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* Department of Physics, University of Missouri, Columbia, Missouri 65211; National Institute of Neurosurgery, Budapest, Hungary H-1145; Department of Obstetrics, Gynecology and Women's Health, University of Missouri, Columbia, Missouri 65211; and Department of Biology, University of Missouri, Columbia, Missouri 65211
Correspondence: Address reprint requests to Gabor Forgacs, Dept. of Physics and Dept. of Biology, University of Missouri, Columbia, MO 65211. E-mail: forgacsg{at}missouri.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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). Migrating cells in general exert forces and reorganize their three-dimensional (3D) extracellular matrices (ECM) (2–4). The details of local matrix reorganization in collagen I by invading brain tumor cells have recently been reported (5,6). Degradation of ECM, a hallmark of malignant invasion, takes place through the complex activity of metalloproteinases (MMP) (7–9) and their inhibitors (TIMP) (9,10). The predominant adhesion molecule in brain tissue is a member of the cadherin family of cell adhesion molecules (11,12), N-cadherin (13,14), although the presence of other types, such as T-cadherin (15,16) and E-cadherin (17), has also been reported. The role of N-cadherin in the acquisition of invasive phenotype is still controversial. In contrast to the well-established tumor suppressor role of E-cadherin, N-cadherin might influence the process in a positive or negative manner. In the prostate, during malignant transformation, cells switch from E-cadherin to N-cadherin expression (18). N-cadherin expression in breast cancer cell lines correlates with greater invasive potential (19). Higher N-cadherin level was observed with increasing tumor grade in astrocytic tumors (20), whereas another study found no correlation between N-cadherin expression and the in vitro wound-filling capacity of glioblastoma cell lines (21). The invasive potential of certain glioblastoma cell lines was found to decrease with increasing overall tissue cohesivity but not with decreasing N-cadherin expression (22). However, N-cadherin was downregulated in recurrent malignant astrocytic tumors as compared to the primary lesions and also correlated with the dissemination of tumor cells into the cerebrospinal fluid (23). A further complication stems from the fact that invasion assays have not typically been performed in true three-dimensional matrices, whereas it has been shown that such environment is crucial to the understanding of tumor progression (24).
Motivated by the limited information on the simultaneous effect of cell-cell and cell-matrix interaction in malignant cell invasion (25), we performed a comprehensive study on the migration pattern of nine brain tumor cell lines (along with normal astrocytes) in 3D collagen I matrices. Multicellular aggregates (a widely used model in invasion studies (6,26–29), as representation of the corresponding tissues, were used to quantify cell-cell and cell-matrix interactions. Because tissues composed of adhesive and motile cells are known to mimic the behavior of liquids (30,31), cell-cell adhesion was parameterized in terms of apparent tissue surface tension, a measure of overall tissue cohesivity (32). Measurements were correlated with aggregation assays, N-cadherin expression, and cell morphology. Cell-matrix affinity was assessed by the extent of invasion as a function of time and collagen concentration as well as by quantification of MMP and TIMP expression.
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MATERIALS AND METHODS |
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). The cell line GBM1 is identical to HB, a cell line described previously (33,34). All cell cultures used in this study had passage numbers higher than 10. U87 and U373 are standard cell lines from American Type Culture Collection (ATCC, Manassas, VA). Cells were grown on 75 cm2 tissue culture dishes in medium consisting of DMEM (GIBCO/BRL, Gaithersburg, MD) supplemented with 10% FBS (U.S. Bio-Technologies, Pottstown, PA), 10 µg/ml of penicillin-streptomycin, gentamicin, kanamycin sulfate (Sigma-Aldrich, St Louis, MO). Control rat primary astrocytes were obtained as described in Xu et al. (35).
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Western blot
Confluent cell cultures were lysed in NP40 lysis buffer for 20 min on ice. Cell lysates were centrifuged at 14,000 rpm for 10 min. Protein measurement was performed using the BCA protein assay kit (Pierce, Rockford, IL). An aliquot with 30 µg of total protein was loaded for each lane on an 8% SDS-PAGE gel. Blots were blocked in 5% dry milk in Tris-buffered saline (TBS) and incubated with anti-N-cadherin primary antibody (C-2542, Sigma-Aldrich; 1:1000) and anti-mouse peroxidase-conjugated secondary antibody (A 5906, Sigma-Aldrich; 1:3000).
Flow cytometry
For flow cytometry cells were harvested from confluent tissue culture dishes with trypsin and washed in phosphate-buffered saline (PBS). After fixation in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS, cells were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich). Following extensive washing with ice-cold HBSS containing 1% FBS, cells were incubated with the primary anti-pancadherin antibody (Ab 6529, Ab Cam, Cambridge, MA) for 30 min on ice. The primary antibody was coupled to phycoerythrin-conjugated antirabbit antibody (Ab 7007). After each step, cells were washed at least twice with ice-cold PBS containing 2% FBS. Flow cytometry was performed on a FACScan cell analyzer (Becton Dickinson, Franklin Lakes, NJ). For each cell line, three measurements were recorded from three independent experiments.
