An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The overall goal of the following experiment is to apply this in vitro dormancy model to study molecular and cellular mechanisms and to generate hypotheses for testing in more biologically complex in vivo models. This is achieved by culturing estrogen positive breast cancer cells in fibrin neck encoded tissue culture plates at congenic density where their interactions are primarily with the substratum and not each other. As a second step, a dormant state is activated in some of the estrogen positive cells supported and maintained by FGF two and fibronectin representative of breast cancer cells in the bone marrow.

Next, the system is perturbed by addition of antibodies, inhibitors, peptides, or nucleic acids in order to disrupt various phenotypic and molecular aspects of the dormant cells and determine their role in the maintenance of the dormant state. The results show the number and appearance of growing and dormant colonies that are the results of treatment of incubated cells with FGF two on fibronectin coated plates. This method can help answer which signaling pathways adhesion molecules, or other specific molecular mechanisms are responsible for development and maintenance of dormancy in estrogen dependent breast cancer cells located in the bone marrow microenvironment.

To begin aspirate the culture medium from plates that are approximately 50%confluent with CF seven cell lines and RINs them with prewarm PBS. Then add one milliliter of point 25 trypsin EDTA solution dissolved in DMEM, high glucose medium without fetal calf serum. Incubate the plates for one to four minutes at 37 degrees Celsius while the cells begin to detach.

Once the cell detach from the plate and begin forming a single cell suspension, break up any remaining cell clumps by pipetting up and down several times with a two milliliter pipette. If cell clumps remain after four minutes of trypsin, discard the cells and prepare new culture cells remaining adhering to each other after four minutes will likely introduce error when counting colony numbers Palate the tryps inized cells and resuspend them at 1, 500 cells per milliliter. In culture medium, use a five milliliter pipette to mix the cell suspension by pipetting gently up and down count the cells and verify that the suspension consists of primarily single cells using an automated cell counter.

This can also be done manually using a counting chamber under a phase contrast microscope. Then draw up three milliliters of the cell suspension and disperse one milliliter into two of the wells from a 24 well fibronectin coated plate at a time. To optimize the spatial distribution of the cells, be sure to pipette the suspension into the middle of the well and do not subject the plate to further movement before the cells settle and attach.

Return the remaining cell suspension to the master cell mixture and again, mix the cells before drawing up another three milliliters. This procedure will reduce cell number variability as the cells continuously sediment in the tube and the pipette work quickly to distribute the large volumes of cells because allowing cells to sit in suspension at room temperature and CO2 concentration will modulate their congenic potential incubate cells at 37 degrees Celsius 5%CO2. This is considered day minus one.

In the timeline of the experiment. On day zero of the assay, replace the medium with one milliliter of fresh, medium, or fresh medium containing 10 nanograms per milliliter of fibroblast growth. Factor two, the small number of cells in a well after six days will not significantly impact the nutrient or cytokine composition nor the pH of the original medium.

On day three, add 100 microliters of a solution containing 10 x of the final intended concentration of a perturbing agent, such as a PI three kinase inhibitor, LY 2 9 4 0 0 2 negative control LY 3 0 3 5 1 1 or media control to the one milliliter of medium already in the wells, and let the solutions mix by diffusion. Set the plate back in the incubator for an additional three days on day six dissolve. 0.1%crystal violet into a solution of two percent's ethanol and 10 millimolar sodium borate.

At pH nine, then aspirate the media from the wells and add one milliliter of the crystal violet solution to each well for 20 minutes. Set arin station by placing an ice bucket in the sink and running water continuously into the bucket wash plates by immersing them in the water with the well openings facing down at an acute angle into the bucket. Tilt the plate to a horizontal angle once underwater so that the well openings face downward.

Then tilt the plate back to an acute angle and remove it in one gentle flowing motion. Repeat the immersion two or three times until the water at the bottom of the wells no longer appear blue. Then place the plates upside down on towels on the bench top to dry overnight.

Once dry, place the plate under an inverted phase contrast microscope and count the number of growing and dormant colonies in each well. At 40 x magnification count colonies of greater than 30 cells as growing and colonies of less than 12 or less cells as dormant.Shown. Here is the typical appearance of a growing and a dormant colony.

Growing colonies contain more than 30 cells and dormant colonies contain 12 or less cells, which are many times larger than growing cells with large cytoplasm to nucleus ratios without addition of FGF two to culture. The colonies are predominantly represented by growing clones. While in the presence of FGF two, the vast majority of clones are dormant, the effect of additional agents can also be evaluated using the system Here, two perturbing agents at previously determined concentrations were added to the wells on day three of the Congenic assay.

The figure represents a preferential inhibition of dormant clones by the PI three kinase inhibitor LY 2 9 4 0 2, but a lack of effect by the negative control LY 3 0 3 511. These experiments can be used to help understand the elements that govern dormancy and resistance to therapy. Here, the surviving dormant cells treated with the PI i three kinase inhibitor lost their spread appearance, became small dendritic and appeared distressed.

After watching this video, you should have a good understanding of how to establish this congenic assay for growing in dormant cells, how to perturb the cells by adding specific inhibitors or perturbing agents after establishing dormancy and how to quantify the effect of the perturbing agents, this assay can be used to both assess quantitative and qualitative outcomes by counting dormant colonies or measuring any number of ultra structural immuno phenotypic or molecular events in the dormant and perturbed cells. Thanks for watching and good luck with your experiments.