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Characterizing Epithelial-Mesenchymal Transition in

Normal Human Bronchial Epithelial cells

Vanshika Agarwal

Dr. Venkataramana Sidhaye

Johns Hopkins Bloomberg School of Public Health


Introduction:

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the

United States (15, 8, 1, 9, 10, 14). COPD is characterized by progressive, destruction of the lung

that impairs airflow and interferes with breathing (11). Patients with COPD have coughing with

excessive production of mucus leading to shortness of breath and wheezing. Cigarette smoking is

a major cause of COPD (8). However, genetic mutations and family history in smoking can also

be contributing factors of COPD. A recent study shows that smokers are more likely to have

more respiratory issues that do their non-smoking counterparts. About 75% of people who have

COPD have either used cigarettes or presently smoke.

In the condition of COPD, two major sub-phenotypes occur: Emphysema, with the

destruction of alveoli, and chronic bronchitis, the inflammation of the airway, are the two main

conditions of COPD. Although the current treatments are ineffective at reversing disease

progression, doctors and scientists have been working on procedures that may comfort their

patients (11).

Fibrous thickening of the small airways, as in COPD, has also been observed in tissue

resected for cancer, suggesting similarities between cancer metastasis and COPD. Changes in the

epithelial cell structure occur with cancer metastasis, where the cells undergo epithelial to

mesenchymal transition (EMT). During the process of EMT, fully differentiated epithelial cells

acquire mesenchymal characteristics such as loss of intercellular adhesion and cell polarity, and

enhanced migratory capacity (4, 5, 13),. Even though most scientists relate EMT to cancer,

researchers have found increases in EMT markers in COPD patients by protein and RNA

analysis suggesting a strong correlation between EMT and COPD (12,2). In vitro studies have
identified a correlation between EMT markers in airway epithelium and cigarette smoke

exposure. Furthermore, many of the in vitro studies start from non-confluent, undifferentiated

cells to show that specific cytokines can lead to the expression of mesenchymal markers. Our

research attempts to better understand this EMT by determining if a complete monolayer of fully

differentiated normal human bronchial epithelial (NHBE) cells can undergo EMT and moreover

characterizing the in vitro changes that occur with EMT induction.

We hypothesize that COPD is on the spectrum of EMT. To elucidate the relationship

between COPD and EMT, we have been working on understanding how cigarette smoke (CS)

effects NHBE cells and where CS exposed cells lie on the spectrum. Multiple studies have

prominently illustrated CS induces functional and morphological changes. To understand if CS

exposed cells undergo basic epithelial plasticity that are indicative of EMT, the positive control

of EMT induction is required. In this document, I attempt to characterize EMT in NHBE cells.

Previous studies have worked on characterizing EMT but at a sub-confluent monolayer,

which is not representative of the human body. In the human body, the monolayers of cells are

100% confluent and well differentiated. Some characteristics of the EMT induced NHBEs

established over a course of three similar experiments shall be discussed in this article.

Materials and Methods:

Cell Culture:

The primary normal human bronchial epithelial (NHBE) (Lonza and Mattek) cells collected from

normal patients, are grown in a collagen coated (rat tail collagen I), T75 flask in BEGM (Lonza)

at 37 °C with 5% CO2. Once the cell monolayer reaches about 60-80% confluency, they are

trypsinized, counted by a hemocytometer, and split into multiple T75 flasks at a density of
500,000 to 1,000,000 cells per flask. Once at 60-80% confluency, the cells are plated on collagen

coated in 6, 12, or 24 well 0.4 micron permeable transwell polyethylene terephthalate inserts

with BALI media (BEBM+BEGM+ATRA). The cells continue to grow on the inserts with apical

and basolateral media. At 100% confluency, the apical media is removed and they are placed at

air liquid interface (ALI). The BALI media is changed three times each week. Cells used for

experiments are at least 6 weeks ALI and well differentiated with tight monolayers as

determined by trans electrical epithelial resistance (TEER) measurements. In addition to the lab

differentiated cells, well differentiated NHBE cells obtained from Epithelix were also used for

specific studies.

EMT treatment:

After a minimum of 6 weeks of differentiation, NHBE cells were treated with 2x EMT media

supplement (100x EMT R&D systems) or PBS in normal NHBE media in the basolateral

compartment. The induction is performed every third day, at the same time the cells are fed.

Experiments are performed on EMT induction day 10.

Transepithelial electrical resistance (TEER):

The resistance of NHBE inserts is checked using a World Precision Instruments voltmeter to

measure movement of ions through the cell monolayer indicating monolayer permeability. 1 ml

of warmed apical media is added to maintain passage for charge. The probe is washed in 1x

autoclaved, sterile PBS before and after each measurement and performed three times per insert.

TEER measurements are averaged from the three measurements. Apical media is aspirated

before the cells are returned to the incubator.

