I have recently establish a cancer cell line, which is now passage 12, and the fibroblast has been removed. The question is whether It's necessary to pick the monoclone of the cancer cells for further use. It seems that picking a monoclone is not always mentioned in the published articles.
That will largely depend on how your current population behaves. Maybe your selection has already selected for the best growing clone.
1. Characterize your cell population - at low passage number - for cell markers, biochemical properties, immunohistochemistry, DNA fingerprinting etc.. See what percent of the population appears to be "homogenous". If you are convinced it is >85% then maybe you don't need to clone. Also, see how close they represent the primary tumor.
2. If you see population drift - change it phenotype over time - then you have to ask yourself (a). Do I let the population grow over several generations till it stabilizes, or (b). Do I use the current population but limit the number of generations I grow them (200 clones and propogate all of them and treat them as different clone numbers. Then you could decide which clone best represents the original cancer. This may require large number of clones or just a few clones, depending on what you have now.
Hope this helps ! Good luck.
That will largely depend on how your current population behaves. Maybe your selection has already selected for the best growing clone.
1. Characterize your cell population - at low passage number - for cell markers, biochemical properties, immunohistochemistry, DNA fingerprinting etc.. See what percent of the population appears to be "homogenous". If you are convinced it is >85% then maybe you don't need to clone. Also, see how close they represent the primary tumor.
2. If you see population drift - change it phenotype over time - then you have to ask yourself (a). Do I let the population grow over several generations till it stabilizes, or (b). Do I use the current population but limit the number of generations I grow them (200 clones and propogate all of them and treat them as different clone numbers. Then you could decide which clone best represents the original cancer. This may require large number of clones or just a few clones, depending on what you have now.
Hope this helps ! Good luck.
As Prem noted above, it is likely that your cell population is a collection of few clonal population. Therefore, you need to use certain criteria to classify the population and then clone the desirable population. Most cancer cell lines exhibit a heterogeneity in due course. Therefore, you need to characterize a low passage population to move forward.
To make sure the clonal status, the most important information is the karyotype. This is especially important for cancer cell lines where most of them are highly unstable. According to our systematic analyses, there are two phases of cancer evolution: stochastic punctuated phase and stepwise Darwinian phase. In punctuate phase, you cannot clonal the population at the karyotype level, even though by they share the same DNA fingerprinting profile. Since the karyotype represents the “system inheritance”, which is more important than individual gene defined “parts inheritance”, it is essential to monitor the genome level clonally.
Following papers have discussed this issue in details.
Horne SD, Pollick SA, Heng HH. (2015). Evolutionary mechanism unifies the hallmarks of cancer. Int J Cancer, 136.9: 2012-2021. PubMed
Abdallah BY, Horne SD, Stevens JB, Liu G, Ying AY, Vanderhyden B, Krawetz SA, Gorelick R, Heng HH. (2013). Single cell heterogeneity: Why unstable genomes are incompatible with average profiles. Cell Cycle, 12.23: 3640-3649. PubMed
Heng HH, Stevens JB, Liu G, Bremer SW, Ye KJ, Reddy PV, Wu GS, Wang YA, Tainsky MA, Ye CJ. (2006). Stochastic cancer progression driven by non-clonal chromosome aberrations. J Cell Physiol, 208.2: 461-472. PubMed
For more info, please check out the book “Debating Cancer”
Debating Cancer: The paradox of cancer research
http://www.worldscientific.com/worldscibooks/10.1142/8879
As Prem pointed out, the behaviour of your current population is cardinal in making this decision, I would suggest you characterize a low passage population and take decision depending on the results you get
As mentioned by Divaker, p53 is mutated in almost all human cancers including colorectal cancer where it (p53) has been reported to be mutated in >50% of cases. May be because of its central role in several cellular transductions, Remember that the wild type p53 protein is highly unstable due to MDM2-mediated ubiquitinization but with the mutant type, p53-MDM2 Interaction is lost which ultimately gives rise to increased expression of dysfunctional p53 protein. However, you can try to assess the phosphorylated p53 and compare with your control to see what you get. You can repeat same using several other cancer lines.
Hi
It may not be possible to grow out single cell clones for some cancers. Even if you do how will you know it represents the initial tumour and not just one clone that grew well in the culture conditions used unless you can keep some of the biopsy to use as a reference for testing and this is not always possible.
If you are making a cell line and using it for experiments, make sure you keep track of the passage number and use only low passage number cells going back to your original freezes to maintain work with low passage number lines. Often a chromosome count or ploidy will give an indication of instability. Where the many chromosomal changes different clones are likely to have advantages in tissue culture and may out grow the initial population. We have seen this happen with Merkel Cell Carcinoma lines.
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