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Establishment of a matrix-associated transepithelial resistance invasion assay to precisely measure the invasive potential of synovial fibroblasts.

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ARTHRITIS & RHEUMATISM
Vol. 60, No. 9, September 2009, pp 2606–2611
DOI 10.1002/art.24782
© 2009, American College of Rheumatology
Establishment of a
Matrix-Associated Transepithelial Resistance Invasion Assay to
Precisely Measure the Invasive Potential of Synovial Fibroblasts
Christina Wunrau,1 Eva-Maria Schnaeker,2 Katharina Freyth,1 Noreen Pundt,1
Doreen Wendholt,1 Katja Neugebauer,1 Uwe Hansen,1 Thomas Pap,1 and Berno Dankbar1
led to a measurable decrease and subsequent breakdown of electrical resistance. Unlike in the assay with
OASFs, which did not achieve a breakdown of resistance
up to 72 hours, RASFs exhibited a pronounced invasiveness in this assay, with a 50% breakdown after 42 hours.
Treatment of fibroblasts with either a matrix metalloproteinase inhibitor or antibodies against ␤1 integrin
significantly reduced the invasiveness of RASFs.
Conclusion. The MATRIN assay is a valuable and
sensitive biologic assay system that can be used to
determine precisely the invasive potential of RASFs in
vitro, and thus would be suitable for screening antiinvasion compounds.
Objective. Synovial fibroblasts (SFs) contribute to
several aspects of the pathogenesis of rheumatoid arthritis (RA) and have been implicated most prominently
in the progressive destruction of articular cartilage.
Targeting the invasive phenotype of RASFs has therefore gained increasing attention, but the precise measurement of their invasive capacity and the evaluation
of potential treatment effects constitute a challenge that
needs to be addressed. This study used a novel in vitro
invasion assay based on the breakdown of transepithelial electrical resistance to determine the course of
fibroblast invasion into extracellular matrix.
Methods. A matrix-associated transepithelial resistance invasion (MATRIN) assay was used to assess
SFs from patients with RA in comparison with SFs from
patients with osteoarthritis (OA). The SFs were grown
on a commercially available collagen mix that was
placed onto the upper side of a Transwell polycarbonate
membrane. In addition, freshly isolated cartilage extracts were studied to assess the conditions in vivo.
Under this membrane, a monolayer of MDCK-C7 cells
was seeded to create a high electrical resistance.
Results. Invasion of fibroblasts into the matrix
affected the integrity of the MDCK-C7 monolayer and
The extracellular matrix (ECM) is a network of
structural proteins, including collagens, glycoproteins,
and proteoglycans. The excessive proteolysis of the
ECM plays an essential role in conditions such as
rheumatoid arthritis (RA). The hyperplastic synovial
membrane is a key feature of this chronic systemic
inflammatory disease (1), and synovial fibroblasts (SFs)
of the lining layer are key effectors of cartilage degradation (2,3). RASFs exhibit features of stable cellular
activation, resulting in up-regulation of both adhesion
molecules and matrix-degrading enzymes, especially matrix metalloproteinases (MMPs) (4). Quantification of
the invasive potential of RASFs, as well as the assessment of treatment strategies with respect to matrix
destruction, has been a major challenge. Until now,
invasion has been measured by methods such as the
Boyden chamber assay or in vivo in the SCID mouse
model (5,6). However, all of these assays have several
disadvantages, the most striking of which is the imprecise nature of the readout data that serve to quantify the
invasive potential of RASFs.
Supported in part by SET Stiftung, Germany. Dr. Schnaeker’s
work was supported by DFG SCHN 1071/1-1.
1
Christina Wunrau, MSc, Katharina Freyth, Dipl Biol, Noreen Pundt, MSc, Doreen Wendholt, PhD, Katja Neugebauer, PhD,
Uwe Hansen, PhD, Thomas Pap, MD, Berno Dankbar, PhD: University Hospital Muenster, Muenster, Germany; 2Eva-Maria Schnaeker,
MD: St. Josef Hospital, Cloppenburg, Germany.