FITC-dextran (4kD) paracellular permeability assay:


Paracellular permeability is measured using .5 mg/ ml 4kD FITC-dextran in PBS. All apical and

basolateral media is removed. Basolateral PBS is added and apical FITC-dextran solution is

added. The insert is then, placed in the incubator at 37 °C with 5% CO2 for 15-30 minutes. The

apical FITC-dextran is removed and 200ul of basal solution is added in triplicate to a 96 well

plate. The 96 well plate is analyzed on the Synergy HT microplate fluorometer.

Ciliary Beat Frequency (CBF):

CBF videos are collected on a Leica Spinning Disc Confocal microscope using a 40x water

objective in the Differential interference contrast (DIC) channel. Random areas within the center,

right, left, top and bottom of the insert are selected with at least 6 different areas total per insert.

At each area, multiple 834 frame videos are taken using the high speed 160 frames per second

camera. A Matlab script, designed to calculate cilia beat frequency, is used to quantify mean

ciliary beat frequency across each video. Data is shown as mean CBF per insert.

Western blotting:

Protein abundance changes are analyzed by western blot. The respective cells are removed from

their inserts and lysed using Radioimmunoprecipitation assay buffer (RIPA) with protease and

phosphatase inhibitors (Sigma). Samples are centrifuged for 10 mins at 4C and full speed to

obtain protein supernatant. In order to determine protein concentration, a bicinchoninic acid

(BCA) assay is performed by creating a standard curve using albumin standards (Pierce). The

plate is placed in the Synergy HT microplate reader to analyze the protein absorbance. The

samples are normalized to the same concentration with RIPA and 4x loading dye (5% BME) and

boiled for 10 mins. The western blot apparatus is set and samples are loaded; the samples are run

at 80-120 volts. The blot is wet transferred overnight at 40 volts for 8 hours or 90 volts for 90
minutes. Ponceau stain is performed to check equal quantities of protein throughout the blot. The

blots are blocked with 5% milk in PBS + 0.2% tween for 30-60 minutes then incubated in

primary antibody (E-cadherin, N-cadherin, Vimentin, ZO-1, Actin, GAPDH) at recommended

concentration overnight at 4C. The blots are washed 3 times in 1X PBS + 0.2% Tween-20

(PBST), and incubated in appropriate secondary antibody (LiCor) at recommended 1:20,000

dilution for 1 hour. The blots are washed 3 times in PBST and imaged on a LiCor imager. The

images are analyzed using image studio lite software.

Scanning Electron Microscopy (SEM):

Samples were fixed in 2.5% glutaraldehyde, 3mM MgCl2, in 0.1 M sodium cacodylate buffer,

pH 7.2 overnight at 4oC. After buffer rinse, samples were post-fixed in 1% osmium tetroxide in

buffer (1 hour) on ice in the dark followed by two distilled water rinses before dehydration in

ethanol. Samples were dried for SEM with HMDS and mounted on carbon coated stubs, coated

with 20 nm AuPd and imaged on a Leo FE-SEM at 1 kV.

Results:

EMT induction increases paracellular permeability (Figure 1)

A TEER measurement performed before and after the 10-day EMT induction showed a decrease

in the resistance of the cell monolayer suggesting an increase in permeability. To ensure the

accuracy of our assertion, a FITC-dextran permeability assay was performed. When the EMT-

induced cells were compared to control cells a significant increase in permeability was recorded.
Figure 1: Paracellular permeability increases in EMT-induced cells. (a) A FITC-dextran setting with the FITC-
dextran solution on the apical side and PBS on the basal side of the insert. (b) In Normal Human Bronchial
Epithelial cells, paracellular permeability increased under continuous EMT induction for 10 days as measured by
FITC-dextran permeability assay, shown by the higher fold fluorescence. (c) A TEER apparatus setting with media
(nutrients and growth serum) on apical and basal sides of the insert with one point of the fork in apical and the other
in the basal media. (d) The paracellular permeability increased under continuous 10 day EMT induction as measured
by TEER, showing lower resistance in EMT induced cells than normal.

EMT induction causes changes in membrane proteins (Figure 2)

In the Normal Human Bronchial Epithelial cells induced with EMT, changes in protein

quantities were analyzing through western blotting. These protein quantifications show an

increase in EMT markers such as N-cadherin, Vimentin and a decrease of epithelial markers: E-

cadherin and ZO-1 in EMT-induced cells. This change corresponds with the onset of Epithelial

Mesenchymal Transition in epithelial cells.


Figure 2: Change in epithelial marker E-cadherin in in EMT-induced cells. (a) In 10 day EMT induced cells, a
change in the epithelial marker - E-cadherin are visible through the western blot analysis when compared to normal
cells. The blot has the samples, in sequence from left to right, normal NHBEs, CS exposed, non-EMT treated, and
EMT treated. (b) Under a 10 day induction of EMT and CS exposure, a significant decrease in E-Cadherin is visible
when normalized to GAPDH.

