Abstract

Introduction Collective cell migration is essential for normal development and functioning of the body. However, it is also involved in a multitude of pathologies, including cancer metastasis. To get more insight into collective cell migration, the scratch (or wound healing) assay is commonly used. In this assay, a scratch is made in a confluent monolayer of cells and images of the scratch are taken at several timepoints to analyze scratch closure. Several disadvantages of this method are that only a small part of the scratch is analyzed and it is difficult to find the exact spot after placing the samples back in the incubator. Automated live-cell imaging and analysis could provide a solution to overcome these issues. The CytoSMART Omni is an automated live-cell imager that is capable of imaging an entire well plate at
regular intervals inside an incubator. Using this device, the complete scratch area can be analyzed, eliminating the difficulty to find the same part of the scratch and increasing data accuracy by analyzing the whole area.

This study provides a proof-of-principle of scratch assay imaging and analysis using the CytoSMART Omni. C6 (rat glial tumor) cells were treated with different concentrations of Paclitaxel (PX) to investigate its effect on collective tumor cell migration.

Material and methods A 24-well plate was seeded with C6 cells at a density of 50,000 cells/cm². After 24 h, a scratch was made and medium supplemented with 0, 1, 10 or 100 nM of PX was added. A high-resolution image was made every hour for 23 h with the CytoSMART Omni. The scratch area, percentage of scratch closure and migration speed were determined using the cloud-based CytoSMART scratch algorithm.

Results Immediately after making the scratch, cells started to migrate in all four groups treated with different concentrations of PX. The scratches of the 0 and 1 nM PX treated groups were (almost) closed after 23 h, while the scratch areas of the 10 and 100 nM PX treated groups were 1.3·10⁶ μm² and 4.5·10⁶ μm², respectively. The migration speed of the samples treated with 100 nM of PX was significantly lower compared to the other groups. The migration speed of the 10 nM PX group was significantly lower compared to the untreated samples and the 1 nM PX treated samples.

Discussion In this study, scratch closure and migration
speed were significantly decreased upon treatment with 10 or 100 nM of PX. PX inhibits collective cell migration by stabilizing microtubules. Normally, dynamic microtubules push the lamellipodia forward and help regulate actin polymerization and focal adhesion formation at the leading edge of the cell. In case the microtubules are stabilized by PX, these processes are inhibited, which in turn inhibits cell migration.

Since the CytoSMART Omni is placed inside an incubator and the well plate is not translocated during the experiment, the exact spot can be monitored over time, increasing the accuracy of the data. Data accuracy is also increased by analysis of the entire scratch instead of analysis of only a (few) part(s) of the scratch. Lastly, since all wells of the well plate are imaged and analyzed automatically, the common cumbersome and labor-intensive manual imaging and analysis process is eliminated, which makes this new method of scratch assay
imaging and analysis very time-efficient.

Conclusion In this study, we have shown that PX inhibits collective cell migration of C6 cells in a dose dependent manner, starting from 10 nM of PX. Furthermore, the CytoSMART Omni and its automated, cloud-based, CytoSMART scratch algorithm provided time-efficient and accurate scratch analysis. Next to this, the CytoSMART Omni also provided the benefit of monitoring the scratch closure at optimal culture conditions.