Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling
© Fisher et al; licensee BioMed Central Ltd. 2006
Received: 04 August 2006
Accepted: 08 December 2006
Published: 08 December 2006
Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-MMP) is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S1P-regulated tumor cell migration in two dimensions (2D) and invasion of three-dimensional (3D) collagen matrices and, in particular, evaluated the role of MT1-MMP in this process.
LPA stimulated while S1P inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT1080 fibrosarcoma cells migrated in response to both lipids. HT1080 cells also markedly invaded 3D collagen matrices (~700 μm over 48 hours) in response to either lipid. siRNA targeting of LPA1 and Rac1, or S1P1, Rac1, and Cdc42 specifically inhibited LPA- or S1P-induced HT1080 invasion, respectively. Analysis of LPA-induced HT1080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (~125 μm vs. ~45 μm) and velocity of invasion (~0.09 μm/min vs. ~0.03 μm/min) only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs)-2, -3, and -4, but not TIMP-1, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT)-MMPs. Furthermore, MT1-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT1-MMP nor invade in the presence of LPA, were transfected with MT1-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT1-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT1-MMP in this process.
LPA is a fundamental regulator of MT1-MMP-dependent tumor cell invasion of 3D collagen matrices. In contrast, S1P appears to act as an inhibitory stimulus in most cases, while stimulating only select tumor lines. MT1-MMP is required only when tumor cells navigate 3D barriers and not when cells migrate on 2D substrata. We demonstrate that tumor cells require coordinate regulation of LPA/S1P receptors and Rho GTPases to migrate, and additionally, require MT1-MMP in order to invade collagen matrices during neoplastic progression.
Tumor cell invasion is a complex process involving genetic and cellular alterations which lead to proteolysis and dispersion through three-dimensional biological barriers [1–4]. Type I collagen is the most abundant component of the extracellular matrix (ECM), and is therefore a significant obstacle for tumor cell dissemination into the lymphatics, vasculature, and surrounding areas [5, 6]. Thus, in most cases, collagen must be degraded in order for tumor cells to spread into surrounding anatomic structures and metastasize . Cell migration, governed by polarity and reorganization of the cellular cytoskeleton, is also an integral aspect of tumor cell invasion [8, 9]. Dissecting the molecular requirements of tumor cell migration and invasion is necessary because the latter, in conjunction with metastasis, is a significant cause of morbidity and mortality in cancer patients .
Recent reports have identified two lipid signaling molecules, lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P), in many critical biological events such as development, angiogenesis, inflammation, and wound repair [11–15]. LPA and S1P function as extracellular lipid agonists which activate a subfamily of G protein-coupled receptors (GPCRs) and subsequent downstream effectors such as the small GTPases RhoA, Rac1, and Cdc42 [16–20]. In addition to fundamental cellular signaling, LPA, particularly in ovarian cancer, and S1P have been implicated in tumor cell proliferation, anti-apoptosis, cytoskeletal rearrangement and migration, and invasion [21–26]. LPA1–3 receptors are thought to be involved in cell motility and are aberrantly expressed in cancer cells [25, 27]. S1P1–3 are also involved in the regulation of cell migration and play important roles in the vascular system [11, 15].
Additional reports have linked LPA and S1P to the matrix metalloproteinases (MMPs) [24, 28, 29]. The role of MMPs in tumor invasion has been well documented [30–33] and clinical cancer therapeutic trials have sought to target these molecules albeit with disappointing results [34, 35]. Membrane-type matrix metalloproteinase 1 (MT1-MMP), also known as MMP-14, is a membrane-bound collagenase that has been shown to localize to the leading edge of invading cells, degrade surrounding extracellular matrix, and play a pivotal role in cancer cell dissemination [36–39]. The objective of the current study, therefore, is to further characterize the roles of LPA, S1P, and MMPs (specifically MT1-MMP) in the processes of tumor cell migration and invasion using both 2D migration analysis and 3D type I collagen invasion assays.
