You are on page 1of 24

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
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,

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

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

M e ch a
Fo rc e


Adhesions mDia

Myosin ROCK G-actin F-actin

MLC-2 pMLC-2 Actomyosin


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 ultimately circulate in the bloodstream until they
exist regarding the efficacy of G1 on limiting reattach along the wall of a capillary near a
PDAC progression, recent work has indicated that secondary organ site. During the metastatic
GPER deficiency enhanced breast and liver process, cancer cells must undergo numerous
tumourigenesis.29,30 Figure 1 provides an intracellular changes to evade apoptosis, resist
illustration of the mechanosensitive signalling high amounts of shear stress, and adapt to
pathways associated with cancer progression unfavourable tissue conditions.31 Importantly,
described above. pancreatic cancer cells (10 – 20 µm) must also
withstand compression as they traverse through
The dismally low survival rate of PDAC can narrow constrictions in compressed capillaries (4-
be partially explained by its ability to rapidly 10 µm).32 Although the cytoplasm of migrating
disseminate through the vasculature to distant cells can smoothly deform in response to
organ sites. Indeed, it is estimated that metastasis mechanical force, growing evidence suggests that
is responsible for approximately 90% of cancer the enlarged nuclei of cancer cells are susceptible
deaths.7 The PDAC metastatic response initiates to mechanical rupture, which can lead to the
as malignant cells detach from the primary

uncontrolled transport of proteins between the fabricated according to a modified protocol
nucleus and cytoplasm.33,34 Previous studies have described in Tse et al.36 1 kPa gels mimicking
indicated that direct compression of tumour cells healthy tissue, and 25 kPa gels mimicking fibrotic
enable coordinated migration by enhancing cell- tissue were prepared by mixing TEMED (N, N,
substrate adhesion, however the direct N’, N’-tetramethylethylenediamine), phosphate
intracellular signalling pathways are not yet fully buffered saline (PBS), and ammonium
understood.25,32,33 Moreover, it remains unclear persulphate solution (APS) at varying
whether compression of circulating PDAC cells concentrations. A table containing exact volumes
through capillaries post-intravasation influences of reagents can be found in Appendix A. Glass
cell phenotype. coverslips were immersed in a hydrophilic 3-
propyl methacrylate solution for 5 minutes,
In this study, we seek to characterize the washed in 100% ethanol, and allowed to dry. 10
mechanosensory changes and mechano- µL of PA solution were then applied to the glass
mutations that arise in circulating tumor cells as coverslip and allowed to set for 45 minutes.
they traverse narrow constrictions and migrate
through stiff tissue. Importantly, we aim to YAP Translocation on Polyacrylamide Gels The
elucidate the effects of nuclear compression on surface of the PA gels were photoactivated with
YAP nuclear translocation, focal adhesion sulpho-SANPAH (sulfosuccinimidyl 6-(4’-azido-
formation, and cancer cell migration. To do this, 2’-nitrophenylamino) hexanoate) diluted in PBS
we will utilize polyacrylamide (PA) gels and high to facilitate fibronectin crosslinking, which is
throughput microfluidic devices with channels necessary for cell attachment to occur. Gels were
that mimic capillary diameter. Finally, we seek to then washed 3 times with PBS for 5 minutes each.
understand whether treatment of PDAC cells Finally, gels were incubated overnight at 4°C in a
with a G1 agonist limits the downstream human fibronectin (FN) PBS suspension. To
phenotypic response of PDAC cells. determine if G1 impedes tumourigenic
mechanotransduction pathways of PDAC, cells
METHODS were seeded onto the PA gels into three distinct
groups: Control SUIT-2 cells on 1 kPa PA gels,
Cell Culture In this study, SUIT-2 (S2-007) cells
Control SUIT-2 cells on 25 kPa gels, and G1
derived from the human PDAC cancer cell line
treated SUIT-2 cells on 25 kPa gels. Cells were
were used. SUIT-2 cells were cultured in 37°C, 5%
incubated for 24 hours on the gels and then fixed
CO2, in sterile, filtered Dulbecco’s modified eagle’s
using a 4% paraformaldehyde (PFA) solution.
medium high glucose (DMEM). Cell cultures
were supplemented with 10% foetal bovine Immunofluorescence Staining The ABCAM
serum, 1% fungizone amphotericin, and 1% immunofluorescence protocol was followed to tag
penicillin streptomycin. Additionally, for each the position of YAP within the seeded cells.37 Cells
experiment one subset of cells were treated with were permeabilized with 0.5% Saponin in PBS for
10 µm G1 in 10 mL of media and then incubated 10 minutes to allow the antibody to enter the cell.
for 24 hours. Cells were washed and incubated in 22.52mg/mL
glycine and 1% BSA in PBST for 30 minutes to
Polyacrylamide Gel Fabrication PA gels were
inhibit unspecific antibody binding. The primary
designed with varying rigidity to mimic the
antibody, YAP, was diluted in 1% BSA in PBS at
stiffness of healthy and fibrotic tissue, as
1:100 dilution and then used to incubate
described in Pelham et al.35 PA gels were
coverslips overnight at 4°C. Coverslips were then