Drs. Pap and Dankbar contributed equally to this work.
Address correspondence and reprint requests to Thomas Pap,
MD, Institute of Experimental Musculoskeletal Medicine, University
Hospital Muenster, Domagkstrasse 3, D-48149 Muenster, Germany.
E-mail: thomas.pap@uni-muenster.de.
Submitted for publication August 15, 2008; accepted in
revised form June 1, 2009.
2606
MATRIN ASSAY FOR SYNOVIAL FIBROBLAST INVASIVENESS
The purpose of this study was to establish an in
vitro assay that determines precisely the invasive potential of SFs as well as the kinetics of matrix degradation of
SFs. The assay is based on the detection of a decrease in
and subsequent breakdown of the transepithelial electrical resistance (TEER) generated by an epithelial
monolayer. Presently, TEER assays are used to measure
the invasive potential of tumor cells, especially melanoma cells (7–9). In these assays, the breakdown of
electrical resistance indicates the opening of tight junctions prior to cell invasion (9). This system was recently
modified by adding a collagen matrix between the cell
layer and the invasive RASFs, in order to obtain an assay
that comes closer to the in vivo situation of human RA.
This matrix-associated transepithelial resistance invasion (MATRIN) assay offers the possibility of comparing precisely the invasive potential of RASFs with that of
other cells, and facilitates the measurement of effects of
pharmacologic modifications in a low volume with a high
throughput and high reproducibility.
PATIENTS AND METHODS
Tissue preparation and cell culture. Samples of synovial membrane tissue were obtained from patients with RA
whose diagnosis met the American College of Rheumatology
(formerly, the American Rheumatism Association) criteria
(10) and from patients with osteoarthritis (OA) at the time of
joint replacement surgery. Fibroblasts were isolated by enzymatic digestion of synovial tissue, and the cells were then
cultured in Dulbecco’s modified Eagle’s medium. The human
melanoma cell line A7 was grown in RPMI 1640. The highresistance C7 subclone of Madin-Darby canine kidney cells
(MDCK-C7) was cultured in minimum essential medium. The
properties of these cells have been described in detail previously (11). All media (PAA Laboratories, Pasching, Austria)
were supplemented with 10% fetal calf serum (Biochrom,
Berlin, Germany), 100 units/ml penicillin, and 10 ␮g/ml streptomycin (PAA Laboratories).
MATRIN assay. A collagen matrix consisting of 97%
type I and 3% type III collagen (Inamed Biomaterials, Fremont, CA) was coated onto the upper side of a polycarbonate
membrane of a filter cup (area 4.2 cm2, pore diameter 0.4 ␮m)
mounted in a 6-well dish (BD, Heidelberg, Germany). In an
additional experimental setting, pieces of human cartilage
sections were homogenized and the suprastructural cartilage
components were extracted. Cartilage extracts (200 ␮l; protein
concentration 0.25 mg/ml) were coated onto the membranes
with a heterobifunctional crosslinker (2 mM sulfo-SANPAH;
Pierce, Rockford, IL) and ultraviolet light (2 lamps at 4 watts
each). In all settings, MDCK-C7 cells were seeded onto the
basal side of the coated filter cups. Both the upper and lower
chambers contained 2.5 ml of medium, and 1.0 ml from each
compartment was replaced by fresh medium every day to
maintain stable metabolic conditions.
2607
Electrical resistance across the MDCK-C7 monolayer
was measured with a set of 2 circular Ag/AgCl electrodes
connected with an ohm-meter (World Precision Instruments,
Sarasota, FL). These electrodes were assembled in a flexible
clamp, which facilitates the positioning of one electrode into
the insert and of the other electrode into the well, always with
the same immersion depth into the medium. Following full
establishment of the MDCK-C7 monolayer, 7 ⫻ 105 fibroblasts
were seeded onto the collagen matrices. In some experiments,
RASFs were treated with 200 ng/ml MMP Inhibitor III (Calbiochem, Nottingham, UK) or incubated with 20 ␮g/ml ␤1
integrin function–blocking antibody (Chemicon, Billerica,
MA) to specify measurable alterations of the invasive behavior.
