Department of Bioengineering

Mechanosensory Characterization of Pancreatic

Ductal Adenocarcinoma in Response to Capillary-
Induced Compression
Walker, Cameron CID: 01451619

Supervisor: Armando del Río Hernández

Submitted in partial fulfilment of the requirements for the

Award of the MRes in Biomedical Engineering and the Diploma of Imperial College.

Date: 10 September 2018 Word count: 5142

 Mechanosensory Characterization of Pancreatic Ductal Adenocarcinoma in
Response to Capillary-Induced Compression
Cameron Walker1, Gulcen Yeldag1, Armando del-Río Hernández1
1
Department of Bioengineering, Imperial College London, London, United Kingdom

Abstract - Pancreatic Ductal Adenocarcinoma (PDAC) is the most common malignancy in the pancreas
and remains extremely resistant to modern forms of cancer therapies and treatment. The extremely low
survival rate of PDAC can be attributed to the lack of early symptoms and its ability to rapidly spread
throughout the body. During the metastatic process, PDAC cells (10 – 20 µm) must withstand
compression and exposure to stiff tissue as they traverse through narrow constrictions in capillaries (4-10
µm) towards secondary sites. Although the cytoplasm of migrating cells can smoothly deform in
response to mechanical force, growing evidence suggests that the enlarged nuclei of cancer cells are
susceptible to mechanical squeezing, which can lead to the uncontrolled transport of cancer-associated
proteins, such as yes-associated protein (YAP), between the cytoplasm and nucleus. Microfluidic devices
recapitulating capillary-sized narrow constrictions were utilized to examine YAP nuclear translocation
and focal adhesion dynamics in response to mechanical constriction. An upstream regulator of YAP,
GPER was constitutively activated to determine whether the effects of mechanical compression could
biochemically abrogated. On a stiff substrate, activation of GPER inhibited YAP nuclear translocation
and abrogated actomyosin contractility. Although imaging revealed that narrow constrictions
significantly flattened PDAC nuclei, mechanical compression of PDAC did not produce significant
changes in focal adhesion size, length, or quantity. Microfluidic device designs and immunostaining
techniques must be improved before conducting further focal adhesion analysis. Activation of GPER
represents a potential novel therapeutic target for the prevention of PDAC cell migration.

INTRODUCTION the initiation of the disease and the diagnosis,

Pancreatic Ductal Adenocarcinoma (PDAC)
is the most common malignancy in the pancreas
and is the fourth leading cause of cancer related
deaths worldwide.1 This gastrointestinal
malignancy arises from the lining of the
pancreatic duct and remains extremely resistant
to modern forms of cancer therapy and treatment.
Indeed, PDAC is currently viewed as a death
sentence due to its extremely low 5-year survival
rate of 5% and mean survival rate of 6 months,
figures that have not wavered for the previous
four decades.1,2 This low survival rate can be
partially attributed to the rapid progression of the
disease and lack of symptoms until late stages of
the cancer.2 However, years often pass between
leaving a large window for potential early
detection of the disease.3 Notably, PDAC is the
only reported form of cancer in which metastasis,
the development of tumours at distant organ
sites, can precede the formation of the primary
tumour.4 At advanced stages, PDAC has typically
progressed to distant organs, which dramatically
decreases the viability of most treatments.
Currently, the only effective treatment is the
surgical removal of the tumour, however fewer
than 20% of patients have operable tumours, and
only 30% of those receiving surgery survive.5,6
PDAC is a disease that is intrinsically linked
to its surrounding environment. PDAC tumours
are generally characterized by the elevated

