Hi, to investigate the EMT transition you should envisage exploring a wide array of markers, keeping in mind that a pivotal role is sustained by the following "hubs": a) PI3K-Akt dependent pathways. Activation of this biochemical cascade leads to inhibition of GSK3beta and NF-kB up-regulation. In turn, NF-kB increase COX-2 and PGE2 release and increased activation of both alpha-SMA and SNAI release: these latter findings (together with changes in beta-catenin expression) should be linked to the activation of the Wnt-related pathways; b) activation of gamma-secretase (usually through increase in presenilin-1 activity). Presenilin cuts E-cadherin transmembrane residues (enabling the dissociation of E-cadherin/beta-catenin comeplexes behind cell membranes) and induces increased Notch-1 release. Concomitantly beta-catenin translocates in the nucleus and FAK were down-regulated. While E-cadherin is reduced (and fragmented), cells re-express N-cadherin, another membrane marker deemed to be a classical mesenchymal marker. The cells also lost their epithelial cell morphology and acquire

fibroblast-like properties: they exhibited reduced cell-cell adhesion, increased motility on fibronectin-coated surfaces, and increased invasiveness in animals. These findings imply that vimentin, Rock and phosphorylated myosin light chain increase. Concomitantly metalloproteinases levels (MMP2 and 9) were shown to increase, in order to facilitate matix remodelling and cell invasiveness. Cytoskeleton remodelling was eventually associated to reduced expression of cytokeratins 8/18. Confocal microscopy aimed at evaluating cytoskeleton rearrangements (involving actin, fasci, tubulin and vimentin) should also be considered.

In conclusion, to investigate EMT you have to take into consideration a wide range of biochemical markers. In addition, I would suggest you to investigate some behavioural parameters, i.e. motility and invasiveness, by means of a wound healing assay.

Bests,

Mariano

I would like to add Dr. Mariano Bizzarri's great comment and give you the important points you should keep in mind. Several signal pathways are involved in EMT process, but it depends on cancer cell context as to whether or not canonical Wnt/beta-catenin signal is essential for EMT. EMT is characterized by the loss of E-cadherin. Surely cancer cells highly expressing membrane beta-catenin associated with E-cadherin exhibit nuclear translocation of beta-catenin upon the loss of E-cadherin. Further, beta-catenin in the nucleus is often difficult to detect only with fluorescent immunostaining. Intranuclear beta-catenin activates the transcriptional level of c-Myc and cyclinD1, both of which enhance cellular proliferation and invasion potential. However, some of the cancer cells express relatively low level of membrane beta-catenin and E-cadherin at the steady state, so that ectopic expression of N-cadherin, vimentin, and fibronectin should be evaluated. By striking contrast, alpha-SMA is one of the cellular markers of cancer-associated fibroblasts and myofibroblasts with high migratory potential, but cancer cells do not tend to acquire the alpha-SMA expression. That is why I strongly recommend you to stain with both E-caadherin and vimentin. After all, few antibodies against N-cadherin are available for specific reaction in the staining experiment.

Are there any agreed on positive markers for EMTs, like there are for CTCs?

Thank you so much for the very informative answer and explanations. I agree with E-cadherin and I will also use various other EMT biomarkers including alpha-SMA and vimentin.