Our data demonstrate that LPA stimulated and S1P inhibited migration of most tumor lines tested. In contrast, HT1080 fibrosarcoma cells migrated in response to both lipids. Invasion of 3D collagen matrices of HT1080 cells, but not migration in either Boyden chambers or on collagen-coated plastic, was blocked in the presence of synthetic MMP inhibitors, and TIMP-2, -3, or -4. This indicates that lipid-induced invasion of 3D collagen matrices is an MT-MMP-dependent event. Transfection of HT1080 cells with siRNAs identified LPA1, S1P1, Rac1, Cdc42, and MT1-MMP as key components of the invasion response. Additional experiments revealed that SKOV3 and HEK293 cell lines, which express low levels of MT1-MMP, do not invade LPA-containing 3D collagen matrices despite a marked ability to undergo LPA-induced migration. When HEK293 were transfected with cDNA encoding MT1-MMP, LPA-induced invasion of 3D collagen matrices occurred. Our results demonstrate that lipid agonist-induced tumor cell invasion requires MT1-MMP, and its signaling occurs via LPA1, S1P1, Rac1, and Cdc42. We propose that coordinated signaling events between LPA and S1P receptors, their effectors, and MT1-MMP are required during tumor cell invasion of 3D collagen matrices, but MT1-MMP is not required for tumor motility on 2D collagen substrates.
LPA stimulates while S1P either inhibits or stimulates migration of tumor cells
Four cell lines were selected for further study due to their marked LPA-migration response namely A2058 melanoma, SKOV3 ovarian carcinoma, human embryonic kidney (HEK) 293, and HT1080 fibrosarcoma. We have previously shown that S1P strongly influences invasive behavior of human endothelial cells (another highly invasive cell type) in 3D collagen matrices [28, 40]. The effects of S1P were therefore also evaluated in these cell lines. LPA produced a strong migratory response for these cell lines, and in all but one case (HT1080) S1P inhibited migration. Interestingly, HT1080 cells exhibited a strong migratory response in the presence of both LPA and S1P, contrary to the trend of the other cell lines. This result is particularly intriguing because HT1080 cells are among the most invasive tumor cell lines in both in vivo and in vitro settings [41, 42], suggesting a role for S1P responsiveness in highly invasive tumor cells.
Tumor cell invasion of 3D collagen matrices mimics the lipid agonist migration profile in select cell lines
Time-lapse analysis of LPA and S1P-induced tumor invasion of collagen matrices
LPA and S1P accelerate MT1-MMP-mediated HT1080 invasion in 3D collagen matrices
Rate of Invasion (μm/hr)
LPA + S1P
LPA + GM6001
S1P and LPA stimulate HT1080 invasion of 3D type I collagen matrices through S1P1, LPA1, Rac1, and Cdc42
MMPs are required for lipid agonist-induced tumor cell motility in 3D collagen matrices but not on 2D collagen substrates
The synthetic MMP inhibitor GM6001 decreases distance and reduces velocity of lipid-induced HT1080 motility in 3D collagen matrices but not on 2D collagen substrates.
Condition (over 24 hours)
Avg. Distance μm (2D)
Avg. Distance μm (3D)
Avg. Velocity μm/min (2D)
Avg. Velocity μm/min (3D)
303.68 ± 71.1
73.07 ± 31.74
0.21 ± 0.049
0.050 ± 0.022
DMEM + GM6001
277.38 ± 29.38
45.06 ± 15.17
0.19 ± 0.020
0.031 ± 0.010
298.58 ± 41.18
128.00 ± 56.98
0.21 ± 0.029
0.088 ± 0.039
LPA + GM6001
303.18 ± 55.41
40.43 ± 6.08
0.21 ± 0.038
0.028 ± 0.0042
303.05 ± 33.78
116.24 ± 25.39
0.21 ± 0.028
0.080 ± 0.017
S1P + GM6001
322.93 ± 69.05
44.598 ± 9.25
0.23 ± 0.049
0.031 ± 0.0057
138.43 ± 57.71
0.096 ± 0.040
92.15 ± 35.90
0.064 ± 0.025
MT1-MMP is a key proteinase required for lipid-induced tumor cell invasion of 3D collagen matrices
To investigate further the role of MT1-MMP in LPA-induced invasion, HEK293 cells (which do not invade in response to LPA) were transfected with MT1-MMP cDNA and allowed to invade in response to LPA for 48 hours. Once transfected, HEK293 cells invaded in the presence of LPA (Fig. 10B). Transfection with a control plasmid (pAdTrack-CMV), or plasmids encoding human MT2-MMP or MT3-MMP did not induce invasion despite appropriate expression of the various proteins (Fig. 10C). These data illustrate that MT1-MMP is required for LPA-induced invasion of HEK293 cells. The HEK293 cells also represent an interesting new model to assess tumor cell invasion due to their inability to invade unless genes such as MT1-MMP are supplied by transfection.
