Hi Vimalathithan, 

MDA-MB-468 cells express high levels of EGFR and EGF is a well-characterised inducer of EMT in this cell line. I suggest you refer to the following references for information on developing an in vitro model of EGF-induced EMT using 468 cells:

http://www.ncbi.nlm.nih.gov/pubmed/23686305 

http://www.ncbi.nlm.nih.gov/pubmed/17909010

Depending on the marker(s) you are interested in, you can see induction (at the mRNA level) by 6 h e.g. Twist, Snail, vimentin... and by 24 h at the protein level for vimentin (possibly earlier?). For these time points you wouldn't need to change media. 

I'm not experienced in using TGFb, but from what I understand it does take longer to induce phenotypic changes compared to treating with EGF.

Hope this helps.

Teneale

Thank you very much Teneale. Before moving into studying markers, I want to make sure that EMT has occurred by observing the morphology. To observe morphological change I think it would take 4+ days, so can I replenish media for every 2 days with the respective growth factor? And also what would be the ideal seeding density in 96 well plate to observe morphological change?

Hi Vimalathithan,

(Please note: the below advice refers to treating with EGF only)

As a basic timeline I would suggest: Day 1 – plate cells and leave 24 h; Day 2 – serum deprive (i.e. 0.5% FBS) 24 h; Day 3 – treat with EGF 24 h (refer to papers for conc (e.g. 50 ng/mL) or perform a concentration response study looking at induction of a specific maker of EMT i.e. vimentin); Day 4 – image or isolate RNA/protein. For this timeline I would suggest starting with 2.0x10^4 cells/well (96-well plate) – but encourage you to optimize plating density as cells may grow differently depending on your cell culture conditions etc.

You should see changes in morphology by 24 h, but suggest looking at 24, 48 and 72 h post EGF-stimulation (you will need to optimize plating density for later time points). For 24 and 48 h you won’t need to change media. I’ve never treated for 72 h, so you could have wells where you don’t change media, and others where you change after 48 h (though I’m not 100% sure this is necessary) and decide which is best. Note, you won’t see 100% of your cells transition to a mesenchymal morphology at one time (at least I haven’t observed this), likely as cells will be transitioning at different times between an epithelial and mesenchymal state. Also, I recommend looking at classic EMT marker mRNA and/or protein levels in addition to morphological changes to fully validate induction of an EMT in the 468 cells (you can refer to the papers I mentioned in the previous post for more details).

Hope this helps.

Teneale

Thanks a lot Teneale for your valuable suggestion!!!

Hello Teneale,

After 24 hrs treatment with EGF on MDA MB 468, all the cells got separated from each other. They look like as if they were trypsinised. But only few cells turned to spindle shape after 72 hrs. Most of the cells remain separated and circular. Is this the usual case? I haven't tried the markers yet. And also is there any way quantify EMT?

 Hi Vimalathithan, 

After treating with EGF you should notice a more rounded morphology and loss of cell-cell adhesions; however, I don't recall my cells ever looking like they had been 'trypsinised'. Maybe you need to optimise the EGF concentration that you are using? It's correct that you wont see all of your cells become mesenchymal, and I think it's normal that some cells will still look rounded after 72 h (a result of epithelial-mesenchymal plasticity?). I always serum deprive (i.e. 0.5% FBS) before treating with EGF, and suggest treating with EGF in serum reduced media, in case you haven't tried this (minimise interference from GFs etc... present in your serum).

You can look at markers of EMT to get an idea of whether induction has taken place e.g. vimentin, Twist, Snail, which should increase and epithelial markers e.g. E-cadherin, which should decrease. If possible, you could look at vimentin and E-cadherin using IF microscopy and use MCF7 (low/no vim and high E-cad) and MDA-MB-231 (low/no E-cad and high vim) breast cancer cell lines as reference/controls. (Note: there are a number of ways to validate your model and confirm induction of an EMT, which you can find in the literature. Above are just some suggestions).

Teneale

Hi Teneale,

Thank you very much!!

I've treated with EGF in serum media containing 0.5% FBS. Is that what you meant by serum reduced media?

Vimal

Yes, that's correct (as opposed to serum-deprived media, which would be 0% FBS).

Hi Teneale,

I maintain MDA-MB-468 cells in DMEM+10%FBS+1xantibiotic. Most of the paper says that 4mM L- glutamine is supplemented. Is it necessary? Also I observe lots of floating cells along with healthy cells. Could you please share your experience in maintaining MDA-MB-468 cell line

With regards,

Vimal

Hi Vimal,

L-glutamine is an amino acid required for cell culture. From my understanding it’s quite unstable in cell culture media (temp, pH), which is why you’ll often see it supplemented in media that you can then aliquot and use within a defined period of time. You can find more info on the links below.

http://www.sigmaaldrich.com/life-science/cell-culture/learning-center/media-expert/glutamine.html

https://www.thermofisher.com/order/catalog/product/25030081

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/t075.pdf

It’s been a little while since I’ve cultured the 468s, but I do remember them being quite ‘floaty’. They also don’t like to be split too thin. I fed my cells every second day and would avoid using them for experiments after passage 10.

I hope this helps.

Teneale

Hi Teneale,

Thank you.

I tried to get a perfect system for EMT. With the help of your research papers I could able to reproduce your system. Thank you very much. But I am facing a problem. When I keep the cells with EGF for long duration (72hrs), I could see lots of cell loss. Is it due to cell death? or since they get transformed to mesenchymal state, they acquire mobility and get lost while changing media? Have you ever faced these kind of problems?

With regards,

Vimal

Hi Vimal, 

Sorry, it's been a while since I've cultured the 468s, and I never treated for greater than 48 h. It's possible cells are detaching from the plate as a consequence of undergoing an EMT - this is something you will have to optimise and trouble shoot I'm afraid. Maybe more gentle washing would help? 

All the best.

Teneale

Hello Vimal,

Discussion is a bit old so I m not sure if my advice can help, but here I go.

If you are seeing loss of cells (getting round and floating), it may be due to :

- handling (gentle wash is suggested for example on side of the dish to avoid to detach cells

- medium not adapted. For instance, for MDAMB468 I use high glucose DMEM for better survival and growth. Addition of glutamine helps, but I don't add it in EMT experiment to avoid quick growth.

- also, if you tried to grow the cells on glass bottom dish for immunofluorescence check, then you need to coat the dishes prior to experiment, using for example 0.1% gelatin solution (1h at 37°C then remove), a collagen solution or Matrigel.

Teneale gave you a nice guideline for EMT experiments, starting with 24h adaptation time (in standard medium). This time is to allow the cell to attach. If cells are not attached after the first 24h of adaptation time (check) then they will die during your treatment -serum reduced and egf treatment.

Best regards,

Arno

Plate the cells in low density (upto 50% confluence) for 16 h. Serum starve for overnight, treat with 50ng/ml (or 100ng/ml) EGF in low serum (0.5%) medium for 3-5 days. You will see a clear morphological transformation.