and eosin-stained tumour sections. Three permanently growing cell
clones were established from the mammary gland tumours of a neu transgenic mouse and
termed 4Neu, 5Neu and 8Neu. Two permanently growing cell clones were established from
the mammary gland tumours of a bitransgenic neu/S100A4 mouse and termed PN1 and PN2.
The stable transfection of the 4Neu and 8Neu clonal cell lines isolated from the neu
transgenic mouse with the rat pSV2neo-p9Ka (S100A4) expression vector yielded two
further cell lines obtained from the pooled transfectant clones, termed 4Neup9Ka and
8Neup9Ka, respectively, which grew in Geneticin-containing medium. Parallel mock transfections
without the expression vector yielded no colonies after incubation of cells in Geneticin-containing
medium. All of the isolated cell lines were epithelial in nature as determined by
staining for epithelial lineage markers such as Cytokeratin 18 and peanut lectin-binding
protein (not shown) (Jenkinson, 2001).
Each of the cell lines had a different expression profile for S100A4 and Neu proteins,
as determined by Western blotting (Table 1
Table 1
Expression levels of Neu, S100A4 proteins and invasive and motile behaviour of cell
lines
Cell linea
Neu proteinb (arbitrary units)
S100A4 proteinc (arbitrary units)
Adhesiond (%)
Growth ratee (cells h−1)
Motilityf (cells per field)
Invasiong (cells per field)
5Neu
2.83±0.42
1.13±0.37
50.6±3.4
546±18
N/D
N/D
4Neu
3.24±0.34
1.59±0.37
30.8±2.8
2111±13
40.8±14.4
8.08±3.52
4Neup9Ka
2.75±0.45
3.12±0.31h
43.5±5.3
1058±47
129±19.8h
25.8±2.21h
8Neu
3.64±0.70
0.09±0.04
24.2±1.6
1104±34
6.95±3.71
3.21±0.68
8Neup9Ka
5.55±0.85
1.13±0.18h
32.9±5.2
2830±183
29.5±6.6h
10.7±1.92h
PN1
5.80±1.19
1.66±0.18
56.1±1.3
2096±49
75.0±21.6
13.2±6.43
PN2
4.20±0.54
1.88±0.09
48.2±0.8
1730±88
111±46.0
23.8±1.98i
a
Cell lines were isolated as described in Materials and Methods.
b
Relative protein values for Neu and S100A4 are normalised to the low Neu-, low S100A4-expressing
cell line Rama 25, after correcting for differences in the Coomassie blue-stained
protein gel, mean±s.e. of three experiments (Materials and Methods).
c
Relative protein values for Neu and S100A4 are normalised to the low Neu-, low S100A4-expressing
cell line Rama 25, after correcting for differences in the Coomassie blue-stained
protein gel, mean±s.e. of three experiments (Materials and Methods).
d
Cell substrate-adhesion was measured at 0.5 h and is expressed as a mean percentage
of cells that had adhered±s.e. of three experiments for 2 × 105 cells plated.
e
Cell growth rate in cells per hour is the mean±s.e. of three experiments for 1 × 104
cells plated.
f
Cell motility, average cells/field for 4 experiments±s.e. (Materials and Methods).
Each experiment consisted of three filters per cell line and 10 fields per filter
were counted.
g
Cell invasion through Matrigel, average cells per field for three experiments±s.e.
(Materials and Methods). Each experiment consisted of three filters per cell line
and 10 fields per filter were counted.
h
Significantly higher for the S100A4-transfected cell line compared to its parental
cell line (Student's t-test, P⩽0.03).
i
Significantly higher for the cell lines isolated from the double transgenic neu/S100A4
mice than the single transgenic neu mice (Student's t-test, P⩽0.02). ND=not determined,
since this cell line had no S100A4-transfected derivative with which to compare motile
and invasive abilities.
