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Page 4 of 9 Mokhamatam et al. J Cancer Metastasis Treat 2020;6:28 I http://dx.doi.org/10.20517/2394-4722.2020.38

MEK, and SRC inhibitors worked well as they are associated with signalling pathways that can activate
[49]
[48]
EMT . Huang et al. made a cell line-based screening system for EMT inhibitors using 43 ovarian cancer
cell lines. They categorized these cell lines into four subgroups, based on their levels of expression of EMT
markers: epithelial, intermediate E, intermediate M, and mesenchymal types. Based on these types, the
EMT inhibiting drugs can be promoters of epithelial markers in the epithelial and intermediate E groups,
or inhibitors of mesenchymal markers in the other two groups. Using this approach, they identified an
Src kinase inhibitor, Saracatinib (AZD0530) which reversed E-cadherin expression in the intermediate
[50]
[49]
M subgroup . Zhang et al. developed a microfluid-based high throughput screening system, named
mesenchymal migration chip. With this, many drugs can be screened for their inhibitory potential of
mesenchymal migration, which might lead to the reversal of EMT. The migration velocity of individual
[50]
cells and the total percentage of migrated cells can be quantifiedusing this assay .

The inhibition of E-cadherin expression is necessary for EMT progression. Using this feature, a
bioluminescence-based epithelial marker promoter induction screening system was developed, whereby the
promoter of E-cadherin or epithelial-specific epidermal growth factor receptor family member ERBB3was
[51]
cloned in a luciferase vector. Several HDAC inhibitors were identified using this system . A further high
throughput study also utilized E-cadherin expression analysis with immunofluorescence in pancreatic
cancer. It also identified a novel HDAC inhibitor 1-(benzylsulfonyl) indoline among 17 other compounds
that worked in a dose-dependent manner. Positive hits were also validated for inhibiting tumorsphere
[52]
formation .
All the models discussed above can test drugs for EMT inhibition only when EMT is induced by ligands
such as TGF-β, epidermal growth factor (EGF), and hepatocyte growth factor. However, none of them
can measure the effect of physical and mechanical forces due to tumour growth which can also induce
[53]
EMT. Nakanishi et al. recently developed a better assay for solving this problem. Here they used
photoactivatable gold substrate which can change from non-cell-adhesive to cell-adhesive upon treatment
with UV light. First, single irradiation with a specific pattern is performed, and cells will be seeded to
confluency. Cells will grow tightly only in those irradiated regions. After the second irradiation for the
remaining areas is given, cells can move into the surrounding regions, because of the mechanical force
induced on the surrounding cells by the central cells. If EMT is successful, the spot size will increase and if
the inhibitors were able to suppress EMT, the spot size will not increase. If the drugs can kill, then the spot
size will decrease. By employing this assay, the group discovered nanaomycin H as a novel EMT inhibitor
[53]
which can specifically kill EMT-induced cells .

3D models
Notwithstanding the advantages with 2D models, they cannot completely mimic the 3-dimensional nature
of the tumour. These 2D cultures are known to induce certain cellular features, which are different from
the cells grown in vivo. 2D cultures in polystyrene plates enhance integrin signalling, as the cells are
dependent on the surface attachment for growth. Because of this, growth factors like EGF and TGF-α
[54]
cannot induce further growth, but induce proliferation in 3D and in-vivo models . Only 3D cultures can
efficiently induce EMT-related transcription factors when compared with 2D cultures. This is achieved by
the activation of nuclear factor-κB in 3D cultures. The EMT-induced cells were able to form successful
[56]
[55]
metastases . The 3D culture was first shown by Sutherland et al. in 1971 as multi-cell spheroids and
it was suggested that the growth properties of these spheroids are more similar to in vivo tumours. Later,
in 1990, the Bjerkvig group showed the growth of multicellular organotypic spheroids to be similar to
[57]
transplanted mouse tumours . It was subsequently discovered that a whole cancer can be regenerated
[58]
using one cell type, which is termed CSC . This led to the development of tumorsphere cultures in almost
[59]
all types of cancers and the development of drug screening systems for CSCs . EMT plays a crucial role in
the development and maintenance of CSCs. Mesenchymal traits are common for normal stem cells as well
as for CSCs .
[60]