MT1-MMP is required for lipid-induced invasion of tumor cells embedded within 3D collagen matrices
LPA and S1P are collaborative regulators of tumor cell migration and invasion
LPA receptors are classic GPCRs, and have been shown to be abnormally expressed in cancer cells [25, 27]. LPA is also elevated in the plasma of patients with ovarian carcinoma, indicating a link between LPA and early intraperitoneal dissemination [24, 56]. Our data strongly indicate that LPA is a potent tumor cell motility factor when compared to other factors such as cytokines or chemokines. The recent discovery of autotaxin, an enzyme that generates LPA or S1P and is expressed by tumor cells, highlights the importance of LPA and S1P in tumor cell motility [22, 57, 58]. Intriguingly, our data suggest that LPA and S1P exert opposing effects on most tumor cell types (Fig. 1). Previous studies establish that lipid agonist signaling pathways may be particularly important in this context [21, 27, 59–62]. By identifying differential receptors involved in LPA and S1P signaling (i.e. LPA1 and S1P1) and the various downstream GTPase effectors (e.g. Rac1 and Cdc42), we hypothesize that motility and invasion are regulated by a balance between these pro- and anti-migratory signals. Contrary to other tumor cell lines, HT1080 cells are not inhibited by S1P; in fact, S1P actually stimulates HT1080 migration and invasion (Figs. 1 and 2). This cell line is also one of the most invasive tumor types known [41, 42, 63]. Thus, the loss of S1P inhibition, or the ability of S1P to stimulate a tumor cell, may directly contribute to metastatic potential. Studies investigating the molecular differences in lipid signaling and their relevance to tumor cell invasion and metastasis in vivo are underway  and should be the subject of future work.
LPA and S1P regulate tumor cell invasion of 3D collagen matrices via MT1-MMP
When invasion of tumor cells was assessed, we found that an ability to migrate in response to LPA did not necessarily confer an invasive phenotype in 3D collagen matrices. In fact, only a small subset of tumor cell lines screened in our models show strong invasive behavior (i.e. HT1080 cells). By far the majority of cell lines do not substantially invade in 3D collagen matrices under the conditions described in this report (Fisher and Davis, unpublished observations). One possible explanation for this result is that Western blot analysis revealed that non-invasive cell types (HEK293 or SKOV3) expressed low levels of MT1-MMP, a proteinase known to be involved in tumor cell invasion (Fig. 10A) [5, 6, 37]. When HEK293 cells were transfected with cDNA encoding MT1-MMP, cells were able to invade collagen matrices containing LPA (Fig. 9B). Using HT1080 cells, we were able to again distinguish MT1-MMP as a critical molecule involved in lipid-induced invasion using siRNA transfection targeting this proteinase (Figs. 12, 13, 14) or the addition of chemical or protein inhibitors of this proteinase (Fig. 9). A critical question that remains unanswered is the mechanism by which LPA stimulates MT1-MMP-dependent invasion of collagen matrices. One possibility is that LPA is involved in localizing or stabilizing MT1-MMP to critical areas of the cell surface required for matrix proteolysis during 3D locomotion.
