A modified and convenient method for assessing tumor cell invasion and migration and its application to screening for inhibitors.

In order to screen potent inhibitors of tumor invasion and metastasis, we here devised a simple and reproducible in vitro assay for tumor invasion and migration. A conventional cell-counting assay using a Transwell chamber with a microporous membrane filter is troublesome and time-consuming, involving visually counting the cells under a microscope, and the invaded or migrated cells are sometimes distributed unevenly in predetermined fields on the lower surface of the filter. Therefore, it is difficult to evaluate the invasive and migratory abilities of tumor cells easily and quantitatively by the cell counting method. In the present study, crystal violet dye was used for staining the invaded cells and colorimetrically assessing the invasive ability per filter as an absorbance. In this crystal violet assay, tumor cell invasion into a reconstituted basement membrane Matrigel was proportional to both the cell number added into the chamber and the incubation period, and inversely proportional to the amount of Matrigel barrier on the upper surface of filter. The results obtained by this dye-uptake method were highly consistent with those of a conventional cell-counting assay. Using this crystal violet assay, the anti-invasive effect of doxorubicin (DOX) was detected more easily and found to be highly proportional to that by the conventional cell-counting method. We therefore applied this convenient assay method to screen anti-invasive and anti-metastatic compounds. As a result, caffeic acid was found to be more active in the inhibition of both tumor cell invasion and migration without showing direct cytotoxicity in vitro than other related compounds.