washed and incubated with Alexa Fluor 488- Microfluidic Device Preparation Microfluidic
conjugated secondary antibody and phalloidin- compression device master silicon masks were
Alexa Fluor 546 in 1% BSA at room temperature obtained from Au laboratory and can be seen
for 1 hour. Finally, coverslips were washed, below in Figure 2.38 The microfluidic master chip
incubated with DAPI nuclear stain, and mounted fabrication protocol can be found in Appendix B.
onto glass slides. 18.0 g of PDMS and 2.0 g of cross-linker were
thoroughly mixed and poured onto the silicon
Image Acquisition Epifluorescent images of cells master mask within a petri dish. The PDMS and
were taken using the Nikon Eclipse Ti-E at 40x mask were placed into a vacuum for 1 hour to
magnification. Nuclear to cytosolic ratio of YAP remove air bubbles. The device was then heated
mean fluorescence intensity was determined at 70°C for 12 hours. Upon removal, individual
using the Fiji package on ImageJ. After PDMS chambers were sliced and carefully
subtracting background signal, the nucleus and removed from the silicon mask. 5 mm biopsy
cytoplasm were segmented. The nuclear to punches were used to cut inlets into the chamber,
cytosolic ratio of YAP was obtained by dividing while 1.5 mm punches were used to cut the outlet
the mean fluorescent intensity of the nucleus by of the device. The PDMS chips were sealed to
the mean fluorescent intensity of the cytoplasm. glass slides using via plasma-activated bonding
The mean fluorescent intensity of the cytoplasm and heated on a hot plate at 100°C for 1 hour.
was defined as the mean fluorescent intensity of Before introduction of cells, devices were cleaned
the nucleus subtracted from the mean fluorescent using 70% ethanol, rinsed with PBS, and treated
intensity of entire cell. 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
Figure 2. Microfluidic Compression Chamber Design. translocation and nuclear compression ratios
5mm inlets lead to hundreds of 7µm diameter channels. upon constriction. Mean fluorescence intensity of
7µm channels converge at 1.5mm outlet. Each silicon nuclear YAP and cytoplasmic YAP were obtained
mask contains 5 unique chambers that are cut from the
silicon master to create 4 devices.

to determine the nuclear to cytosolic ratio of YAP adhesion adaptor protein, Paxillin. Images were
upon constriction. obtained using total internal reflection
fluorescence (TIRF) microscopy at 60x
Focal Adhesion Characterization Upon magnification to determine the area, length, and
Compression To determine the effect of capillary- number of focal adhesions within each cell.
induced compression on PDAC migration and re-
attachment at a secondary site, focal adhesion Focal Adhesion Quantification and Analysis
dynamics were assessed. SUIT-2 cells were ImageJ was used to analyse area, length, and
cultured on 1kPA PA gels for 24 hours and then quantity of focal adhesions in each cell. For 60x
split into four subgroups: control-compressed, magnification, a scale of 9.30 pixels/µm was
control-uncompressed, G1-treated compressed, implemented. Image files were then converted to
and G1-treated uncompressed. Control grayscale. Image contrast was then enhanced to
uncompressed SUIT-2 and G1-treated clearly illustrate the boundary of cell so that cell
uncompressed SUIT-2 cells were collected from area could be determined. The image was then
the 1 kPa gel, seeded onto glass, and fixed using manually thresholded and made binary using the
4% PFA. Meanwhile, control and G1-treated cells Threshold tool. Finally, the analyse particles tool
were passed through the compression device, was utilized to determine the area, number, and
collected, seeded onto glass, and fixed with 4% Feret diameter of each focal adhesion. A lower
PFA. To visualize focal adhesion complexes upon limit of 0.11µm2 was set for focal adhesion area as
compression, all groups were immunostained for previous studies have indicated that focal
adhesions must exceed this area to generate
A B 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.