, Figure 1
Figure 1
Detection of S100A4 and Neu protein in cell lines isolated from mammary tumours of
transgenic mice. Cellular protein extracts were analysed by Western blotting for (A)
S100A4 and (C) Neu protein on SDS–polyacrylamide gels. Lanes 1, 2 and 3 contain 10 μg
of protein from the cell lines isolated independently from a neu transgenic mammary
gland tumour: 4Neu, 5Neu and 8Neu. Lanes 4 and 5 contain 10 μg of protein from S100A4-transfected,
neu-transgenic-derived cell lines: 4Neup9Ka and 8Neup9Ka. Lanes 6 and 7 contain 10 μg
of protein from cell lines isolated from a neu/S100A4 bitransgenic mammary gland tumour:
PN1 and PN2. Lane 8 contains 10 μg of protein from a low S100A4, low Neu-expressing
rat cell line: Rama 25. Coomassie blue staining of the protein on the SDS–polyacrylamide
gels is shown for the same gels as those immunocytochemically stained for (B) S100A4
and for (D) c-erbB-2.
). The S100A4 content of the mouse cell lines varied from about a tenth to over three
times that of the known low S100A4-expressing control Rama 25 cell line isolated from
a dimethyl benzanthracene-induced rat mammary tumour. Cell lines transfected with
the S100A4 cDNA, 4Neup9Ka and 8Neup9Ka showed a two to 12-fold increase in S100A4
compared to their parental 4Neu and 8Neu cell lines (Student's t-test, P<0.05). The
correct sized protein of apparent molecular weight 9 kDa was seen for all cell lines
(Figure 1A). The Neu protein was expressed in all of the mouse cell lines, there being
about a two-fold difference between the highest PN1 and lowest 4Neup9Ka cell lines.
The control Rama 25 cell line showed a level of endogenous Neu gene expression significantly
lower than all of the mouse cell lines including 4Neup9Ka (Student's t-test, P<0.05).
The correct sized protein of 185 kDa was seen for all the cell lines, but an additional
band at 135 kDa was sometimes seen; this may have been due to crossreactivity with
the EGF receptor (Figure 1C). The neu transgene retained its activating transmembrane
point mutation at nucleotide position 2012, as determined by sequencing of PCR-amplified
mRNA isolated from the cell lines (Jenkinson, 2001).
Comparison of cell substrate-adhesion and proliferation with S100A4 levels
Preliminary experiments showed that 0.5 h after plating, 25–50% of the total cells
in the assay had adhered to the plastic substratum and therefore 0.5 h was taken as
an appropriate time period over which to measure rate of adhesion. The substrate adhesion
of the different cell lines varied. The percentage of total cells adhering at 0.5 h
was plotted against the levels of S100A4 protein (Figure 2A
Figure 2
Relationship between the levels of S100A4 protein, growth rate, substrate adhesion,
cell motility, invasion and metastatic potential of the cell lines. (A) substrate
adhesion (adhesion); (B) growth rate; (C) motility and (D) invasion of each cell line
are plotted against the relative levels of S100A4 protein. (E) Motility of each cell
line is also plotted against its invasion. The data for the cell lines (4Neu, 5Neu,
8Neu, 4Neup9Ka, 8Neup9Ka, PN1, PN2) are taken from Table 1. (F) Relative levels of
S100A4 protein are plotted against the percentage of tumour-bearing mice with lung
metastases for each of the cell lines: 8Neu, 8Neup9Ka and PN2 (data taken from Table
2). Least-squares regression analysis of a fit of the points to a straight line yields
for (A) regression coefficient R
2=0.23, probability P=0.27; (B) R2=0.0002, P=0.97; (C) R
2=0.822, P=0.012; (D) R
2=0.765, P=0.0022; (E) R
2=0.950, P=0.00096; and (F) R
2=0.996, P=0.038.
), as determined by Western blotting, for each cell line. Cell adhesion to plastic
was found not to correlate significantly by least-squares regression analysis with
S100A4 protein levels (correlation coefficient R
2=0.23; probability points lie on a straight line, P=0.27) (Figure 2A), nor with Neu
(R
2=0.036; P=0.71) protein levels (Table 1).
The rate of cell proliferation measured during the exponential phase of growth varied
for each cell line. To examine whether cell proliferation was associated with S100A4
expression, the rate of cell proliferation was plotted against the protein levels
of S100A4 as determined by Western blotting for each cell line (Figure 2B). Cell proliferation
rates did not correlate with levels of S100A4 protein by least-squares regression
analysis (R
2=0.002; P=0.97) (Figure 2B), although they did with levels of the Neu protein (R
2=0.6; P=0.04) (data in Table 1).