1
deposition of stiff connective tissue and
extracellular matrix (ECM) proteins within the
adjacent microenvironment.7 In healthy tissue, the
ECM undergoes dynamic remodelling to provide
critical biochemical and structural support for
resident cells.8 However, in malignant tissues,
ECM-secreting pancreatic stellate cells (PSCs)
become activated, adopt a contractile morphology,
and dramatically stiffen adjacent tissue.8
Increased deposition of matrix proteins directly
facilitates tumour growth by increasing cell
contractility, interfering with cell-cell adhesion,
and amplifying growth factor signalling.8,9
Moreover, an increase in ECM stiffness is
associated with an increase in cancer motility, as
cells sense the rigidity of their surroundings and
migrate through dense tissues towards
capillaries.10,11 This process, known as durotaxis,
provides evidence that cancer cell behaviour and
phenotype are directly influenced by mechanical
force.12,13
The process by which cells convert external
mechanical stimuli into downstream intracellular
biochemical signals is known as
mechanotransduction. Numerous studies have
indicated that critical cell functions including cell
differentiation, cell proliferation, and cell-cell
communication can be regulated by mechanical
force.14,15,16 One protein crucial for
mechanotransduction is Rho-A (Ras homolog
gene family member A), which is known to
directly regulate cell adhesion and migration
through the formation of focal adhesions, protein
complexes that physically connect the cell to
surrounding ECM.17,18 During the metastatic
process, circulating PDAC cells utilize these focal
adhesions to reattach to vasculature walls and
extravasate directly to secondary sites. Rho-A is a
member of the GTPase family of proteins that
function as molecular switches, cycling between
an active GTP-bound and inactive GDP-bound
state to regulate signalling pathway activation.19
Rho-A has numerous downstream effectors,
including two key proteins associated with cancer
progression: Rho-associated protein kinase
(ROCK) and yes-associated protein (YAP). ROCK
activation by Rho-A has been shown to facilitate
actomyosin contractility and stress fibre
formation, which are critical for cell migration.20
YAP serves as a transcriptional co-activator
of many cancer associated genes pertaining to cell
proliferation and survival by translocating from
the cytoplasm to the nucleus upon activation.21
While YAP is typically activated as a downstream
effector of Rho-A, it has been demonstrated that
both mechanical force and rigidity are sufficient
for YAP translocation to the nucleus, independent
of biochemical activation.22 On stiff substrates,
cells spread as Rho-A activates the formation of
actin bundles, which directly facilitate the
translocation of YAP to the nucleus.23 However, a
recent study demonstrates that a direct
application of force to the nucleus on a soft
substrate alone is sufficient to drive YAP nuclear
translocation and activation.24,25 The roles of both
YAP and ROCK as mechanosensitive regulators of
cell adhesion and proliferation suggest Rho-A
inhibition as a potential target to limit metastasis.
G protein-coupled receptors (GPCRs)
constitute the largest family of signalling
molecules in humans. Within this group of
transmembrane proteins is G protein coupled
estrogen receptor 1 (GPER), which is expressed in
most human tissues, including the pancreas.
Importantly, Rho-A and its downstream effectors
can be inhibited by the activation of the GPER.26
Recent work in our group has demonstrated that
activation of GPER by a GPER-1 agonist (G1)
mechanically deactivates PSCs via suppression of
Rho-A actomyosin contractility, YAP deactivation,
and suppression of LOX-L2 and MMP-2 (crucial
ECM remodelling proteins).27, 28 Our preliminary
results also indicate GPER activation could inhibit
PDAC’s ability to mechanosense external stimuli,
2
 M e ch a
nical
Fo rc e

G1 GPER YAP

Rho-A
Focal
Adhesions mDia

Nucleus
Myosin ROCK G-actin F-actin
Phosphatase

MLC-2 pMLC-2 Actomyosin

Contractility

Figure 1. Illustration of mechanosensitive signalling pathways associated with cancer progression. Blue arrows
indicate activation. Red lines indicate inhibition. Rho-A facilitates actomyosin contractility via ROCK and mDia and
aids focal adhesion maturation. YAP nuclear translocation can be activated biochemically via F-actin formation or
through mechanical force. Mechanical force applied to cell membranes also can result in direct Rho-A activation.17

blocking mechanotransduction pathways tumour, migrate towards the vasculature, and

associated with metastasis. Although no studies
exist regarding the efficacy of G1 on limiting
PDAC progression, recent work has indicated that
GPER deficiency enhanced breast and liver
tumourigenesis.29,30 Figure 1 provides an
illustration of the mechanosensitive signalling
pathways associated with cancer progression
described above.
The dismally low survival rate of PDAC can
be partially explained by its ability to rapidly
disseminate through the vasculature to distant
organ sites. Indeed, it is estimated that metastasis
is responsible for approximately 90% of cancer
deaths.7 The PDAC metastatic response initiates
as malignant cells detach from the primary
ultimately circulate in the bloodstream until they
reattach along the wall of a capillary near a
secondary organ site. During the metastatic
process, cancer cells must undergo numerous
intracellular changes to evade apoptosis, resist
high amounts of shear stress, and adapt to
unfavourable tissue conditions.31 Importantly,
pancreatic cancer cells (10 – 20 µm) must also
withstand compression as they traverse through
narrow constrictions in compressed capillaries (4-
10 µm).32 Although the cytoplasm of migrating
cells can smoothly deform in response to
mechanical force, growing evidence suggests that
the enlarged nuclei of cancer cells are susceptible
to mechanical rupture, which can lead to the