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
Figure 3. Thresholding and Quantification of Focal rounded shape, while cells cultured on a stiff 25
Adhesions. (A) Immunofluorescence images of Paxillin kPa substrate are spread and spindle shaped.
(green) in G1-treated uncompressed SUIT-2 cells at 60x Notably, treatment of cells cultured on 25 kPa
magnification. (B) Grayscale converted image. (C) substrates with G1 reduced actomyosin
Manually thresholded binary image. (D) Automated focal contractility and reverted cell morphology back to
adhesion regions determined by Analyse Particle tool. a rounded shape.


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.
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
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-

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

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.
areas for each cell were summed and divided by that GPER serves as a suitable target to limit the
the cell area to create a normalized focal adhesion activation of Rho-A, hence abrogating actomyosin
area, which accounted for the relative size of the contractility and focal adhesion formation in
cell (Figure 7D). The control group exhibited response to mechanical force.
nearly no change in percentage focal adhesion of
cell area, rising only from 12.99% to 13.28% post- Immunofluorescence staining of SUIT-2
compression. Meanwhile, G1-treated cells saw a cells cultured on varying rigidity substrates was
significant increase in percentage focal adhesion also performed to elucidate whether YAP
of cell area, from 12.88% to 16.11% (p < 0.05). translocation is mediated via GPER signalling.
Cells cultured on 1 kPa substrate exhibited
DISCUSSION primarily cytoplasmic YAP concentrations (Figure
5A, D), while cells seeded on a 25 kPa
The aim of this study was to determine demonstrated a significant influx of YAP into the
how mechanical compression of PDAC cells by nucleus (Figure 5B, D). These results confirm
narrow capillary constrictions influences previous reports that YAP nuclear translocation is
downstream Rho-A pathways associated with cell indeed mechanosensitive.21,23,24 Previous studies
migration and adhesion. Moreover, we sought to have hypothesized that Rho-A-mediated F-actin
understand whether activation of GPER by its G1 formation directly flattens the nucleus by forming
agonist abrogated these downstream intracellular a mechanical link between the nucleus and the
changes by characterizing changes in YAP nuclear cell membrane in response to elevated tissue
translocation and focal adhesion dynamics post- rigidity.25 Elosegui-Artola et al. posit that this
compression. nuclear membrane flattening widens the exterior
of nuclear pores, which could enhance nuclear
Cell cultures on stiff (25 kPa) and soft (1
import and inhibit nuclear export of large
kPa) substrates confirmed previous results that
cytoplasmic proteins, including YAP.25
cell morphology is directly mediated by substrate
Importantly, the introduction of G1 resulted in a
rigidity.16,18,38 As cells probe their mechanical
significant decrease in YAP nuclear translocation
environment, they continuously adjust their
(Figure 5C, D), suggesting that YAP nuclear
internally developed forces to match the
translocation is biochemically dependent on
resistance offered by the ECM. During this
GPER activation. It is likely that GPER activation
process, Rho-A becomes activated to facilitate the
inhibits Rho-A and F-actin formation, blocking
formation of filamentous actin (F-actin) via the
the cytoskeletal link to the nuclear membrane.
formin, mDia-1.34 Rho-A activation mediates the
However, whether GPER activation can inhibit
phosphorylation of myosin light chain-2 (MLC-2),
YAP nuclear translocation when the nucleus is
a protein kinase necessary for actin contractility.39
mechanically compressed remains unclear.
The conjunction of excess F-actin and MLC-2
significantly enhances actomyosin contractility of To investigate whether mechanical
the cell, which leads the cell to adopt a spread squeezing of the nucleus by capillaries elevates
morphology when exposed to a stiff matrix, as YAP nuclear translocation, cells were transfected
shown in Figure 4B.35 However, introduction of with a fluorescent YAP-GFP plasmid to visualize
the GPER-1 agonist, G1, to SUIT-2 cells cultured live YAP location as cells passed through the
on 25 kPa substrate, likely deactivated Rho-A, compression microfluidic device. Unfortunately,
reducing the formation of F-actin and decreasing YAP-GFP transfections were largely unsuccessful,
actomyosin contractility. These results indicate and YAP nuclear translocation could not be