Cell motility in vitro and levels of S100A4
Chemotactic motility was measured as described in Materials and Methods. Motility
was determined over a 24 h period, a time period, which was shown to ensure that all
motile cells remained attached to the lower side of the filter (Jenkinson, 2001).
Cell motility varied from about seven to nearly 130 migrating cells per field (Table
1). Motility of the S100A4-transfected cell lines 4Neup9Ka, 8Neup9Ka was significantly
higher than their respective parental cell lines 4Neu and 8Neu (Student's t-test,
P<0.03) (Table 1). Moreover, the motilities of cell lines isolated from the neu/S100A4
transgenic mice (PN1, PN2) were higher than those isolated from neu transgenic mice
(4Neu, 8Neu) (Table 1).
Comparison of S100A4 levels with cell motility for the different cell lines showed
that S100A4 levels and motility were closely associated (Table 1). This difference
in cell motility and S100A4 protein levels is significant when 8Neu is compared individually
to all the other cell lines (Student's t-test, P<0.05). When S100A4 protein levels
are plotted against motility, there is a significant linear relationship when assessed
by a least-squares regression analysis (R
2=0.822; P=0.012) (Figure 2C). In the case of Neu, no correlation was found between
cell motility and Neu protein levels for the different cell lines, by least-squares
regression analysis (R
2=0.0061; P=0.88) (Table 1).
Cell invasion in vitro and levels of S100A4
To allow for direct comparison between cell invasion and cell motility, the invasive
abilities of the cell lines were measured using the assay conditions optimised for
the motility assay but with a coating of Matrigel on the filter separating the upper
and lower compartment of the Boyden chamber. Using this assay, cell invasion for the
different cell lines varied from about three to nearly 26 invading cells per field
(Table 1). The invasive ability was significantly higher in the S100A4 transfected
cell lines 4Neup9Ka, 8Neup9Ka than their corresponding Neu parental cell lines 4Neu,
8Neu (Student's t-test, P<0.03) (Table 1). Moreover, the invasive abilities of cell
lines isolated from the neu/S100A4 transgenic mice (PN1, PN2) were higher than those
isolated from neu transgenic (4Neu, 8Neu), that for PN2 was significantly so (Student's
t-test, P<0.02).
A close association was also observed between increasing S100A4 protein levels and
increasing invasive ability of the cell lines (Table 1). All of the cell lines expressed
a significantly higher level of S100A4 protein than the 8Neu cell line (Student's
t-test, P<0.01) and demonstrated a significantly higher invasive ability when compared
individually to 8Neu (Student's t-test, P<0.01). A significant linear correlation
between S100A4 protein levels and invasive ability was demonstrated by least-squares
regression analysis for each cell line (R
2=0.765; P=0.022) (Figure 2D). However, the level of Neu protein was unrelated to
the invasive abilities of the cell lines, since increasing levels of Neu protein did
not correlate with increasing cell invasion (R
2=0.0006; P=0.96) (Table 1).
To examine whether the invasive abilities of the cell lines correlated with those
of their motile properties, these two parameters were compared for each cell line.
A plot of cell motility against cell invasion for six cell lines approximated to a
straight line and showed a strong correlation between these two parameters using least-squares
regression analysis (R
2=0.95; P=0.00096) (Figure 2E). Interestingly, the 4Neu cell line and its S100A4-transfected
derivative cell line 4Neup9Ka showed the same five-fold difference between motility
and invasion, and the 8Neu cell line and its derivative transfectant 8Neup9Ka showed
a similar difference between motility and invasion of 2.2- and 2.7-fold, respectively
(Table 1). It would therefore seem that the invasive abilities of the cell lines through
Matrigel are directly related to their motile abilities in vitro.
Matrix metalloproteinase production and levels of S100A4
Matrix metalloproteinase (MMP) activities were measured by gelatin zymography of conditioned
media harvested from cell lines grown in Routine Medium supplemented with 2% (v v−1)
FCS, the same medium as that used in the upper chamber of the Boyden system during
the invasion assay. To analyse any effect of cell confluency on MMP production, medium
was harvested from sparse cultures of cells, grown to 40–50% confluency and from dense
cultures of cells grown to 80–90% confluency.
Bands of lysed gelatin were detected in 2 and 20 μg samples of the conditioned medium
of sparsely grown cultures (Figure 3A, B
Figure 3
Zymogram analysis of matrix metalloproteinase production by cell lines. Conditioned
medium harvested from each cell line plated at a low density for (A) 2 μg protein,
(B) 20 μg protein, and at a high density for (C) 20 μg protein was analysed by gelatin
zymography for the presence of both pro and active MMPs, as described in Materials
and Methods.