3
uncontrolled transport of proteins between the
nucleus and cytoplasm.33,34 Previous studies have
indicated that direct compression of tumour cells
enable coordinated migration by enhancing cell-
substrate adhesion, however the direct
intracellular signalling pathways are not yet fully
understood.25,32,33 Moreover, it remains unclear
whether compression of circulating PDAC cells
through capillaries post-intravasation influences
cell phenotype.
In this study, we seek to characterize the
mechanosensory changes and mechano-
mutations that arise in circulating tumor cells as
they traverse narrow constrictions and migrate
through stiff tissue. Importantly, we aim to
elucidate the effects of nuclear compression on
YAP nuclear translocation, focal adhesion
formation, and cancer cell migration. To do this,
we will utilize polyacrylamide (PA) gels and high
throughput microfluidic devices with channels
that mimic capillary diameter. Finally, we seek to
understand whether treatment of PDAC cells
with a G1 agonist limits the downstream
phenotypic response of PDAC cells.
METHODS
Cell Culture In this study, SUIT-2 (S2-007) cells
derived from the human PDAC cancer cell line
were used. SUIT-2 cells were cultured in 37°C, 5%
CO2, in sterile, filtered Dulbecco’s modified eagle’s
medium high glucose (DMEM). Cell cultures
were supplemented with 10% foetal bovine
serum, 1% fungizone amphotericin, and 1%
penicillin streptomycin. Additionally, for each
experiment one subset of cells were treated with
10 µm G1 in 10 mL of media and then incubated
for 24 hours.
Polyacrylamide Gel Fabrication PA gels were
designed with varying rigidity to mimic the
stiffness of healthy and fibrotic tissue, as
described in Pelham et al.35 PA gels were
fabricated according to a modified protocol
described in Tse et al.36 1 kPa gels mimicking
healthy tissue, and 25 kPa gels mimicking fibrotic
tissue were prepared by mixing TEMED (N, N,
N’, N’-tetramethylethylenediamine), phosphate
buffered saline (PBS), and ammonium
persulphate solution (APS) at varying
concentrations. A table containing exact volumes
of reagents can be found in Appendix A. Glass
coverslips were immersed in a hydrophilic 3-
propyl methacrylate solution for 5 minutes,
washed in 100% ethanol, and allowed to dry. 10
µL of PA solution were then applied to the glass
coverslip and allowed to set for 45 minutes.
YAP Translocation on Polyacrylamide Gels The
surface of the PA gels were photoactivated with
sulpho-SANPAH (sulfosuccinimidyl 6-(4’-azido-
2’-nitrophenylamino) hexanoate) diluted in PBS
to facilitate fibronectin crosslinking, which is
necessary for cell attachment to occur. Gels were
then washed 3 times with PBS for 5 minutes each.
Finally, gels were incubated overnight at 4°C in a
human fibronectin (FN) PBS suspension. To
determine if G1 impedes tumourigenic
mechanotransduction pathways of PDAC, cells
were seeded onto the PA gels into three distinct
groups: Control SUIT-2 cells on 1 kPa PA gels,
Control SUIT-2 cells on 25 kPa gels, and G1
treated SUIT-2 cells on 25 kPa gels. Cells were
incubated for 24 hours on the gels and then fixed
using a 4% paraformaldehyde (PFA) solution.
Immunofluorescence Staining The ABCAM
immunofluorescence protocol was followed to tag
the position of YAP within the seeded cells.37 Cells
were permeabilized with 0.5% Saponin in PBS for
10 minutes to allow the antibody to enter the cell.
Cells were washed and incubated in 22.52mg/mL
glycine and 1% BSA in PBST for 30 minutes to
inhibit unspecific antibody binding. The primary
antibody, YAP, was diluted in 1% BSA in PBS at
1:100 dilution and then used to incubate
coverslips overnight at 4°C. Coverslips were then
4
 washed and incubated with Alexa Fluor 488-
conjugated secondary antibody and phalloidin-
Alexa Fluor 546 in 1% BSA at room temperature
for 1 hour. Finally, coverslips were washed,
incubated with DAPI nuclear stain, and mounted
onto glass slides.
Image Acquisition Epifluorescent images of cells
were taken using the Nikon Eclipse Ti-E at 40x
magnification. Nuclear to cytosolic ratio of YAP
mean fluorescence intensity was determined
using the Fiji package on ImageJ. After
subtracting background signal, the nucleus and
cytoplasm were segmented. The nuclear to
cytosolic ratio of YAP was obtained by dividing
the mean fluorescent intensity of the nucleus by
the mean fluorescent intensity of the cytoplasm.
The mean fluorescent intensity of the cytoplasm
was defined as the mean fluorescent intensity of
the nucleus subtracted from the mean fluorescent
intensity of entire cell.
Figure 2. Microfluidic Compression Chamber Design.
5mm inlets lead to hundreds of 7µm diameter channels.
7µm channels converge at 1.5mm outlet. Each silicon
mask contains 5 unique chambers that are cut from the
silicon master to create 4 devices.
Microfluidic Device Preparation Microfluidic
compression device master silicon masks were
obtained from Au laboratory and can be seen
below in Figure 2.38 The microfluidic master chip
fabrication protocol can be found in Appendix B.
18.0 g of PDMS and 2.0 g of cross-linker were
thoroughly mixed and poured onto the silicon
master mask within a petri dish. The PDMS and
mask were placed into a vacuum for 1 hour to
remove air bubbles. The device was then heated
at 70°C for 12 hours. Upon removal, individual
PDMS chambers were sliced and carefully
removed from the silicon mask. 5 mm biopsy
punches were used to cut inlets into the chamber,
while 1.5 mm punches were used to cut the outlet
of the device. The PDMS chips were sealed to
glass slides using via plasma-activated bonding
and heated on a hot plate at 100°C for 1 hour.
Before introduction of cells, devices were cleaned
using 70% ethanol, rinsed with PBS, and treated
with 1% BSA to limit cell adhesion to the PDMS.
YAP Translocation During Compression SUIT-2
cells were transfected with fluorescent-tagged
YAP (YAP-GFP) and cultured onto 1 kPa PA gels
for 24 hours to ensure YAP remained initially
cytoplasmic. These cells were then collected,
stained with Hoechst nuclear dye, and split into
two groups. One group was then treated with the
G1 agonist while the other served as a control.
Cells were suspended in media and inserted
directly into the 5mm inlets of the compression
device. A syringe connected to 10 cm length, 1.5
mm diameter tubing was partially filled with PBS
and attached to the outlet of the device. Cells
were slowly drawn from the inlet through the
narrow channels upon pulling of the syringe. Live
epifluorescence microscopy at 60x magnification
was used to visualize YAP-GFP nuclear
translocation and nuclear compression ratios
upon constriction. Mean fluorescence intensity of
nuclear YAP and cytoplasmic YAP were obtained
5
to determine the nuclear to cytosolic ratio of YAP
upon constriction.
Focal Adhesion Characterization Upon
Compression To determine the effect of capillary-
induced compression on PDAC migration and re-
attachment at a secondary site, focal adhesion
dynamics were assessed. SUIT-2 cells were
cultured on 1kPA PA gels for 24 hours and then
split into four subgroups: control-compressed,
control-uncompressed, G1-treated compressed,
and G1-treated uncompressed. Control
uncompressed SUIT-2 and G1-treated
uncompressed SUIT-2 cells were collected from
the 1 kPa gel, seeded onto glass, and fixed using
4% PFA. Meanwhile, control and G1-treated cells
were passed through the compression device,
collected, seeded onto glass, and fixed with 4%
PFA. To visualize focal adhesion complexes upon
compression, all groups were immunostained for
A B
C D
Figure 3. Thresholding and Quantification of Focal
Adhesions. (A) Immunofluorescence images of Paxillin
(green) in G1-treated uncompressed SUIT-2 cells at 60x
magnification. (B) Grayscale converted image. (C)
Manually thresholded binary image. (D) Automated focal
adhesion regions determined by Analyse Particle tool.
adhesion adaptor protein, Paxillin. Images were
obtained using total internal reflection
fluorescence (TIRF) microscopy at 60x
magnification to determine the area, length, and
number of focal adhesions within each cell.
Focal Adhesion Quantification and Analysis
ImageJ was used to analyse area, length, and
quantity of focal adhesions in each cell. For 60x
magnification, a scale of 9.30 pixels/µm was
implemented. Image files were then converted to
grayscale. Image contrast was then enhanced to
clearly illustrate the boundary of cell so that cell
area could be determined. The image was then
manually thresholded and made binary using the
Threshold tool. Finally, the analyse particles tool
was utilized to determine the area, number, and
Feret diameter of each focal adhesion. A lower
limit of 0.11µm2 was set for focal adhesion area as
previous studies have indicated that focal
adhesions must exceed this area to generate
adhesive force.9 An example of image
thresholding and focal adhesion quantification
can be seen in Figure 3. It is important to note that
automated analysis and manually thresholding
occasionally failed to differentiate between
smaller focal adhesions. In these instances,
manual area calculation supplemented automated
area calculation.
RESULTS
Morphology on Polyacrylamide Gels Bright field
images of SUIT-2 cells cultured on PA gels with
varying rigidities (1kPa and 25 kPa) confirm
previous reports that cell morphology is
dependent on substrate rigidity (Figure 4). Cells
cultured on soft 1 kPa substrates exhibit a
rounded shape, while cells cultured on a stiff 25
kPa substrate are spread and spindle shaped.
Notably, treatment of cells cultured on 25 kPa
substrates with G1 reduced actomyosin
contractility and reverted cell morphology back to
a rounded shape.
6
A B C