The work presented here shows that LPA can both stimulate tumor cell motility on 2D substrates (Figs. 1, 6, and 8) and tumor cell invasion of 3D collagen matrices (Figs. 2, 8, 10, and 12). Importantly, only the latter process depends on the MT1-MMP proteinase (Figs. 6, 8, 10, 14, and Table 2). Blockade of MT1-MMP using siRNA suppression or proteinase inhibitors strongly suppressed HT1080 movement in 3D collagen matrices and not on 2D collagen substrates. Also, HT1080 random cell motility on 2D substrates was not affected by LPA, while LPA clearly stimulated tumor cell movement/invasion in 3D collagen matrices. This data demonstrates that LPA strongly stimulates MT1-MMP-dependent tumor cell invasion behavior, and thus, is a critical co-factor along with matrix proteolysis in tumor cell invasion. An important unanswered question remains: how does LPA or S1P affect the ability of MT1-MMP to coordinate these invasive events? This central issue is fundamental to our understanding of how tumor and other cells such as endothelial cells invade 3D matrix environments. We hypothesize that LPA facilitates the localization of MT1-MMP to specialized areas of the cell membrane that are necessary to drive tumor cell locomotion through 3D matrices. Other possibilities include that LPA stimulates the co-association of MT1-MMP with critical co-regulatory molecules such as collagen-binding integrins (e.g. α2β1, α1β1, α10β1, α11β1). Preliminary experiments using siRNAs directed to MT1-MMP and the β1 integrin subunit show that suppressing both of these genes at the same time completely inhibits HT1080 invasion of 3D collagen matrices (Fisher and Davis, unpublished observations).
Previous studies have implicated other MMPs (i.e. MMP-2 and MMP-9) in tumor cell invasion and progression, and that LPA triggers upregulation or activation of MMP-2 [24, 63, 65–67]. However, in our system, siRNAs targeting MMP-2 or -9 or other MT-MMPs did not influence tumor invasion of collagen matrices (Fig. 12). Our data indicate that MT1-MMP is the predominant proteinase involved in lipid-induced invasion of collagen matrices. These conclusions are similar to a recent study showing the selective ability of MT-MMPs to stimulate tumor invasion with no evidence presented for a role of soluble MMPs . Additionally, MT1-MMP acts upstream and can activate MMP-2. Thus, MT1-MMP appears to be the principle regulator of LPA-induced invasion. However, markedly decreasing levels of MT1-MMP via targeted siRNA transfection did not completely block completely invasion in HT1080 cells as effectively as the addition of GM6001 (Figs. 3, 8, and 12, 13, 14). In addition, the depth and numbers of invading MT1-MMP-transfected HEK293 cells were not as extensive as seen in HT1080 cells (Fig. 10). These results may indicate a role for additional molecules (e.g. collagen-binding integrins or multiprotein complexes involving MT1-MMP) in our invasion system. It is also plausible that other cell surface expressed metalloproteinases (i.e. other MT-MMPs or ADAMs) could contribute to degradation of matrix components and host/tumor interactions (i.e. tumor-driven angiogenesis) not addressed here . Additional studies are needed to identify and address the role of key complementary molecules in tumor invasion and how this relates to lipid-agonist induced invasion.
It is clear that tumor cell migration and invasion are multifaceted events that require a myriad of cellular processes to act in concert to achieve cell movement and propulsion through natural barriers. The systems described here illustrate innovative ways to study the molecules involved in tumor locomotion. Using our systems, we identified molecular components (e.g. LPA1, S1P1, Rac1, Cdc42, and MT1-MMP) involved in lipid-induced HT1080 invasion (Figs. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14). We also quantitated the effects of LPA, S1P, or reduced MT1-MMP protein expression on the distance and rate of HT1080 invasion using a new time-lapse analysis system (Figs. 3, 8, 13, 14, and Tables 1 and 2). We believe that these in vitro 3D microassay collagen invasion systems represent an important and attractive approach to study the molecular requirements for tumor invasion and the mechanisms underlying the ability of single tumor cells to invade through 3D collagen matrices. Identifying the key molecules involved in tumor migration and invasion will provide better comprehension of these processes and ultimately, may yield more specific cancer therapeutics, particularly those that target lipid agonist-induced migration and MT1-MMP-mediated invasion.