), but no bands were present in the same amounts of conditioned medium isolated from
dense cell cultures (Figure 3C). Analysis of 2 μg of protein from conditioned medium
showed clear bands at a size corresponding to proMMP9 (M
r 92 kDa) and MMP9 (M
r 78 kDa) (Figure 3A). A single band was seen at 67 kDa, which corresponds to the
size of MMP2 (Figure 3A). When 20 μg of protein from conditioned medium was analysed,
the separation between proMMP9 and MMP9 bands was lost and a large band of lysis was
also seen at a size corresponding to MMP2 (Figure 3B). In sparse cultures, the amount
of digestion was dependent on the amount of total protein (compare Figure 3A and B).
The molecular mass of the lysed bands and the intensity of the bands of digestion
of the gelatin substrate was, however, the same for each cell line for either 2 or
20 μg of protein from conditioned medium. This result provided semiquantitative evidence
that each of the cell lines from these sparse cultures produced approximately the
same amount of both pro and active MMPs. In contrast, conditioned medium from the
dense cultures contained no detectable pro or active MMPs (Figure 3C). No attempt
was made to measure MMP activity in cells or cell membranes.
Incidence, metastatic ability and pathology of tumours produced by selected cell lines
In all, one million cells from the low S100A4-expressing 8Neu cell line, from the
moderate S100A4 expressing 8Neup9Ka and from the high S100A4-expressing PN2 cell line
were injected into the mammary fat pads of female nude mice. All mice produced mammary
gland tumours after a period of 2–3 weeks (100% incidence) (Table 2
Table 2
Incidence of mammary gland tumours and lung metastases in nude mice injected with
cell lines
Cell line injecteda
Total number of miceb
Number of mice with mammary gland tumoursc (%)
Number of mice with metastasesd (%)
8Neu
19
19 (100%)
0 (0%)
8Neup9Ka
20
20 (100%
6e,f (30%)
PN2
19
19 (100%)
9f,g (48%)
a
Cell lines representative of three different cell types, those isolated from neu transgenic
mice (8Neu), their S100A4-transfected derivatives (8Neup9Ka) and those isolated from
neu/S100A4 double transgenic mice (PN2) were injected into the mammary fat pad of
female nude mice.
b
Total number of mice injected for each cell line.
c
Number of mice with palpable mammary gland tumours 3 weeks after inoculation with
cell lines; percentage of mice bearing tumours (%) is also shown.
d
Number of mice with metastases in the lungs; percentage of tumour-bearing mice with
metastases (%) is also shown.
e
All six of the lung metastases were classified as micrometastases, <0.1 mm diameter.
f
Significantly higher number of metastases than the 8Neu cell line (Fisher's exact
test; 8Neup9Ka, P=0.028; PN2, P=0.005).
g
Two of the lung metastases were classified as macrometastases >1 mm diameter, and
the remaining seven lung metastases were classified as micrometastases <0.1 mm diameter.
).
The primary tumours consisted of chord-like structures (Figure 4A
Figure 4
Histopathological appearance of cell lines growing as tumours in nude mice. (A) Mammary
tumour produced by PN2, showing chord-like structures (c), surrounded by spindle cells
(arrows). (B) Mammary tumour produced by PN2, showing a predominant spindle cell component.
(C) Mammary tumour produced by 8Neup9Ka, showing invasion of muscle (arrows). (D)
Lung (l) macrometastasis produced by a PN2-derived tumour, showing chord-like structures.
(E) Higher magnification of PN2-derived lung macrometastasis in (D) above, showing
chord-like structures with central necrosis (n). (F) Lymph node (l) metastasis produced
by an 8Neup9Ka-derived tumour, showing chord-like structures with central necrosis
(n). Magnification, (A, B, C) × 220; (D) × 22; (E) × 175; (F) × 55. Bars, (A, B, C,
E) 50 μm; (D, F) 200 μm.
