Reviewer #1: Cancer mechanics are important for tumor biology and progression, but
the effects of mechanical forces on cancer cells are poorly understood. The authors
present methodology for applying compressive forces to cancer cells using a microfabricated
platform. They show that cyclic compression affects the intensity of actin-GFP and
nuclear morphology in ovarian cancer cells. Although this is an interesting system,
there are a number of issues related to novelty, biological relevance and interpretation
that need to be addressed.
Specific comments:
1) In the introduction, it is mentioned that "...cells are exposed to chronic compressive
stress from different sources such as tumour growth, displacement within stromal tissue
and hydrostatic pressure out of ascitic fluid in peritoneal cavity." This is not completely
accurate, as hydrostatic pressure will not deform the cell in the same way as anisotropic
compression. The forces would be isotropic for fluid pressure.
Asem et al. (2020) Scientific Reports, show that ascites-induced compression exist
in the peritoneal microenvironment due to the ascites volume that can reach >2L and
cause intraperitoneal pressure (IPP) to rise from normal values of 5 mmHg to as high
as 22 mmHg (~3 kPa). Ovarian cancer cell adhesion to peritoneum increased due to the
impact of ascites-induced compression, shown in in vivo assay.
Asem et al. also applied a pressure of ∼3 kPa on OVCAR5 or OVCAR8 cells added atop
the mesothelial surface of murine peritoneal explants ex vivo for 24 hours in a bulk
compression system. As a result, they observed enhanced interaction between peritoneal
mesothelial cells and cancer cells via induction of tunneling nanotubes (TNT), which
later lead to metastatic ovarian cancer progression. These compression-induced TNTs
are actin based and aid in trafficking of mitochondria between ovarian cancer cells
and mesothelial cells.
The other consequence of the ascites-induced compression through hydrostatic pressure
in the peritoneal cavity has been a more linear anisotropic alignment of peritoneal
collagen fibers. Such alignment of collagen is a common indication for enhanced invasion
of solid tumors.
Overall, Asem et al. have found that ascites-induced compression promotes metastatic
ovarian cancer progression.
This information has been summarized and added in the first paragraph of the Introduction:
“…….Ascites-induced compression exists in the peritoneal microenvironment due to the
ascites volume that can reach >2L and increases ovarian cancer cell adhesion to peritoneum,
shown by Asem et al. using in vivo assay [7]. This peritoneal microenvironment is
continuously affected by the movements of the surrounding organs, musculoskeletal
dynamics, and gravity [6].…… ”
2) The rationale for using matrigel is not clear. Collagen and/or fibronectin would
be more representative.
In the Materials and methods section “Cell culture, Matrigel coating and chip loading
with cells” it was noted that “…… Matrigel was previously shown to facilitate cancer
cell adhesion and mimic the extracellular matrix (ECM) microenvironment in comparison
to other substrates ….. .”
It has been also added into this section and further referenced that “Matrigel is
commonly used basement membrane extracellular matrix for providing improved microenvironmental
and physiological conditions in in vitro culture assays of cancer cells by its composition
[20-23].”, as shown by such as Kleinman et al (1982) Biochemistry, Mullen et al (1996)
Int J Cancer, Kleinman et al (2005) Seminars in Cancer Biology, and Vaillant et al
(2011) Breast Cancer.
Major components of Matrigel are laminin, collagen IV, heparan sulphate proteoglycans,
entactin and nidogen (Kleinman et al.,1982, Biochemistry). It also contains various
growth factors such as TGF-�, epidermal growth factor, fibroblast growth factor and tissue plasminogen activator,
insulin like growth factor which are naturally occurring in tumor microenvironment
(Corning Inc.). Thus, Matrigel is rich in extracellular matrix proteins and polymerises
under normal physiological conditions to produce a reconstituted, biologically active
matrix material which is effective for the attachment and differentiation of cellular
material as shown by Kleinman et al (1982) Biochemistry.
It has been also added in Conclusions “Similar to Matrigel coating used in this study,
our platform and established chip loading method would allow coatings of other hydrogel
types such as collagen and fibronectin”, which could be tried as in the method shown
in Figure 1(b).
3) Does the disruption of the cytoskeleton depend on the rate of application of the
force? If the piston is applied more slowly, do the cells have time to adjust and
avoid damage?
Regarding deformation and disruption of actin cytoskeleton, Figure 3 and Figure 4
show how live cell actin cytoskeleton behave under dynamic compression applied over
time in cyclic mode at a physiological value and sequentially in varying applied pressures
in ascending order, and are out of at least 3 independent experiments. In text, while
explaining the associated figures, the representative pressure profile for sequential
cyclic compression process was indicated to be in Figure 1(d): “Similar as to the
sequence shown in Fig 1(d), first, a pressure amount ladder was formed up to a pressure
in the mild compression range, represented by an externally applied pressure of 350
mbar and resulting in a piston contact pressure of 15.6 kPa, during which the piston
reached the cells at the bottom surface and cells started to distinctly deform.”
We think that this pressure application is gradual enough for cells to adjust to the
varying pressures during sequential cyclic compression application. Mechanical cell
damage that we see at higher pressures is intrinsic to cell response to higher amounts
of applied pressure. The following explanation has been added at the end of the 1st
paragraph in section “Live cell actin profiles of cancer cells during sequential cyclic
compression”:
“Throughout the application of sequential cyclic compression, cells were allowed to
adjust to each next higher applied pressure. As shown in the pressure amount ladder
in Fig 1(d), pressure was applied gradually in 30 s among rising steps of the ladder
until the piston reached the focal plane of the cells and started cell deformation.
After observing that cells were distinctly deformed at a mild compression level but
not to a disruption or lysis level, compression was set to be in cyclic mode. Although
the pressure was changing suddenly along the cycles as per the settings of the selected
cyclic mode in the pressure controller, cells were not damaged when compressed as
they resulted in being resistant at this physiological value. Next, pressure application
was continued with gradual increase from 350 to 370 mbar, from 370 to 400 mbar and
from 400 to 640 mbar in 30 s for each, as shown in Fig 1(d). Thus, cells could have
the time to adjust during the pressure increase and show their intrinsic response
to the pressurized stage of each compression level.”