To avoid an artificial decrease in resistance due to alterations
in temperature or CO2, the 6-well plate was measured within 3
minutes, allowing measurements of kinetics. All measurements
were performed in quadruplicate and were terminated after
total breakdown of the electrical resistance.
MMP enzyme-linked immunosorbent assays
(ELISAs). Supernatants were collected from the MATRIN
assay at appropriate time intervals. MMP ELISAs were performed according to the manufacturer’s instructions (R&D
Systems, Minneapolis, MN).
Statistical analysis. Data are expressed as the mean ⫾
SD. Statistical analysis was performed using GraphPad Prism
software (version 4.0; GraphPad Software, San Diego, CA).
Correlations were determined by Spearman’s rank correlation
test for bivariate analysis. Differences between groups were
examined for statistical significance using the Mann-Whitney
test. P values less than 0.05 were considered statistically
significant.
RESULTS
The experimental setting and procedure for the
MATRIN assay are illustrated in Figure 1A. After the
MDCK-C7 cells had reached confluence (Figure 1B)
and had established an electrical resistance up to
3,500⍀, the SFs were added onto the collagen matrix in
the upper compartment of the Transwell system. The
decrease and subsequent breakdown of the electrical
resistance in the course of invasion were determined as
a way to evaluate the invasiveness of the fibroblasts.
The first set of experiments determined the invasive potential of RASFs in comparison with that of
OASFs (using invasive A7 melanoma cells as a positive
control). As shown in Figure 2A, MDCK-C7 cells were
able to keep the initially established electrical resistance
for more than 144 hours, demonstrating that the lack of
cells in the upper compartment preserved the integrity
of the monolayer over prolonged periods of time. Addition of RASFs led to a 50% breakdown of the TEER
within 46 hours, followed by a complete breakdown after
⬃72 hours. In contrast, addition of OASFs resulted in a
50% breakdown of the TEER only after 112 hours
2608
WUNRAU ET AL
Figure 1. Matrix-associated transepithelial resistance invasion (MATRIN) assay for measurement of synovial fibroblast (SF) invasion.
A, The schematic representation of the MATRIN assay shows a filter cup coated apical with collagen, which was mounted in a culture
dish. An MDCK-C7 monolayer was grown basal on the insert, and rheumatoid arthritis SFs or control cells were added on top of the
collagen matrix. Electrical resistance was measured with a set of electrodes. B, Assessment of the monolayer of MDCK-C7 cells shows
that MDCK-C7 cells constitute a cell clone with particularly “tight” junctional complexes. The high transepithelial electrical resistance
of the C7 subclone of MDCK cells can be measured.
(⌬t50 ⫽ 66 hours; P ⬍ 0.01 versus RASFs), indicating
that RASFs exhibit a significantly higher invasive capacity than OASFs. As expected, A7 cells, which were used
as a positive control, displayed a high degree of invasiveness, as demonstrated by a 50% breakdown of the
electrical resistance within 18 hours and a subsequent
complete breakdown after ⬃24 hours.
In order to determine whether the MATRIN
assay involving coating with a type I and type III
collagen matrix is an accurate and reliable setting for
measuring fibroblast invasion, we compared the results
in this matrix with the observations in isolated human
cartilage extracts. Neither the human cartilage extracts
nor the crosslinking procedure itself had effects on the
TEER. Moreover, there were no differences with respect to the invasive potential of RASFs between the
different coating components (Figure 2B).
To understand this process in further detail, we
assessed whether matrix degradation by RASFs, as
measured by the extent of breakdown of electrical
resistance, was associated with the secretion of MMPs.
