Tumor cells try to spread throughout the body. In order to do so, these cells need Rho and ROCK or Rac to signal their movement. There are at least two types of morphology (shapes/forms) that need these signals for cell movement. One type is the elongated morphology, which needs to use Rac for signalling, and pericellular proteolysis (an enzyme) for movement. The other type is the rounded (amoeboid) morphology, which uses RhoA and ROCK signalling. Initially, tumor cells were known to take the elongated form. It is now known that the rounded shape also exists and possibly other morphologies as well. This research is focused on tumor cells that have rounded morphology, elongated morphology, and those that can utilize both forms.
There has been research in attempts to inhibit, or block, these signals in order to stop tumor cells from spreading. However, this may cause a problem for other cells that may need to move to certain places of the body to function. Researchers have experimented with the use of inhibitors, such as Y27632, C3, and a combination of Y27632 and C3, on different types of tumor cells to see what percentage of cells still invaded to a certain distance. This information is compared to the percentage of untreated cells that invaded the same set distance. The effectiveness of these inhibitors are then related to the type of morphology (elongated or rounded or both) to see if certain types of inhibitors work better in a type of morphology. More research has been conducted to find the percentage of different tumor cells that have invaded three different depths. This is also used to analyze the effectiveness of the inhibitors on the varying types of morphologies.
The basic procedure for this type of experiment is discussed below with an overview covering the main points of each of the steps.
Step 1: See how far tumor cells are able to move when Rho and ROCK are inhibited.
For this step, lines of tumor cells were placed in Matrigel. Matrigel is basically a layer of material that contains the same things as normal cell membranes (like collagen) as well as growth factors.
How deep into the layer of Matrigel do each of the cells go?
Step 2: See how the different lines of cells change shape as they move.
As the different cells moved through the Matrigel layer, the researchers observed that the different cell lines had different shapes. To do this, they placed four different kinds of tumor cells on a thick layer of Matrigel. After 24-36 hours, the cells were imaged using confocal microscopy, and their 3D shapes, or morphologies, were reconstructed.
Various cells types
Step 3: Find out if the different cell shapes that were observed in Matrigel are present in live cells.
To do this, two different kinds of cells, BE and A375m2, were labeled with a green fluorescent protein marker. Pools of these cells were injected into mice and grown as subcutaneous (under the skin) tumors.
Once the tumors reached 1 cm in diameter,...