4) In Figure 3, it looks like some labeled cells are actively moving away from the
area of the piston. Did this affect the measurements? Related to this, the authors
might refer to doi.org/10.1073/pnas.1118910109.
For CTCF calculations as shown in Eq (1) (format of the equation has been revised)
in methods section “Imaging and data analysis”, while analysing the cells under micro-piston
we must draw an ROI and set it as threshold that separates the analysis area (Area
of micro-piston ROI) from the rest which is control group. We set an ROI from the
beginning and then we do not change its boundaries during the frames taken along the
stages of the dynamic compression process. The way we decided the micro-piston ROI
is shown as in the ROI (yellow frame) drawn the videos in the links, we accept slightly
larger area which is drawn based on the pressurized from of the micro-piston.
Phase contrast with micro-piston ROI: https://www.dropbox.com/s/sixcpmtw2abteth/S300um_3pistonSKOV-3%20actin-GFP_phasecontrast_withROI.avi?dl=0
Actin-GFP with micro-piston ROI: https://www.dropbox.com/s/1ww7p90el7t3ej1/S300um_3pistonSKOV-3%20actin-GFP_withROI.avi?dl=0
Cells are partially compressed partially not under the micro-piston periphery (boundaries
of the ROI), so in CTCF calculations the compressed and then buckled back parts of
the cells were taken into account. This was explained as in the last paragraph in
section “Live cell actin profiles of cancer cells during sequential cyclic compression”:
“The current device also allowed the response of individual cells at the interface
between compressed and non-compressed regions in the periphery of the micro-piston
to be studied, thus providing a degree of control of the localization of compression
(see S2 Movie). As depicted in the video, parts of cells at the interface were subject
to compression, while the rest remained uncompressed. Thus, the cell actin was deformed
and damaged only partially in the compressed part depending on the magnitude of the
applied pressure, whereas it remained more intact in the non-compressed part. This
functionality which is unique to our platform by having control and compressed groups
in the same microdevice, is useful to mimic partial cellular deformation and damage
that can occur in vivo in presence of a localized mechanical loading.”
Cells under the periphery of the micro-piston are mainly changing shape in partial
and some are being pushed away at compressed stage due to the physical presence of
the micro-piston edges and accumulated cell debris towards the end of process when
higher pressures were applied. For the cells that might be moving away from the area
of the micro-piston, such as the cell at the top right corner of the representative
actin-GFP video in S2 Movie (in the links here as well), which looks pulled its body
part and changed its shape to move away from the contacting piston, the reviewer is
right, such cells might get impacted by the pressurized pressure and sensing the mechanical
stress loading and move away from the stress. However, to better understand this sensing
and movement of the cells away from the stress (whether this is a mechanism for the
cell to protect itself from the mechanical stress) we need to observe more cells doing
that. On the other hand, we are more confident on that majority of the cells were
deforming, being pushed when the piston was contacting and then buckling back when
the stress was removed and remained there as partially compressed and damaged (at
high pressures towards the end of the process) and partially intact at the control
region (that was outside the micro-piston ROI as threshold area).
Regarding movement of the cells away from under the micro-piston periphery, we suspect
that confluency would be important as well. We expect that if the culture is confluent
enough cells might not find enough space to move away, while in the less confluent
cultures cells would be able to show their response better to decide whether to stay
partially under the pressure or move away to a potentially available empty space nearby.
We see the reviewer’s observation invaluable to be further investigated in such confluency-controlled
samples under compression with tracking of the cells at the periphery of the micro-piston
in the future work.
5) Are the differences in cell response (and death) seen in a given compression experiment
related to cell cycle?
We have not looked at the impact of compression on cell cycle in our experiments in
this manuscript. However, we have extensively studied impact of compression on cell
viability, running multiple assays in our previous reports (e.g. Onal et al 2021 Frontiers
in Physics). Pressures that cause cell death and thus decrease in cell viability were
well characterized there and thus these were referenced here at the beginning of the
section “Live cell actin profiles of cancer cells during sequential cyclic compression”:
“For this, the actin-GFP transduced cancer cells were dynamically stimulated and compressed
with various intermediate actuation pressures in a sequential cyclic manner, namely
following a sequence from Mild (15.6–15.9 kPa) to Intermediate 1 (23.8–26.8 kPa),
Intermediate 2 (37.8–41.7 kPa) and Severe (127.8–140 kPa) piston contact pressures.
The first two ranges of this sequence fell within physiological and upper physiological
pressure values and cell viability was high (on average 94 % for Mild and 77 % for
Intermediate 1), while Intermediate 2 and Severe levels constitute hyperphysiological
values and cell viability decreased (on average 43 % for Intermediate 2 and 20 % for
Severe) [5].”
Thus, our previous report showed mechanical stress induced cell damage and death towards
higher pressures so categorized as Intermediate 2 and Severe pressures, providing
the degree of the damage in detail in Onal et al 2021 Frontiers in Physics. Cell viability
results for the applied pressure ranges were reminded from Onal et al, 2021, Frontiers
in Physics to give an idea at what viability states the cells were at these pressure
ranges while investigating live cell actin cytoskeleton under dynamic compression
in the current manuscript.
For the cell cycle itself e.g. impact of mechanical compression on mitosis, or other
cell cycle phases, this looks beyond the scope of current work, yet, worth looking
at in future work.
6) In Fig 6 b, it appears that there is a difference between the two "0 (CR)" values.
Was this significant, and can the authors discuss the details of these samples?
All the statistical significance values for nuclear deformations in compressed and
recovered cells after compression are noted in S2_File, newly added as supplementary
file.
S2 File caption “Statistical significance levels among all groups, obtained with Student's
t-test run for nuclear deformations in compressed and recovered cells after compression
presented in Fig 5 and Fig 6.” has been added. Fig 5 and Fig 6 captions have been
also updated.
We added the following result and discussion for the difference between the two "0
(CR)" values: “There is statistically significant change in nuclei area between the
control groups of zero-time and of 24 h-recovery after compression (see S2 File).
