Dear Zubair,

I handled several cultures called A549 - two originals and three multidrug-resistant derivatives, and even original lines looked very different from each other. If you've got a new stock, cultivated them simultaneously with other cultures and see signal in other cultures unlikely it can be micoplasma alterations. What I would try is to feed them better for 2-3 passages. F12 is very poor medium, practically it is almost buffer saline. You can add MEM (minimal essential aminoacids), NEAA (non-essential aminoacids) solutions, more glutamine, serum extract for hybridomas survival (if you have one), more FCS up to 15 percent or another better FCS. I've never seen cell culture suffering from exccess of aminoacids. Contrarywise, if cell culture is recovering poorly after it was thawed in thin medium you will not have declared levels of expression for all proteins, for E-cadherin also.

If you add some insulin (not much, 2-3 nM instead of 10) proliferation of culture will be reduced and all proteins expression increases significantly after 3-6 weeks of cultivation. Only I don't know whether it is good to alter the culture. We take these cancer cells in vitro models to simulate original tumor, after all.

A549 which were delivered to me once with recommendation to use DMEM for cultivation were approximately two times bigger than the other culture from ATCC bank.

It is also possible to try other lung carcinoma cell lines, several are available. When we were developing our test sistem for breast cancer I had collected 14 cultures and have chosen 4 only from them which worked perfectly as controls for my target gene positive-negative expression. In any case to spend the whole summer or longer for fishing expression is too long!

http://www.lgcstandards-atcc.org/Search_Results.aspx?dsNav=Ntk:PrimarySearch|lung+carcinoma|3|,Ny:True,Ro:0,N:1000552&searchTerms=lung+carcinoma&redir=1

I would also consider the possibility that the cells used in some of the previous reports were not A549. Cell cross-contamination and misidentification is still a common problem (see attached). If you purchased your cells from a reliable supplier, such as ATCC, which verifies the identity of cell lines, I would trust them more than published reports.

sorry, missed the link in my first answer….

http://discovermagazine.com/2014/nov/20-trial-and-error

In addition to cells identity, the passage number and the age of cancerous cells could be a matter. It has been clarified that during the epithelial to mesenchymal transition (EMT), cancerous cells loss their content of E-cadherin. I think along with what explained in the prior cherished answers, your cells may have experienced the EMT.

I have no experiance with this particular cell line but "transdifferentiation" of cell lines with passage number and change of culture condition is well known (middle of the 70ies, as a student I had to report on one such paper) and there are thousands of papers on this topic since then.

Beside the above mentioned contamination of your cell line with Hela local phenomena also influence expression of particular gene.

Also be aware that in other labs people also passage their cells. So after 10 passages "your" cell line and theirs may be quite different.

I was actually using the cell line to measure if a drug I'm working with would modulate EMT. The cells, even at a very early passage (and prior to treatment with TGF-Beta or the drug), do not express E-cadherin and instead express vimentin, which suggests that they've drifted genetically and are beginning to undergo EMT (even as early as passage 1). If it's not genetic drift, then I'm not sure what else would cause induction of EMT.

Dear Zubair and Yuri,

There are many more dirty secrets in cancer research, and they are not so little like in vitro culture contamination or its genetic drift. Rivalry for fundamental cancer biology funds is a triffle compared to those "secrets" concealed in pre-clinical GLP assays that bring forvard up to 95-98 percent of not-working at the II-III phases medicines. The wast majority of specialists not working with animals themselves cannot even imagine where controls should have been put  in vivo assays with many respectful-IPs publications.

Protein expression level can alter 2-3 times in many cancer cell cultures with stimulation of proliferation or differentiation within 3 weeks easily, and I think this is yet without genetic drift.

If you ask GLP staff whether they are inoculating in vivo human tumor line (it is 3rd of further passages in vivo) in nude mice anti-tumor agents testing - 85 percent won't understand the question, another 10 percent will assure you that yes, of course, 3rd passage... And in reality as there were in vitro cultures - here we come to 95 percent that I mentioned in the first paragraph.

The positive thing is if you question your own research instead of repeating mistakes of measurements for years like too many people do, you have room to move forward. However many questions are not improving career in research...

Greetings 

Thanks, Elena! You are right about the consequences of not keeping one's mouth shut, I also have learned them, but not asking questions can also be ruinous or outright deadly, as the recent story about the acid washed stem cells demonstrated.

Thanks for explaining the situation in preclinical trials. Why should the 3d passage be used? Thanks. Yuri

Aren't all living things subject to the driving forces of evolution, i.e., genetic drift, random mutations, natural selections etc?

Zubair, I work with A549 cells quite often and several reports (EMT literature) has identified them as "semi-mesenchymal". In my hands (purchased from ATCC) they express very little E-cadherin and also express a variety of mesenchymal markers (vimentin/fibronectin). There is a definite possibility of genetic drift although I tend to think that this subset of NSCLC is rather heterogeneous as there is certainly a debate over the 'status' of this cell model. Hope this helps in any way. ~Andrew

Did you resolve this?

As one person said here - it is entirely possible other publications were produced using misidentified A549.

You might find that even publications using authentic cells will not be consistent on (a) the cultivation medium and (b) the state of differentiation of the cells. Remember this is a cancer derived cell line with all sorts of weird stem cell like characteristics and its culture environment will have a significant effect on the gene expression.

In my experience to get A549 to perform anything like an epithelium you have to allow these cells to differentiate in a complex medium (like F12 or F12 K) with 10% FBS for 20-25 days (long term culture at confluence with regular medium changes). The problem with this cell line is that there are probably dozens of clones of the cells at various passages being used by labs all over the world. And it it a popular cell line with a finite resource of low passage material so even cells from trusted suppliers will eventually be supplied at increasing passages.

Jim