Supernatants obtained from the MATRIN assay were
analyzed for the levels of MMPs 1, 3, and 13. As shown
in Figure 3A, the expression of all investigated MMPs
was found to be increased more than 4-fold over the
time course of 72 hours. Analysis of the invasion of
Figure 2. Invasive capacity of rheumatoid arthritis synovial fibroblasts (RASFs) in the matrix-associated transepithelial
resistance invasion (MATRIN) assay, and testing of surface coatings in the MATRIN assay. A, RASFs from 4 patients exhibited
a strong invasiveness in the MATRIN assay, as demonstrated by the percent decrease in the transepithelial electrical resistance
(TEER) over a total of 144 hours, whereas osteoarthritis SFs (OASFs) from 5 patients showed a significantly reduced
invasiveness. The A7 cells and the MDCK-C7 layer alone served as internal assay controls, and the broken line indicates the
50% resistance breakdown level. B, The invasive capacity of RASFs was similar regardless of whether the membrane was coated
with fresh human cartilage extract (indicated by asterisk) or commercial collagen (indicated by pound sign). An untreated
MDCK-C7 monolayer served as the control in each culture condition, and a layer treated with crosslinker also served as a
control. Bars show the mean ⫾ SD of triplicate cultures.
MATRIN ASSAY FOR SYNOVIAL FIBROBLAST INVASIVENESS
Figure 3. Detection of treatment effects by matrix-associated transepithelial resistance invasion (MATRIN) assay. A, Secretion of matrix
metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 was detected in
the MATRIN assay, in supernatants of 700,000 rheumatoid arthritis
synovial fibroblasts (RASFs) from 4 different patients. A stepwise
increase in the levels of MMPs 1 and 3 is evident after 24, 48, and 72
hours. Bars show the mean and SD. B, MMP inhibition experiments
were carried out by treating RASFs with 200 ng/ml MMP inhibitor III,
and the invasive potential of the RASFs was then examined by
measuring the percent transepithelial electrical resistance (TEER) in
RASFs treated with inhibitor in comparison with that of untreated
RASFs. The effect was particularly evident at early phases of invasion.
An untreated MDCK-C7 monolayer and a MDCK-C7 layer treated
with inhibitor served as assay controls. Results are the mean of
quadruplicate experiments. Broken lines indicate the 50% and 90%
resistance breakdown levels. C, Fibroblast adhesion was assessed by
treating RASFs with 20 ␮g/ml of a function-blocking antibody for
human ␤1 integrin, which reduced the invasive potential (the percent
TEER) of the RASFs as compared with that of untreated RASFs. An
untreated MDCK-C7 monolayer served as assay control. Results are
the mean ⫾ SD of quadruplicate experiments. Broken line indicates
the 50% resistance breakdown level.
RASFs showed a significant correlation with the levels
of all MMPs measured. For MMP-1, the correlation
coefficient was 0.9940 (P ⬍ 0.05), while that for MMP-3
was 0.9715 (P ⬍ 0.05) and that for MMP-13 was 0.9824
(P ⬍ 0.05).
To confirm the involvement of MMPs in RASF-
2609
mediated breakdown of resistance, as well as to assess
the applicability of this method for the measurement of
treatment effects, we incubated RASFs with the broadspectrum MMP inhibitor III, which blocks collagenases
(MMPs 1 and 13) as well as gelatinase A (MMP-2),
stromelysin (MMP-3), and matrilysin (MMP-7). As
demonstrated in Figure 3B, this inhibitor had no effects
on the integrity of the MDCK-C7 monolayer itself, but
caused a significant delay in the resistance breakdown by
the treated RASFs. This effect was particularly evident
early after the addition of the inhibitor. The 20%
breakdown value was delayed by 14 hours (⌬t80 ⫽ 14
hours; P ⬍ 0.01 versus untreated RASFs). The inhibitory effect decreased somewhat over time and resulted
in a difference of 9 hours before breakdown occurred at
the 50% resistance level (⌬t50 ⫽ 9 hours; P ⬍ 0.01 versus
untreated RASFs).
To further determine the mechanisms by which
breakdown of the resistance occurs, we performed experiments in which we blocked the adhesion of RASFs
to the ECM with the use of anti–␤1 integrin antibodies.
These experiments revealed that blocking of ␤1 integrin
resulted in a significant delay in matrix invasion by
RASFs. As shown in Figure 3C, blocking of ␤1 integrin
caused a delay of 11 hours before breakdown at 50%
resistance (⌬t50 ⫽ 11 hours; P ⬍ 0.01 versus untreated
RASFs).
