There are a number of reports for and against isolating CSCs from established cancer cell lines. One of the best work from Prof. Max Wicha lab, where they isolated CSCs from 33 breast cancer cell lines is attached. However, a number of my colleagues in North America, particularly in pharmaceutical industry, prefer CSCs from primary tumors for generating reliable "Hit-Lead Molecules" for further preclinical testing in appropriate animal models? Some of the prestigious journals' reviewers quite often questions the authenticity of CSCs isolated from cell lines? On the other hand, for obvious reasons, getting primary tumors and isolating CSCs are laborious, time consuming and resource intensive? What are my best options for studying the role of breast CSCs in metastasis?
HI Kulvinder there are probably a number of reasons why the pharmacological industry prefer to obtain CSCs from the primary tumour as opposed to cell lines.
1. Often cancer cell lines are produced from caner metastases (secondaries) and there has been some discussion as to whether they are the same as the primary tumour with regards treatment/drugs (as often the cells undergo EMT to migrate to the secondary site or express different proteins to the primary tumour)
2. The second reason, and probably the greatest reason, is that all cells that have been grown or expanded in vitro are under a "selective pressure" caused by growing in 2D/on plastic/under mono-culture conditions etc (for example cells that gain mutations in vitro that make them grow faster will come to dominate the culture in time, cells grown at too low a density may select for cells capable of density independent growth etc). One example of this is HeLa cells. These have been around for many years and during this time have been expanded by an extraordinary amount. The genomes of these cells are now completely scrambled (I have even read that some people now want to re-classify them as "non-human"). These cells probably bear very little resemblance to the original cervical cancer that killed this poor woman, hence any drug that works on these cells may have no effect or a different effect on a "normal" cervical cancer.
Unfortunately a lot of cancers that will grow in a human are difficult to cultivate in the lab - for example I have had problems growing triple-negative breast cancer lines in vitro - despite the fact that these are some of the most aggressive cancers in humans. There are probably several reasons for this - for example I mentioned the term "mono-culture" above. This refers to the fact that cells in the lab are generally grown in isolation from other cells (as a single cell type) In the body a lot of cancers are fed, and made more aggressive by, normal surrounding cell types (i.e. CAFs). Not to mention the fact that cells in culture do not have access to a vascularised environment.
Although I do not work in this field if I did want to study CSCs from breast cancers (especially in terms of secondary cancer formation) I would either look at the circulating Tumour Cells (CTC) in the blood (which can be enriched in a number of ways for study) and see f I can detect CSC from these cells.
Alternatively you might want to look at blood drawn from the sentinel lymph node of breast cancer patients as these can often be enriched for cancer cells/CTC.
Both these procedures involve a needle biopsy which is less invasive to the patient than a tissue biopsy. Also as these cells are already in the circulation these are probably the best candidates to look at for the formation of secondary metastases.
I have worked with various cell lines and primary cells for many years and one of the most important things to know about these cells is that they are not 100% representative of the original tissue/cancer. Growing cells in culture, in 2D is abnormal so at best cell lines are a "model" of the original tissue so experiments in the lab can only answer biological questions in a limited way.
I hope this helps!
Gary
HI Kulvinder there are probably a number of reasons why the pharmacological industry prefer to obtain CSCs from the primary tumour as opposed to cell lines.
1. Often cancer cell lines are produced from caner metastases (secondaries) and there has been some discussion as to whether they are the same as the primary tumour with regards treatment/drugs (as often the cells undergo EMT to migrate to the secondary site or express different proteins to the primary tumour)
2. The second reason, and probably the greatest reason, is that all cells that have been grown or expanded in vitro are under a "selective pressure" caused by growing in 2D/on plastic/under mono-culture conditions etc (for example cells that gain mutations in vitro that make them grow faster will come to dominate the culture in time, cells grown at too low a density may select for cells capable of density independent growth etc). One example of this is HeLa cells. These have been around for many years and during this time have been expanded by an extraordinary amount. The genomes of these cells are now completely scrambled (I have even read that some people now want to re-classify them as "non-human"). These cells probably bear very little resemblance to the original cervical cancer that killed this poor woman, hence any drug that works on these cells may have no effect or a different effect on a "normal" cervical cancer.
Unfortunately a lot of cancers that will grow in a human are difficult to cultivate in the lab - for example I have had problems growing triple-negative breast cancer lines in vitro - despite the fact that these are some of the most aggressive cancers in humans. There are probably several reasons for this - for example I mentioned the term "mono-culture" above. This refers to the fact that cells in the lab are generally grown in isolation from other cells (as a single cell type) In the body a lot of cancers are fed, and made more aggressive by, normal surrounding cell types (i.e. CAFs). Not to mention the fact that cells in culture do not have access to a vascularised environment.
Although I do not work in this field if I did want to study CSCs from breast cancers (especially in terms of secondary cancer formation) I would either look at the circulating Tumour Cells (CTC) in the blood (which can be enriched in a number of ways for study) and see f I can detect CSC from these cells.
Alternatively you might want to look at blood drawn from the sentinel lymph node of breast cancer patients as these can often be enriched for cancer cells/CTC.
Both these procedures involve a needle biopsy which is less invasive to the patient than a tissue biopsy. Also as these cells are already in the circulation these are probably the best candidates to look at for the formation of secondary metastases.
I have worked with various cell lines and primary cells for many years and one of the most important things to know about these cells is that they are not 100% representative of the original tissue/cancer. Growing cells in culture, in 2D is abnormal so at best cell lines are a "model" of the original tissue so experiments in the lab can only answer biological questions in a limited way.
I hope this helps!
Gary
Hello Kulvinder, hi everybody,
Really great comments of Gary, a short but pretty comprehensive review of the 'cons' you will encounter when trying the in vitro approach. I just like to add or emphasize a few things in this debate.
For 'spontaneous' metastasis specific niches are presumably required at the eventual metastatic sites.There is good evidence that circulating exosomes e.g. from tumor cells are involved in 'conditioning'. Certainly, many migrating tumor cells undergo EMT, though this can be reversed in CTCs (by MET) at the metastatic site, generating tumors like the initial phenotype.
By definition, stem cells are normally slow cycling. When establishing tumor cell lines CSCs may not at all or only poorly grow in culture (getting lost eventually) or loose their properties/stemness. In cases it may be possible to create in vivo like conditions by using complex 3D/ organotypic cocultures as done previously with skin or breast cancer cells for example - not an easy task.
Concerning the microenvironment, apart from a huge number of other cell types (e.g. TAFs/CAFs, perivascular and vascular or inflammatory cells) also the nature or changes of the ECM (extracellular matrix) play an important role.
As mentioned above (HeLa cells) genetic instability is a major problem. Though tumor cells are prone to that in vivo, this is enhanced in vitro in addition to a quite different kind of selection pressure.
Finally, I also agree completely that cell lines may provide useful models for studying a wide range of mechanisms such as signaling cascades in cancer research, but this will never truely reflect tumor cell biology including the CSC issue.
Isolating CTCs from patients makes a lot of sense to get the relevant part of the tumor cell population, but to get sufficient quantities and the right growth conditions may be extremely difficult.
Good luck!
Dirk
Thanks very much-Gary and Dirk. I am indebted to both of you for extremely valuable comments/suggestions, appreciate "additional insights and challenges" to this new & evolving field of CSCs-Ex-vivo and in vitro biology, and how to leverage this knowledge for discovering "personalized medicines" and optimizing new drug discovery research screening parameters......Best regards.
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