I am trying to induce EMT in breast cancer cell lines using LPS (10 ug /mL) as per other literature suggestions (MCF-7 and MDA-MB-231) but I am not able to get significant reduction in E-cad in MCF-7 ? although 10ug being a very un-physiological amount, is that alright to treat cells in this high amount ? Is semiquantitative PCR right choice or should I go for real time study ?
Also I have come across the information that MDA-MB-231 being highly metastatic cells they have very low expression of E-cad, Can that be the reason why I am not able to detect E-cad in gene expression studies ?
Can anyone help me in these regard !
MCF-7 can behave differently than the MDA-MB cell lines, and using a designated transfection reagent (https://altogen.com/product/mcf-7-transfection-reagent-breast-cancer-htb-22/) can help you keep the transfection efficiency high. If the cargo you are transfecting is large, then you should shrink it down with a complex condenser, and that will keep your transfection efficiency high.
Hi Manoj
You can use TGF-beta 10 ng/ml for 72 hs or more like control positive. In MCF7 cells you will see E-Cad protein reduction.
Cynthia
Hi Manoj, personally I have problems accepting this EMT idea.
You have transformed epithelial cells that revert to mesenchymal cells that migrate and when they find a niche, they transform back to epithelial cells?
And then they do this transformation and retransformation all over again to establish other niches?
Doesn't this sound far fetched?
And as far as I know there are no positive markers for this EMT phenotype, only loss of epithelial markers.
Can somebody set me straight here and show that this EMT is real and not a theoretical model to explain away how transformed epithelial cells can migrate?
Hi Manoj
I would recommend you to go through this paper of my colleague where we have shown significant reduction of Ecad in MDA-MB-231. Ecad is regulated by wnt/beta catenin pathway, so try out treating the cells with Wnt3a.
https://breast-cancer-research.biomedcentral.com/articles/10.1186/s13058-014-0496-5
Hello Cynthia,
I am actually trying to mimic the inflammatory milieu which is responsible for driving metastasis in cancer cells .Although I am aware that TGF-beta is the most important factor responsible for driving EMT, I assume using just one cytokine won't be able to mimic the inflammatory condition. One other option I think, i can use is combination of different cytokines. Please correct me , if i am wrong.
MDA-MB-231 cells practically have no cell-cell adhesions, so the level of E-cadherine is really low. MCF-7 line is better for basal level of Ecad. You can enhance the E-cad by lower % of FCS, which is good also to add any cytokines and work with these conditions. You can use EGF (or combinations) to induce EMT in breast cancer cell lines like MCF7.
Hello Dr Steingrimur,
I really like your idea about EMT and i totally agree to it but then practically speaking we dont have any cell based model for studying EMT other than in vivo experiment. Besides I am interested in checking the inflammatory causes responsible for the transition. Is there any other way I can carry out the study other than relying solely on animal models ?
Hi Manoj, you and others are talking about different things. 1. MCF7 is epithelial BC cell line with all characteristics of that, like expression of the CDH1+, ER+, HER2+ and no EMT markers as CD10, AXL, MMPs and EMT transcription factors Twist, Zep and so on. If you wanted induce MCF7 EMT it is not so simple. You need driving oncogene there.
2. 231 BC cell line is fully EMT cells. You can't induce EMT, all markers are there.
For checking inflammatory in EMT transition think about MCF7+oncogenes, or CRISP CAS9, or shRNA inhibition of the CDH1. This must works very well.
There are tons of publication about CDH1 knockout in MCF7 and others epithelial cell lines.
Hello Manoj,
Talking about trying to mimic the inflammatory milieu which is responsible for driving metastasis in cancer cells maybe you can treat endothelial cells with LPS and use the supernatant or make a co-culture of endothelial cells with the tumoral cells and check EMT markers .
Cynthia
I happened to be in this space. Are there any works on genomic data of E & M? Just curious.
Hi Manoj, There are tons of assays for cell migration, cell differentiation, phenotype changes due to epigenic modifications, viral infections and inflammatory modulation of cell behavior.
Yet, as far as I know, there is no verifiable assay of transformed differentiated epithelial cells reverting to undifferentiated mesenchymal cells that migrate and then do a U-turn to revert back to differentiated epithelial cells?
And then do this over and over again to metastasize to other loci?
Am I the only one that finds this improbable?
Are there any other non-hematopoietic cells that can de-differentiate and re-differentiate without having any positive surface markers to follow this process?
(Just added now-are there animal models for this EMT thing?)
Hi Cynthia, what are the positive EMT markers that can be used to differentiate epithelial cells from the EMT cells in cancer patient blood samples?
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