Cell aggregate preparation
Confluent cultures grown on 75 cm2 TC dishes were washed twice with Hanks' balanced salt solution (HBSS) containing 2 mM CaCl2, then treated for 10 min with 0.1% trypsin (GIBCO/BRL). Depleted cells were centrifuged at 905 x g for 3 min. The resulting pellet was transferred into capillary micropipettes of 500 µm diameter. Following a 15-min incubation in medium containing 2 mM CaCl2 at 37°C with 5% CO2, the firm cylinders of cells extruded from the pipettes were cut into 500-µm fragments. For invasion assays, the cylindrical aggregates were immediately embedded into collagen gel. For scanning electron microscopy and cohesivity studies, spheroidal aggregates were used. These were obtained from the cylindrical aggregates, which, on further incubation in 10-ml tissue culture flasks (Bellco Glass, Vineland, NJ) with 3 ml DMEM on a gyratory shaker at 120 rpm with 5% CO2 at 37°C, in most cases reproducibly rounded into spheroids in 24–36 h.
Scanning electron microscopy of aggregates' surface
Cell morphology on the surface of aggregates was analyzed by field emission scanning electron microscopy (FESEM). Spherical aggregates were fixed in 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA) in PBS on a low-speed shaker. Subsequently, samples were rinsed three times in PBS. Dehydration was performed by an increasing concentration series of ethanol as follows: 10%, 25%, 50%, 75%, and 95% for 30 min each and finally in 100% ethanol overnight. After critical point drying with CO2 in a Samdri-PVT-3B (Tousimis, Rockville, MD), aggregates were mounted on a holder with double-sided carbon tape and sputter coated with platinum to a nominal thickness of 2 nm. Aggregates were examined with a Hitachi S4700 cold-cathode field-emission scanning electron microscope at 5 kV accelerating voltage.
Measurement of tissue surface tension
The compression apparatus used in this work to measure liquid tissue properties is shown in Fig. 1. Modified from previously used similar devices (36), it is monitored by Labview software (National Instruments, Austin, TX) to record the entire force relaxation following uniaxial compression of a spherical aggregate between the compression plates. To minimize adhesion to the plates, these were coated with poly (2-hydroxyethylmethacrylate) (polyHEMA) (37). A typical measurement was performed as follows. The initially spheroidal aggregate was placed on the lower plate of the apparatus in CO2-independent medium (GIBCO/BRL) with antibiotics (as described above) and rapidly compressed with the help of a stepping motor to produce a deformation of a definite magnitude. To avoid irreversible damage to cells, aggregates were compressed to a maximum of 30% of their original diameter. The relaxation process was followed until the compressive force reached a constant equilibrium value (45–60 min), at which point the plates were separated, and the aggregate left to regain its original shape. Measurements in the rare cases when the aggregate did not regain its precompressed shape were discarded. The shape of the aggregate before, during, and after compression was recorded by a Spot Insight CCD camera (Diagnostic Instruments, Sterling Heights, MI) fitted to a horizontally positioned dissecting microscope (SZ60, Olympus). The surface tension of the model tissue was evaluated using the Laplace equation, 
. Here, 
is the tissue's apparent surface tension (i.e., interfacial tension with the surrounding tissue culture medium), 
is the equilibrium value of the compressive force, 
is the radius of the circular contact area of the compressed aggregate with the plates. 
and 
are the radii of curvature of the aggregate's surface, respectively along its equatorial plane, and its peripheral contour, which is assumed to be circular (see inset in Fig. 1). The geometric parameters were determined by an in-house built tracking program with a precision of 3 µm. The program evaluates the aggregate's recorded contour on the basis of variation in gray-scale values in its vicinity. To check tissue liquidity (i.e., the independence of on the compressive force), aggregates were compressed two or three times with varying force with 60- min recovery in the uncompressed state. Average values were calculated from at least six independent measurements.
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Quantitative real-time PCR
Total RNA was isolated from confluent cell cultures with TriZOL (Invitrogen, Carlsbad, CA), following manufacturer's instructions. Purity and final concentrations of mRNA were determined by spectrophotometric analysis at A280 and A260. Complementary DNAs were constructed from 1 µg of total RNA from each sample using the Roche Diagnostics First Strand Synthesis Kit (Indianapolis, IN) per manufacturer's instructions. Q-RT-PCR was performed with an ABI 7700 system (Applied Biosystems, Foster City, CA). Gene-specific TaqMan Gene Expression Assays (ABI) with manufacturer designed and validated probe and primer sets and universal cycling conditions were used for quantitative gene expression studies of human MMPs, TIMP, and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. The PCR reaction mixtures included TaqMan Universal PCR Master Mix, 0.1 mM of each gene-specific primer and 1.25 units of Taq DNA polymerase. The primer sets did not work with normal rat astrocytes; thus, these cells were not included in the PCR study.