quantified. However, Hoechst nuclear staining Focal adhesion dynamics were assessed
did reveal that nuclear membranes were post-compression to determine whether
significantly flattened in control cells during mechanical compression altered the ability of
transport through narrow constrictions. Nuclear PDAC cells to reattach to capillary walls and
flattening must also be analysed in G1-treated extravasate toward secondary organ sites. Data
cells to determine if acytomyosin contractility is shown in Figure 6 illustrate no significant
necessary for sufficient nuclear constriction. relationship between mechanical compression
Furthermore, testing must be conducted to and focal adhesion area, length, or number.
confirm whether nuclear squeezing facilitates Although we hypothesized that mechanical
YAP nuclear translocation in the absence of compression would increase focal adhesion
biochemical activation. production due to the mechanical activation of
Rho-A, the data presented a significant decrease
Should future testing confirm that YAP in focal adhesion quantity for control cells
indeed translocates to the nucleus upon exposed to mechanical compression. This would
compression, additional testing must be suggest that mechanical squeezing of the nucleus
conducted to determine the mechanism of action. actually hinders the ability of PDAC to reattach
While previous studies have suggested nuclear and migrate to secondary organ sites. The results
flattening as a potential mechanism for YAP also suggest that percentage focal adhesion area
translocation, others have indicated that nuclei of cell area increased significantly for G1-treated
are mechanically ruptured during transport cells post compression. This runs counter to our
through narrow constrictions.25,33,34 During nuclear hypothesis that GPER activation would inhibit
ruptures, proteins, such as YAP, are able to shuttle Rho-A-mediated formation of focal adhesions.
unregulated between the nucleus and cytoplasm.
The nuclear membrane reseals rapidly during There are a number of possible reasons
interphase, however, assisted by ESCRT why the results did not illustrate a significant
machinery (endosomal sorting complexes relationship between mechanical compression of
required for transport).40 Analysis of ESCRT levels circulating PDAC cells and focal adhesion
post-compression could elucidate whether the dynamics. First, the quantification of focal
nuclear membrane is ruptured during capillary adhesions proved to be difficult and subjective.
transport. Additionally, knocking down genes As shown in Figure 3, a manual threshold was
directly associated with YAP activation will used to determine what level of Paxillin
elucidate the effect of each protein on YAP fluorescence qualified as a focal adhesion. By
translocation. Constitutively activating or manually establishing the fluorescence threshold,
inhibiting factors within the YAP signalling quantification of focal adhesion number and size
pathway provides insight into how mechanical became subjective. Indeed, the analyse particle
force influences the role of each factor in the tool would struggle to differentiate between a
signalling pathway. A table illustrating the conglomerate of smaller focal adhesions and one
functions of each mutant YAP plasmid and its large focal adhesion. This inability to differentiate
respective predicted outcome can be found in between small and large focal adhesions was
Appendix D. These findings may provide insight partially responsible for the large variation in
into potential therapeutic targets to mitigate focal adhesion size and length. For future
mechano-mutations associated with metastasis. quantification of focal adhesion dynamics, image
quality and immunostaining techniques must be

improved to more accurately assess focal It would be interesting to explore how capillary-
adhesion dynamics. induced compression influences cancer cell
reattachment and collective migration. It would
It also likely that PDAC cells were also be beneficial to explore whether introduction
compressed for an insufficient duration while of the G1-agonist influences invasive behaviour.
passing through the narrow constrictions. As Furthermore, studies should be conducted to
cells were manually drawn through the device via explore the relationship between PDAC and
syringe, it was difficult to control flow velocity. ECM-secreting PSCs as they migrate through
While some cells passed slowly through the capillaries. Recent work in our group confirmed
device, others travelled rapidly. Thus, time of that mechanical rigidity is sufficient for PSC
compression was inconsistent across cells. This activation. Future studies should explore
issue could be solved via the introduction of an intracellular changes and mechano-mutations
automated pump which consistently controls the within PSCs due to mechanical compression
flow of fluid through the device. It would also be through narrow constrictions. Moreover, it would
beneficial to quantify fluid shear stress be valuable to evaluate the relationship between
experienced by PDAC cells as they traverse mechanically compressed PDAC cells and PSCs
channels using computational fluid dynamics as they migrate to secondary organ sites.
models. Shear stress could be controlled by
altering flow rates to ensure consistent stress is CONCLUSION
applied to cells as they pass through the device.
Additionally, the length of channels should be This study has helped to elucidate the
extended to ensure that all cells travelling relationship between substrate rigidity and
through the device experience sufficient intracellular changes associated with cell
compression. Novel compression device designs adhesion and cell proliferation. Importantly, it
were prepared in which the length of each demonstrated that GPER activation directly
channel was doubled from 1.4 mm to inhibits YAP nuclear transport in the presence of a
approximately 2.8 mm. These devices were also stiff substrate. These results suggest that GPER
designed to minimize spacing issues associated serves as an attractive potential therapeutic target
with punching inlets during fabrication. for inhibiting cancer-associated Rho-A
Implementation of these devices would downstream effectors. Unfortunately, the results
significantly increase compressions times of cells did not clearly indicate whether mechanical
and reduce errors associated with fabrication. constriction of PDAC cells enhanced nuclear YAP
These device designs are shown in Appendix E. translocation or significantly altered focal
adhesion dynamics. Further experiments must be
Future work should explore the invasive conducted to understand whether constriction of
behaviour of PDAC cells post-compression. PDAC cells during metastasis influences
Although focal adhesions give important insight downstream cancer-associated signalling
into a cell’s reattachment ability, microfluidic pathways and cell behaviour.
devices should be implemented to explore
PDAC’s ability to extravasate post-compression. Acknowledgement
Novel microfluidic device designs in our group I would like to thank Dr. Armando Del Río
have been implemented to explore the dynamics Hernández, Dr. Sam Au, Gulcen Yeldag, Julian
of pancreatic cancer extravasation from capillaries Ashby, and Carlos Matellan for their assistance
and collective migration to secondary organ sites. and guidance during this experiment.