Although these are not exactly the same control groups in the same conditions and
we intend to compare the test group (e.g. zero-time or 24 h-recovery after compression)
with their own control groups, we consider that the observed significant difference
could be due to cell-cell signaling between the cells in compressed and control groups
during that 24 h-recovery period after compression. In the groups of zero-time after
compression, we did not allow control and compressed cells enough time to further
interact with each other as they were fixed right after the compression.”
We further added the following discussion for the absence of statistical change in
nucleus area between the control and compressed groups at zero-time after compression,
in the associated paragraph: “While we expect a statistically significant change between
the compressed and its control group at zero-time after compression at 20.8 kPa similar
to the results observed in our previous report [5], we attribute the absence of such
a significant change to cells being cultured on Matrigel-coating here as opposed to
PLL-coating used in the previous experiments. Since Matrigel is expected to better
represent the cancer cell microenvironment, it thus aided in cell growth into denser
monolayers and formation of supporting extracellular matrix layer, that can alter
sensing of the applied stress for the compressed cells.”
7) The meaning of "plasticity" should be defined more clearly. Does plasticity refer
to mechanical lack of elasticity? If so, why would we expect this to be related to
loss of actin-GFP? Also, plasticity would be reflected by a lack of recovery of the
cell to its original state. Or are the authors simply analyzing biological processes
that restore the cell after stress?
For analysis of actin-GFP signal we had done CTCF calculations and have now improved
the graph to show the statical significances in Fig 4 in the revised manuscript. When
there was statistically significant loss in actin-GFP signal and as seen on S2 Movie,
actin disruption happens and that indicates for plastic response of the cells at those
higher pressures. We added a discussion in the last paragraph of section “Live cell
actin profiles of cancer cells during sequential cyclic compression”: “The observed
disruption of actin cytoskeleton in these experiments indicates for cell plasticity
during a cyclic compression in ovarian cancer when varying higher pressures occur
in the microenvironment. There is need for more studies in literature to compare our
results on cell plasticity after cyclic or dynamic compression applied sequentially
up to high pressures. For cell actin deformation at milder pressures, however, a distinct
change in actin cytoskeleton morphology was observed by Asem et al. for mesothelial
cells, interacting with ovarian cancer cells, under compression that is statically
applied at ∼3 kPa for 24 h in a bulk compression system. As a result, the interaction
between ovarian cancer cells and mesothelial cells by enhanced formation of actin-based
tunneling nanotubes (TNTs), and hence metastatic progression in ovarian cancer, was
promoted under compression [7].”
In the section Conclusions it has been also added that “While actin deformation at
mild pressures (e.g. 15.6 kPa) in cyclic mode indicates for cell elasticity, the resultant
actin disruption indicates for cell plasticity that can emerge when ovarian cancer
cells are exposed to varying and higher pressures in a microenvironment.”
The reviewer is right on that plasticity refers to that mechanical lack of elasticity.
If there is a lack of recovery of the cell to its original state due to, for instance,
actin disruption, we think this is because of cell plasticity (plastic response of
cells), as well. However, the cell being not recovering to its original state does
not always refer to cell plasticity (mechanical lack of elasticity), especially at
lower intermediate pressure values such as 20.8 kPa. The cell could be adapting to
this pressure level and acquire a new phenotype while it is still elastic despite
that there is an incomplete shape recovery to its original state.
We have gone through the entire manuscript and checked the parts where elasticity,
plasticity and recovery words are used and revised the associated parts accordingly
to prevent confusions in case of that the terms had been used interchangeably.
Thus, wherever further slight changes were done, those are also highlighted in the
revised sentences as in the following.
The following sentence in the last paragraph of Results and discussion has been revised:
“Furthermore, the data obtained in this work suggest that ovarian and likely other
cancer cells can exhibit a plastic cell response to applied sequential cyclic compression.”
revised to
“Furthermore, the data obtained in this work suggest that ovarian and likely other
cancer cells can exhibit a varying cell response to applied sequential cyclic compression.”
In the paragraph starting as in the following in Introduction:
“In the presence of a mechanical stress, cells strive to restore their shape to maintain
cell integrity after the removal of mechanical loading. However, cells may show a
plastic response in the form of cytoskeletal bond ruptures and an incomplete cell
shape recovery [1]. Meanwhile, cells produce an adaptive mechanism via these bond
ruptures to reduce the mechanical cell stress and thus protect themselves against
mechanical damage, while subsequently deforming under the mechanical load [1,8].”
3rd sentence has been revised to
“Meanwhile, cells produce an adaptive mechanism to reduce the mechanical cell stress
and thus protect themselves against mechanical damage, while subsequently deforming
under the mechanical load [1,10].”
Plastic word has been removed in the following sentence in Introduction:
“Using a single cell microfluidic compression platform, their application resulted
in full recovery and thus no permanent plastic deformation in MCF10A normal breast
epithelial cells after compression [2].”
Plastic word has been removed in the following sentence in Results and discussion
section “Actin cytoskeleton and nuclei profiles of cancer cells after compression
and recovery”:
“No statistical difference in cell height before and after compression was observed,
indicating that the breast epithelial cells did not exhibit permanent plastic deformation.”
8) Similarly, the meaning "recovery" should be defined more clearly. The authors write
"The extent of recovery of compressed cells, inferred by imaging cell membrane bulges
and actin cytoskeleton and measuring the shape descriptors of cell nuclei, can give
insights into the plasticity of cancer cells." However, there are no measurements
of cell membrane bulges, and no spatial analyses of actin organization. How do we
know the cells have "recovered"?
Our response to this question is in agreement with the response to previous (7th)
question of the reviewer.
Indeed, we used the recovery word in this manuscript for the investigation of cell
response after compression, whether they fully restore their shape or not, allowing
cells enough time. Thus, it refers to a process to investigate what happens to cells
after being exposed to compression.
