This study investigates lung metastasis in a breast cancer mouse model using 4T1 cells. The methodology involves culturing these cells and assessing their ability to survive in the presence of a toxic nucleotide.
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.
Bring the volume of the tube up to 10 milliliters with 1X HBSS. Then, pour the contents over a 70-micrometer cell strainer into the 50-milliliter conical tube. Use the plunger of a 1-milliliter syringe to gently grind the sample through the strainer.
Centrifuge the tube for 5 minutes at 350 times g, and discard the supernatant. Then wash the pellet twice with 10 milliliters of 1X HBSS. Resuspend the pellet in 10 milliliters of 60 micromolars 6-Thioguanine complete culture media, and plate the samples in the 10-centimeter cell culture plates using a dilution scheme, if desired.
Incubate the plates at 37 degrees Celsius and 5% carbon dioxide for 5 days. Pour culture media off of the plates into the appropriate waste container. To fix the cells, add 5 milliliters of undiluted methanol to each plate, and incubate for 5 minutes at room temperature, making sure to swirl the methanol so that it covers the entire plate.
Pour the methanol off the plates into the appropriate waste container, then, rinse each plate with 5 milliliters of distilled water. Add 5 milliliters of 0.03% methylene blue per plate, and incubate it for 5 minutes at room temperature, making sure to swirl the methylene blue solution so that it covers the entire plate. Pour the methylene blue into the appropriate waste container, and rinse each plate again with 5 milliliters of distilled water.
Turn the plate upside down and blot it against a paper towel to remove excess liquid. Place the plate on its lid, and let it air dry overnight. Metastatic colonies will be blue. Once the plates have dried, they can be stored at room temperature indefinitely.
繁體中文