) with varying amounts of spindle cells (Figure 4B), but only the 8Neup9Ka and PN2
cell lines showed local invasion of the muscle (Figure 4C) and produced lung metastases
(Figure 4D) (Table 2). For the moderate S100A4-expressing 8Neup9Ka cell line, all
of the lesions in the lung were classified as micrometastases, less than 0.1 mm in
diameter. For the PN2 cell line, two mice produced macrometastases, greater than 1 mm
in diameter, and the remaining seven mice, which produced lesions in the lung, were
classified as micrometastases, as above (Table 2). The histology of the lung metastases
was similar to the primary with chords of cells (Figure 4E), often with central necrosis
for the larger lesions (Figure 4E). Both the moderate S100A4-expressing 8Neup9Ka cell
line and the high S100A4-expressing PN2 cell line produced a statistically significant
greater number of lung metastases than the low S100A4-expressing 8Neu cell line (Fisher's
exact test, P<0.03). The incidence of metastasis in the high S100A4-expressing PN2
cell line was also greater than that in the moderate S100A4-expressing 8Neup9Ka cell
line, although not statistically significant (Fisher's exact test, P=0.18). Furthermore,
when the level of S100A4 protein in the cultured cells was plotted against the percentage
of tumour-bearing mice with lung metastases, a straight line could be drawn through
the three points (least-squares regression analysis R
2=0.9965; P=0.038) (Figure 2F). In contrast, no such linear relationship occurred
between the levels of Neu protein and the percentage of mice with metastases (R
2=0.18; P=0.72; graph not shown). Metastases were also observed in the inguinal lymph
nodes of some of the mice injected with the 8Neup9Ka and PN2 cell lines; however,
these were not included in any statistical analyses, since lymph node tissue was not
available from all of the injected mice.
Immunocytochemical staining for Neu showed that all three cell lines 8Neu (Figure
5A
Figure 5
Immunocytochemical staining of mammary tumours and metastases produced in nude mice.
(A) Mammary tumour produced by 8Neu, showing strong cell surface staining of carcinoma
cells for Neu (arrows). (B) Mammary tumour produced by PN2, showing strong staining
of chords of epithelial cells for Neu (e), the stromal fibroblasts (f) were unstained.
(C) Mammary tumour produced by PN2, showing strong staining of spindle cells for Neu.
(D) Lymph node metastasis produced by an 8Neup9Ka-derived tumour, showing strong staining
for Neu (m), but no staining of lymphoid cells (l). (E) Lung micrometastasis produced
by an 8Neup9Ka-derived tumour, showing strong staining for Neu (m), but no staining
of parenchymal lung tissue (l). (F) Lung metastasis produced by a PN2-derived tumour,
showing strong staining for Neu (m), but no staining of lung parenchymal tissue (l).
(G) Mammary tumour produced by 8Neu, showing no staining above background for S100A4.
(H) Mammary tumour produced by 8Neup9Ka showing low/moderate staining for S100A4.
(I) Mammary tumour produced by PN2, showing moderate staining of spindle cells for
S100A4. (J) Higher magnification of a mammary tumour produced by PN2 showing moderate/strong
cytoplasmic staining for S100A4. (K) Lymph node metastasis produced by an 8Neup9Ka-derived
tumour, showing strong staining for S100A4 (m), but no staining of lymphocytes (l).
(L) Lung micrometastasis, showing strong staining for S100A4 (m), but no staining
of lung parenchymal tissue (l). Magnification, (A, E, J) × 550; (B, C, F, G, H, I,
K, L) × 220; (D) × 22. Bars, 50 μm; (A, E, J) 25 μm; 50 μm; (B, C, F, G, H, I, K,
L); (D) 200 μm.