To prevent confusion the sentence in the last paragraph of Introduction has been revised
to a clear form: “The extent of recovery of compressed cells can give insights into
the cell integrity and adaptation of cancer cells to restore their shape or acquire
new one when exposed to mild or upper mild compressive stress at physiological levels.”
Cell membrane bulges formed at compressed stages are distinct in S1 Movie which include
huge amount of information per time-lapse imaging video. While membrane bulges in
cells observed in phase contrast live cell imaging are fixed directly after compression
and distinctly appeared as increased actin signal at cell edges when fluorescently
stained, their further analysis out of time-lapse videos would require an advanced
and automated image analysis method as label-free and high-content. Spatial analyses
of actin organization (in addition to CTCF calculations) would require imaging data
taken at higher resolution, via a super resolution microscopy for instance.
9) Other groups have published similar pressure-actuated devices for compressing cells,
so there should be more discussion of these studies and comparison with the presented
technology. In general, a more extensive literature review on in vitro analyses of
cancer cell solid stress would be useful.
The following paragraph that was in Results and discussion has been moved to Introduction
and slightly revised:
“To date, dynamic compression experiments in literature include frequencies of 0.1
– 30 Hz with an applied stress of 5.1, 9.3, 12.9 and 18.7 kPa on breast cancer cells
over short durations (30 – 300 s), as used by Takao et al., who observed a mixed mode
of apoptosis and necrosis-dominant cell death in mechanically compressed monolayers
in a bulk compression platform [18]. In contrast, work by Novak et al. used a frequency
of 0.05 Hz for cyclic compression applied at 3.9 – 6.5 kPa for 24 hours [3]. They
observed an increase in proliferation capacity and decrease in apoptosis for OVCAR3
and OVSAHO ovarian cancer cells cultured in 3D hydrogel components in a bioreactor
at millimeter scales. In another example, Ho et al. applied cyclic compression by
alternating between external pressures of 10 and 15 psi (68.9 and 103.4 kPa) at 0.5
Hz for 6 minutes [2]. Using a single cell microfluidic compression platform, their
application resulted in full recovery and thus no permanent plastic deformation in
MCF10A normal breast epithelial cells after compression [2]. In our work we add to
these examples by using the capabilities of our compression platform to apply cyclic
compression with a low frequency in microfluidic settings. In particular, we utilize
this functionality to provide a first investigation of the effects of cell compression,
deformation and recovery after compression on SKOV-3 ovarian cancer cells. As part
of this we provide evidence that the amount of the pressure and duration of the application
clearly affects morphology during cell compression and recovery. While compression
frequencies in an in vivo tumor microenvironment are not yet fully known, cyclic compression
is important for maintenance of cell culture during mechanical compressions in an
in vitro experimental setup, in particular as it enhances media flow underneath the
micro-pistons.”
10) Given the relatively rapid application and removal of compressive forces, there
must be some interesting fluid dynamics happening between the cells and the piston.
Can the authors estimate the changes in fluid pressure and shear stress on the cells?
This could also affect the cell biology.
This is very good point. We have not particularly looked at the shear stress effect.
We do not have horizontal, continuous flow on the cells in the channel, which means
the cultures were in static media, but while moving the piston at vertical direction,
the media also displace and that might create a negligible shear stress. However,
we believe that the solid stress that we applied with physically sharply contacting
surface of the micro-piston dominates the effect of compressive stress on cells, not
that of shear stress. Considering other stresses from the very beginning, we applied
the pressure gradually on the cells as demonstrated at the beginning of S1 Movie while
increasing the pressure at a gradual rate shown on the pressure ladder on sensor reading
graphs. Cells were experiencing the changing pressure mostly at vertical directions
in the form of compression.
There could be hydrostatic pressure which also translate into compressive stress while
deflecting the membrane, so we expect that hydrostatic pressure to be all around the
channel. Comparing the viability results among the different shapes and among different
compression states, studied extensively in Onal et al. 2021 Frontiers in Physics,
the cells in the control regions around the micro-piston were intact and alive throughout
the applications as well as that the cells under micro-piston responded according
to the applied pressure. This shows that other stress factors seem to not have an
influence on cells in terms of viability.
If we had a horizontal cell compression setup as Paggi et al. (2020, Sensors Actuators
B: Chemical) had in their design with a vertical membrane adjacent to the cell culture
chamber that is deflected at horizontal direction, we would need to be looking at
multi-modality of the forces and the combination of compressive and shear stress as
they did in their work. However, in our design, the membrane is located horizontal
to the cell culture chamber, and we have a piston to physically contact the cells
once compressed. Thus, we deflect the membrane vertically and the forces are mainly
in compressing mode at the vertical direction in the cell culture chamber.
Nevertheless, we believe that the reviewer made an interesting point here, worth looking
at combination of shear stress and compressive stress effects on cells further in
the scope of a next piece of work within cell compression field.
11) Further justification should be provided for the cyclic stress schedule chosen.
Musculo-skeletal tissues would be expected to experience such forces, but this is
not clear for ovarian cancer.
The cyclic stress schedule (i.e. 5-min compressed stage followed by 5-min rest stage
in each cycle) was chosen to allow cells enough time during each stage to complete
bulge formation or recovery, processes which were considered important for the investigation
of cell deformation and recovery after compression. Membrane bulge formation and recovery
processes are clearly observable in S1 Movie. This was explained in the following
paragraph in section “Comparison of the experimental and computational results in
micro-piston device”
“While inferring these results, the cell compression process was imaged in detail
in Matrigel-coated microchannels at a speed of 1 frame per 100 ms, as shown in S1
Movie. As illustrated by this time-lapse video, the platform allowed for the temporal
evolution of the dynamic cell compression to be investigated in each cycle while the
piston was contacting cells and applying compression. Distinct cell membrane bulges
could be observed to form during such compression at piston contact pressure of 15.6
kPa and recovery of cell membranes from these bulges was visible when pistons were
lifted off during rest stages. Although the platform is capable of temporal control
of cell compression (e.g. to decrease, increase or remove the pressure at any certain
time interval), duration of each stage in a cycle was set to 5 minutes, corresponding
to a 5-min compressed stage followed by 5-min rest stage in each cycle. This was chosen
to allow cells enough time during each stage to complete bulge formation or recovery,
processes which were considered important for the investigation of cell deformation
and recovery after compression. Sensor readings in S1 Movie showed that the micro-piston
actuated according to externally applied pressures with reliable control and could
be operated in short durations gradually or suddenly when needed.”
