A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.

Our method is a modified version of a standard circular wound-healing assay with an added matrix barrier component (Matrigel™), which better mimics those physiological conditions present in vivo . We examined 3 cancer cell lines (MCF-7, SCOV-3, and MDA-MB-231), each with a different established degree of aggressiveness, to test our assay's ability to detect diverse levels of invasiveness. Percent wound closure (or invasion) was measured using time-lapse microscopy and advanced image analysis techniques. We also applied the CIA technique to DLD-1 cells in the presence of lysophosphatidic acid (LPA), a bioactive lipid that was recently shown to stimulate cancer cell colony dispersal into single migratory cells, in order to validate our method's ability to detect collective and individual motility.

Methods Materials All cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). McCoy's 5a Modified Medium was also purchased from ATCC. L-α-lysophosphatidic acid (oleoyl sodium salt, LPA 18:1) was purchased from Avanti Polar Lipids (Alabaster, AL) and used at a concentration of 2 μM. Phosphate buffered saline solution (PBS), Dulbecco's Modified Eagle's Media (DMEM), fetal bovine serum (FBS), penicillin-streptomycin antibiotics, and L-glutamine were obtained from GIBCO BRL (Carlsbad, CA). Matrigel™ Matrix Growth Factor Reduced was purchased from BD Biosciences (San Jose, CA) and used at 50% concentration (in DMEM). The optimal concentrations of these reagents were determined in previously performed dose-dependent experiments, taking concentrations given in the literature as baseline values.

Cell culture

We examined four cancer cell lines that have various established levels of invasiveness both in vitro and in vivo in order to test the applicability of our method to different stages of cancer progression. DLD-1 (CCL-221), a human colorectal adenocarcinoma cell line that is tumorigenic in nude mice [ 30 , 31 ], MCF-7 (HTB-22), a human mammary epithelial cell line found to be nonaggressive and noninvasive in mice [ 32 ], and MDA-MB-231 (HTB-26), a human mammary epithelial cell line found to be highly aggressive and known to rapidly progress to extensive and well-vascularized metastatic lesions [ 32 , 33 ], were routinely cultured and maintained in DMEM supplemented with 10% heat-inactivated FBS, 1% penicillin-streptomycin antibiotics, 1% L-glutamine, and kept in a humidified atmosphere of 5% CO 2 at 37°C. SKOV-3 (HTB-77), a human ovarian epithelial cell line found to be moderately aggressive and tumorigenic in nude mice [ 34 ], was regularly maintained in McCoy's 5a Modified Medium also supplemented with 10% FBS, 1% antibiotics, 1% L-glutamine, and grown in the same incubator conditions. All cell lines were seeded, in sterile conditions, at a density of 1.5–2 × 10 6 on polystyrene, 35-mm, tissue-culture treated Petri dishes (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ) for 18–24 hours or until confluent. For indicated experiments (those involving LPA treatment), cells were serum-starved by incubation in DMEM in the absence of FBS for 18–24 h after monolayers reached confluence. Circular wound-healing assay (CWA) Uniform, circular-shaped "wounds" (1.5 – 2 mm diameter; 8 per dish) were generated using a rotating drill press (Delta Shopmaster, Type 1, Model DP200) fit with a custom-shaped silicon tip (Home Depot; manually cut down to rounded shape with approximately 1.5 mm diameter and rounded bottom, using razor blade) as seen in Figure 1A . The optimal size and shape of the "wounds", their spacing, and other parameters were established in preliminary experiments (results not shown). The silicon tip was regularly washed with 70% ethanol between "wounding" of monolayers in individual dishes. Cell debris created by "wounding" was removed from each dish by manual pipetting, and intact cells were gently washed twice more with PBS. Two ml of growth media with 10% FBS was added to each dish for the remainder of incubation. For those experiments examining LPA effects, LPA or PBS in DMEM (2 ml total volume; without FBS) was directly applied into each dish and allowed to incubate for up to 24 h. Circular invasion assay (CIA) For the novel CIA method, "wounds" were created as described above in the CWA method (8 per dish). Additionally, 50% Matrigel™ in growth media (600 μl total) was overlaid onto the "wounded" cell monolayer to create a matrix barrier against the cellular surface and allowed to polymerize for 15 min prior to imaging the original time point (Figure 1B ). For those experiments examining LPA effects, LPA or PBS was added directly to Matrigel™ overlay prior to polymerization. Two ml of growth media with 10% FBS was added to each dish (LPA experiments used DMEM without FBS). "Wounded" monolayers, with fabricated matrix, were then incubated in a humidified atmosphere of 5% CO 2 at 37°C for 24 h.