C
Figure 4. Cell Morphology on Varying Rigidity Substrates. Brightfield images of SUIT-2 cells on varying
rigidity substrates. (A) Control 1 kPa cell exhibits round morphology (B) Control 25 kPa cell spreads due to
high actomyosin contractility (C) G1-treated 25 kPa reverts to rounded morphology. Magnification 40x.

Figure 5. Nuclear to Cytosolic YAP Ratio in Response to Substrate Stiffness. Immunofluorescence images of
SUIT-2 cells cultured on (A) 1 kPa and (B) 25 kPa PA gels, respectively. YAP (green) translocates to nucleus (blue)
upon introduction to stiff substrate. (C) Introduction of G1 agonist inhibits YAP nuclear translocation. (D)
Quantification of nuclear: cytoplasmic YAP ratio. SUIT-2 cells cultured on 25 kPa have significantly higher nuclear
YAP ratio than 1 kPa and 25 kPa + G1 cultured cells. Introduction of G1 nearly returns nuclear YAP ratio to 1 kPa
levels. N=25 cells. Scale bar signifies 20 µm.
7
 Nuclear: Cytoplasmic YAP Ratio in Response to
Substrate Stiffness Immunofluorescence images
revealed that cells seeded on 1 kPa gels exhibited
round morphology and significantly reduced
nuclear YAP (green) signal of 0.478 (Figure 5A,
D). Cells seeded on 25 kPa PA gels present spread
morphology, indicative of strong cytoskeletal
tension and actomyosin contractility (Figure 3B).
Additionally, cells seeded on 25 kPa had a
significantly higher nuclear: cytosolic YAP ratio of
0.731 (p<0.01) (Figure 3D). Upon introduction of
the G1 agonist, cells cultured on 25 kPa PA gels
displayed a significantly reduced YAP clear:
cytosolic ratio of 0.518 (p<0.01) (Figure 3D), nearly
returning to levels of cells seeded on 1 kPa gels.
Due to the normal distribution of data across all
subgroups, a student’s t-test was used to illustrate Figure 6. Narrow Constriction Compresses Nuclear
statistically significant differences between Membrane Hoecsht nuclear staining of PDAC cell
nuclear YAP ratios. traversing 5 µm channel. Nuclear membrane diameter
is reduced by 72.40%.
Microfluidic Device Successfully Compresses
PDAC Nucleus Hoechst nuclear staining and live compression. No apparent trends in focal
epifluorescence imaging revealed high adhesion quantity were exhibited across
throughput of SUIT-2 cells through the device. treatment groups (Figure 7A). Control cells
Measurements of nuclear membranes of SUIT-2 exhibited a statistically significant (p<0.05)
cells traversing narrow channels reveal a 30.59% decrease from 49.0 focal adhesions/cell to 36.1
B
A
reduction in nuclear diameter for 7 µm channels focal adhesions/cell post compression while G1-
and a 72.40% reduction for 5 µm channels (Figure treated cells displayed a slight increase in focal
6). Bright field images of cells undergoing adhesion quantity from 49.7 focal adhesions/cell
compression can be found in Appendix C. to 57.1 focal adhesions/cell post-compression. A
Unfortunately, because repeated attempts to large variance in focal adhesion quantity was
transfect SUIT-2 cells with the YAP-GFP plasmid observed across cells. No significant changes in
were unsuccessful, there was an insufficient focal adhesion length were observed post
number of transfected cells to obtain the nuclear: compression for either treatment or control
cytoplasmic ratio of YAP during compression. groups (Figure 7B). Measurements of focal
Focal Adhesion Dynamics To investigate the role adhesion area of the control group indicated a
of capillary compression on PDAC reattachment slight decrease in average focal adhesion area
ability, focal adhesion dynamics were assessed for from 1.997µm2 to 1.368 µm2 post compression,
G1-treated and control cells pre and post however this change is not statistically significant
compression. As all subgroups demonstrated an (Figure 7C). G1 treated groups displayed nearly
approximate normal distribution in length, area, no change in mean focal adhesion area post
and quantity, a student’s t-test was used to assess compression (Figure 7C). Total focal adhesion
differences across groups for pre and post-

8
 A Focal Adhesion Quantity B Average Focal Adhesion Length

Average Focal Adhesion Length (µm)

# of Focal Adhesions per Cell

Control Control G1 G1
Control Control G1 G1
Pre-Channel Post- Pre-Channel Post-
Pre-Channel Post- Pre-Channel Post-
Channel Channel
Channel Channel

C Average Focal Adhesion Area D Percentage Focal Adhesion

of Cell Area
% Focal Adhesion Area of Cell Area
Average Focal Adhesion Area
(µm2)

Control Control G1 G1 Control Control G1 G1

Pre-Channel Post-Channel Pre-Channel Post-Channel Pre-Channel Post- Pre-Channel Post-
Channel Channel

