Epithelial-Mesenchymal Transition - Science topic

Hi guys, did you test this point in HEK293. I wonder to know if HEK293 can undergo EMT induced by TGFb.

Thanks,

Jiamin

Good day.

H&E is a routine stain and I am not sure that it can show the expression of one protein. May be you need IHC protocol?

Dear Alexandr Chernov

We tried EGF alone & in combination with insulin to undergo transformation for 72 hrs. But, we did not observe any change in morphology.

I did not use IL-1b to induce EMT. It has been reported that A549 cells would undergo EMT upon 48-hour IL-1β (1 ng/ml) exposure - E-Cad protein reduction was very clear after 3~7 days of IL-1b exposure. You might use TGF-b as a positive control. Good luck.

I suggest you use at least 50ng/ml EGF (even 100ng/ml can be used) in serum 0.5 % for 3 days atleast.

Legitemedly, sure it would not be expressed, because as you knock-out the first exon probably by frameshift, all you get is residual of proteins which are not functional and decays swift. Sometimes, however, there are secondary starting codons in different part of exons which can produce sort of pseudogenes but rearly functional.

that is an interesting perspective

Hi Go, I have no idea what you're talking about, but thanks for your continuous participation to the study group and for your effort in studying science related to our disease! Say hi to Reina for me!

Cancer cells have inherent misregulation of gene networks/programs. Though one cannot exclude the possibility of mutations in the Ecad gene body that may affect transcript or protein levels/trafficking/function, I would most readily believe that epigenetic regulation is the most common cause of Ecad suppression. Further, at the epigenetic level it is likely more complex than promoter methylation and involves other cis regulatory modules.

if are cancerous cells somwhere there is a report about a intermediate phenotype beetween ephitelial and mesenchymal during EMT. A double positive cells have been reported actually.

Hi Maayan was wondering if your endothelial cells actually adhere well without any kind of coating( gelatin, etc) on the coverslip?

You can use hypoxic culture conditions to induce EMT in MCF-7 cell.

Hi, Sonali! 

Tumor biology is always complex. For this reason it is not easy to talk about "opposite biology". Of course, biology of primary tumour is at least partially different from its metastatic form, but we have to remember that primary toumours have often an intrinsic capacity to undergo EMT. I hope it will be helpfull.

Silvia

Hi, I analyzed CTC in breast cancer blood sample. I used pankeratin to identify CTCs and Vimentin to assign a mesenchymal phenotype (and Dapi, to be sure that is a cell). And it was tricky, for me you can just say: it is positif or negatif but it's really tricky to use only two marker to assign an intermediate phenotype (E/M). I think that you need more markers (See paper of Yu 2013: Circulating breast tumor....). And if a cell is negative for CK and positive for vimentin, it doesn't mean that is a mesenchymal CTC, it could be a leucocyte. And neg for Vim but positive for CK, it could be a contaminating epithelial cell.

For me, we can't only use Mcf7 and MDA-MB-231 to define your scale, you need a cell line with an "intermediate" phenotype to validate your scale. Because, the mcf7 are really bright for CK (and negative for vimentin) and 231 for vimentin, you will never find the same stainning for CTC.

It's tricky but interesting! Good luck.

Morgane

Hello

Yes it's a good model and the papers here will help you .

Good Luck 

I have tried inducing EMT with TGFB-1 in MCF-10A cell, which are epithelial. 5ng/ml of tgfb is shown to induce EMT in this cell line if you treat them for 5 days. This happens if you do not start with a high dense culture. Because normally if your cells are packed, they might revert back to epithelial phenotype. So, I would reduce starting cell density, adjust TGFB concentration, and treatment duration. Hope it helps!  

I am including part of an unpublished paper that may help you answer your question:

Cancer stem cells metabolism differs from cancer “bulk” cells metabolism?

There is no clear answer to this question.

Ciavardelli et al (246) found that breast cancer stem cells (CSC) rely on Warburg effect (aerobic glycolisis).

Four seminal works by the team headed by Lisanti and Sotgia paved the way for a different concept.

In the first of these works (247) they used breast cancer cell lines in suspension and in attached monolayer. The mammospheres formed in suspension represent mainly CSCs, while those growing in monolayer represent “bulk” cancer cells.

(Mammospheres  origin comes from a tiny group of cells that exhibits stem cell phenotype and is able to grow in suspension culture and may originate tumors in mice.  Mammosphere grows in a three-dimensional shape and it does not grow in monolayer, while “bulk” cancer cell grows in monolayers).

