The overall goal is to determine the effect of experimental alterations on the cell extension repertoire and how it relates to cell adhesion function in cancer. This method can help identify key roles of the cell membrane extension repertoire in modulating cell behavior such as cell adhesion, motility and invasion. The main advantage of this technique is that it is relatively simple to perform and can be easily adapted to various experimental conditions.
The implications of this technique extend to our therapy or diagnosis of locally advanced or metastatic cancers because it helps to accurately quantify changes in cell extension repertoire associated with alterations in tumor cell in adhesive and invasive capacities. It can also be applied to other systems such as ion channel regulation which is known to play an important role in cell extension formation and normal physiological processes. Visual demonstration and recording of this method is critical as the counting of cell extension steps are difficult to learn because of the subtleties of their appearance at times.
A day before imaging, count SW 13, SW Neo and SW DKK3 cells grown in a 37 degrees Celsius and five percent carbon dioxide incubator using a hemocytometer. Place sterile glass coverslips into individual wells of three six well plates. Use separate plate for each cell line and plate the three cell lines at a density of 5, 000 cells per well into these six well plates.
Place the cells in an incubator at 37 degrees Celsius and five percent carbon dioxide overnight. The next day, from each well aspirate the growth medium. Then, wash cells in one milliliter of warm PBS.
Subsequently, add one milliliter of 3.7 percent formaldehyde into each well to fix the cells. Leave the cells in the fixative for 10 minutes. After fixation is done, aspirate the formaldehyde.
Add one milliliter of 0.05 percent crystal violet into each well to stain the cells for 30 minutes. Next, clear the excess stain by washing cells with one milliliter of deionized water per well three times. Next, place a drop of clear mounting reagent on a labeled glass slide.
Then, using fine tipped forceps, gently retrieve the coverslips and place them face down over the mounting reagents. After mounting all the stained coverslips on slides, under a light microscope choose 20 random views of non overlapping cells from each coverslip. Take photo micrographs of 400 strength magnification for each field of view.
Then, observe and count the number of extension types in each of the 20 representative cells. Use the definitions and the text protocol to guide your classification of the membrane extension as filopodia, lobopodia or lamellipodia. When first counting cell extension types, it may be helpful to keep a reference image available for review.
If possible, counting in a blinded fashion with respect to their experimental samples will provide additional objectivity. Determine the average number of extensions of each type per cell in each of the three cell lines examined. Use one way anova to test of the difference in the average percentage of cell membrane extensions among the different cell types is statistically significant.
A day before the assay, using a hemocytometer, count SW 13, SW Neo and SW DKK3 cells. Plate these cells at density of 100, 000 cells per well into six well plates using one plate for each of the six designated time points that will be tested. Place the cells in an incubator at 37 degrees Celsius and five percent carbon dioxide overnight.
The next day, remove the plate from the incubator and wash cells once with warm PBS. Then, add 0.5 milliliter of single strength non enzymatic cell dissociation solution into each well. Gently swirl the plates to even spread the added solution.
Incubate the plates with the solution for defined times. Aspirate the designated plate at the end of each time point. Soon after each aspiration, wash the cells with one milliliter of warm PBS.
Then gently tap the plate to detach loosely attached cells. Repeat the wash along with the intermittent tapping two more times. Finally, aspirate any remaining PBS and fix the cells that have remained in the plate with one milliliter of 3.7 percent formaldehyde.
After fixation is done, aspirate the formaldehyde. Then, add one milliliter of 0.05 percent crystal violet into each well to stain the cells. Once the staining is complete, clear the excess stain by washing cells with one milliliter of deionized water per well three times.
Using a light microscope at 100 strength magnification, count the remaining attached cells in each well for each plate. Then determine the percentage of cells that have remained detached during the different time points for the three cell lines that were tested. Using a two way anova, test of the differences and the rates of cell detachment between the three cell lines are statistically significant.
Crystal violet stained control SW 13 and SW Neo cells formed mostly filopodia and lamellipodia. In contrast, DKK3 expressing cells formed more lobopodia with near complete absence of lamellipodia and very few filopodia. Also, note the differences in polarity between the DKK3 expressing cells and the control cell lines.
The graph shows the proportion of different cell extensions in the three cell lines tested. Note, the significantly higher proportion of lobopodia in DKK3 expressing cells. Line graphs show the percentage of cells that remained attached to the plate after treatment with the dissociation solution for the time points indicated in the X axis.
At all time points up to 10 minutes, DKK3 expressing cells showed significantly higher cell attachment strength compared to the control cells. Once mastered, this technique can be done in one to two day time period depending on your experimental set up if it is performed properly. While performing this procedure it is important to remember to assess how changes in cell extension morphology affect adhesive capacity.
After watching this video, you should have a good understanding of how to accurately quantify cell extensions in relation to changes in cancer cell adhesion characteristics. Good luck and thanks for watching.