48-well micro-chemotaxis chambers and polycarbonate membranes (8 μm pores) were purchased from Neuro Probe, Inc. (Gaithersburg, MD). D-Erythro-sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) 1-oleoyl-2-hydroxy-SN-glycero-3-phosphate, sodium salt were purchased from Avanti Polar Lipids (Alabaster, AL). Rat tail collagen type I was prepared at 7.1 mg/ml using the method previously described . Tumor cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA). Dulbecco's Modified Eagle Medium (DMEM), Lipofectamine 2000, and fetal bovine serum (FBS) were from Invitrogen Life Technologies (Rockville, MD). Bovine TIMP-1 and TIMP-2 were obtained from Chemicon Corp (Temecula, CA). Recombinant TIMP-3 and TIMP-4 were from R&D Systems (Minneapolis, MN). A rabbit polyclonal antibody was prepared against recombinant green fluorescent protein (GFP) as described . Human MT1-MMP (AF918, R&D Systems, Minneapolis, MN), MT3-MMP (RP1-MMP-16, Triple Point Biologics, Forest Grove, OR), and Rac1 (ARC01, Cytoskeleton, Denver, CO), and monoclonal antibodies directed against human MT2-MMP (MAB916, R&D Systems), Lamin A/C (MAB3211, Chemicon Corp), Actin (JLA-20, Calbiochem, San Diego, CA), RhoA (ARH01, Cytoskeleton), and Cdc42 (610929, BD Tansduction, San Jose, CA) were used for Western blot analysis as described previously . siGENOME SMART pool® reagents were products of Dharmacon (Lafayette, CO). Plasmids encoding human MT1-MMP (EX-M0327-M02) and MT3-MMP (EX-Q0300-M02) were obtained from Genecopoeia (Germantown, MD). A plasmid encoding human MT2-MMP (TC118648) was purchased from Origene (Rockville, MD). Other reagents were obtained from Sigma (St. Louis, MO) unless noted.
Tumor cells were cultured in T-75 flasks and supplied with DMEM supplemented with 10% FBS containing 100 μg/ml gentamicin (Invitrogen), 100 μg/ml of streptomycin, 100 Units/ml of penicillin, and 0.25 μg/ml Amphotericin B [Fungizone®, Invitrogen, Carlsbad, CA), and incubated at 37°C with 5.0% CO2. The stable HT1080 cell line expressing nuclear-GFP was cultured in media containing 10 μg/ml Blasticidin (Invitrogen). HEK293 cells were cultured on flasks pre-coated with 20 μg/ml type I collagen in PBS. Prior to use in experiments, cells were washed with phosphate buffered saline (PBS), removed from monolayer with trypsin-EDTA, neutralized with fetal bovine serum, and centrifuged. Cells were then washed in two, 10 ml volumes of DMEM to remove residual serum.
Generation of stable HT1080s expressing nuclear-localized GFP
A vector expressing nuclear-localized GFP (Nuc-GFP) (vector pACGFP1-Nuc, 632431) was obtained from BD Biosciences-Clontech (Mountain View, CA). The Nuc-GFP insert was amplified via PCR using the following primers: Nuc-GFP UP: 5'-CACCATGGTGAGCAAGGGCGCCGAG-3' and Nuc-GFP DN: 5'-AGCAGTTATCTAGATCCGGTGGATC-3'. PCR product size was verified via agarose gel electrophoresis, and following PCR purification (Qiagen, Valencia, CA), was TOPO cloned into the pLenti6/V5-D-TOPO lentiviral vector (Invitrogen). Following TOPO cloning, One Shot Stbl3 Chemically Competent E. coli were transformed with DNA. Individual clones containing the Nuc-GFP insert were verified via PCR and sequence analysis. The 293FT lentiviral packaging cell line was obtained from the ViraPower Lentiviral Gateway Expression Kit (Invitrogen) and were cultured in 500 μg/ml G418. Nuc-GFP lentivirus was produced following the Invitrogen lentivirus protocol. HT1080s were infected with Nuc-GFP lentivirus and were incubated for 72 hours prior selection using 10 μg/ml of Blasticidin. HT1080s were cultured in Blasticidin for 10 days to ensure stable cell line selection, and Nuc-GFP expression was verified via fluorescent microscopy.
