Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

The overall goal of this procedure is to rapidly quantify in real time ovarian cancer cell invasion in asteroid mesothelial cell co-culture model of metastasis. This is accomplished by firstly generating ovarian cancer from cell lines by culturing the cells under non-adherent conditions in methyl cellulose. The second step is to prepare a real-time cell analyzer, CIM plate by coding the upper chamber of the two chambered well with matrigel to mimic the basement membrane that underlies the mesothelium of the peritoneal cavity and the mesothelial lining itself.

Next, the ovarian cancer steroids are harvested and added to the upper chambers of the realtime analyzer plate wells. The final step is to program and initiate the real-time cell analyzer, which will take periodic readings at predefined intervals over a prolonged period of assay. Ultimately, the results obtained depict the continuous invasion process, which is used to determine the key factors regulating ovarian cancer cells as they invade cellular and matrix barriers.

The main advantage of this technique over existing methods like standard transworld assays of cancer cell invasion, is that this approach allows for continuous measurements of cancer cell invasion over extended periods of time. Unlike a single endpoint assay, which doesn't fully capture the dynamic nature of ovarian cancer metastases, Though this method is used to provide insight into ovarian cancer can also be applied to study different types of metastatic processes such as breast cancer or the ation of cancer cells through an endothelial layer. Likewise, it can be used to study normal cell invasion such as trophoblast invasion during embryo implantation To prepare ovarian cancer cells for fluorescent cell labeling.

First harvest cells at 75%co fluency and resus suspend cells at a final concentration of 1 million cells per milliliter in one milliliter of prewarm PBS per 0.1%BSA Adding the optimal amount of steroid per well and handling steroid carefully are critical to the success of this procedure. Next, add two microliters of a five millimolar stock of cell trace CSFE solution to cells at a final concentration of 10 micromolar and incubate at 37 degrees Celsius for 10 to 15 minutes In a water bath, quench the reaction with one milliliter of ice cold medium containing 10%FBS and re pellet by centrifugation.Reus. Spend in 10 milliliters of the appropriate prewarm growth medium and seed cells into a 75 square centimeter filter capped flask culture at 37 degrees Celsius, 5%carbon dioxide until the level of fluorescence labeling is adequate for detection.

Begin this procedure by measuring out the volume of culture medium appropriate for each cell line, which is 15 milliliters minus the volume of cells needed for sphe generation. Add three milliliters of methyl cellulose stock solution to the culture medium to achieve a final methyl cellulose concentration of 20%and mix thoroughly by gentle inversion. Add 300, 000 cells to the Methylcellulose containing medium and mixed thoroughly by gentle inversion pipette 150 microliters of the cell Methylcellulose media mix into each well of a 96 well concave bottom culture plate.

This will result in a total of 3000 cells per well culture in 37 degrees Celsius, 5%carbon dioxide for one to four days or until uniform PHE form. Typically one steroid per well is observed the realtime cell analyzer or RTCA cell invasion assay utilizes a 16 well RTCA two chambered CIM plate. Working in groups of four wells at a time, add 50 microliters of matrix to each well of the upper chamber of the plate to ensure that the total surface area is covered.

Remove 30 microliters of matrix immediately but slowly to get rid of any excess fluid. Incubate the plate at 37 degrees Celsius for four hours prior to use in a tissue culture incubator. After four hours, add 30 microliters of the serum free, medium appropriate for each cancer cell line to the upper chamber and 160 microliters of media with or without serum.

As per the experimental design to the lower chamber, assemble the CIM plate by clicking the lower chamber into the upper chamber and equilibrate in a 37 degrees Celsius incubator. For one hour in a tissue culture incubator, open the RTCA program and select the layout tab. Highlight all experimental wells, right click on wells and select turn on wells.

Then fill in experimental conditions and sell names as desired. To add steps or substeps to the program, select the schedule tab and right click on add a step. The first step is a pre-programmed background sweep, which is automatically incorporated into the program.

Once add a step is chosen, enter the desired program details for step two of the RTCA program, which will record readings during the establishment of the LP nine human mesothelial cell monolayer. Add the time intervals between impedance readings by selecting the interval tab and entering 15 minutes. Add experimental duration by selecting the duration tab and entering a time between 12 to 24 hours.

Subsequent steps are added in the same way. Step three of the RTCA program will direct the instrument to take readings during frid invasion. Enter five minutes under the interval tab and 48 hours under the duration tab.

