I performed a cell invasion assay to asses invasive capability of two different tumor population by using matrigel coated inserts. After experiment, I found that:
No. of stained cells on the bottom of the membrane of population A (around 510 cells) is higher than the population B (around 440 cells), about 1.2 fold.
However, for population B, there are so many cells started to grow on the bottom of carrier plate (after going through the membrane and these cells eventually fall to the bottom of carrier plate). Around 60 cells found for population B while there is only 1 or 2 or even no cells found for population A, that is like 30 fold or more.
I repeated a few times, found similar results. It seems that population B are more easily de-attaching from the membrane than population A.
So which result I should use, the no. of cells stained or focus on the falling off cells? If the tumor cells are more likely to fall off from the membrane, does it mean the cells are less likely to attach to the extracellular matrix and easier to enter into a new matrix environment? Any biological significance for this? like invasive tumor cells go through the blood vessel and fall into the blood, instead of stick on the membrane of blood vessel?
Thanks for your insight!
Hi Xi. For quantifying the invasion assay, if I was a reviewer, I'd suggest you use less cells so that much fewer detach and migrate to the bottom of the well (sounds like you may be using a saturating number of cells). You may find a big difference between how many cells cross the matrigel in such a case. If they continue to pool at the bottom of the well, maybe they have less adhesion molecules, more proteases, etc. This woudl be interesting and maybe worthy of following up for biological significance. However, until you do the assay with less cells (maybe three different dilutions), it seems you won't know if it is due to the number of cells crossing the membrane (population B having more cells cross the membrane and therefore require more to slough off in order to make room for more to cross) or whether there actually is some other reason for their detachment from the bottom of the transwell insert. Your results sound intriguing.
Hi Xi, I echo Erik's comments about the number of cells you are using for your assay. If you are using epithelial-type cancer cells, they migrate on ECM but should not drop to the bottom of the well unless they are crowded.
That being said, there are epithelial circulating tumor cells (CTCs) found in cancer patients. These cells are shed off from the primary tumor(s) into the circulation.
There might be a paper in this for you if you can estimate at what point the epithelial cells are being "shed" from the underside of the chamber to reside at the bottom of the well.
How much percent of matrigel used. Make caoting less diluted, hard matrigel.
also 0.5% media used at top (insert) and 10% at bottom in well.
Do imaging of atleast 10 field and divide in quadrant for count.
Thanks
I hope it will help.
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