The HC11 and EpH4 cells are mouse breast epithelial cell lines which can differentiate in culture. At the same time, these cells can be transformed by a number of oncogenes. These cells are ideal for the study of the interrelationship between differentiation and the neoplastic transformation.
These techniques can be used in signal transduction studies to discover targets for cancer therapy. For example, the small GTPase Rac and other molecules may trigger transformation of breast epithelial cells when expressed to high levels, but surprisingly at low levels, they may induce differentiation. Other signal transducers may behave in a similar manner.
The principles may apply to other types of differentiation as well where cell confluence plays an important role, for example differentiation of adipocytes or myotubes. Someone performing this technique for the first time should keep in mind that plating of the HC11 cells is important. This is because cell-to-cell contact is important for differentiation.
Another point to watch out is that proper extraction of EpH4 cells from the matrix is critical for protein quantitation because any residues will affect protein determination. Visual demonstration of this method is useful because some details of the protocol are difficult to describe on paper. Start by preparing a bottle of 50 milliliters of HC11 cell medium according to manuscript directions.
To plate the cells, aspirate the medium from five six centimeter 50%confluent Petri dishes and wash the cells with approximately 1.8 milliliters of trypsin using a sterile nine inch cotton plugged Pasteur pipette. Then add 200 microliters of trypsin per plate and swirl the plate to dislodge the attached cells. Observe the cells under phase contrast microscopy with a 4X objective to make sure they have started to dislodge.
Then aspirate approximately 1.5 milliliters of HC11 medium with a sterile nine inch Pasteur pipette and squirt it vertically against the cells while rotating the dish making sure to avoid splashing. Transfer all the cells to the bottle with HC11 medium and swirl to disperse evenly in the bottle. Then aliquot the cell suspension into 20 three centimeter Petri dishes by pipetting two milliliters of cells per dish.
Rock the Petri dishes to spread the cells and incubate them at 37 degrees Celsius and 5%carbon dioxide overnight. On the next day when the cells are 90 to 100%confluent, aspirate the medium and replace it with medium with FBS but without EGF. After growing the cells in medium without EGF for 24 hours, add the differentiation medium to 10 dishes and grow the cells for up to 10 days keeping the other 10 dishes as controls.
Change the medium every two to three days with both HIP treated and control cells. To monitor the differentiation, observe the cells with phase contrast microscopy. Use fluorescence microscopy if the cells are expressing green fluorescent protein.
Additionally, quantify the degree of differentiation by extracting proteins from the HIP treated and control cells once per day and performing Western Blot analysis. Gather and place the EpH4 growth medium, the EHS matrix, and two conical 50 milliliter tubes on ice. Prechill the tissue culture plates with pipette tips at minus 20 degrees Celsius and prepare EpH4 growth medium supplemented with 10 or 20%matrix in two 50 milliliter conical tubes and keep them on ice until ready to use.
After retrieving the prechilled tissue culture plates at minus 20 degrees Celsius, coat 10 wells of the 24-well plate with 150 microliters of undiluted matrix by spreading the matrix with a pipette. Avoid creating bubbles when spreading the matrix. Then gently tap the sides of the plate and incubate the plate at 37 degrees Celsius for one hour to allow the matrix to solidify.
Meanwhile, prepare for the differentiation by trypsinizing the EpH4 cells well as previously described and ensuring single cell suspensions after resuspension of trypsinized cells. Next, count the cells in suspension with a hemocytometer. Transfer five times 10 to the four cells per well to a 1.5 milliliter sterile conical centrifuge tube and spin them at 250 times g for five minutes.
Carefully aspirate the supernatant medium and place the tube on ice. Use a one milliliter pipette tip to resuspend the cells in 350 microliters of EpH4 growth medium supplemented with 20%matrix while keeping the tubes on ice making sure to avoid bubbles. Once the bottom layer has solidified, add the 350 microliters of cell suspension to each coated well and place it in a carbon dioxide incubator at 37 degrees Celsius for one hour to allow the 20%matrix layer to solidify.
Observe the cells with phase contrast microscopy to make sure single cells are visible. Then add 200 microliters of EpH4 medium with 10%matrix on top and incubate the plate at 37 degrees Celsius. Begin differentiation induction one day after plating the cells in the matrix.
Carefully remove 150 microliters of top 10%matrix medium and add 200 microliters of EpH4 medium containing HIP and 10%matrix and for controls add a 10%matrix medium without HIP. Replace the medium every two days for up to 10 days monitoring mammosphere formation with phase contrast microscopy. To quantify the differentiation, carefully pipette off the 10%matrix HIP medium from the wells and rinse the 20%matrix layer with 350 microliters of ice cold PBS.
Then add 70 to 100 microliters of ice cold PBS with one millimolar EDTA directly into the wells and gently detach the bottom layer of 100%matrix with a pipette tip. Shake the plate gently at four degrees Celsius for 30 minutes. Carefully transfer the spheroid suspension to a conical tube and rinse the wells with 500 microliters of PBS EDTA to recover any remaining spheroids.
Rock the tube on ice for an additional 30 minutes making sure that the matrix fully dissolves. If visible matrix clumps are seen, add more PBS EDTA or shake longer. Centrifuge the solution at 350 times g to pellet the spheroids.
Then aspirate the supernatant, lyse the spheroids, and probe the cells for beta-casein, cyclin D1, and p120RasGAP by Western blotting. There is a striking dependence of differentiation upon the strength of the Rac signal in HC11 cells. While endogenous CRAC1 is required for differentiation and low levels of mutationally activated RacV12 cause an increase in differentiation capacity, high RacV12 levels trigger a block of differentiation and induce neoplasia.
When the differentiation properties of EpH4 cells were explored in a 3D matrix culture, it was found that beta-casein production peaked at eight to 10 days and cyclin D1 expression was maximal at four to six days after HIP stimulation. To determine the positioning of individual cells, they were stained with DAPI and imaged with confocal microscopy. Inner cell death and formation of hollow lumens was observed in the mammospheres while addition of HGF resulted in the formation of tubular structures.
Someone performing this technique for the first time should keep in mind that the plating of the HC11 cells is important. Also proper extraction of the EpH4 cells from the matrix is critical for protein quantitation because any residues will affect protein determination. This technique can be used to investigate other signal transducers that may behave in a similar manner.
If there are driver mutations in a cancer, then their complete inhibition will not be necessary since low levels remaining would actually induce differentiation.
