The overall goal of the following procedure is to evaluate the invasive ability of cancer cells using a three dimensional invasion assay. This is accomplished by first culturing cancer cells in a basement membrane matrix. The cellular invasion of the matrix is then observed for five or more days via light microscopy.
In the final step, the cells are labeled with fluorescent antibodies for localization of the proteins of interest. Ultimately, the rate of cancer cell invasion and the resulting changes in protein expression can be analyzed by immunofluorescent microscopy. The main advantage of this technique over existing techniques such as the Transwell chamber invasion assay, is that the 3D major gel invasion assay better mimics the in vivo environment that cancer solves growing.
The implication of this technique is towards cancer therapy, where new proteins can be identified in study, which are involved in cancer invasion and metastasis. Before beginning the culture procedure, place the basement membrane matrix a P 200 pipette and pipette tips on ice overnight at four degrees Celsius. The next day, use the tip of the ice cold 200 microliter pipette to spread 50 microliters of the matrix in a spiral pattern over the bottom of a confocal number one glass bottom dish.
Then place the dish in a cell culture incubator at 37 degrees Celsius with 5%carbon dioxide for at least 30 minutes while the matrix is undergoing solidification trypsin eyes, a 70 to 80%confluent 100 millimeter plate of cells once the cells have begun to detach. Inactivate the trypsin with 10 milliliters of medium, and then transfer the cell suspension into a 15 milliliter conical tube. Centrifuge the cells for three minutes at 100 Gs and four degree Celsius while the cells are spinning down.
Aliquot 50 microliters of matrix into one one milliliter micro centrifuge tube per dish of basement membrane matrix, and place the tubes on ice when the cells have finished spinning. Aspirate the supernatant without disturbing the pellet, and then resus. Suspend the cells in one milliliter of media and count them next.
Transfer 2.5 times 10 to the fourth cells into a new micro centrifuge tube topping off the cell suspension with media to a final volume of 50 microliters. Then add the 50 microliters of ice cold basement membrane matrix to the cells at a one-to-one ratio for a final volume of 100 microliters. Gently plate the matrix to cell mixture onto the solidified basement membrane matrix, and allow the cells to become embedded in the matrix in the cell culture incubator.
After 30 minutes, cover the matrix with two milliliters of media and place the dish back into the incubator, changing the media every day for the duration of the experiment, use the 10 x objective of a light microscope once every day for the duration of the experiment to take 20 differential interference. Contrast images of the colonies suspended in the basement membrane matrix. Analyze the images blindly to determine the cell colony stellate formation.
A colony is deemed to be stellate if one or more projections from the steroid of cells are observed. To examine the morphogenic features of the 3D colonies, remove the cells from the incubator and place them on a tray of ice. Aspirate the media and then wash the colony three times with two milliliters of cold PBS.
After the last wash, fix the cells in two milliliters of 20%acetone, 80%methanol solution for 20 minutes at four degrees Celsius, and then bring the dishes back to room temperature. Once the basement membrane matrix is at room temperature, aspirate the fixative and wash the plate three times with PBS as just demonstrated. After the final wash, block any non-specific binding on the cells with two milliliters of 3%BSA for at least 30 minutes at room temperature.
Then incubate the cells in the primary antibodies of interest diluted in 3%BSA at room temperature. After an hour, remove the primary antibody solution after washing away the unbound primary antibody three times with PBS, add the secondary antibody dissolved in 3%BSA at the appropriate dilution and incubate the cells for one hour at room temperature in the dark. After washing away the unbound secondary antibody with PBS stain the nuclei of the cells in two milliliters of hext 3, 3, 2, 5 8 in PBS for five minutes in the dark.
Then wash the unbound hext from the cells five times with PBS. After the fifth wash, add mounting medium directly onto the matrix to cell mixture and carefully cover the mounting medium with a glass cover slip to prevent any disruptions to the integrity of the basement membrane matrix to cell mixture. Dry the dish overnight at room temperature, then acquire images with a fluorescent microscope at the appropriate laser wavelengths for the antibodies used in these images.
MDA MB 2 3 1 cells invading a 3D matrix are illustrated. The cells embedded into the matrix on day one and started forming invasive stellate structures by day three. By day five, a complete invasion of the matrix could be observed.
The number of stellate colonies formed was then counted and expressed as a percentage of the total number of the invasive and non-invasive colonies per dish. Additionally, since the measurements were completed daily for five days, the rate of invasion could also be evaluated in these immunofluorescent images. Representative invasive stellate colonies of MDA MB 2 3 1 cells displaying a loss of membrane integrity and diffuse localization of the basement membrane protein laminin five are shown in stark contrast to what is observed in the MDA MB 2 31 breast cancer cells.
The laminin five was localized to an intact basement membrane layer enclosing the memory asinine of the untreated non-malignant MCF 10 A cells. Following this procedure, co-culture with stromal cells can be performed to answer additional questions such as do cancer cells crosstalk with stromal cells in the microenvironment after its development. This technique paved way for researchers in the field of oncology for exploring cancer invasion and migration, both of which are required for metastatic spread of cancer cells.
