The overall goal of the following experiment is to determine whether genetic lesions that predisposed towards breast cancer alter the behavior of pineal plastic memory cells in primary culture. This is achieved by isolating primary mammary epithelial cells from genetically engineered mice. As a second step live cell time-lapse imaging is performed over five to seven days, creating a movie to analyze cell behavior.
Then a software program that tracks single cells is utilized to quantify and compare the behavior of cells isolated from different genetically engineered mouse models of breast cancer. The results show that specific genetic lesions induce different behaviors in the pre neoplastic memory. Epithelial cells based on quantitative behavioral analyses performed using single cell tracking.
Though this method can provide insight into how different genetic lesions alter behavior of preneoplastic mam mammary epithelial cells. It can also be applied to other systems such as preneoplastic, cells from other organs, cancer cells from any organs, including the study of possible therapy induced behavioral changes, as well as comparisons of stromal cell behavior from normal and cancerous tissue demonstrating the procedure. Today will be Dr.Priscilla Firth, senior author of this publication, and Pete Johnson, manager of the Microscopy and Imaging shared resource Center at the Lombardi Cancer Research Center at Georgetown University.
Euthanize a mouse and immediately proceed with the necropsy. Place the mouse supine on a styrofoam necropsy platform and secure all four limbs so the ventral skin is taut. Saturate the ventral skin and hair, including the skin on the limbs with 70%ethanol.
Expose the number two and three and number four and five mammary glands with a midline incision through the skin. Extend the incision with two y incisions through medial skin of the four limbs. Be careful to not enter the peritoneum using a blunt dissection.
Separate the skin from underlying peritoneum. Pull back on both sides of the skin until it is taut and pin it to the platform beginning at the outer side. Use the scissors to make a blunt dissection that isolates the intact inal and or thoracic mammary glands from the underlying connective tissue and muscle.
Place one or two mammary glands in a 10 centimeter Petri dish and move to a tissue culture hood. Now, examine the glands for mammary lymph nodes. Small well circumscribed nodules with a yellowish color.
Using a scalpel and forceps, remove them from the surrounding gland. Mince the mammary tissue into millimeter sized cubes using two scalpels. Place the minced mammary tissue in a 50 milliliter conical tube with five milliliters of dissociation media.
Mix it with gentle tation and incubate it overnight. Keep the cap loose the next day. Isolate and culture the primary epithelial cells.
Immediately after plating the cells. Position the six well plate securely on a microscope stage within an incubation chamber. Allow the plate to equilibrate for a minimum of 15 minutes.
Adjust the condenser for Kohler illumination and center the phase rings. Open the image acquisition software such as velocity and create and name a library for the time-lapse images and save to a location with sufficient space for large files. Lower the lenses to avoid stage lens interference.
Under the stage heading, select calibrate stage reacquire the focal plane set Z equals zero under the X, Y, Z tab. And mark imaging points by selecting add point under the stage. Heading place imaging points in the middle of each well.
Phase contrast imaging may be distorted near the periphery of plastic wells. Arrange points in a square in four by four fields each with an approximately 5%overlap. Save the selected points under stage.
Under stage select make focus map and follow the prompt to set the focus of each point. Then set the image acquisition timing so it captures the right number of pictures per hour. Now save the focus map under stage.
Proceed by right clicking image acquisition on the right side toolbar. Then save the imaging settings. Press the record button to begin the imaging process after an hour, check the focus of the images to see if any adjustment is needed.
Monitor and continue the live imaging for five days when the cells become confluent. It is critical to monitor the acquired digital images and adjust the focus map frequently during the first 24 hours because the cells will be attaching to the plate thereafter at least twice daily. Check that the cells are in focus and the cultures are not contaminated.
To determine the mechanism of initial colony formation, start with a single image stack representing one imaging site on the plate. In the initial frames, the cells will be floating, epithelial and fibroblast cells can be distinguished by cell morphology. Epithelial cells have a cuboidal shape and form cell colonies.
Fibroblasts a type of stromal cell have an elongated morphology. Follow the serial images to determine when the first epithelial cell adheres to the plate. At this step, epithelial cells can be identified and differentiated from fibroblasts by their more cuboidal morphology.
Follow this single epithelial cell through serial images and follow its fate over the subsequent 24 hours. Make note, if the cell becomes surrounded by additional epithelial cells, undergoes cell division or undergoes apoptosis If surrounded by epithelial cells, the mechanism of colony formation can be determined as from the aggregation of surrounding or from cell division. Record the number of colonies formed by cell aggregation versus cell division during the first 24 hours to determine the number of initially adherent cells that undergo apoptosis over a specific time period.
Follow the serial images for the appearance of classic features of apoptosis developing in a cell that has been adherent over time. Epithelial cells in culture alter their morphology from an initial cuboidal shape to a more elongated appearance. Consistent with EMT.
Follow the serial images to determine the day and hour after plating When this occurs, begin by starting the TTT program. Select user initials and click continue. Press set NAS, select the user created TTT export folder and click okay.
In the browser, select an experiment folder. Select a position folder, and then press the load position button. Send the ocular factor to 10 times.
Load the number of images required, which is typically all of the images. When using this analysis. For the first time in the cell editor window, select new colony under the file menu in the movie window.
Press the track cell button or press F two. To begin tracking a cell. Identify a cell in the movie window and place the cursor over the cell.
A circle with the number of the cell should appear after F two has been clicked. Use the zero key on the number pad to track the placement of the cell and advance to the next picture. Move the cursor to follow the placement of each cell through each frame.
To delete a track and return to the previous image, press delete on the number pad to move forward frames without tracking the cell, press three. And to move backward without tracking, press one. To mark a cell division in the movie window, click the division button to mark cell death in the movie window, click cell death.
Once a division has occurred, the daughter cells can be tracked in the same cell fate map. To do this, go to the cell editor window and right click on the circle symbol of the designated dotter cell. This starts tracking mode automatically now press F 10 to save the tree.
Each tree will save in the designated output folder. To start a new sulfate map in the cell editor window, select the open menu, click file and click. New colony cells were rounded and floating at the onset of imaging.
The size bars equal 200 microns After attachment to the plate, they became flat and demonstrated a cuboidal type appearance. By four days of culture, the majority of epithelial cells elongated into an EMT like morphology. This change occurred with the same chronology in all genotypes studied the floating cells developed into defined individual epithelial cell colonies.
By 24 hours after plating, serial image analysis revealed that these colonies were generated by cell aggregation rather than cell division. While disruption of BRCA one by itself did not alter the number of colonies formed. Addition of the TP 53 haplo insufficiency significantly reduced the number of colonies formed.
Likewise, addition of ER alpha over expression significantly increased the number of colonies formed Following this procedure. Other methods that employ the use of time-lapse imaging combined with single cell tracking can be performed in order to answer additional questions like how specific therapeutic approaches or additional genetic lesions alter cell behavior and may influence either therapeutic response or cancer progression.