REFERENCES cytoskeleton tension. J Cell Sci., 125, 5110-
5123 (2012).
1. Adamska, A., Domenichini, A. & Falsca,
M. Pancreatic Ductal Adenocarcinoma: 10. Rice, AJ.; Cortes, E; Del Rio Hernandez, A.
Current and Evolving Therapies. Int. J. Matrix Stiffness Induces Epithelial-
Mol. Sci. 18, 1338 (2017). Mesenchymal Transition and Promotes
Chemoresistance in Pancreatic Cancer
2. Smit, V.T., Boot, A. KRAS codon 12 Cells. Oncogenesis, 6 (2017).
mutations occur very frequently in
pancreatic adenocarcinomas. Nucleic Acids 11. Liu, C.; Liu, Y.; Xie, H.G.; Zhao, S.; Xu,
Res. 16, 7773-7782 (1988). X.X.; Fan, L.X. Role of three-dimensional
matrix stiffness in regulating the
3. Lin, W.C.; Wagner, K.U. Cancer cell chemoresistance of hepatocellular
dormancy in novel mouse models for carcinoma cells. Biotechnol Appl Biochem,
reversible pancreatic cancer: a lingering 62, 556–562 (2015).
challenge in the development of targeted
therapies. Cancer Res., 74, 2138-2143 (2014). 12. Cox, T.R; Erler, J.T. Remodeling and
homeostasis of the extracellular matrix:
4. Rhim, A.D., EMT and dissemination implications for fibrotic diseases and
precede pancreatic tumor formation. Cell cancer. Dis Model Mech, 4, 165-178 (2011).
148, 349-361 (2012).
13. Friedl, P. and K. Wolf, Tube travel: the role
5. Carrato, A., Falcone, A., Ducreux, M. A of proteases in individual and collective
systematic review of the burden of cancer cell invasion. Cancer Res, 68, 7247-
pancreatic cancer in Europe: Real-world 72419 (2008).
impact on survival, quality of life, and
costs. J. Gastrointest. Cancer, 46, 201-211 14. Yusko, E., Asbury, C., Force is a signal that
(2017). cells cannot ignore. Mol Biol Cell, 15, 3717-
3725 (2014).
6. Stark, A.; Guido, E. Pancreatic Ductal
Acarcinoma. Pancreapedia, 1, 399-408 15. Goodwin, K., Ellis, SJ., Basal Cell-
(2015). Extracellular Matrix Adhesion Regulates
Force Transmission during Tissue
7. Paszek, M.J., Zahir, N., Tensional Morphogenesis. Dev Cell, 39, 611-625
homeostasis and the malignant (2016).
phenotype. Cancer Cell, 8, 241-254 (2005).
16. Lesman, A., Notbohm, J., Contractile
8. Seyfried, T., Huysentruyt, L., On the forces regulate cell division in three-
Origin of Cancer Metastasis. Crit Rev dimensional environments. J Cell Biol. 205,
Oncog, 18, 43-73 (2014). 155-162 (2014).
9. Coyer, SR., Singh, A., Nanopatterning 17. Lessey, E., Guiluy, C., From mechanical
reveals an ECM area threshold for focal force to RhoA activation. Biochemistry. 51,
adhesion assembly and force transmission 7420-7432 (2012).
that is regulated by integrin activation and

