Appnote-Chemotactic Cancer Cell Migration
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Accurate tracking of chemotactic cancer cell migration – basic insights in metastasis Marc van Vijven, MSc 1; Lieke Stemkens, MSc 1; Nathalie van de Laar, MSc 1 1 CytoSMART® Technologies B.V., Eindhoven, The Netherlands; Introduction Worldwide, cancer is responsible for around 10 million mortalities cells in these vessels can provide practical issues. Currently, the every year. The main cause for cancer-related death is not the most commonly-used live-cell imaging setup is a microscope primary tumor, but cancer metastasis: malignant cells invade tissues with sufficient magnification and a stage-top incubation box other than where the primary tumor is located and spread in those to regulate the culture conditions. Although these microscopes tissues. Fundamental cancer research is one of the key factors in the have the required optical properties for accurate imaging and World Health Organization’s strategy to reduce cancer mortality , cell tracking, there are practical issues when using such a setup and investigating the underlying mechanisms for metastasis can for live-cell imaging. The regulation of the culture conditions in provide important information for the main clinical problem. the incubation box is more sensitive to variations compared to a dedicated incubator.13,14 Besides that, images are only captured In cancer metastasis, the migration of single cells is largely at certain time points, but the microscope is unavailable for (end- affected by chemical cues, which provide direction for point) imaging by other users during the entire live imaging migration to the cells via chemotaxis : the single cells migrate experiment. A system that could overcome these issues is the along a concentration gradient of a chemoattractant. Various CytoSMART® Lux3 BR Duo Kit. This dedicated system for live-cell imaging fits in a regular incubator, and therefore enables culture investigated cell lines. HeLa cells (cervical cancer cells) have been monitoring in a constant and optimal culture environment. The reported to migrate towards higher concentrations of for instance optical properties of the devices provide high-quality imaging, fibronectin and stromal cell-derived factor 1α. The rat glioma as well as accurate cell tracking. By connecting two devices cell line C6 has been described to be attracted by bradykinin. A within the same incubator to a single laptop, two cultures can be commonly-used supplement in cell culturing that contains all of monitored simultaneously and compared side-by-side, without the aforementioned chemoattractant molecules is fetal bovine occupying a microscope for other lab members. serum (FBS; the blood serum of an unborn calf). provides an interesting and straightforward model system for the In this proof-of-concept study, we determine the effect of an FBS chemical cues provided to cancer cells. gradient on directed migration of cancer cell lines, using sideby-side high-quality live-cell imaging. The CytoSMART® Lux3 However, in order to study the behavior of cancer cells in response BR Duo Kit was used to monitor two cancer cell lines exposed to the chemical cues, accurate cell tracking in an environment to an FBS gradient or constant FBS concentration. Cells were with a chemoattractant gradient is required. Multiple cell culture tracked over time, and this provided fundamental insight into vessel designs have been made to provide a chemoattractant the chemotactic migration of cancer cells, which may ultimately be related to cancer metastasis. Figure 1: Experimental setup. Cancer cell lines were seeded in Ibidi µ-Slide Chemotaxis and culture medium was added, either resulting in an FBS gradient or a constant FBS concentration. Cultures were monitored for 24 h using the CytoSMART® Lux3 BR Duo Kit, with an imaging interval of

Accurate tracking of chemotactic cancer cell migration – basic insights in metastasis Materials and methods HeLa cells (Innoprot P20107) and C6 cells (ATCC CCL-107) were 0% FBS in the other or 10% FBS in both reservoirs (Fig. 1). High- cultured to sub-confluency in DMEM (Gibco) supplemented quality images of the cultures were made using the CytoSMART® with 10% FBS (Gibco) and 1% pen-strep (Gibco), under standard Lux3 BR Duo Kit (37° 5% CO2): cultures were monitored for C; culture conditions (37° 5% CO2). Each of the cancer cell lines C; 24 h, taking a snapshot every 5 min. Afterwards,...

Accurate tracking of chemotactic cancer cell migration – basic insights in metastasis Figure 3: C6 cells display no clear chemotactic migration in an FBS gradient. A) Raw images of C6 cells made using CytoSMART® Lux3 BR Duo Kit, single cell detection (purple) and path detection (yellow) with FIJI-plugin TrackMate. B) Chemotactic displacements of C6 cells visualized using FIJI-plugin Chemotaxis and Migration Tool, with longitudinal and lateral displacements defined with respect to the FBS gradient. Discussion Cancer metastasis is the main cause of cancer-related Cervical cancer is amongst...

Accurate tracking of chemotactic cancer cell migration – basic insights in metastasis The FBS that was applied as chemoattractant in this research using individual chemoattractants can provide more detailed provided an experimentally straightforward model system. information on the cellular behavior. Besides that, directed migration towards blood vessels was mimicked with this setup. However, FBS has a variable and The high-quality images made by the CytoSMART® Lux3 BR Duo complex composition , therefore providing little insight in the Kit were directly suitable for the TrackMate plugin....

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