Figure 7. Focal Adhesion Dynamics Analysis Post Device Compression. (A) Average focal adhesion quantities
were determined for each cell in four treatment groups: i) control pre-compression ii) control post-compression iii)
G1 pre-compression iv) G1 post-compression. There is a significant decrease in focal adhesion quantity post-
compression for the control group. (B) Maximum Feret diameter of each cell was obtained and averaged across
treatment groups. (C) Focal adhesion areas were quantified across all cells and averaged. Large variance across
focal adhesion areas resulted in no statistically significant differences across treatment groups. (D) Focal adhesion
areas for each cell were summed and divided by total cell area to determine a normalized focal adhesion area. G1-
treated cells post compression saw a significant increase in normalized focal adhesion area. N = 31, 25, 40, 14 for
pre-control, post-control, pre-G1, and post-G1 groups, respectively.
9
areas for each cell were summed and divided by
the cell area to create a normalized focal adhesion
area, which accounted for the relative size of the
cell (Figure 7D). The control group exhibited
nearly no change in percentage focal adhesion of
cell area, rising only from 12.99% to 13.28% post-
compression. Meanwhile, G1-treated cells saw a
significant increase in percentage focal adhesion
of cell area, from 12.88% to 16.11% (p < 0.05).
DISCUSSION
The aim of this study was to determine
how mechanical compression of PDAC cells by
narrow capillary constrictions influences
downstream Rho-A pathways associated with cell
migration and adhesion. Moreover, we sought to
understand whether activation of GPER by its G1
agonist abrogated these downstream intracellular
changes by characterizing changes in YAP nuclear
translocation and focal adhesion dynamics post-
compression.
Cell cultures on stiff (25 kPa) and soft (1
kPa) substrates confirmed previous results that
cell morphology is directly mediated by substrate
rigidity.16,18,38 As cells probe their mechanical
environment, they continuously adjust their
internally developed forces to match the
resistance offered by the ECM. During this
process, Rho-A becomes activated to facilitate the
formation of filamentous actin (F-actin) via the
formin, mDia-1.34 Rho-A activation mediates the
phosphorylation of myosin light chain-2 (MLC-2),
a protein kinase necessary for actin contractility.39
The conjunction of excess F-actin and MLC-2
significantly enhances actomyosin contractility of
the cell, which leads the cell to adopt a spread
morphology when exposed to a stiff matrix, as
shown in Figure 4B.35 However, introduction of
the GPER-1 agonist, G1, to SUIT-2 cells cultured
on 25 kPa substrate, likely deactivated Rho-A,
reducing the formation of F-actin and decreasing
actomyosin contractility. These results indicate
that GPER serves as a suitable target to limit the
activation of Rho-A, hence abrogating actomyosin
contractility and focal adhesion formation in
response to mechanical force.
Immunofluorescence staining of SUIT-2
cells cultured on varying rigidity substrates was
also performed to elucidate whether YAP
translocation is mediated via GPER signalling.
Cells cultured on 1 kPa substrate exhibited
primarily cytoplasmic YAP concentrations (Figure
5A, D), while cells seeded on a 25 kPa
demonstrated a significant influx of YAP into the
nucleus (Figure 5B, D). These results confirm
previous reports that YAP nuclear translocation is
indeed mechanosensitive.21,23,24 Previous studies
have hypothesized that Rho-A-mediated F-actin
formation directly flattens the nucleus by forming
a mechanical link between the nucleus and the
cell membrane in response to elevated tissue
rigidity.25 Elosegui-Artola et al. posit that this
nuclear membrane flattening widens the exterior
of nuclear pores, which could enhance nuclear
import and inhibit nuclear export of large
cytoplasmic proteins, including YAP.25
Importantly, the introduction of G1 resulted in a
significant decrease in YAP nuclear translocation
(Figure 5C, D), suggesting that YAP nuclear
translocation is biochemically dependent on
GPER activation. It is likely that GPER activation
inhibits Rho-A and F-actin formation, blocking
the cytoskeletal link to the nuclear membrane.
However, whether GPER activation can inhibit
YAP nuclear translocation when the nucleus is
mechanically compressed remains unclear.
To investigate whether mechanical
squeezing of the nucleus by capillaries elevates
YAP nuclear translocation, cells were transfected
with a fluorescent YAP-GFP plasmid to visualize
live YAP location as cells passed through the
compression microfluidic device. Unfortunately,
YAP-GFP transfections were largely unsuccessful,
and YAP nuclear translocation could not be
10
quantified. However, Hoechst nuclear staining
did reveal that nuclear membranes were
significantly flattened in control cells during
transport through narrow constrictions. Nuclear
flattening must also be analysed in G1-treated
cells to determine if acytomyosin contractility is
necessary for sufficient nuclear constriction.
Furthermore, testing must be conducted to
confirm whether nuclear squeezing facilitates
YAP nuclear translocation in the absence of
biochemical activation.
Should future testing confirm that YAP
indeed translocates to the nucleus upon
compression, additional testing must be
conducted to determine the mechanism of action.
While previous studies have suggested nuclear
flattening as a potential mechanism for YAP
translocation, others have indicated that nuclei
are mechanically ruptured during transport
through narrow constrictions.25,33,34 During nuclear
ruptures, proteins, such as YAP, are able to shuttle
unregulated between the nucleus and cytoplasm.
The nuclear membrane reseals rapidly during
interphase, however, assisted by ESCRT
machinery (endosomal sorting complexes
required for transport).40 Analysis of ESCRT levels
post-compression could elucidate whether the
nuclear membrane is ruptured during capillary
transport. Additionally, knocking down genes
directly associated with YAP activation will
elucidate the effect of each protein on YAP
