A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The overall goal of this cancer cell steroid invasion assay is to allow for the monitoring of cancer cell invasion out of a cellular bolus and into a surrounding three dimensional matrix. This method can help answer key questions in cancer biology, like the identification of conditions that promote or inhibit cell invasion. The main advantage of this technique is that the various nuances of how cells invade can be evaluated under a more physiologically relevant setting in vitro.

To begin, remove a cultured dish containing adherent cancer cells from the incubator. Wash the cells once with PBS and add 1.5 milliliters of 0.05%tripsin EDTA. Then incubate the cells for three minutes at 37 degrees Celsius.

Once the cells have detached, pass the tryps inized cells through a five milliliter pipette to generate a single cell suspension, and then add 8.5 milliliters of cell culture media to the flask. Count the cell suspension using a cell counter. Dilute the cell suspension to 500 to 1000 cells per 20 microliters.

In culture medium, use a multi-channel pipette to form 20 microliter droplets on the inner surface of a 10 centimeter dish lid In all pipette, five rows of eight drops for total of 40 drops. Next, pipette five milliliters of sterile PBS into the bottom of the 10 centimeter dish. Carefully invert the lid and place it on the 10 centimeter dish.

Then incubate the hanging drop cultures at 37 degrees Celsius and 5%carbon dioxide for up to 72 hours to form steroids. To generate the 3D matrix, thaw an aliquot of growth factor, reduced basement membrane solution at four degrees Celsius overnight. Then collect the steroids by tilting the lid of the 10 centimeter dish to pool the droplets.

Transfer the media to a 1.5 milliliter centrifuge tube and leave the tube for 10 minutes to allow the OIDs to settle to the bottom. Next, mix 100 microliters of the thawed basement membrane solution with 100 microliters of 2.3 milligrams per milliliter cold. Type one collagen solution in a pre chilled 1.5 milliliter centrifuge tube.

Incubate the extracellular matrix or ECM mixture on ice. Pipette 40 microliters of cancer cells steroids from the bottom of the 1.5 milliliter centrifuge tube, and combined with the ECM mixture, now pipette 40 microliter drops of this viscous mixture into the center of individual wells in a 24 well cell culture plate. Keeping the plate level, place it into a 37 degree Celsius cell culture incubator and leave it undisturbed for 30 minutes.

Once the 3D cultures have polymerized, slowly pipette one milliliter of warm cell culture medium into each well to submerge the culture. Then return the 3D cultures to the incubator using an inverted microscope image, the invading cancer cells. Using the 20 x objective at predetermined time points.

Incubate the dish at 37 degrees Celsius. In between imaging time points for data analysis, quantitate the invasive ability of the cells using image analysis software such as Image J to determine cell distance from the edge of the spheroid. Use the straight draw tool to mark the radius.

Then click analyze in the top menu, followed by measure to display the length measurement. To determine the invasive area of the spheroid, use the freehand draw tool to trace the border of the spheroid and then the area of invasive cells. Again, click analyze in the top menu, followed by measure to display the area measurement.

In testing a panel of cancer cell lines, most were observed to form steroids using the hanging drop culture method. Several cell lines, however, were unable to form steroids when using this culture method. Additionally, there was variability within the group of sphere forming cancer cell lines regarding their ability to invade the surrounding ECM.

These data were confirmed visually with cellular invasion becoming pronounced by 24 hours for several of the tested cell lines Once mastered, this technique can be done with just a few hours of work that occurs over the course of five days. While attempting this procedure, it is important to remember to work quickly and avoid trapping air bubbles in the mixture that will comprise the 3D culture. This technique can be modified to examine the impact of drugs, cellular co-culture, or different ECM components on cancer cell invasion.

After watching this video, you should have a good understanding of how to evaluate and quantitate cancer cell invasion in a 3D setting.