Mammosphere Assay Reveals Api5-Induced Stemness in Non-Tumorigenic Breast Epithelial Cell Lines

We are investigating how overexpression of a protein in a non-immunogenic cell line leads to the acquisition of cancer stem cell-like properties. This protocol uses a minimal medium supplemented with B-27 and EGF, making it more cost effective. To begin, coat each well of a standard six-well tissue culture plate with one milliliter of 1.2%poly 2-hydroxyethyl methacrylate solution, and place the plate in an oven set at 40 degrees Celsius to dry under sterile conditions.

Expose the dried plate to ultraviolet light for 15 minutes. Then, wash each well three times with Dulbecco's PBS before using the plate to set up the mammosphere formation assay. Obtain a previously cultured 3D dissociated MCF-10A cell culture dish and remove the spent medium.

Wash the cells with one milliliter of Dulbecco's PBS. Then, add 500 microliters of 0.05%trypsin-EDTA and incubate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for 12 to 15 minutes until the cells are trypsinized. Once the cells are dislodged, add two milliliters of resuspension medium to neutralize the trypsin activity.

Spin the cell suspension at 112 G for 10 minutes at room temperature. Aspirate the supernatant and resuspend the resulting cell pellet in one milliliter of mammosphere medium. Pass the cell suspension through a 25-gauge needle to create a single-cell suspension.

Next, using a hemocytometer, count the number of cells. Calculate the required volume of the cell suspension to seed 2000 cells per well. Dilute the calculated volume of cell suspension in two milliliters of mammosphere medium per well, and seed the diluted suspension into each well of a six-well plate, preparing three replicate wells for each condition.

Incubate the plate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for seven days. After seven days, manually count the number of mammospheres larger than 50 micrometers in each well. Image the mammospheres using a digital camera attached to a light microscope at 20 x magnification and calculate the mammosphere formation efficiency using the given formula.

Collect the first generation mammospheres in a 15-milliliter tube and centrifuge at 300 G for 10 minutes at room temperature. Aspirate the supernatant carefully, leaving the mammosphere pellet intact at the bottom of the tube. Wash the pellet with Dulbecco's PBS.

Centrifuge the cell suspension to obtain a mammosphere pellet, then pipette out the Dulbecco's PBS without disturbing the pellet. Now, add 200 microliters of 0.05%trypsin-EDTA to the washed pellet and incubate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for two to three minutes. Then, pass the mammospheres in trypsin solution through a 25-gauge needle three times to obtain a single-cell suspension.

Then, add 0.5 milliliters of resuspension medium to neutralize the trypsin activity. Centrifuge the cell suspension at 112 G for 10 minutes at room temperature. Aspirate the supernatant and resuspend the cell pellet in 0.5 milliliters of mammosphere medium.

Using a hemocytometer, count the number of cells and calculate the required volume of cell suspension to seed 2000 cells per well. Dilute the required cell suspension volume in two milliliters of mammosphere medium per well, and seed into each well of a six-well plate, preparing three replicate wells for each condition. Incubate the plate at 37 degrees Celsius in a humidified 5%carbon dioxide incubator for seven days.

After seven days, count the number of mammospheres with a diameter greater than 50 micrometers in each well. Image the mammospheres under 20 x magnification and calculate the self-renewal capacity using the given formula. Collect the first generation mammospheres into a 15-milliliter tube and centrifuge at 300 G for 10 minutes at room temperature.

Aspirate the supernatant carefully, ensuring the mammosphere pellet remains undisturbed at the bottom of the tube. Wash the pellet gently with Dulbecco's PBS. After removing the PBS, collect the washed pellet in ice-cold RIPA buffer to obtain the protein lysate and perform immunoblotting using the standard protocol.

Finally, quantify the protein levels using densitometric analysis in image J.Select the region of interest on the western blot image and generate a density profile for each lane and the pluripotency marker. Normalize the relative densities of the protein of interest to those of GAPDH for each lane, then calculate the fold change for each protein. API5 over expressing MCF-10A cells showed a 1.58-fold increase in mean mammosphere formation efficiency compared to control cells under non-adherent, serum-deprived conditions.

These cells formed second generation mammospheres with a self-renewal capacity nearly 2.5 times higher than control cells. Immunoblotting showed increased expression of Oct 3/4, Nanog, Sox 2, and BMI1 in API5 over expressing primary mammospheres compared to controls. Densitometric analysis showed a 1.83-fold increase in Oct 3/4, a 1.65-fold increase in Nanog, a 1.68-fold increase in Sox 2, and a 1.62-fold increase in BMI1 levels in API5 over expressing mammospheres compared to controls.

A challenge in the protocol is standardizing the cell count to ensure sufficient mammosphere formation without cell aggregation. Mammospheres generated by this protocol can also be used to visualize protein localization in a three-dimensional context using immunofluorescence.