Further explanation and discussion have been also added in this section as in the
following paragraph:
“Cyclic compression in the study of Ho et al. was applied only for 6 minutes in total
[2], significantly shorter than the entire duration of a cyclic compression in this
work, and results presented here suggest that cell deformation at compression stage
and recovery at rest stage depends on the temporal length of the compression on cells.
Particularly observable in S1 Movie, cell membrane bulge formation slowly developed
during the compression stage while cells were responding to contact pressure at the
interface with the PDMS piston. This was despite the fact that the piston was actuated
onto the cells rapidly during cyclic compression as directed by the external pressure
controller. As shown by the pressure sensor readings in the same movie, PDMS membrane
and piston were consistently responding to amount, duration and mode of the applied
pressure. Thus, the gradual deformation of cells and bulge formation during compression
stages must originate in the response type of the cells to mechanical load when in
solid contact with the PDMS piston. During rest stages, where the compression was
lifted, cell bulges did not fully recover right after compression was removed, suggesting
that the cells require a certain amount of time to recover compressed cellular parts
(S1 Movie). Based on these observations of temporal evolution of the cell compression
application, and comparison of application durations in this study with the study
by Ho et al. [2], we hypothesize that this recovery also depends on the duration and
number of the cycles.”
Regarding sources of compression ovarian cancer cells are exposed to, further information
has been added in Introduction:
“While some cells experience permanent plastic deformation after a repetitive mechanical
tensile loading and unloading, the impact of such repetitive compression on deformation
of cells remains largely unknown [1,2]. As such, the ability to apply cyclic compression
is crucial for any experimental setup aimed at the study of mechanical compression
taking place in cell and tissue microenvironments [3–5]. For instance, in ovarian
cancer, cells are exposed to chronic compressive stress from different sources such
as tumor growth, displacement within stromal tissue and hydrostatic pressure out of
ascitic fluid in peritoneal cavity [3,6]. Ascites-induced compression exists in the
peritoneal microenvironment due to the ascites volume that can reach >2L and increases
ovarian cancer cell adhesion to peritoneum, shown by Asem et al. using in vivo assay
[7]. This peritoneal microenvironment is continuously affected by the movements of
the surrounding organs, musculoskeletal dynamics, and gravity [6]. Thus, ovarian cancer
cells might experience cyclic compression profiles during chronic exposure to compression
from different sources and due to anatomical location of the ovary. Mimicking such
compression profiles and physiological pressure values in an in vitro dynamic compression
system is necessary to further study impact of compressive stress in cancer.
Compressive stress is estimated to reach 18.9 kPa for human tumors and can exceed
20 kPa based on the experimental data from murine tumors, while those values can be
even higher in situ with the impact of the surrounding extracellular matrix in tumor
microenvironment [3,8]. Compared to other cancer types such as breast and metastatic
bone niches, compression studies have been limited for ovarian cancer, including for
investigating the impact of various amounts of applied pressures [6]. Pressure values
existing in literature that are applied for ovarian cancer have been in a range from
∼3 kPa to 6.5 kPa [3,7,9]. Ovarian cancer cell response at higher applied pressures
that can occur in the body need further investigation.”
Furthermore, it was noted in our manuscript that “While compression frequencies in
an in vivo tumor microenvironment are not yet fully known, cyclic compression is important
for maintenance of cell culture during mechanical compressions in an in vitro experimental
setup, in particular as it enhances media flow underneath the micro-pistons.”
Reviewer #2: The following are the attached Comments for Authors.
In this manuscript, authors characterized changes and recovery in actin fluorescence
signal and nucleus shape after applying cyclic compression to ovarian cancer cells.
However, there are several major issues with the current manuscript: 1) the introduction
lacks information and citations of previous work and does not address the importance
of this work, i.e., why apply cyclic compression on ovarian cancer cells; 2) Key parts
of the experimental design lack rationale, i.e. why 5 min pressure 5 min recovery,
how large is the piston/cell contact area, variability of the pressure within contact
area, etc. 3) Several results are questionable, as detailed the following comments;
and 4) Several results lack information on significance tests and number of independent
studies. As such, the reviewer recommends rejection.
1. The introduction is not clear what the similarities and differences are between
chronic and cyclic compression, and why cyclic compression is important to study in
ovarian and other cancer cells.
Chronic compression in this manuscript and in literature was meant to be that ovarian
cancer cells are under a dynamic compression from different sources.
Chronic word was removed from the following sentence to prevent confusion, as it was
meant to be that higher pressure amounts (total perceived stress which can originate
from chronic exposure to compression from different sources) and profiles that can
be experienced by ovarian cancer cells need to be mimicked in an in vitro dynamic
compression system and the impact on cells need to be studied further:
“Mimicking such chronic compression profiles and physiological pressure values in
an in vitro dynamic compression system is necessary to further study impact of compressive
stress in cancer.”
The first two paragraphs of the Introduction have been revised as follows:
“While some cells experience permanent plastic deformation after a repetitive mechanical
tensile loading and unloading, the impact of such repetitive compression on deformation
of cells remains largely unknown [1,2]. As such, the ability to apply cyclic compression
is crucial for any experimental setup aimed at the study of mechanical compression
taking place in cell and tissue microenvironments [3–5]. For instance, in ovarian
cancer, cells are exposed to chronic compressive stress from different sources such
as tumor growth, displacement within stromal tissue and hydrostatic pressure out of
ascitic fluid in peritoneal cavity [3,6]. Ascites-induced compression exists in the
peritoneal microenvironment due to the ascites volume that can reach >2L and increases
ovarian cancer cell adhesion to peritoneum, shown by Asem et al. using in vivo assay
[7]. This peritoneal microenvironment is continuously affected by the movements of
the surrounding organs, musculoskeletal dynamics, and gravity [6]. Thus, ovarian cancer
cells might experience cyclic compression profiles during chronic exposure to compression
from different sources and due to anatomical location of the ovary. Mimicking such
compression profiles and physiological pressure values in an in vitro dynamic compression
system is necessary to further study impact of compressive stress in cancer.