EMT induction causes changes in ciliary beat frequency (Figure 3)

As previously known that mesenchymal cells do not have cilia, a further study was performed to

check changes in the movement of cilia. From the collected data, a significant difference

between the EMT induced and normal cells, specifically a decrease in the CBF in EMT induced

cells was observed. The Matlab script allowed for the analyzation of the collected videos to

attain quantifiable data showing the significance.

Figure 3: EMT-induced cells have decreased Ciliary Beat Frequency. (a) The ciliary beat frequency decreased
in 10 day 2x EMT-induced cells with a probability of 0.000978 showing a significant difference between the control
and EMT induced. (b) The mean frequency of cilia, in an average normal monolayer, is similar and spread
throughout the entire area imaged. (c) The mean frequency of cilia, in an average EMT-induced monolayer, is
patched with each patch having a similar ciliary beat frequency. (d) The number of pixels moving in the CBF videos
Figure 3: EMT-induced cells have decreased Ciliary Beat Frequency. (a) The ciliary beat frequency decreased
in 10 day 2x EMT-induced cells with a probability of 0.000978 showing a significant difference between the control
and EMT induced. (b) The mean frequency of cilia, in an average normal monolayer, is similar and spread
throughout the entire area imaged. (c) The mean frequency of cilia, in an average EMT-induced monolayer, is
patched with each patch having a similar ciliary beat frequency. (d) The number of pixels moving in the CBF videos
in fold change shows no significant difference between the quantity of cilia moving. However, the difference has a
13.5 percent chance of occurrence.

EMT induction changes the quantity of cilia in NHBEs (Figure 4)

Using scanning electron microscope (SEM), to quantity cilia, there is a significant decrease in

the number of cilia seen on the epithelium. In addition to an overall change in number, there is

also a decrease in clumping of the cilia. NHBE cells have clumps of cilia on the epithelium

under normal conditions. After EMT induction, these clumps were decreased, and on occasion, a

single cilium was seen on the cells.


Figure 4: Decrease in overall cilia count occurs in EMT-induced cells. (a) In the normal cells, the cilia is
thoroughly spread or forms definite clumps as visible using SEM at 800X magnification. (b) In the EMT-treated
cells, the cilia count decreases and the cilia is spread but not through the entire surface, also there are many locations
where single cilia is observable at 800X.

Discussion:

In this study, we describe the changes in important adherens junction proteins and, even more

crucial changes in the physical and chemical properties of Normal Human Bronchial Epithelial

cells in response to 10-day induction of EMT. In response to EMT induction, we found a

disruption in the monolayer integrity, a decrease in number of cilia present on the cells and a

reduction in ciliary beat frequency. These studies indicate that a confluent monolayer can be

transitioned into more mesenchymal type cells with evidence of altered cellular structure and

function.

As shown by TEER and FITC-dextran results, EMT induction increases paracellular

permeability, suggesting looser contacts between the cells and changes in the structure of

adherens junctions. As the cells change to form a more circular cell body, gaps form between
the cells leading to increased permeability. Since FITC-dextran can only transport paracellularly,

an increase in quantities of transport establishes the intracellular junctional proteins. These

changes in permeability relate to the reduction of the adherens junction protein E-cadherin and

increase in N-cadherin. The lowering E-cadherin equates to the loss of adhesion among cells

thereby allowing substances to pass between the cells.

While obtaining videos pertaining to CBF, a visual difference in the quantity of cilia

movement was recorded. Investigating further we performed electron microscopy (EM) to look

at cilia abundance and morphology. SEM showed differences in cilia abundance suggesting that

during the transition from epithelial to mesenchymal type cells, the cells lose cilia.

The quantifiable changes in EMT markers such as N-cadherin, Vimentin, and epithelial markers

such as E-cadherin, in the EMT induced cells shows that the cells undergo the change from

epithelial to mesenchymal cells (13). These also reaffirm that during the process of EMT,

changes in cell junction proteins such as Cadherins, Vimentin, and ZO-1 occur (6).

Since the quantifications of permeability performed on CS cells are similar to the changes

in permeability in EMT induced cells, we will perform further analysis to better understand the

similarities. They will also continue to analyze changes in EMT markers in CS cells as they

compare to normal cells. It has also been recorded that COPD suffer from goblet cell hyperplasia

urging for further study of cell population (3), specifically in that of ciliated cells to goblet cells

in EMT induction and CS. Using this knowledge, we plan to continue working on understanding

the process of EMT and its relation to COPD to convert the process to MET, similar to that in

fetuses, to cure COPD (7).

Acknowledgments:
I thank Dr. Venkataramana Sidhaye for her time in allowing me to be a part of her lab by

supporting and encouraging my efforts in conducting a research project as well as guiding me

throughout my research as a mentor. I thank Kristine Nishida because she aided in my

development as a lab member by pushing me as I grew to perform the different parts of my

project. I thank Allison and Mia for taking the time to teach me to better perform as a group

member and challenge my abilities motivating my growth. I thank the rest of the lab for their

continual guidance throughout my research project.

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