), 8Neup9Ka and PN2 (Figure 5B) produced strongly-staining primary tumours, even when
they were predominantly composed of spindle cells (Figure 5C). Lymph node metastases
(Figure 5D), the micrometastases (Figure 5E) and the macrometastases (Figure 5F) in
the lungs were also stained strongly for Neu. In contrast, immunocytochemical staining
of the same lesions for S100A4 depended on the identity of the cell line injected
into the nude mice. Thus, no staining for S100A4 was seen in the tumours produced
by the 8Neu cell line (Figure 5G). The tumours produced by the 8Neup9Ka cell line
showed a low to moderate staining for S100A4 (Figure 5H), which varied within each
tumour. Tumours produced by the PN2 cell line showed higher levels of staining for
S100A4 than the tumours produced by the other two cell lines, particularly for the
spindle cell component (Figure 5I, J). Immunocytochemical staining of the macrometastases
for S100A4 usually showed a stronger intensity of staining than was present in the
primary tumours (compare Figures 5K and I), and all the micrometastases in the lungs
were stained strongly (Figure 5L). There was no immunocytochemical staining of lymphocytes
observed in the lymph nodes of the nude mice (Figure 5K). Although no immunocytochemical
staining for Neu or S100A4 was found in the lung tissue of the nude mice bearing mammary
gland tumours produced by the 8Neu cell line (not shown), clusters of stained cells
forming micrometastases and even single cells were readily discernable in the lungs
of mice with tumours produced by the 8Neup9Ka and PN2 cell lines (Figure 5E, L). These
results confirmed that the tumour cells observed in the lungs were derived from the
injected cell lines and not from the parenchymal lung tissue, and that these cells
had arisen from the primary tumours and metastasised to the lungs.
DISCUSSION
Cell lines expressing elevated levels of S100A4 have been obtained either by direct
transfection of cell lines from mice carrying the neu transgene or by isolation of
cell lines from mice carrying both the neu and the S100A4 transgenes. The former mice
produce only nonmetastatic tumours, while the latter mice produce metastatic tumours
(Davies et al, 1996). The establishment of these cell lines expressing different levels
of Neu and S100A4 has provided the opportunity to examine the effects of different
levels of these proteins on cell behaviour in vitro, which may be related to metastasis
in vivo. There was no correlation between the levels of S100A4 and cell-substrate
adhesion to plastic substrata or proliferation rates for this particular set of cell
lines; cell–cell adhesion was not measured. These data are the first to demonstrate
in a transgenic murine-derived tumour system that S100A4 levels correlate positively
in a linear fashion with cell invasive ability through Matrigel in response to an
FCS density gradient. This result demonstrates also that S100A4 causes, either directly
or indirectly, the enhanced invasive ability in at least the S100A4-transfected cell
lines. In contrast, correlation of levels of Neu protein with only proliferation rates
and not with substrate adhesion, motility or invasion is consistent with the expression
of the neu transgene causing the largely proliferative breast lesions and noninvasive
breast tumours observed in transgenic mice (Davies et al, 1996) and rats (Davies et
al, 1999). To exclude the possibility that any correlations were obtained by chance
with the cell lines reported here (Table 1), the analyses were expanded to include
a further two cell lines with differing levels of Neu and S100A4 from another neu
transgenic mouse tumour. These analyses still yielded the same significant correlations
as before (Jenkinson, 2001).
Whether production of S100A4 correlates with invasive ability has remained equivocal.
Previously, S100A4 had been shown to correlate with the invasive ability in vitro
of Lewis lung carcinoma cell lines (Takenaga et al, 1994). However, in a previous
murine mammary gland-derived cell line system, S100A4 levels were found not to correlate
with invasive ability through Matrigel (Ford et al, 1995). In that system, invasive
ability was observed for only one S100A4-expressing cell line. Comparison of this
invasive cell line to the other S100A4-expressing, noninvasive cell lines in that
study, demonstrated that the invasive cell line was the only one to possess proteolytic
activity, at least in vitro (Ford et al, 1995). Zymogram analysis of proteolytic activity
in the cell lines isolated here showed that MMP2 and MMP9 were secreted in both pro
and active forms at similar levels, in all of the cell lines, independently of levels
of S100A4. Thus, in our present system, S100A4 would appear not to regulate cell invasion
by increasing proteolytic activity either in low- or high-density cultures. Since
MMP activity in membranous fractions was not measured, we cannot exclude the possibility
that differences in these activities occurred in our systems and that these differences
could be regulated by S100A4.