translocation. Constitutively activating or
inhibiting factors within the YAP signalling
pathway provides insight into how mechanical
force influences the role of each factor in the
signalling pathway. A table illustrating the
functions of each mutant YAP plasmid and its
respective predicted outcome can be found in
Appendix D. These findings may provide insight
into potential therapeutic targets to mitigate
mechano-mutations associated with metastasis.
Focal adhesion dynamics were assessed
post-compression to determine whether
mechanical compression altered the ability of
PDAC cells to reattach to capillary walls and
extravasate toward secondary organ sites. Data
shown in Figure 6 illustrate no significant
relationship between mechanical compression
and focal adhesion area, length, or number.
Although we hypothesized that mechanical
compression would increase focal adhesion
production due to the mechanical activation of
Rho-A, the data presented a significant decrease
in focal adhesion quantity for control cells
exposed to mechanical compression. This would
suggest that mechanical squeezing of the nucleus
actually hinders the ability of PDAC to reattach
and migrate to secondary organ sites. The results
also suggest that percentage focal adhesion area
of cell area increased significantly for G1-treated
cells post compression. This runs counter to our
hypothesis that GPER activation would inhibit
Rho-A-mediated formation of focal adhesions.
There are a number of possible reasons
why the results did not illustrate a significant
relationship between mechanical compression of
circulating PDAC cells and focal adhesion
dynamics. First, the quantification of focal
adhesions proved to be difficult and subjective.
As shown in Figure 3, a manual threshold was
used to determine what level of Paxillin
fluorescence qualified as a focal adhesion. By
manually establishing the fluorescence threshold,
quantification of focal adhesion number and size
became subjective. Indeed, the analyse particle
tool would struggle to differentiate between a
conglomerate of smaller focal adhesions and one
large focal adhesion. This inability to differentiate
between small and large focal adhesions was
partially responsible for the large variation in
focal adhesion size and length. For future
quantification of focal adhesion dynamics, image
quality and immunostaining techniques must be
11
improved to more accurately assess focal
adhesion dynamics.
It also likely that PDAC cells were
compressed for an insufficient duration while
passing through the narrow constrictions. As
cells were manually drawn through the device via
syringe, it was difficult to control flow velocity.
While some cells passed slowly through the
device, others travelled rapidly. Thus, time of
compression was inconsistent across cells. This
issue could be solved via the introduction of an
automated pump which consistently controls the
flow of fluid through the device. It would also be
beneficial to quantify fluid shear stress
experienced by PDAC cells as they traverse
channels using computational fluid dynamics
models. Shear stress could be controlled by
altering flow rates to ensure consistent stress is
applied to cells as they pass through the device.
Additionally, the length of channels should be
extended to ensure that all cells travelling
through the device experience sufficient
compression. Novel compression device designs
were prepared in which the length of each
channel was doubled from 1.4 mm to
approximately 2.8 mm. These devices were also
designed to minimize spacing issues associated
with punching inlets during fabrication.
Implementation of these devices would
significantly increase compressions times of cells
and reduce errors associated with fabrication.
These device designs are shown in Appendix E.
Future work should explore the invasive
behaviour of PDAC cells post-compression.
Although focal adhesions give important insight
into a cell’s reattachment ability, microfluidic
devices should be implemented to explore
PDAC’s ability to extravasate post-compression.
Novel microfluidic device designs in our group
have been implemented to explore the dynamics
of pancreatic cancer extravasation from capillaries
and collective migration to secondary organ sites.
It would be interesting to explore how capillary-
induced compression influences cancer cell
reattachment and collective migration. It would
also be beneficial to explore whether introduction
of the G1-agonist influences invasive behaviour.
Furthermore, studies should be conducted to
explore the relationship between PDAC and
ECM-secreting PSCs as they migrate through
capillaries. Recent work in our group confirmed
that mechanical rigidity is sufficient for PSC
activation. Future studies should explore
intracellular changes and mechano-mutations
within PSCs due to mechanical compression
through narrow constrictions. Moreover, it would
be valuable to evaluate the relationship between
mechanically compressed PDAC cells and PSCs
as they migrate to secondary organ sites.
CONCLUSION
This study has helped to elucidate the
relationship between substrate rigidity and
intracellular changes associated with cell
adhesion and cell proliferation. Importantly, it
demonstrated that GPER activation directly
inhibits YAP nuclear transport in the presence of a
stiff substrate. These results suggest that GPER
serves as an attractive potential therapeutic target
for inhibiting cancer-associated Rho-A
downstream effectors. Unfortunately, the results
did not clearly indicate whether mechanical
constriction of PDAC cells enhanced nuclear YAP
translocation or significantly altered focal
adhesion dynamics. Further experiments must be
conducted to understand whether constriction of
PDAC cells during metastasis influences
downstream cancer-associated signalling
pathways and cell behaviour.
Acknowledgement
I would like to thank Dr. Armando Del Río
Hernández, Dr. Sam Au, Gulcen Yeldag, Julian
Ashby, and Carlos Matellan for their assistance
and guidance during this experiment.
12
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15
 APPENDIX A