Compressive stress is estimated to reach 18.9 kPa for human tumors and can exceed
20 kPa based on the experimental data from murine tumors, while those values can be
even higher in situ with the impact of the surrounding extracellular matrix in tumor
microenvironment [3,8]. Compared to other cancer types such as breast and metastatic
bone niches, compression studies have been limited for ovarian cancer, including for
investigating the impact of various amounts of applied pressures [6]. Pressure values
existing in literature that are applied for ovarian cancer have been in a range from
∼3 kPa to 6.5 kPa [3,7,9]. Ovarian cancer cell response at higher applied pressures
that can occur in the body need further investigation.”
The following sentence that was in Results and discussion has been moved to Introduction:
“While compression frequencies in an in vivo tumor microenvironment are not yet fully
known, cyclic compression is important for maintenance of cell culture during mechanical
compressions in an in vitro experimental setup, in particular as it enhances media
flow underneath the micro-pistons.”
2. Page 2, line 9-11: “Such compression is estimated to reach 18.9 kPa for human tumours
and can exceed 20 kPa based on the experimental data from murine tumours [3, 7].”
Given the wide range of compressive stresses in different tumor models & species,
the authors should specify the physiological range of compression for ovarian cancer.
Regarding ovarian cancer, compression sources and current studies in literature and
expected compression values are now explained in the first two paragraphs of the Introduction
in our revised manuscript. Further compression values and findings for ovarian cancer
cell compression based on the ovarian cancer cell response to the applied pressures
in our study are presented in Results and discussion in this manuscript.
3. The introduction is not clear about the similarities and differences between chronic
and cyclic compression, and why cyclic compression is important to study in ovarian
and other cancer cells.
Same as the 1st question.
4. The innovations in the microdevice authors developed are not clear. What has been
developed in the past and what is new should be summarized in the Introduction.
Last two paragraphs of Introduction included information on what has been developed
in past and what is new. Now the two paragraphs are revised, and the last paragraph
has been further improved to better reflect on what is new.
“Thus, with the purpose of applying compression in a cyclic, dynamic and controlled
manner for the investigation of cell deformation and recovery, we have recently developed
a robust cyclic compression microfluidic method based on a flexible microdevice. This
platform provides extensive control of the amount, duration, and mode within a pressure
profile. Fabrication and characterization of the multilayer microfluidic platform,
and the observation of directional growth of cells, viability and mechanical cell
lysis as end point assays under static state and pressurized states within this platform
were demonstrated previously [4,5].”
“In this paper, we further extend the reproducibility and repeatability validation
of our flexible microdevice-based compression method via comparisons of experimental
and computational piston actuations for independent microdevices with different membrane
thicknesses. We then use the microfluidic platform for the in vitro application of
cyclic cell compressions that mimic biologically relevant compression profiles occurring
in cellular microenvironments. In particular, we demonstrate applicability of the
platform for the chronic exposure of ovarian cancer cells to repetitive compressive
stress. As such, the current work extends the use of the platform to the application
of cyclic compressions at and beyond physiological pressure values in a sequential
fashion to study dynamic biomechanical processes by recording GFP-tagged actin dynamics
of live cells under compression. In relation to our previous work which discussed
initial findings on cellular deformations observed at cyclically applied ascending
pressures, the current work also expands applicability by providing a more physiologically
relevant setting in form of a hydrogel coating within micro-piston device. We showcase
data of cellular deformations and recovery during and after cyclic compression at
mild (e.g. 15.6 kPa) and upper mild (e.g. 20.8 kPa) physiological pressure values,
obtained via end point assays of actin and nuclei deformations in compressed cells
at zero time or at 24 h-recovery after compression, showing flexibility and novel
use of our microdevice for the applications of not only cell compression but also
recovery. We demonstrate that the platform can control the strength and duration of
cyclic compression, thus providing a powerful new tool for the study of mechanobiological
processes, by which cell deformation, cytoskeletal and nuclear changes and recovery
in cells exposed to such compression can be readily captured.”
Revised to
“Thus, with the purpose of applying compression in a cyclic, dynamic and controlled
manner for the investigation of cell deformation and recovery, we have recently developed
a robust cyclic compression microfluidic method based on a flexible microdevice. Fabrication
and characterization of the multilayer microfluidic platform, and the observation
of directional growth of cells, viability and mechanical cell lysis as end point assays
under static state and pressurized states within this platform were demonstrated previously
[4,5]. The developed platform was characterized to provide extensive control of the
amount, duration, and mode within a pressure profile [5].
In this paper, we further extend the reproducibility and repeatability validation
of our flexible microdevice-based compression method via comparisons of experimental
and computational piston actuations for independent microdevices with different membrane
thicknesses. We then extend the use of the platform to the application of cyclic compressions
at and beyond physiological pressure values in a sequential fashion to study dynamic
biomechanical processes by recording GFP-tagged actin dynamics of live cells under
compression. To the best of our knowledge, this is the first demonstration of how
live cell actin behave under dynamic compression applied over time in cyclic mode
and sequentially at varying pressures in ascending order. Using time-lapse imaging,
we thus show a sequence of actin deformation and disruption events based on the amount
of the applied pressure in a flexible microdevice. The current work also expands applicability
by providing a more physiologically relevant setting in form of a hydrogel coating
within micro-piston device as shown in Fig 1. We showcase data of cellular deformations
and recovery during and after cyclic compression at mild (e.g. 15.6 kPa) and upper
mild (e.g. 20.8 kPa) physiological pressure values, obtained via end point assays
of actin and nuclei deformations in compressed cells at zero time or at 24 h-recovery
after compression. These experiments further prove flexibility and novel use of our
microdevice for the applications of not only cell compression but also recovery. Differences
observed between compressed cells fixed at zero time or after 24 h-recovery suggest
that SKOV-3 cells exhibit deformations at the time of the compression, a proposed
mechanism cells use to prevent mechanical damage. The extent of recovery of compressed
cells can give insights into the cell integrity and adaptation of cancer cells to
restore their shape or acquire new one when exposed to mild or upper mild compressive
stress at physiological levels. As demonstrated with SKOV-3 ovarian cancer cells,
biomechanical responses of cells to sequential cyclic compression and during recovery
after compression can be revealed in a flexible microdevice where cell deformation,
cytoskeletal and nuclear changes and recovery in cells exposed to such compression
can be readily captured.”