They compared the proteomics of cells grown in mammospheres with those grown in monolayers, and they identified proteins that were selectively over-expressed in mammospheres. They found three groups of proteins that were highly over-expressed in mammosphere cells:

1)    mitochondrial enzymes

2)    proteins related with mitochondrial biogenesis

3)    proteins related with inhibition of autophagy or mitophagy

Based on these findings, the authors concluded that CSCs require accumulation of mitochondrial mass.

To test this idea they treated the cancer cells with inhibitors of monocarboxilate transporter 1 (MCT-1) and monocarboxilate transporter 2 (MCT-2) inhibitors. The inhibition of the transporters down-regulated the entrance of two mitochondrial fuels in cells: lactate and ketone bodies. The cells treated with MCT-1 and MCT-2 inhibitors showed a significant decrease in mammosphere formation which actually meant less CSCs.

These findings confirms previous publications by  the team headed by Lisanti and Sotgia regarding the “parasitic” metabolism in cancer (134), the second work we are considering here, where tumor cells have the capacity to obtain nutrients from normal host cells, such as fibroblasts. So that there is a group of cells in tumor stroma that predominantly uses aerobic glycolisis (Warburg effect) as source of energy and produces lactate that exits these cells. This lactate is incorporated by cancer cells generating energy through mitochondrial oxidative phosphorylation.  CSCs belong apparently to this last group and a decreased transportation of lactates through inhibition of MCT-1 and MCT-2 decrease the viability of CSCs.  

The third work to be considered (248) analysed 4 different groups of FDA approved antibiotics that target mitochondrial protein synthesis against 12 cancer cell lines (breast, prostate, melanoma, ovary, pancreas, brain, lung and DCIS). The antibiotics tested were azithromycin,  doxycycline (DOXY), chloramfenicol, and tigecyclin.

DOXY and tigecyclin showed dose dependent reduction in mammosphere production. DOXY inhibited tumor sphere formation with an IC50 between 2 and 10μM in breast cancer cell lines (MCF7 and T47D). At 200μM concentration of DOXY no mamospheres were formed. (2 to 5μM are clinically achievable concentrations, 200 μM is not ). When DOXY was tested with other tumor cell lines, it was also effective in all the cases, but the concentration used was 50μM which is higher than clinically achievable.

After a 100-mg IV dose, tigecycline serum concentrations are ∼1.5 μg/ml (Cuhna BA). With a high dose like 400-mg IV, a concentration of 6 μg/ml is attainable (Cuhna BA). The concentrations tested with this antibiotic in the experimental setting start with 10μM (approximately 5 μg/ml) which shows reduction in sphere formation but for an important reduction it is necessary to achieve a concentration of 50 μM/ml which cannot be obtained in the clinical setting.

In the fourth publication (249) they found that mitochondrial biogenesis is necessary for CSCs survival and propagation.

Tetracyclines  by interfering with oxidative phosphorylation and mitochondrial biogenesis may be useful tools against CSCs.

Metformin probably works in the same direction (134) and it may represent a synergistic pharmaceutical with tetracyclines.  There is strong evidence of metformin´s activity against CSCs (250- 258) and the mechanism of action is probably similar in this sense to that of tetracyclines: down-regulation of oxidative phosphorylation. This possible synergy deserves further experimental research.

Yang B et al (259) described the anti-CSCs activity of DOXY. Stem cell markers decreased with the treatment of these cells with DOXY and also invasion, migration, proliferation and colony formation were decreased. The importance of this research lays in a new method  to obtain these HeLa stem cells. HeLa-CSCs were treated with 20 μg/ml concentration of DOXY (this concentration is above clinically achievable concentrations). EMT (epithelial-mesenchymal transition) and all the stem cell markers were reduced in the treated cells. The expression of Snail and Twist were also significantly reduced.

The conclusion from the Lisanti and Sotgia group research, is that CSCs

1)   require an important mitochondrial mass,

2)   that the prevailing metabolic pathway is oxidative phosphorilation,

3)   that they can use lactate and other ketone bodies as energy supply (parasitic enslaving of associated fibroblasts) and

4)   down-regulation of phosphorylative oxidation (metformin or tetracyclines) or decreased fuelling of lactates (MCT1 and MCT2 inhibition) decreases proliferation and survival of CSCs. 

Possible explanations for these discrepancies:

1)   There are two different phenotypic cancer stem cells.

2)   There is a switch from an oxidative phosphorylation phenotype to a fermentative one at some point of cancer progression in the same way as in the “bulk” cancer cell.

3)   CSCs´phenotype is tissue or cancer specific.

4)   There are environmental factors, not identified yet, that may influence one or another phenotype.

Those are the characterisitic dome morphology of Club cells, which used to called Clara cells.

