we analyse the presence of CSCs by proper CDs and the use of Flow cytometry
why we need to confirm the presence of CSCs by colonic assay or another one
The CDs and their combinations help to enrich for CSCs, but DO NOT identify them; i.e., most of the selected cells are not CSCs, but a few may be. It is critical to recognize that when cells are injected in a suspension, the number of cell injected follow a Poisson distribution. So, when a single CSC is injected, 37% will recieve no cell and hence develop no cancer, while 63% will receive greater than or equal to 1 CSC and so develop a cancer. Thus, when breast cancer cells were sorted by flowcytometery (or magnetic bead separations) 1 - 2% of the initial tumor cells were CD24 neg CD44 pos and CD326 (ESA or EpCam) positive. However, injection of 100 such cells into the "humanized" breast fat pads of NOD/SCID (now would use NSG) mice, yielding about 63% of mice getting the tumor, showed that only 1% of the cells in this 2% of the tumor were CSCs, i.e., 1-2 per 10,000 of the original tumor cells were CSCs.
The point is that a functional assay is needed to measure the number of CSCs. One such option is our patented Hybrid Spheroid Assay.
I agree with Dr. Lange, you need to demonstrate functionality. As for marker selection, we have had very good correlation when using SmartFlare to sort Nanog/Sox2 positive cells from the fresh tumor. See that paper in my profile. But you still need to show function!
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