5. References supporting nucleus as mechanosensory are lacking. Kirby (2018) Nature
Cell Biology, Fu (2012) Lab on a Chip, etc. Additionally, past findings on nucleus
deformation in ovarian cancer cells and its functional effects should be introduced
as background to this study
We thank the reviewer for letting us know about these references. We have looked at
and cited them in the respective part of the Introduction “In itself, the nucleus
is a mechanosensitive organelle [11-13] …..”, along with Nava et al (2020) Cell.
We are not aware of ovarian cancer cell-specific cell nucleus deformation by mechanical
compression and its functional affects if such study results exist in literature.
However, we have added a literature search from various cancer types, supporting background
and motivation of this study:
“Morphological changes in nuclear structure, such as increased nuclear size, irregular
shape by grooving, convolutions and invaginations of the nuclear envelope and altered
organization by disturbed chromatin distribution, are commonly used cancer markers
by pathologists [15]. Mechanical compression through confinement aid in mechanical
adaptation of the cell as shown with Hela-Kyoto cancer cells in which stretching of
the nuclear envelope and upregulation of actomyosin contractility were observed [16,17].
Furthermore, irregular nuclear morphology of cancer cells aligns with altered expression
of nuclear envelope proteins such as lamins A/C or lamin B in cancers as detailed
by Denais et al., which has the capacity to promote metastatic processes [15]. Deformation
of the large and stiff nucleus is required during the passage of metastatic cancer
cells through the tight interstitial space and narrow capillaries in the microenvironment.
Thus, mechanical properties of the cell nucleus, such as deformability and adaptation
under compression, and its connection to the cytoskeleton may play a major role in
cancer metastasis [15].”
6. Page 7, Fig 2(b): it is not clear which are experimental data and which are simulated
pressures on the graph, and how well they match quantitatively.
Fig 2(b) was plotted for membrane thickness (on x-axis) vs. pressure sensor readings
(on y-axis). Those values (both on x- and y-axis) are from experiments that resulted
in a similar piston contact pressure. Before experiments those were computationally
simulated as shown in Fig 2(a) to predict what external pressures need to be applied
at different thicknesses to be able to get a similar piston contact pressure. Thus,
while Fig 2(a) shows simulations, Fig 2(b) shows comparison of experimental values
that were applied based on simulations. Because R2 resulted to be high among independent
experiments, giving a good correlation along different thicknesses and applied external
pressures, to obtain a similar piston contact pressure calculated based on simulations,
we think the model is robust to predict what external pressures should be applied
when there is a variation in membrane thickness.
Caption of Fig 2(b) has been revised as in the following:
“(b) Correlation of independent experiments with the pressure sensor read for the
externally applied pressure based on simulations run for the corresponding membrane
thicknesses, resulting in a similar piston contact pressure.”
Revised to
“(b) Correlation among independent experiments with the pressure sensor read for the
externally applied pressures applied based on simulations in (a) run for the corresponding
membrane thicknesses, resulting in a similar piston contact pressure.”
The 2nd sentence in the following part at the end of the 1st paragraph in section
“Comparison of the experimental and computational results in micro-piston device”
has been revised to make it clear that the calculated R2 was among independent experiments
with different membrane thicknesses operated under different amounts of externally
applied pressures that were predicted with simulations:
“A summary of the compression applied to cancer cells via different membrane thicknesses
for externally applied pressures and the resulting maximum piston contact pressures
is shown in Table 1. Fig 2b further shows that comparable piston contact pressures
were achieved by mildly compressing cells with applied external pressures, which were
experimentally matching the pressure amounts in simulations (R2 = 0.9967). ……”
Revised to
“……. Fig 2(b) further shows that comparable piston contact pressures were achieved
by mildly compressing cells with applied external pressures (R2 = 0.9967), which were
experimentally applied according to the pressure amounts in simulations Fig 2(a).
…….”
7. Page 6, line 198: “maximum piston contact pressure”…the authors should include
how much variation in pressure exists for the entire area under the piston, and address
how this variation may affect results
New supplementary figure, S2 Fig in the revised manuscript, has been added to illustrate
the distribution of contact pressure under piston. It is referenced in the manuscript
text in “Computational modelling used to calculate the maximum piston contact pressure
(S2 Fig) was further validated experimentally using independent microdevices with
different membrane thicknesses to enhance reproducibility and repeatability of our
flexible microdevice-based compression method.”
Explanation for the piston contact pressure illustrations has been added in figure
caption as follows: “S2 Fig. Illustrations of the piston contact pressure area by
the symmetrical half-cell of the piston and cell-culture chamber in the computational
model. They show the distribution of contact pressure (in N/m2) under piston for the
selected applied external pressures (in mbar). Red dashed line of model symmetry drawn
on the illustrations at different pressures point out the middle side of the piston.
The contact pressure is slightly higher at the edges of the piston towards the side
channel walls where the attached membrane distance between piston and side wall is
shorter. Thus, there seem to be some variation due to how the piston bottom gets squashed
in these sides of the piston, but it is minimal compared to the overall area. This
is demonstrated by the color being relatively even over the whole area, especially
for externally applied lower pressures such as 0-420 mbar.”
8. Page 8, line 234: It is not clear why authors chose 5 min pressure stage followed
by 5 min rest stage. Experimental characterization of nucleus deformation recovery
time in relationship to pressure AND/OR theoretical support is needed.
