Characterization of a Cell Invasion Assay for High-Content Screening Using a Laser Microplate Cytometer

Cancer invasion from a primary tumor site is one of the critical factors in disease prognosis,1 and together with metastasis is the major cause of morbidity and mortality. The molecular and cellular processes that govern tumor invasion and metastasis are complex and involve multiple pathogenic steps, including changes to the adhesive and migratory capabilities of tumor cells and the tumor microenvironment, and invasion of the surrounding extracellular matrix (ECM).2,3 In order to delineate these complex and precise mechanisms, in vitro models are needed that reliably and accurately recreate the metastatic process.

While cell culture is a valuable tool for studying cell behavior, plastic substrates are two dimensional and generally promote cellular proliferation and inhibit differentiation.4 It is now widely recognized that a more physiologically relevant model is necessary. A 3-D cell culture system could provide such a model since the cells have a more differentiated in vivo-like morphology, and thus can elicit more physiological responses, e.g., the production of specific proteins, when compared to 2-D cell culture.4 This is exemplified by recent advances in the understanding of the mechanisms of cell invasion.

Originally, it was believed that cell invasion relied on the ability of a cell to use proteases, e.g., matrix metalloproteases (MMP), to digest through 3-D biological barriers.5–8 However, preclinical studies with MMP inhibitors did not translate into efficacy as cancer therapy in human clinical trials.9,10 These findings raised the question of the relevance and importance of the protease-dependent invasion model in the in vivo environment. It has since been shown, using 3-D culture techniques, that there is also a proteolysis-independent invasion mode.11 During this process, cells adopt an amoeboid phenotype and use actomyosin-based mechanical forces to deform collagen fibers and push through the ECM.11,12 This mechanism, which is dependent on Rho/ROCK signaling, has only been observed in 3-D cultures.12 Given that such cell invasion models are recognized as being more physiologically relevant, this mechanism may be a more accurate representation of the in vivo situation.

One of the most common in vitro methods to explore cell invasiveness is the Boyden chamber, a two-chamber system that uses a filter coated with a basement membrane extract (BME) preparation as the ECM barrier, with conditioned tissue culture media as a chemoattractant.13,14 In this system, cells are allowed to invade the ECM, and those that pass to the other side of the filter are counted.15,16 While this assay is rapid and permits quantitative measurements, the results obtained can be misleading, since only the end-point cell invasion can be measured. However, cells that successfully invade only account for a small proportion of the population seeded on top of the filter and may not accurately reflect phenotypic characteristics of the seeded population. Thus, the ability to observe morphological phenotypes and use multiplexed immunostaining (i.e., to visualize differences between invading and noninvading cells) could circumvent these limitations.

Figure 1 - Oris cell invasion assay schematic.

Oris™ cell invasion assays (Platypus Technologies, Madison, WI) were developed as an alternative and more versatile method for quantifying and imaging cells invading through ECM in real time compared to Boyden chamber devices. An assay schematic is shown in Figure 1. The utility of the assay was illustrated in a paper that investigated signal transduction mechanisms underlying induced epithelial-to-mesenchymal transition (EMT) in ras-transformed human (HaCaT II-4) keratinocytes.17

Laser scanning technologies enable the rapid detection and quantification of multiparametric data from individual cells. The Acumen eX3 fluorescence laser scanning microplate cytometer (TTP Labtech Ltd., Melbourn, Royston, U.K.) offers a highly flexible approach to cell-based assay screening. The platform uses three lasers (405, 488, and 633 nm) for fluorescence excitation, in conjunction with highly sensitive photomultiplier tubes for the detection of fluorescently labeled cells. The combination of high-throughput laser scanning data acquisition and the simple cytometric approach to data analysis permits the generation of high-content and highly multiplexed data that are applicable for a broad range of biological assays. The cytometer is capable of whole well scanning, which allows the generation of statistically robust data from representative cell populations and the normalization of biological responses to total cell number.

This article describes the use of Oris cell invasion assays in combination with the Acumen eX3 microplate cytometer to characterize the relative invasive properties of two widely studied cancer cell lines, HT-1080 fibrosarcoma and MDA-MB-231 breast carcinoma, through varying concentrations of BME or collagen I.

Methods

MDA-MB-231 breast cancer epithelial cells were cultured in low-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) and 1% penicillin-streptomycin. The HT-1080 fibrosarcoma cell line was grown in Eagle minimal essential medium (EMEM) containing 10% FBS and 1% penicillin-streptomycin, and supplemented with 1.0 mM sodium pyruvate and 1.5 g/L sodium bicarbonate. Both cell lines were maintained at 37 ºC in a humidified atmosphere with 5% CO2. All cell culture reagents were obtained from Lonza (Allendale, NJ) unless otherwise stated.

For the BME invasion assay, 96-well Oris-compatible microplates (Platypus Technologies) were used. A 3.5-mg/mL solution of BME (Trevigen, Gaithersburg, MD) was prepared in phosphate-buffered saline (PBS) and kept on ice. Wells were coated by dispensing and immediately withdrawing 100 μL of the BME solution. Following an incubation of 30–60 min at 37 ºC in a humidified atmosphere with 5% CO2, Oris cell seeding stoppers were inserted. For the collagen I invasion assay, Oris collagen-I coated plates, prepopulated with Oris cell seeding stoppers, were utilized.

The plates were seeded with MDA-MB-231 cells (30,000 cells/well) or HT-1080 cells (35,000 cells/well) and incubated at 37 ºC in a humidified atmosphere with 5% CO2 for 2–4 hr to permit attachment. Stoppers were then removed and the wells were washed with either PBS or serum-free media. The cells were overlaid with 40 μL of varying concentrations of BME (10, 12.5, or 15 mg/mL) containing 10% FBS or collagen I (2, 3, or 4 mg/mL), and the plates were incubated for 1 hr to allow the overlay to gel. Cell culture media (100 μL) containing 10% FBS was added to each well. In certain experiments, cytochalasin D (0.01–3 μM) in a 0.1% (v/v) or 0.1% (v/v) dimethyl sulfoxide (DMSO) vehicle was added to the cell culture medium above the BME overlay. Stoppers remained in designated preinvasion reference wells (n = 4) and were removed 1 hr prior to termination of the experiment; 40 μL of overlay was added to each well followed by fixation and staining. Plates were incubated for 48 hr to permit invasion into the detection zone, and cells were fixed with 0.25% glutaraldehyde for 15 min and labeled with 4ʹ,6-diamidino-2-phenylindole (DAPI) (1:4000) for 15 min or tetramethylrhodamine isothiocyanate (TRITC)-phalloidin (1:100) for 45 min. Cell invasion was quantified using an Acumen eX3 microplate cytometer, and images were acquired using a Axiovert microscope (Carl Zeiss Microimaging, Thornwood, NY).