The table below illustrates the concentrations of various reagents necessary to create a
polyacrylamide gel with the prescribed stiffness. Acrylamide concentration was steadily
increased in order to increase substrate rigidity.

Table A.1 Polyacrylamide Gel Reagent Volumes and Stiffness

Gel Acrylamide Total PBS APS TEMED 40%

Stiffness Concentration Volume Volume Volume Volume Acrylamide &
(kPa) (%) (µL) (µL) (µL) (µL) Bis-acrylamide
(29:1) Volume
(µL)

1 2.7 500 461.6 2.5 1 34.9

4 4.6 500 437.1 2.5 1 59.4

25 9.7 500 371.2 2.5 1 125.3

16
APPENDIX B

Microfluidic master chips were produced on silicon wafers, which allowed them to be
used as masks for creating numerous identical copies in an efficient, cost-effective manner.
Microfluidic master chips were manufactured according to classic soft lithography methods.
Master templates were initially created by spin coating SU-8 (GM 1070) negative photoresist
onto plasma treated silicon wafers. Spin coating speeds were used according to SU-8
specification charts. Spin coating speeds carried according to target photoresist heights (10, 20,
40 µm). Coated silicon wafers were soft-baked at 65 °C and then 100 °C for 2 and 10 minutes,
respectively. Next, SU-8 coated silicon wafers were exposed to UV light via a mask aligner
(Kruss Suss mask aligner). SU-8 exposure dosage was obtained from the SU-8 specification
charts, while the wafer exposure time was determined using equation B.1.
mJ
desired exposure dose
Exposure time (s) = cm3 (𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 𝐵. 1)
mW
Light Intensity : 3 <
cm

UV exposure on the negative photoresist induces a chemical crosslinking which causes

exposed regions to become insoluble during the SU-8 developing step. A post exposure bake was
then performed on the silicon wafer at 65℃ and 100℃ for 2 minutes and 10 minutes, respectively.
The master chip was then placed into a glass petri dish filled with SU-8 developer for 10 minutes.
The wafer was rinsed with IPA and dried using a nitrogen gun. The master chip was left to hard-
bake at 100℃ for 10 minutes. SU-8 features were checked using a microscope and the heights were
confirmed to be within ±10% of the target heights set using a surface profilometer (AlphaStep).
After baking, the microfluidic device design protruded from the silicon mask, creating an inverse
copy of the device. Devices would be created by covering the master mask with PDMS and cross
linker. After baking, the PDMS device would be carefully removed from the master chip as to
ensure no photoresist protrusions were removed from the silicon master chip.