96-well A/2 vertical collagen invasion assay
Three-dimensional (3D) collagen type I invasion assays were adapted from previously described work . Briefly, collagen gels (n = 3 gels per condition, 25 μl volume) were prepared as described  at a final concentration of 2.0 or 3.75 mg/ml and added to A/2 wells (4.5 mm diameter) of 96-well plates (Costar, Corning, NY). 1 μM S1P and/or LPA were incorporated into each gel prior to polymerization and equilibration at 37°C and 5% CO2 for 45 minutes. After equilibration, 100 μl of media containing a 1:250 dilution of RSII(+) (Transferrin, BSA, oleic acid, and insulin) and 35,000 tumor cells were added to each well. Cells were allowed to invade 3D collagen gels for 48 hours. Culture media was removed and collagen gels containing invading tumor cells were fixed in 3% glutaraldehyde in PBS for 30 minutes. Gels were stained with 0.1% toluidine blue in 30% methanol for 30 minutes prior to destaining with water. Transected gels were imaged using an inverted Nikon Eclipse TE2000-U microscope, camera, and Metamorph® software (Molecular Devices Corporation, Downington, PA) or were acquired using an inverted Olympus microscope, camera, and Kodachrome 64T color film. Kodachrome images were scanned using a Polaroid SprintScan 4000 and PolaColor Insight Software (Polaroid, Cambridge, MA). To quantitate invasion of tumor cells in 3D collagen gels, cells were counted with eyepiece equipped with an ocular grid. For each condition, twenty random fields were selected and the number of invading cells per high power field (HPF) was counted manually at 20× magnification. Data are reported as mean number of invading cells per HPF (± S.D.).
Effects of metalloproteinase inhibitors and pertussis toxin on migration or invasion
GM6001 (Calbiochem) and TAPI-1 (Chemicon) were reconstituted in DMSO at 2.5 mM. TAPI-0 (Chemicon) was reconstituted in DMSO at 0.25 mM. TIMP-1, TIMP-2, TIMP-3, and TIMP-4 were prepared at 100 μg/ml in 0.1% BSA. To determine the effects of MMP inhibitors on tumor cell migration and invasion, GM6001 (5 μM), TAPI-1 (5 μM), TAPI-0 (5 μM), and TIMPs 1–4 (5 μg/ml) were added to upper chambers of migration assays and directly to cell media in invasion assays. Likewise, 100 ng/μl of pertussis toxin was added to cell media where noted. Migration and invasion assays were performed as described.
Transfection of HT1080 cells with siRNA
siGENOME SMART pool® human MMP-2, MMP-9, MT1-MMP, MT2-MMP, MT3-MMP, LPA1–3, S1P1–3, RhoA, Rac1, and Cdc42 were purchased from Dharmacon. A 21-nucleotide custom MT1-MMP siRNA described previously by Weiss and colleagues  was prepared by Dharmacon. A Lamin A/C siGENOME SMART pool® siRNA and a Luciferase GL2 duplex siRNA oligo (Dharmacon) were utilized as controls. All siRNA transfections were performed as described previously ; however, Lipofectamine 2000 and 20% serum were utilized for transfection purposes. Cells were allowed to recover from siRNA transfection for 18 hours prior to initiation of experiments. Cells were allowed to invade for 48 hours as described above, after which conditioned media were collected and stored at -20°C. Invading tumor cells were extracted from collagen matrices and stored at -20°C as described previously . The remaining wells were fixed, stained, quantitated, and imaged as described.
