Analysis of Invasion Dynamics of Matrix-Embedded Cells in a Multisample Format

Abstract

In vitro tests of cancer cell invasion are the "first line" tools of preclinical researchers for screening the multitude of chemical compounds or cell perturbations that may aid in halting or treating cancer malignancy. In order to have predictive value or to contribute to designing personalized treatment regimes, these tests need to take into account the cancer cell environment and measure effects on invasion in sufficient detail. The in vitro invasion assays presented here are a trade-off between feasibility in a multisample format and mimicking the complexity of the tumor microenvironment. They allow testing multiple samples and conditions in parallel using 3D-matrix-embedded cells and deal with the heterogeneous behavior of an invading cell population in time. We describe the steps to take, the technical problems to tackle and useful software tools for the entire workflow: from the experimental setup to the quantification of the invasive capacity of the cells. The protocol is intended to guide researchers to standardize experimental set-ups and to annotate their invasion experiments in sufficient detail. In addition, it provides options for image processing and a solution for storage, visualization, quantitative analysis, and multisample comparison of acquired cell invasion data.

Combination of EGFR Inhibitor Lapatinib and MET Inhibitor Foretinib Inhibits Migration of Triple Negative Breast Cancer Cell Lines

“…These data were obtained using a 3D wound healing-like invasion assay or cell-zone exclusion assay in which a confluent cell population embedded between collagen gel layers invaded a central cell-free collagen gel (ORIS, Platypus Technologies, [ 25 , 26 ]). The velocity was calculated using in-house-developed software tools (i.e., image-analysis using CELLMIA [ 27 ] and CellMissy [ 28 ]). Invasion velocity was significantly lower for the cells treated with inhibitors in comparison to the control cells.…”

“…The 3D collagen matrix was prepared as described in the collagen coating preparation section. 4.5 × 10 3 cells were seeded in 96-well plates on top of a thin layer of collagen gel and around a shielded central zone in the middle of the well, according to the ORIS cell invasion protocol [ 27 ]. The cells and the central cell-free zone were covered with a second collagen gel layer (40 µL volume).…”

“…0.30) on a CellM system with an IX81 microscope (Olympus). Control cells and cells treated with inhibitors were allowed to invade the central zone for 48 h. Image and data analyses were done using CELLMIA image processing software [ 27 ] and the data management and analysis software CellMissy [ 28 ], respectively. The cell invasion velocity is based on the increase in areas covered by the cells in a given time.…”

The interaction of p130Cas with PKN3 promotes malignant growth

“…To analyze the effect of PKN3 on cell invasiveness, we utilized a 3D cell‐zone exclusion assay as characterized in Van Troys et al . (). In this assay, a monolayer of cells grown on a thick layer of collagen is wounded, and then, the system is overlaid with another layer of collagen.…”

“…After cell attachment (4 h), the medium was removed and scratch was performed. Immediately after removal of the rest of media in the generated wound, another layer of collagen mix (100 μL) was added (schema of the experiment is shown in Van Troys et al ., ). Collagen was allowed to polymerize for 15 min at RT to prevent formation of bubbles in the collagen interface, then for another 15 min at 37 °C.…”