Select plate on the menu bar and save. To begin this procedure, place the CIM plate in the RTCA instrument and open the program that was saved earlier. Select execute in the menu bar and then start a background sweep will automatically be performed.

Following the background sweep, remove the CIM plate from the instrument and place it in a tissue culture hood plate. 50, 000 LP nine cells suspended in 160 microliters of serum free medium into the upper chamber of each CIM plate. Well place the plate in the RTCA instrument and take readings every 15 minutes while the LP nine monolayer is establishing overnight on the following day, aseptically cut one millimeter off the top of a one milliliter pipette tip and use it to gently retrieve the contents of each well for each cell line.

Pool 10 steroid in a sterile tube centrifuge. Steroids at 120 GS for eight minutes, and then remove Methylcellulose containing medium by gentle aspiration. Wash sphe twice more with PBS centrif for using at 120 GS for eight minutes to pellet steroids after each wash.

For each experimental well Resus spend a total of 10 steroids in 160 microliters of medium without PBS. Pause the RTCA experiment by selecting execute from the menu bar and checking pause. Remove the CIM plate from the instrument and place it in a tissue culture hood.

Aspirate off the media from each well and replace with steroid containing media. Return the plate to the RTCA instrument. Select execute from the menu bar and check abort step.

The program will move to the next step automatically. To resume the experiment, select execute from the menu bar and check start continue. Step three in the RTCA program will initiate in this experiment two epithelial ovarian cancer cell lines OVCA 4 33 and OVCA 4 29 and an ovarian granulosa cell tumor line.

KGN were used to generate steroids following overnight culture and U bottomed dwells suspended in methyl cellulose containing media. All three cell lines formed compact steroid structures of approximately 400 to 500 micrometers in diameter. The bottom panels show the matching cell line grown in a monolayer scale.

Bars represent 100 micrometers once formed. Steroids were harvested and plated on top of an LP nine mesothelial cell monolayer in an R-T-C-A-C-I-M plate. Images under phase contrast microscopy or under fluorescent microscopy of parallel cultures were taken periodically to aid in the interpretation of the RTCA data.

It is informative to assess both the basal layer of invasion of a cancer cell line as well as chemo attracted induced invasion. In this example, basal invasiveness is measured by the addition of serum free medium or SFM to both the upper and lower chambers. Complete media with 10%FBS, which contains many potential chemo attractants is used to examine chemo attractant induced invasion.

Shown here are representative results from a comparison of cell invasion between the kgn cells and the LP nine mesothelial cells. The RTCA invasion assay was conducted with and without FBS in the bottom chamber of A CIM plate. Results are shown as mean positive minus SD cell index from triplicate wells at the 24 hour time point and over an entire two day assay period.

These results confirm that LP nine cells are minimally invasive over two days under either basal or chemoattractant induced conditions, and thus were a good cell type to use in co-culture assays with ovarian cancer cells. In contrast KG and ovarian cancer steroids exhibited the capacity to invade toward a chemo attractant. This graph presents results from a comparison of the invasive capacity of K-G-N-O-V-C-A 4 29 and OVCA 4 33 cell lines depicted over a 24 hour period over the two day assay.

All three cell lines exhibited the capacity to invade toward a chemo attractant as indicated by the increasing cell indices. The cell lines exhibited similar rates of invasion as indicated by the parallel line slopes of the curves. However, the OVCA 4 29 curve exhibit a higher upper asim tote, which indicated a higher maximal level of invasion compared to the other cell lines.

In contrast, basal levels of invasion of all cell lines were low. Furthermore, the cell lines exhibited differences in their times to onset of invasion as shown by a more detailed analysis of invasion data over a 2.5 hour time period. The KGN cells invade the cell and matrix barriers quickly while the OVC 4 33 and OVC 4 29 cells take three and five times longer to invade respectively.

Importantly, their behaviors at the onset of invasion were not predictive of their overall capacity for invasion. This suggests different inherent capabilities of the cancer cell lines, but may also indicate that different factors regulate early and late invasive behaviors of steroids. Once masters, this technique is readily adaptable to study various signaling molecules and cellular pathways within the peritoneal microenvironment that affect the metastatic behavior.

This can be achieved by the addition of exogenous growth factors, cytokines or inhibitors to either the bottom or top chamber of the well. Alternatively, you can genetically manipulate the cancer cells themselves or the peritoneal target cells.