Transfection of HEK293 cells with plasmids encoding human MT-MMPs
Plasmids encoding human MT1-, MT2-, and MT3-MMP cDNA were purified using the Qiagen plasmid MIDI kit (Valencia, CA). T-25 flasks were pre-coated with type I collagen as described above, and HEK293 cells were cultured to 80% confluency in 5 ml of culture media. On the day of transfection, 6 μg of MT1, MT2, or MT3-MMP plasmid DNA or the control vector pAdTrack-CMV encoding GFP , kindly provided by Dr. Bert Vogelstein (Johns Hopkins University College of Medicine, Baltimore, MD), was diluted into Opti-MEM I (Invitrogen) and mixed with Lipofectamine 2000 to a final volume of 1 ml. The plasmid/Lipofectamine 2000/Opti-MEM solution was added to the cells prior to overnight incubation. Cells were washed, trypsinized, and seeded onto 2.0 mg/ml collagen matrices as described above. Transfected HEK293 cells were allowed to invade for 48 hours prior to fixation, staining, and quantification as described above. For each plasmid, protein expression was verified via Western blot analysis.
Real-Time Quantitative PCR (RTQ-PCR)
LPA1 and S1P1 mRNA were quantified using the Applied Biosystem apparatus 7500 Fast Real-Time PCR system. Briefly, total RNA was extracted from siRNA-treated (Luciferase, LPA1, and S1P1) HT1080 cells using the Totally RNA Isolation kit (Ambion, Austin, TX) according to the manufacturer's instructions. RNA (5 μg) was reversed transcribed using StrataScript First Strand Synthesis System (Stratagene, Cedar Creek, TX). RTQ-PCR was performed in a final volume of 20 μl containing 2 μl of a 1:5 dilution of each RNA template, 10 μl of a TaqMan® Fast Universal PCR Master Mix, and 1 μl of a TaqMan® Gene Expression Assays (Applied Biosystems, Foster City, CA). Primers were designed using PrimerExpress sofware (Applied Biosystems), and targeted sequences between Exon 1 and Exon 2 (S1P1), between Exon 2 and Exon 3 (LPA1), and an inventoried eukaryotic 18S rRNA (internal control). Each sample was assayed using three replicates for each primer. The 18S rRNA-specific primer was used as an internal standard. The cycling conditions were as follows: one cycle at 95°C for 20 seconds, followed by 40 cycles of PCR amplification, each consisting of 95°C for 3 seconds and 60°C for 30 seconds. ΔCt values were obtained by subtracting the Ct value of the housekeeping gene 18s rRNA from the Ct value for each indicated gene. The ΔΔC t relative expression method was used to normalize LPA1 and S1P1 to 18s rRNA as a housekeeping gene (comparative C t method). Data are reported as mean expression values normalized to 18s RNA (± S.D.) of three replicates per siRNA treatment.
Gelatin zymography and Western blotting
Gelatin zymography was performed with SDS-PAGE gels containing 8.5% acrylamide and 1 mg/ml porcine gelatin. Conditioned media from the HT1080 siRNA transfected cells were prepared under non-reducing conditions. Gel electrophoresis was performed at 150V for 55 minutes. Zymograms were incubated for three 20 minute periods in 100 mls of 2% Triton X-100 in water, washed twice in distilled water, placed in 25 mM Tris-HCl (pH 7.5) containing 5 mM CaCl2, and incubated overnight at room temperature. The following morning, gels were stained with 0.1% Amido Black in 30% methanol and 10% acetic acid for 15 min and destained in 30% methanol and 10% acetic acid for 1 hour at room temperature. Western blot analysis was performed using 10% or 14% (for RhoA, Rac1, and Cdc42) SDS-PAGE gels as described previously  and probed with antibodies at the following concentrations: GFP, 0.1 μg/ml; MT1-MMP, 2 μg/ml; MT2-MMP, 1 μg/ml; MT3-MMP, 1 μg/ml; Lamin A/C, 2 μg/ml; Actin, 0.1 μg/ml; RhoA, 2 μg/ml; Rac1, 5 μg/ml; and Cdc42, 2 μg/ml
Time-lapse measurements and imaging of HT1080 invasion
