Current methods of isolating CTCs are based on either specific cell surface markers, such as EpCAM (which limits the range of cells one can isolate), on size, density or on screening massive numbers of cells. What, if anything, is missing? What would be the "ideal" CTC isolation instrument?
I think, isolation is the wrong method to deal with CTC. Staining is the answer. You wouldn't isolate cells for a blood picture, too. I was at MD Anderson, working on an isolation method and found out that too many cells get lost by isolation. Now, the maintrac system is much more sensitive than isolation methods. we already did a comparison with Cellsearch, showing the sensitivity cap.
On behalf of Prof Katharina Pachmann
Peter Pachmann
If you want to have more information on CTC staining, please have a look at the file attached.
Indeed a good question. Isolation or staining (Dr. Pachmann) is usually a very one-dimentional view of the cells. If epithelial cells undergo EMT- they are no longer detected by 80% of the methods out there. Size-based enrichment can miss the smaller cells, and depletion methods (usually CD45- cells) are problematic since CTCs have beed identified that do express CD45. Also culturing methods can be problematic as far as purity and of course depend on cell adhesion properties.
So, what method is open enough and yet sensitive enough?
I think that we must revert to PDX-derived CTCs. This method in which humanized mice (patient-derived xenograft models) enable derivation of MNCs from the blood and then unbiased mouse cell depletion, in effect isolating only tumor-derived human cells. Each cell is a potential CTC and can be further characterized downstream to ascertain that it isnt and endothelial cell or a hTIL.
Naturally, you could also monitor changes in CTC populations in response to medication etc.
The downside is that this cannot be utilized for patient-diagnosis directly, but rather a by-proxy approach to the patient's tumor and CTCs.
The question is, what you want to do with the results from CTC analysis:
1. If you analyze the large cells they may comprise the more differentiated cells which probably still proliferate but are not metastatic.
2. If you select for EpCAM expression by any antibody dependent method, you might miss the cells that have undergone EMT with low expression and thus do not bind enough antibody.
3. With respect to density little is known about the impact of density on cell properties
4. It is well known, that nor all CTCs are metastatic, so if you just want to analyze the global properties of the cells that have left the tumor you can do massive screening. However, it may be a minimal number of these cells that a capable of forming metastases and you may miss them.
5. A prerequisite for metastasis formation is the ability of the cells to self-renew. So PDX may be a good system because you will only select cells that do grow in the mouse system. However, as far as I know, although this has been successful for tumor tissue derived cells it has, so far not been very successful for circulating tumor cells and taken up to several months to have tumors grow. This may work for research, but would be way too slow for diagnostic purposes.
6. The question is, to what extent CTCs represent the tumor for diagnostic purposes.
7. We have developed an approach, where we culture the cells for up to three weeks and look for sphere forming cells (not adherent!!). These cells, we think are the most promising ones to tell us about the metastatic properties of the patient’s circulating tumor cells
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