Matrix metalloproteinase production could, however, be regulated in all of the cell
lines by confluency in culture. Matrix metalloproteinase activity was observed only
in the conditioned medium of sparsely plated cells. It would therefore seem that complete
cell–cell contact provides an inhibitory extracellular signal, which prevents MMP
production/secretion in these cells. Previous studies have also demonstrated that
proteolytic activity is dependent on cell density. For example, in the media of sparse
astrocytoma cells, more collagenase activity was seen than in dense cultures (Tamaki
et al, 1996), and in epidermoid carcinoma cells, MMP9 secretion was detected only
in sparse cultures (Xie et al, 1994). Furthermore, MMP2 binding to the cell surface
of breast carcinoma cells was detected only in low-density cultures (Menashi et al,
1998). The most likely explanation for this observation would be self-preservation
of the tumour, as MMP production in a tight mass of tumour cells would possibly lead
to tumour cell degradation. The findings reported here are, therefore, in accordance
with those of the Lewis lung carcinoma cell line model described above, where MMP9
was produced in all of the cell lines, independently of the level of S100A4 (Takenaga
et al, 1994), but contrast with studies in an osteosarcoma cell system which implicated
S100A4 in the control of both the levels of MMPs and of the tissue inhibitors of matrix
metalloproteinases (TIMPs) (Bjornland et al, 1999). In the latter system, the reduction
in S100A4 expression in these cells resulted in downregulation of mRNA levels for
MMP2, MT-MMP1 and TIMP-1, and upregulation of TIMP-2. The levels of TIMPs produced
in the cell system isolated and characterised in this study have not been examined.
Thus, it is still possible that TIMPs may be differentially expressed in our cell
lines and thereby inhibit MMP activity to a different extent in each of the cell lines,
although their role in vivo is more complex (Hayakawa et al, 1992; Sato et al, 1994;
Ree et al, 1997; Yoshiji et al, 1998; Remacle et al, 2000).
In contrast to gelatinase activity, motility, a second major component of invasion
examined in vitro, showed a strong linear correlation with levels of S100A4 protein
in the different cell lines. The stimulus for motility did not need to be directional,
since chemotactic and chemokinetic stimuli provided by 10% FCS, induced the same degree
of motility (Jenkinson, 2001). However, 2% FCS was only able to stimulate minimal
motility of the cell lines (Jenkinson, 2001), suggesting that a threshold level of
motility-stimulating molecules are probably required for cell motility. These results
in vitro suggest that an appropriate and sufficient chemical stimulus is probably
required for cell migration in vivo. The results demonstrated here are in accordance
with previous findings in other cell systems that increased S100A4 levels results
in increased cell motility (Takenaga et al, 1994; Ford et al, 1995). A significant
linear correlation between S100A4 and cell motility, however, has only been previously
demonstrated in the Lewis lung carcinoma cell system by measuring the phagocytic tracks
of cells in the absence of a chemotactic stimuli (Takenaga et al, 1994). The results
presented in this study are, therefore, the first to demonstrate in a mammary tumour
cell system, that cell motility correlates with levels of S100A4 in a statistically
significant linear fashion and that, at least in the transfected cell lines, S100A4
is the cause of this change. Furthermore, cell motility was shown to correlate strongly
with cell invasion. The fact that the gradient of the plot of S100A4 against motility
is about five times that of the plot of S100A4 against invasion (Figure 2C, D) suggests
that the same cells are having to penetrate a more resistant barrier. However, since
both plots are linear and the plot of invasion against motility is also linear with
a similar gradient (Figure 4E), the three parameters are likely to be closely related.
It is therefore probable that S100A4-induced increases in invasive ability are a result
of S100A4-driven cell motility in the cell lines under study. The requirement for
proteolytic activity for successful cell invasion should not, however, be underestimated,
since previous reports have shown that high S100A4-expressing mammary tumour cell
lines, which were shown to be motile in vitro, were not invasive in vitro or in vivo,
due to a lack of MMP production (Ford et al, 1995). This difference may reflect differences
between the two mouse mammary systems under study. The transgenic mouse system reported
here may produce sufficient levels of MMPs for motility to be rate limiting for invasion,
whereas for the mouse system of Ford et al (1995) this is not the case.
The mechanisms whereby S100A4 modulates cell motility are unknown. However, S100A4
is known to bind to various components of the actin–myosin cytoskeleton (Watanabe
et al, 1993; Kriajevska et al, 1994; Takenaga et al, 1994). In addition, recent advances
in DNA microarray technology have led to the isolation of several other metastasis-associated
genes, many of the products of which are associated with the cellular cytoskeleton
(Khanna et al, 2001) and its regulation (Clark et al, 2000). The ability of S100A4
to bind and thereby regulate the phosphorylation of nonmuscle myosin heavy chain in
vitro (Kriajevska et al, 2000), inter alia (Chen et al, 2001), may indicate that S100A4
plays a role in regulating cellular dynamics. A precedent has already been set for
myosin phosphorylation as a mechanism of regulating cell motility. The Rho family
of small GTPase proteins regulates motility by regulating actin–myosin dynamics via
phosphorylation of both the myosin heavy and light chains (Amano et al, 1996; Kimura
et al, 1996). The Rho family has, however, been fairly extensively studied in the
context of cell motility, while relatively little is known, as yet, about the effector
mechanisms of S100A4 and other metastasis-related proteins in mediating motility.