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RESULTS |
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).
Cell morphology is affected by cadherin expression
No apparent correlation between the morphology of 2D cell cultures and cadherin expression has been reported. We observed a dramatic variation in the surface morphology of 3D multicellular spheroids with cadherin expression. The three cell lines with the highest cadherin expression formed tight aggregates with smooth surfaces, similar to primary astrocytes. Cells on the surface were flat, displaying stretched cell membrane, akin to morphology in 2D cultures (Fig. 3, EM1). In many instances junctions between the cells were visible at higher magnifications (not shown). In contrast, aggregates of cells with intermediate cadherin expression showed a rougher surface with both flat and round cells (Fig. 3, GBM1). The surface of aggregates composed of the lower cadherin expressers (Fig. 3, ENB) was berry-like. Cells were mostly round and displayed a large number of membrane blebs and processes; adhesion foci were only occasionally visible.
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, even after 96 h, remained in relatively compact clumps (Fig. 4, EM1; Fig. 5, EM2 for collagen concentration 
1 mg/ml; the figures contain at least one cell line from each of the low, intermediate, and high cadherin expresser groups). Those that invaded the matrix displayed characteristic spindle shape. Interestingly, the three cell lines not capable of reproducibly forming spherical aggregates and thus having the lowest (Figs. 4 and 5, U87) showed global invasion patterns not much different from aggregates with high (Fig. 4, EM1; Fig. 5, EM2), although locally the latter appear more dispersed. Tumor cells in aggregates with intermediate , showed peculiar organization. In every experiment they quickly migrated into the gel, forming ring-like structures with digested collagen and small compact clumps within the ring (Fig. 4, GBM1; Fig. 5, GMB1, GMB2). More cohesive aggregates occasionally showed similar behavior but only at low collagen concentration (Fig. 5, EM2). The migration rate of cells in the intermediate-cohesivity group was considerably higher than that of those in the low- and high-cohesivity groups (Fig. 6 A). The number and displacement of migrating cells typically decreased with increasing collagen concentration. The thicker gel also slowed ring formation (Figs. 5 and 6 B). Collagen concentration had little effect on the dispersion of cells with the lowest cadherin expression (U87). These results imply that no strict correlation exists between the magnitude of and the invasion pattern even though the latter does depend on the former.
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)) were chosen because they all are needed for the complete degradation of collagen I (8). Collagenase (MMP-1) cleaves fibrillar collagen, whereas gelatinases (MMP-2 and MMP-9) digest the denatured collagen fragments. We considered here only TIMP-1 for the following reason. It is the most multifunctional of the four known TIMPs with diverse functions (10). It is the direct inhibitor of MMP-1 and most probably of MMP-9. Because it controls TIMP-2 expression, it also acts as inhibitor of MMP-2 (10). (Note the almost perfect correlation between MMP-2 and TIMP-1 expression in Fig. 7.)
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DISCUSSION |
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). However, migration involves movement, which is controlled by global cellular physical quantities (e.g., velocity). The interplay between molecular and physical mechanisms in the spreading of tumors is vital to the understanding of how cancer progresses.
To gain insight into this interplay, we employed a large number of human brain tumor cell lines to quantitatively correlate their biophysical and molecular properties with their invasive characteristics in 3D collagen I matrices. Tissue cohesion was assessed through the measurement of apparent tissue surface tension (), using multicellular spherical aggregates (the analog of liquid drops; Fig. 2 C). Cell-matrix interaction was studied by quantifying the expression level of the relevant matrix metalloproteinases and their inhibitor (Fig. 7).
The cohesivity of a tissue is determined by all the adhesive mechanisms acting between its constituent cells (not only that related to cadherins). Nevertheless, we found linearity between and effective N-cadherin surface density (
) over an almost order-of-magnitude variation in the former quantity. (Winters et al. (29) measured for three glioblastoma cell lines including U87, for which they reported a value of 7 dyne/cm, lower than any of our values listed in Table 1. Together with our flow cytometry results, U87 would still fall on the curve in Fig. 2 C.) The observed linearity, however, must eventually cease with decreasing because the true function () must obey the condition 
(the curve in Fig. 2 C would cross the vertical axis at negative values). Linearity, as shown in Fig. 2 for larger values, suggests that at higher N-cadherin expression it is this molecule that dominates the cohesion of the studied brain tissues, whereas at low , other adhesive mechanisms take over or become as important. When results on tissue cohesion alone were compared with the outcome of invasion assays (Figs. 4 and 5), no strict correlation between N-cadherin expression and the spreading patterns could be established.