Time-lapse microscopy

Time-lapse microscopy was conducted using a Zeiss Axiovert 200 M microscope (Zeiss, Thornwood, NY; 2.5× Plan NEOFLUR objective, NA 0.075; 10× Achroplan, NA 0.25, Ph1 objective) equipped with a Hamamatsu ORCA-ER CCD camera and temperature- and CO 2 -controlled chamber. Microscopy was under the control of OpenLab software (Improvision, Lexington, MA). At the beginning of each experiment (0 h), phase-contrast images were captured and microscopically accessed for standard, reproducible "wounds", with irregular outliers thrown out of the data set. Reflecting the precision of the method in creating consistent wounds by shape and size, this subpopulation of "unusable" wounds was negligible, as intra-operator variance was found to be < 3% (results not shown). Images of all "wounds" were then captured at regular time points for 24 h thereafter. The cell-free areas of each monolayer were distinguished from the surrounding intact cells by applying an automatic, software-defined threshold to each image, and pseudo-color applied to these areas using Adobe Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA).

Confocal microscopy analysis

A glass coverslip was coated by incubating in PBS containing 10 μg/ml of collagen I (C8919, Sigma, St. Louis, MO) overnight at 4°C. Circular wounds were made in the DLD-1 cell layer as previously described. Cells were further incubated for 5 hr at 37°C with or without 50% Matrigel™ overlay and fixed by 3.7% formaldehyde. Cortactin (green; lamellipodial marker) and actin (red; cytoskeletal marker) were visualized by immunofluorescence staining using anti-cortactin antibody 4F11 (Upstate Biotechnology Incorporated, Lake Placid, NY) and Alexafluor 568-phalloidin (Invitrogen). Confocal images were obtained with a Zeiss LSM-510 laser scanning confocal microscope equipped with a Plan-NEOFLUAR 40×/1.3 Oil DIC lens (Zeiss, Germany).

Image analysis and quantitation

Time-lapse images were further processed using Java's ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD). Appropriate pairs of corresponding images were overlaid and compared to one another, to determine the difference between the pseudo-color applied areas measured from the original time point (0 h), to the final time point of interest (4, 6, 8, 12, or 24 h). This difference (in pixels) was then calculated and presented in terms of percent wound closure, or invasion measured.

Statistical analysis

Each cell line was sampled at least 8 times for each method (N = 8–32; Power = 0.94–1.00), over the course of 10 days (N = 1–4 days per line). Wound repair data are referenced to time point 0 h, with results presented as mean percent wound closure (out of 100%) after a given period of time ± standard deviation. To avoid confounding problems with multiple analyses along the time-response curve, final differences were only analyzed at 4, 6, 8, 12, and 24 h. Differences between cell lines were examined using Student's t-tests, and were considered significant when P < 0.05. To further compare the two methods, post-hoc analysis (ANOVA) was performed for all parameters (method, treatment, time) using SPSS, Version 16 (SPSS Inc., Chicago, IL). Post-hoc power analyses were also performed for each set of experiments using G*Power 3 (E. Erdfelder, F. Faul and A. Buchner; University of Trier).

Materials All cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). McCoy's 5a Modified Medium was also purchased from ATCC. L-α-lysophosphatidic acid (oleoyl sodium salt, LPA 18:1) was purchased from Avanti Polar Lipids (Alabaster, AL) and used at a concentration of 2 μM. Phosphate buffered saline solution (PBS), Dulbecco's Modified Eagle's Media (DMEM), fetal bovine serum (FBS), penicillin-streptomycin antibiotics, and L-glutamine were obtained from GIBCO BRL (Carlsbad, CA). Matrigel™ Matrix Growth Factor Reduced was purchased from BD Biosciences (San Jose, CA) and used at 50% concentration (in DMEM). The optimal concentrations of these reagents were determined in previously performed dose-dependent experiments, taking concentrations given in the literature as baseline values.

Supplementary Material Additional file 1 Classical wound-healing method versus CIA: Comparison of ECM and cell damage. After DLD-1 cells were grown to confluence on laminin-332 coated Petri dishes, wounds were created using either a standard pipet tip to manually scratch cells similarly to classical assays (top row), or a silicon-tipped drill for the CIA technique (bottom row). Post-wounding, the dishes were stained with anti-laminin-332 polyclonal antibody (2778; green) and actin (red), and imaged with a Zeiss Axiovert 200 M (10× Achroplan, NA 0.25, Ph1 objective; scale bar = 50 μM). Employing a classical scratch method, both the laminin-332 undercoat and cells were damaged when "wounding" with a pipet. In contrast, both components appear to be minimally affected and left intact, by application of the CIA technique. Click here for file Additional file 2 CIA: Cell death and debris. (A) DLD-1 cells were grown to confluence overnight, wounded by a silicon-tipped drill press machine, stained with 0.2% Trypan Blue (TB) solution in DMEM for 5 min to detect disrupted (dead) cells and debris, washed once in PBS, and imaged using a Zeiss Axiovert 200 M (10× Achroplan, NA 0.25 objective; scale bar = 100 μM). (B) The monolayer was subsequently washed three times in PBS to remove additional debris, and again stained with TB. (C) Cells were incubated for an additional 16 h at 37°C and again stained with TB. Clearly, the wounding technique employed causes a minimal level of death and debris (stained blue; converted to gray-scale). Click here for file