The tumorigenic and metastatic abilities of the cell lines were examined in the immunoincompetent,
nude mouse, without the complication of an immune response. The isolation and culture
of tumour cells from both neu and neu/S100A4 transgenic mice increased the efficiency
of mammary tumour production from 44 and 68% as seen after 14 months in neu and neu/S100A4
transgenic mice (Davies et al, 1996), respectively, to 100% in the nude mice for all
of the injected cell lines. This increase in efficiency of production of primary tumours
was probably due to the following reasons. Firstly, constitutive expression of neu
was observed in the cell lines. There was no requirement to add hormones in vitro
or to mate in vivo to stimulate the expression of neu, whereas in the original transgenic
mice the neu transgene requires the hormones of pregnancy to activate its MMTV promoter
(Bouchard et al, 1989; Davies et al, 1996). Secondly, in the neu transgenic mice all
of the epithelial cells express the neu transgene, but only some cells are able to
form tumours. This latter result indicates that a second genetic change is required,
in addition to expression of the activated neu transgene, to allow tumour formation.
One possible explanation is the high level of the loss of heterozygosity, a fact which
may be related to the loss of tumour suppressor genes, which has previously been observed
in these neu transgenic mice (Cool and Jolicoeur, 1999). Since cell–cell adhesion
was not measured, the possible role of E-cadherin as a tumour suppressor in these
systems is unknown. Whatever the change required, this event had clearly taken place
in the tumours from which the cell lines were isolated.
The frequency of tumour metastasis, however, remained the same in the neu/S100A4 tumour-derived
PN2 cell line as in the neu/S100A4 transgenic mice, with only 50% of mice with mammary
gland tumours developing lung metastases. The efficiency of tumour metastasis for
the 8Neup9Ka cell line was lower, with only 30% of mice with mammary gland tumours
developing lung metastases. The 8Neu cell line developed no lung metastases. These
in vivo results follow the same pattern observed for the cell lines in in vitro assays,
where the 8Neu cell line had a low level, the 8Neup9Ka cell line a moderate level,
and the PN2 cell line a high level of both motility and invasion. Confirmation that
these metastases are derived from the neu/S100A4-expressing mammary gland tumours
is obtained by positive staining of these metastases for both Neu and S100A4. The
level of metastasis expressed as a percentage of afflicted animals was found to be
positively correlated with the level of S100A4, but not with the level of Neu protein
in the cell lines, albeit for only three of the cell lines. Metastasis in these Neu-expressing
cell lines is therefore clearly dependent, either directly or indirectly, on the level
of S100A4 protein.
In this transgenic mouse model system, neu acts as an oncogene producing neoplastic
transformation of the targeted mammary epithelial cells in vivo (Davies et al, 1996)
and thereby enables immortalised mammary epithelial cell lines to be established in
culture, as described in this report. Without overexpression of neu these cells remain
normal and cannot be grown in culture (Jenkinson, 2001) without additional immortalising
events. The tumours produced by the neu transgenic mice (Davies et al, 1996) or by
a derived mammary epithelial cell line, when reintroduced into mice, are indolent
and do not metastasise. These results are consistent with the correlation between
the levels of Neu protein in the different cell lines and their growth rates in culture,
but not with other properties measured including metastasising ability in vivo. Tissues
of transgenic mice overexpressing S100A4, on the other hand, fail to undergo neoplastic
transformation and that includes those of the mammary gland (Davies et al, 1995).
However in neu-transformed neoplastic immortalised mammary cell lines, in vitro studies
demonstrate that S100A4 levels correlate well with cell invasion and that S100A4 mediates,
either directly or indirectly, this invasion through its ability to induce cell motility,
at least in the transfected cells. It therefore seems probable that S100A4-driven
cell motility mediates invasion in vivo and thereby metastasis in vivo, at least in
this particular model system.