Spreading tumor cells are able to degrade their surrounding matrix. Thus, to characterize cell-matrix interactions, we concentrated on those metalloproteinases in the studied cell lines that are most relevant for the degradation of collagen I (i.e., MMP-1, MMP-2, and MMP-9) as well as on the most ubiquitous inhibitor of these enzymes (TIMP-1). Our results (Fig. 7) indicate that similarly to tissue cohesion, MMP expression alone does not explain the observed invasion patterns. However, a consistent picture occurs when tissue cohesion (Fig. 2 C) and cell-matrix interaction (Fig. 7) are simultaneously considered.
Fig. 7 reveals a striking interplay of these two decisive factors of invasive potential. Cells with either a low MMP pool and relatively low cohesivity (and thus small ; the three leftmost cell lines, not shown in Fig. 2 C) or strongest cohesivity (the three rightmost cell lines) have similarly limited migratory capacity. (The effect of relatively high MMP-2 expression of the U87 cells seems to be abolished by their equally high TIMP-1 expression.) Cell lines with intermediate cohesion and relatively high MMP expression (the middle three) exhibit complex migratory patterns as a consequence of competitive interactions. (Note that the initial cylindrical aggregates used in the invasion assays were all of same size.) Cells of these tissues simultaneously try to maximize their contact to each other (mostly through their N-cadherins) and to degrade the matrix (through their MMPs). As a consequence of this competition (i.e., between cell-cell adhesion and matrix proteolytic activity), as a compromise, they display ringlike patterns. Within the ring, cells benefit from their adhesion. At the same time, because the interior of the ring is devoid of collagen, they also benefit from their MMPs. (Note that these ring patterns cannot be explained by the differential gene expression of rim and core cells mentioned above (38) because the latter do not exist.) The appearance of these rings also depends on the concentration of matrix molecules (Fig. 5) and thus on the gel's mechanical properties (39). Similar structures, termed pseudopalisades (40,41), have been reported earlier in surgically excised tumors but, to our knowledge, not in vitro.
How a migratory cell moves through an extracellular matrix (ECM) typically depends on integrin-ECM ligand (e.g., collagen, fibronectin) interactions (42). In particular, for nonmalignant cells it was shown that, on two-dimensional ECM substrates, the rate of migration or speed displays a biphasic dependence on ligand concentration (43): at low ligand concentration speed increases, whereas at high ligand concentration speed decreases. Intermediate ligand concentration results in the greatest speed. These findings are consistent with the mechanism of normal cell migration, which is driven by the traction forces such cells exert on the matrix. The magnitude of the traction force increases with the amount of available ligand. At high ligand concentration, however, movement through the dense network of ECM macromolecules hinders motion.
In the case of malignant invasion, besides integrin-ECM ligand binding, cell-matrix interaction also involves matrix degradation through MMPs. Which of these two effects dominates the invasion pattern depends on cell type. Although we have not considered the role of integrins, we believe our data are consistent with the MMP activity being the more dominant in glioblastomas. We found that the higher the expression level of MMPs, the faster is invasion (Fig. 6 A), whereas the denser the matrix, the slower is invasion (because there is more material to degrade), and no biphasic dependence of the speed of migration on ligand concentration is observed (Fig. 6 B). (In transformed NIH3T3 cells, it was found that overexpression of the ß4 integrin subunit increases invasive capacity (44)).
In summary, our results strongly suggest that despite the complex molecular nature of brain tumor spreading, the emerging global cellular patterns can consistently be characterized in terms of two competing effects: cell-cell and cell-matrix interaction. Our findings also suggest specific ways to characterize, control, or engineer cell migratory patterns by the tuning of these interactions. Competitive interactions are known to drive phase transitions in physical systems (e.g., liquid-gas transition). Here they seem to be responsible for the characteristic cell configurations that accompany the epithelial-mesenchymal-like transition during which brain tumor cells change their phenotype from the more adhesive to the more migratory.
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ACKNOWLEDGEMENTS |
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This study was partially supported by grants from the National Aeronautics and Space Administration and the National Science Foundation.
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FOOTNOTES |
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Submitted on November 16, 2005; accepted for publication June 19, 2006.
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500 µm diameter) were placed into 1 mg/ml collagen and imaged at regular time intervals. Note that normal astrocytes practically do not invade the collagen matrix.
was determined at distinct time points as 
, where 
is the measured contiguous area covered by the cells (see 
.