17
APPENDIX C

The following images reveal SUIT-2 cells suspended in media traveling from the inlet,
through 5 µm width channels and collecting towards the outlet. Cells are pulled through an
outlet tube and collect in a syringe filled partially with PBS. Cells are excreted in PBS onto a
glass slip and fixed.

Figure C.1 Cells are pulled via syringe-driven pressure from the inlet (pink) through 5µm
narrow constrictions towards the outlet. Narrow constriction length: 50 µm. Magnification 20x.

Figure C.2 Cells are constricted as they traverse 5 µm channels. Total cell diameter is reduced
~73% during constriction. Magnification 40x.
18
APPENDIX D

The following table illustrates a list of isolated mutant YAP plasmids, their function, and
concentration. These plasmids can be transfected into SUIT-2 cells to better illustrate the
mechanism of YAP nuclear translocation upon nuclear compression.

Table D.1 Function, Outcome, and Concentration of Mutant Plasmids

Plasmid Function Predicted Outcome Concentration

(ng/µL)

GFP-YAP Fluorescently tags YAP Fluorescent YAP 1381

translocates to nucleus
upon capillary
compression

YAP-S94A Mutant YAP plasmid. Decreases YAP nuclear 1432

Prevents YAP from binding to translocation
TEAD domain within nucleus

YAP-S127A Mutant YAP plasmid. Impairs Decreases YAP 724

YAP phosphorylation cytoplasmic retention

Rho-A Q63L Constitutively active Rho-A Enhances YAP nuclear 3126

translocation if actin is
necessary for
translocation

Nesprin KASH 1 Blocks main interaction of Decreases YAP nuclear 1600

LINC complex (protein translocation if
complex that mechanically actin/nuclear membrane
couples nucleus to interaction is necessary
cytoskeleton) for translocation

19
APPENDIX E

Despite its innovative design, the initial microfluidic devices contained a few drawbacks
that made their use difficult. Firstly, inlets on the initial design were placed too close together.
When punching inlet holes into the middle row of devices, holes would often overlap. If a biopsy
punch caused inlets to overlap, neither device could be used due to contamination. Thus, the user
would have to leave an inlet un-punched on one device, or punch deliberately into the channels
of both devices as to ensure no overlap. However, punching into the channels would create
uneven channel lengths, thereby creating inconsistent compression exposure times between
individual cells. Additionally, placement of inlets close to the edge of the device often led to
punches that led off the side of the device. In this case, fluid would leak from the inlet out of the
device and pressure would be significantly reduced. Secondly, initial focal adhesion dynamics
analysis suggests that the length of the narrow constriction within each device was insufficient.
Thus, two novel device designs were prepared using AutoCAD software to address issues
regarding ease of cutting and length of narrow constriction. Both proposed designs should reduce
errors during fabrication and enhance efficiency of testing.

Device 1 Design:

Figure E.1 AutoCAD drawing of microfluidic compression device design 1. Four inlet channels
with a diameter of 5 mm are punched in a rectangular fashion. Channel lengths were extended
from 1.4mm to 2.8mm. All four channels feed into a 1.5mm outlet.

20
Figure E.2 AutoCAD drawing of design 1 finalized silicon master mask. Four separate devices
are placed onto a single master mask design. Placement of the inlets alleviate issues of inlet
overlap and inlet cuts extending over edge of device.

Device design 1 still retains relatively long channels that connect the narrow
constrictions to the outlet. Due to these lengthy channels, it is likely that some cells will adhere
to the channels, slightly reducing the expected output of compressed cells. However, the
orientation of inlets in this fashion greatly reduces risk of error due to the cutting.

21
Device 2 Design:

Figure E.3 AutoCAD configuration of microfluidic compression device design 2. Four inlet
channels with a diameter of 5 mm are punched in a square fashion, with all inlets perpendicular
to one another. Channel lengths were extended from 1.4mm to 2.8mm. All four channels feed
into a 1.5mm outlet. Length between narrow constrictions and outlet is greatly reduced.

22
Figure E.4 AutoCAD configuration of design 2 master mask. Five separate devices are placed
onto a single master mask design. Reduction of channel length between narrow constrictions
and outlets allows for the placement of an extra device onto the silicon mask. However,
proximity of constriction channels to the exterior of the device raises probability of cutting
errors during fabrication.

Device design 2 arranges channel inlets in a compact manner. Reduction of channel

length between 7 µm constrictions and outlets reduces total channel wall area, which should
increase compressed cell output. Additionally, reduction in channel length allows for the
placement of an additional device within the silicon master mask. However, orientation of inlets
places the constriction channels in close proximity with the edge of the device, enhancing the
probability of cutting error during fabrication.

23