Hollow glass casings (3 × 3 × 8 mm) were designed by Wale Apparatus (Hellertown, PA) to accommodate a collagen gel and tumor cells during invasion. Collagen gels were prepared at a final concentration of 3.75 mg/ml. 1 μM of S1P and/or LPA was incorporated into each gel before filling each glass casing with ~20 μl of collagen. Gels were allowed to polymerize and equilibrate at 37°C with 5% CO2 for 45 minutes. After equilibration, 50 μl of clear DMEM (Invitrogen) containing 25,000 HT1080 cells was added to the upper portion of the glass casing above the collagen. Cells were allowed to attach for 30 minutes before the glass casings were secured in a horizontal plane within the well of a 48 well plate (Costar) with sterile silicon grease. Each well was then supplied with 500 μl of media containing a 1:250 dilution RSII (+). Cells were allowed to invade for 48 hours at 37°C with 5% CO2. Digital images were captured directly every ten minutes using an inverted Nikon Eclipse TE2000-U microscope, camera, and Metamorph® software (Molecular Devices Corporation, Downington, PA). Using Metamorph®, images of individual fields of invading tumor cells were arranged in sequential order. Distance of HT1080 invasion was measured using digital pixel measurements and plotted against time using Microsoft Excel®. Rate of invasion was determined using linear regression statistical analysis. Data are reported as the mean distance in micrometers of invading cell fronts (± S.D.). Five measurements for each time point of duplicate or triplicate experiments were performed.
Assessment of chemotactic migration
Modified Boyden chambers were utilized to assess lipid-induced chemotactic directional migration. Polycarbonate filters (8 μm pores) were pre-coated with 1 mg/ml type I collagen or gelatin in PBS. LPA and/or S1P in DMEM containing a 1:250 dilution of reduced serum II supplement [RSII(-)] (Transferrin, BSA, no oleic acid, and insulin) were added to lower wells. Upper wells contained a 1:250 dilution of RSII(-) and lower wells contained 20 μg/ml of fibronectin for gelatin-coated membranes. 5 × 104 tumor cells were added to upper chambers and allowed to migrate for 4 hours. Filters were removed and stained with 0.1% amido black in 30% methanol and 10% acetic acid. Membrane-adherent migrating cells were calculated using Scion® image and Microsoft® Excel. Migration data are represented mean number of migrating cells × 103 (± S.D.) and represent the results of quadruplicate experiments.
Tracking Nuclear-GFP HT1080 cells on collagen-coated plastic (2D) or embedded within in collagen matrices (3D)
To observe the movement of HT1080 cells embedded in 3D collagen matrices, nuc-GFP HT1080s or siRNA treated (Luciferase or custom MT1-MMP) nuc-GFP HT1080s were mixed 1:1 with non-fluorescent HT1080s. Cells were then incorporated into a 3.75 mg/ml collagen matrix (n = 3 cultures per condition) containing 1 μM LPA. 15 μl of collagen and cell mixtures were added to square 384-well tissue culture plates (VWR, West Chester, PA), and allowed to equilibrate for 1 hour prior to addition of 60 μl of media containing a 1:250 dilution of RSII(+). 5 μM GM6001 was added to the media as noted. Cells were allowed to invade the surrounding matrix for 48 hours at 37°C with 5% CO2. Digital fluorescent images were captured directly with Metamorph® as described above. By tracking fluorescent nuclei in chronological frames, Metamorph® generated cell tracings of the movements of individual cells. For 2D tracings, nuclear-GFP HT1080 cells were seeded on 6 well plates pre-coated (30 minutes) with 50 μg/ml of type I collagen and cultured with 2 mls of media containing a 1:250 dilution of RSII (+). GM6001 was added as described. Images and cell tracings were generated by Metamorph® as described.
This work was supported by NIH grants HL59373 and HL79460 (to GED). The authors would like to thank Andrea Nolte, Aaron Mobley, and Jennifer Faske for their excellent technical assistance.
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