Methylene Blue Staining to Assess Metastatic Colony Formation In Vitro: A Staining Procedure to Assess Metastasis by Quantifying Harvested Cancer Cells From Cancer Mouse Model

During cancer metastasis, tumor cells from a primary tumor travel through the circulatory system, extravasate to distant organs, and establish secondary tumors.

To evaluate lung metastasis in a breast cancer mouse model that has been injected with 4T1 cells, highly metastatic breast cancer cells, prepare a cell suspension, including 4T1 cells, isolated from the harvested lungs. Centrifuge the suspension and resuspend the cells in media containing 6-thioguanine, 6-TG - a guanine analog. Seed the cell suspension in a culture plate.

6TG enters normal cells where specific transferase enzyme converts it to 6-TG nucleotide, a toxic nucleotide. These cytotoxic nucleotides get incorporated into DNA, causing DNA damage, and selectively eliminate cells from culture. 4T1 cells have decreased ability to form toxic nucleotides, facilitating their survival and adherence to the plate. Depending on the proliferative ability of cells, they form colonies.

Treat the cells with methanol. Methanol permeabilizes the cells and denatures the cellular proteins. Incubate the cells with methylene blue.

The positively-charged dye enters the cell, interacts with the negatively-charged DNA, intercalating into spaces between the DNA strands’ adjacent base pairs. It also binds to RNA in the cytoplasm, but with lower affinity. The nuclei stain dark blue due to high DNA concentration than the cytoplasm owing to the low RNA concentration. Metastatic colonies appear as blue dots.

Image the plate. Use software to determine the metastatic colonies, quantifying the metastatic burden in the mouse lungs.