reactivated in certain cancer cells [2] stimulating tumor cell migration, invasion, and ultimately metastasis to distant organs. During
this reactivation, different hybrid EMT states are generated and
some of them present the highest metastatic potential [3].
At the molecular level, EMT is a complex multistep process that
involves cellular reprogramming through numerous signaling pathways and alterations in gene expression. Epithelial and mesenchymal phenotypes are characterized by highly regulated expression of
key proteins such as E-cadherin, N-cadherin, vimentin, or
occludin [4].
Furthermore, beyond gene expression and cellular morphology, nuclear structure also reflects cellular activity [5]. Nuclei are
highly spatially organized structures where chromatin domains are
arranged in discrete functional regions [6]. It is now clear that gene
position responds to physiological and pathological changes as it
was reported in the case of breast and prostate cancer cells [7–
9]. Furthermore, it is well recognized that cells that have undergone an EMT process present an altered nuclear structure [10] and
that EMT is characterized by the reorganization of specific chromatin domains across the genome [11]. Undoubtedly, correlating
cellular EMT states (complete or partial) with spatial reorganization
of genomic regions would be a powerful diagnostic tool but is still
in its infancy. Through development of automated methods, artificial intelligence and machine learning, added to improvement of
fluorescence microscopy resolution probing the genome may help
personalized prognosis and treatment.
Here, we provide detailed protocols including a combination of
molecular biology assays to study the genomic localization of
EMT-related genes during transforming growth factor beta
(TGF-β) dependent EMT induction kinetics.
We use the MCF10A human breast epithelial cell line [12]
issued from benign proliferative breast tissue and spontaneously
immortalized without defined factors. These non-tumorigenic
cells have been extensively used to study EMT because they are
capable of undergoing an EMT process upon continuous exposure
(one to several weeks) to TGF-β [13]. Interestingly, the MCF10A
cell line also responds to short (4 h) transient treatment and rapidly
(within 4 days) revert to their initial epithelial phenotype through
Mesenchymal-to-Epithelial Transition (MET) [14].
After induction of EMT in the MCF10A cell line with transient
or continuous TGF-β treatment, cells were harvested and their
physiological state determined using three independent assays:
(a) observation of the phenotypic cellular aspect using phase contrast microscopy (b) determination of the EMT-associated gene
mRNA expression levels by RT-qPCR and (c) determination of
EMT-associated gene protein expression by Western Blotting.
Fluorescence In Situ Hybridization is a commonly used technique to specifically label a genomic region of interest. Unique
DNA-FISH-probes targeting EMT-related genes such as CDH1,
354
Noe ´ mie Kempf et al.
this reactivation, different hybrid EMT states are generated and
some of them present the highest metastatic potential [3].
At the molecular level, EMT is a complex multistep process that
involves cellular reprogramming through numerous signaling pathways and alterations in gene expression. Epithelial and mesenchymal phenotypes are characterized by highly regulated expression of
key proteins such as E-cadherin, N-cadherin, vimentin, or
occludin [4].
Furthermore, beyond gene expression and cellular morphology, nuclear structure also reflects cellular activity [5]. Nuclei are
highly spatially organized structures where chromatin domains are
arranged in discrete functional regions [6]. It is now clear that gene
position responds to physiological and pathological changes as it
was reported in the case of breast and prostate cancer cells [7–
9]. Furthermore, it is well recognized that cells that have undergone an EMT process present an altered nuclear structure [10] and
that EMT is characterized by the reorganization of specific chromatin domains across the genome [11]. Undoubtedly, correlating
cellular EMT states (complete or partial) with spatial reorganization
of genomic regions would be a powerful diagnostic tool but is still
in its infancy. Through development of automated methods, artificial intelligence and machine learning, added to improvement of
fluorescence microscopy resolution probing the genome may help
personalized prognosis and treatment.
Here, we provide detailed protocols including a combination of
molecular biology assays to study the genomic localization of
EMT-related genes during transforming growth factor beta
(TGF-β) dependent EMT induction kinetics.
We use the MCF10A human breast epithelial cell line [12]
issued from benign proliferative breast tissue and spontaneously
immortalized without defined factors. These non-tumorigenic
cells have been extensively used to study EMT because they are
capable of undergoing an EMT process upon continuous exposure
(one to several weeks) to TGF-β [13]. Interestingly, the MCF10A
cell line also responds to short (4 h) transient treatment and rapidly
(within 4 days) revert to their initial epithelial phenotype through
Mesenchymal-to-Epithelial Transition (MET) [14].
After induction of EMT in the MCF10A cell line with transient
or continuous TGF-β treatment, cells were harvested and their
physiological state determined using three independent assays:
(a) observation of the phenotypic cellular aspect using phase contrast microscopy (b) determination of the EMT-associated gene
mRNA expression levels by RT-qPCR and (c) determination of
EMT-associated gene protein expression by Western Blotting.
Fluorescence In Situ Hybridization is a commonly used technique to specifically label a genomic region of interest. Unique
DNA-FISH-probes targeting EMT-related genes such as CDH1,
354
Noe ´ mie Kempf et al.