My opinion is that cells injected directly into the circulation do not require EMT as the early stages of metastasis has been bypassed. There is no reqirement for dissociation of tumor cells from the primary site or invasion or intravasation. They will require to extravasate once they reach the secondary site. Most tumor cells injected iv will die those that survive will implant in the target parenchyma. it is believed that epithelial type cells are more efficient at doing this than mesenchymal like cells, hence the reqirement for MET when cells go through the full metastatic cascade. Iif the tumor cells injected are largely epithelial like anyway MET transformation would not be needed. Poorly differentiated colorectal cancer cells for example tend to be more invasive but less metastatic to the liver when injected intra splenically than the more epithelial like well differentiated cells.  

Honestly, i think that it is not possible define EMT by morphological characteristics, if this is your aim. Basically, you should check the down regulation of  E-cadherin and up-regulation of Vimentin and N-cadherin by PCR, WB or Flow cytometry. If you want to check the intracellular signaling of EMT, expression of Snail and Slug is a good start.

Good luck!!

We obtain bands from 120-170 kDa. ZEB1 can be phosphorylated and also glycosylated depending on the cell. We use H102 SCBT and Sigma. As positive controls, you can use 293T, C2C12, SW480, etc..

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

Hi Martina,

in my experience, working with integrins is very reliable, as in EMT there is a integrin switch, as matrix molecules differ from basal plate to mesenchyme. Furthermore, look for beta1 integrin (alpha3beta1 and alpha5beta1 for example) as mesenchymal markers and aalpha4beta6 as epithelial marker. Also there are other integrins that can be used in this way.

Thank you Moamen! Yes, finally I will use GAPDH!

I think this may be due to trypsin effect. After passaging you should add more medium In order to inhibit trypsin activity and also you should add sufficient amount of cells which helps in better cell-cell communication. 

Well, it depends on what you intend as "totally different". In fact, E- and N-cadherins share similar molecular functions: they form homophilic interactions with same molecules on other cells, thus allowing cell-cell interaction; they are anchored to the microfilaments thanks to the catenins, allowing mechanical coupling of the cytoskeletons of two contiguous cells; they are constituents of the Adherens Junction. That said, they are expressed by different kind of cells and tissues during embryonic development and adult life, so that only tissues that express a particular cadherin are affected by its genetic inactivation. In other words: E-cadherin is different from N-cadherin primarily because they are expressed by different tissues.

I have already worked with that cell line and A549 expressed E-cadherin. I used to grow them in RPMI 1640 medium. To exclude for sure your lysis protocol you can plate and fix the cells (with methanol for a maximum of 10 minutes in cold ice for instance) and do an immunofluorescence for E-cadherin.

HI Adam,

The best method for these experiments is to use Nunc Up-Cell dishes, coated with a temperature reactive polymer, cell adhere at 37"c and float loose at room temp!! Amazing product, BUT each 90mm dish or 6 well plates costs about $40!!!!! I use them for critical experiments. the routine way we do this on my lab is to trypsinize the cells, then put them back into a flask on a rocking platform in the incubator so they can not re-adhere. We usually do this before going home and let them rock overnight. Next morning we do the staining and flow analysis or sorting. I can recommend the exact rocker we use if you want that info. Please see our paper "Expression of pluripotent stem cell reprogramming factors by prostate tumor initiating cells" the PDF is available on this site.

E Cadherin is probably more sensitive than N Cadherin to trypsin, but both are significantly removed and flow to measure them is totally unreliable directly after tripsin treatment. They are also damaged by accutase in my hands.

Kind regards,

Steve

In the mouse, the PGCs originate from few cells that are localized in the proximal part of the epiblast (an epithelial tissue) that can be detected as early as E6.25 (so at the time of gastrulation). At E7.5, these cells are localized at the basis of the allantois (extraembryonic mesoderm), at the posterior, and are about 50. Contrary to the chick, these cells do not enter the blood flow, but migrate (as single cells but as a collective wave) along the dorsal part of the hindgut until they reach the genital ridges that will form the gonads. During gonad differentiation, germ cells are associated with the granulosa (female) or Sertoli (male) cells, but they always remains as single cells. The only exception is during male spermatogeneis where it is known that germ cells are actually connected to each other via cellular bridges, but not as an epithelium. On the other hand, Sertoli cells do form an epithelium.

Interestingly, there was a recent paper in Nature from Mitinori Saitou's lab that show that PGC-like cells can be induced via the forced expression of some transcription factors and that this pathway does not include a mesodermal step. this would imply that even if in real life, EMT is the first step of PGCs individualization, in vitro, EMT is not absolutely required for this process.