18. Liu, Z., Tan, J., Mechanical tugging force 27. Prossnitz, E., Barton, M., The G protein-
regulates the size of cell-cell junctions. coupled estrogen receptor GPER in health
PNAS, 107, 9944-9949 (2010). and disease. Nat Rev Endocrinol, 7, 715-726
19. Somlyo, A., Somlyo, A., Signal
transduction by G-proteins, Rho-kinase 28. Ren, J., Guo, H. GPER in CAFs regulates
and protein phosphatase to smooth hypoxia-driven breast cancer invasion in a
muscle and non-muscle myosin II. J CTGF-dependent manner. Oncol. Rep., 33,
Physiol, 522, 177-185 (2000). 1929-1937 (2015).

20. Amano, M., Nakayama, M., Rho- 29. Ignatov, T., GPER-1 expression decreases
Kinase/ROCK: A Key Regulator of the during breast cancer tumourigenesis.
Cytoskeleton and Cell Polarity. Cancer Invest., 31, 309-315 (2013).
Cytoskeleton, 67, 545-554 (2010).
30. Wei, T., G protein-coupled estrogen
21. Dupont, S., Role of YAP/TAZ in cell- receptor deficiency accelerates liver
matrix adhesion-mediated signaling and tumorigenesis by enhancing inflammation
mechanotransduction, Experimental Cell and fibrosis. Cancer Letters, (2016).
Research, 343, 42-53 (2016).
31. Huang, Q., Hu, X., Fluid shear stress and
22. Dupont, S., Role of YAP/TAZ in tumor metastasis. Am J Cancer Res, 8, 763-
mechanotransduction. Nature, 474, 179-183 777 (2018).
32. Weigelin, B., Bakker, G., Intravital third
23. Piccolo, S.; Cordenonsi, M., The biology of harmonic generation microscopy of
YAP/TAZ: hippo signaling and beyond. collective melanoma cell invasion.
Physiol Rev., 94, 1287-1312 (2014). Principles of interface guidance and
microvesicle dynamics. IntrqVital, 12, 1-32
24. Hong, W.; Guan, K., The YAP and TAZ (2012).
transcription co-activators: Key
downstream effectors of the mammalian 33. Denais, CM., Gilbert, RM., Nuclear
Hippo pathway. Seminars in Cell & envelope rupture and repair during cancer
Developmental Biology, 23, 785-793 (2012). cell migration. Science, 352, 353-358 (2016).

25. Elosegui-Artola, A.; Roca-Cusachs, P., 34. Lim, S., Ryan, Q., Nuclear envelope
Force Triggers YAP Nuclear Entry by rupture drives genome instability in
Regulating Transport across Nuclear cancer. Mol Biol Cell, 12, 3210-3213 (2016).
Pores, Cell, 171, 1397-1410 (2017).
35. Pelham, R., Wang, Y. Cell locomotion and
26. Yu, X., Zhang, Q., Activation of G protein- focal adhesions are regulated by substrate
coupled estrogen receptor 1 induces flexibility. PNAS, 94, 13661-13665 (1997).
coronary artery relaxation via Epac/Rap1-
mediated inhibition of RhoA/Rho kinase 36. Tse, J., Engler, A., Preparation of hydrogel
pathway in parallel with PKA. PLoS One, substrates with tunable mechanical
12 (2017). properties. Curr Protoc Cell Biol, 10 (2010).

37. ABCAM, Immunocytochemistry (ICC)
and Immunofluorescence (IF) Protocol,

38. Au, S., Storey, B., Clusters of circulating

tumor cells traverse capillary-sized
vessels. PNAS, 113, 4947-4952 (2016).

39. Guo, W., Frey, M., Substrate rigidity

regulates the formation and maintenance
of tissues. Biophys J., 90, 2213-2220 (2006).

40. Sah, V., Rho is required for Gaq and a1-

adrenergic receptor signalling in
cardiomyocites. J Biol Chem, 271, 31185-
31190 (1996).

41. Raab, M., Gentili, M., ESCRT III repairs

nuclear envelope ruptures during cell
migration to limit DNA damage and cell
death. Science, 352, 359-362 (2016).


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

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


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.
desired exposure dose
Exposure time (s) = cm3 (𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 𝐵. 1)
Light Intensity : 3 <

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.


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.

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


GFP-YAP Fluorescently tags YAP Fluorescent YAP 1381

translocates to nucleus
upon capillary

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

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


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.

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.

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.

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.


You might also like