DISCUSSION
Cell invasion is a complex process in which the
attachment of cells to the ECM and secretion of MMPs
are key steps, leading to digestion of the surrounding
ECM, cell migration, and penetration of the ECM
(4,12). To date, in vitro measurement of cell invasion,
especially of tumor cells, has been performed mainly by
Boyden chamber experiments in which the migrated
cells were counted at a prespecified end point (13). This
method is generally accepted and widely used, but there
are a number of disadvantages associated with this assay;
e.g., the technique of counting the cells is imprecise, and
determination of the invasiveness is limited to only one
time point in one setting. With our novel MATRIN
assay, we designed a truly quantitative method that
permits us to assess cell invasion in a time-dependent
manner by measuring the same parameter several times.
Moreover, our novel MATRIN assay is highly sensitive,
since minor decreases of the TEER could be measured
at early time points, reflecting marginal collagen degra-
2610
dation. Therefore, this assay is suitable not only for the
evaluation of cells exhibiting a low invasive potential but
also for the analysis of invasion kinetics and treatment
effects.
With the use of the MATRIN assay, we were able
to investigate the invasive potential of SFs, which we had
not been able to evaluate with the Matrigel assay. We
could clearly demonstrate that RASFs exhibit a highly
invasive phenotype that differs markedly from that of
OASFs both in its extent and in its kinetics. This is in
accordance with the notion that invasion of SFs into
articular structures is an important feature of joint
destruction in RA and that activated RASFs are involved prominently in this process through their attachment to the cartilage surface and the release of matrixdegrading enzymes (14).
In accordance with the prominent role of MMPs
in cartilage degradation, we found increased MMP
levels over the time course of invasion and a significant
retardation of RASF invasion in the presence of an
MMP inhibitor. Moreover, we demonstrated that blocking of ␤1 integrin, an integrin subunit that has been
shown to be involved prominently in adhesion of cells to
collagen (15), again results in a delayed invasion, indicating that, in addition to the release of MMPs, adhesion
of RASFs to the collagen matrix is an important step in
cartilage invasion.
From a technical point of view, we could clearly
show that our assay is sensitive enough to investigate
treatment-mediated alterations of the invasive potential
of these cells. Another advantage of our assay is the
separation of the cells by a collagen matrix, which avoids
undesirable cell–cell interactions. Whereas MDCK-C7
cells are cultured on the basal side of the cell culture
insert, serving as a measuring instrument, SFs are
seeded on the apical side of the insert onto the collagen
matrix. Thus, SFs are in direct contact with the collagen
matrix, while, conversely, direct interactions of
MDCK-C7 cells and SFs can be excluded. We could also
clearly demonstrate that there is no measurable difference when the membrane is coated with human cartilage
extract or when the membrane is coated with the
commercially available collagen mixture used in these
experiments, supporting the use of the easily accessible
collagen mixture.
Our MATRIN assay is obviously an improvement
over commonly used invasion assays in terms of sensitivity, precision, and handling. This assay allows assessment of not only the overall invasiveness of the cells, but
WUNRAU ET AL
also the kinetics of the invasive process and, thus, of
distinct molecular events associated with the different
steps of cell invasion. Therefore, the MATRIN assay is a
novel tool for analyzing new therapeutic strategies
aimed at modulating the invasive potential of RASFs.
ACKNOWLEDGMENTS
We thank Borna Truckenbrod and Vera Eckervogt for
technical assistance, and Jennifer Gerding for reading the
manuscript. We also thank the Department of Dermatology
(University Hospital Muenster, Muenster, Germany) for
kindly providing the A7 and MDCK-C7 cells.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Pap had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Wunrau, Schnaeker, Freyth, Neugebauer, Hansen, Pap, Dankbar.
Acquisition of data. Wunrau, Schnaeker, Freyth, Pundt, Dankbar.
Analysis and interpretation of data. Wunrau, Schnaeker, Wendholt,
Pap, Dankbar.
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