The cyclic stress schedule (i.e. 5-min compressed stage followed by 5-min rest stage
in each cycle) was chosen to allow cells enough time during each stage to complete
bulge formation or recovery, processes which were considered important for the investigation
of cell deformation and recovery after compression. Membrane bulge formation and recovery
processes are clearly observable in S1 Movie. This was explained in the following
paragraph in section “Comparison of the experimental and computational results in
micro-piston device”
“While inferring these results, the cell compression process was imaged in detail
in Matrigel-coated microchannels at a speed of 1 frame per 100 ms, as shown in S1
Movie. As illustrated by this time-lapse video, the platform allowed for the temporal
evolution of the dynamic cell compression to be investigated in each cycle while the
piston was contacting cells and applying compression. Distinct cell membrane bulges
could be observed to form during such compression at piston contact pressure of 15.6
kPa and recovery of cell membranes from these bulges was visible when pistons were
lifted off during rest stages. Although the platform is capable of temporal control
of cell compression (e.g. to decrease, increase or remove the pressure at any certain
time interval), duration of each stage in a cycle was set to 5 minutes, corresponding
to a 5-min compressed stage followed by 5-min rest stage in each cycle. This was chosen
to allow cells enough time during each stage to complete bulge formation or recovery,
processes which were considered important for the investigation of cell deformation
and recovery after compression. Sensor readings in S1 Movie showed that the micro-piston
actuated according to externally applied pressures with reliable control and could
be operated in short durations gradually or suddenly when needed.”
Further explanation and discussion have been also added in this section as in the
following paragraph:
“Cyclic compression in the study of Ho et al. was applied only for 6 minutes in total
[2], significantly shorter than the entire duration of a cyclic compression in this
work, and results presented here suggest that cell deformation at compression stage
and recovery at rest stage depends on the temporal length of the compression on cells.
Particularly observable in S1 Movie, cell membrane bulge formation slowly developed
during the compression stage while cells were responding to contact pressure at the
interface with the PDMS piston. This was despite the fact that the piston was actuated
onto the cells rapidly during cyclic compression as directed by the external pressure
controller. As shown by the pressure sensor readings in the same movie, PDMS membrane
and piston were consistently responding to amount, duration and mode of the applied
pressure. Thus, the gradual deformation of cells and bulge formation during compression
stages must originate in the response type of the cells to mechanical load when in
solid contact with the PDMS piston. During rest stages, where the compression was
lifted, cell bulges did not fully recover right after compression was removed, suggesting
that the cells require a certain amount of time to recover compressed cellular parts
(S1 Movie). Based on these observations of temporal evolution of the cell compression
application, and comparison of application durations in this study with the study
by Ho et al. [2], we hypothesize that this recovery also depends on the duration and
number of the cycles.”
9. Page 9: Panels a-c in Figure 3 were not referenced in the text.
Panel a of Figure 3 has been referenced in section “Live cell actin profiles of cancer
cells during sequential cyclic compression”: “……., for these experiments SKOV-3 ovarian
cancer cells were transduced on-chip with CellLight Actin-GFP, BacMam 2.0 baculovirus
to visualize the dynamic biomechanical processes during compression (Fig 3(a)).” and
panels b-c referenced in “…… and actin-GFP signals of the compressed cells were captured
(Fig 3(b-c)).”
10. Page 11, figure 4 should include levels of significance for intended comparison.
Significance levels for intended comparison have been added onto the graph in Figure
4. Rest of all the significance test results (p-values) are in S1_File excel file.
Figure 4 caption has been also updated:
“….. Student's t-test was used to determine the statistical significance of live cell
actin deformation across increasing pressure ranges. Continuous horizontal bars show
significant differences between compressed cell groups under micro-pistons for images
taken at the time of compression at the indicated pressure ranges. Dashed horizontal
bars show significant differences between the cell groups at rest under micro-piston
for images taken after compression stages at the corresponding pressure ranges of
the cycles. Results represent at least three independent experiments.”
11. Page 9, line 295-297: It is not clear the lower actin signal from higher pressure
groups is due to higher pressure or accumulated stress from the sequential testing.
Independent experiments should be carried out for each pressure..
This is very good point. We have not run such independent experiments for each pressure.
From the very beginning we deliberately aimed for sequential cyclic compression profiles
in a dynamic manner that can mimic varying pressures in the same microenvironment
that can be applied on the same sample. The platform is suitable to run individual
steps of sequential cyclic compression.
We revised Fig 4 to show statistical significance levels and those were given in detail
in S1 File for all groups, and further explained and discussed in text as highlighted
in the revised manuscript in section “Live cell actin profiles of cancer cells during
sequential cyclic compression”. Based on these, there are no significant differences
at rest stages (after completion of the compression stages) until Intermediate 2 level
pressures which are considered high. Thus, although cells deform throughout 1-hour
cyclic compression applied at Mild pressures, loss of actin signal was not statistically
significant between 1st cycle (beginning of 1-hour) and last cycle (at the end of
1-hour). Then pressure was risen to Intermediate 1 to Intermediate 2 and Severe pressures
in 30 s at each increase (gradual rate) and cells were compressed for up to 2 min
at each pressure level. Those are relatively gradual and short durations at those
higher pressures applied sequentially, following cyclic mode mild pressure that was
applied for 1 hour. Statistically significant change was seen at rest stage after
Intermediate 2 level of pressures at which we think there was a mechanical impact
due to high pressure. Such an impact is statistically significant again at rest stage
after applied Severe pressures compared to all other levels.
“…….. Similar as to the sequence shown in Fig 1(d), first, a pressure amount ladder
was formed up to a pressure in the mild compression range, represented by an externally
applied pressure of 350 mbar and resulting in a piston contact pressure of 15.6 kPa,
during which the piston reached the cells at the bottom surface and cells started
to distinctly deform. Once this was observed, cyclic compression was applied at mild
pressure for a total of 1 hour, with cells compressed for 5 min at 15.6 kPa, followed
alternately by a rest for 5 min at 0 kPa. To track cell response, actin-GFP signals
of the compressed cells were captured before compression, as well as during the first
and last cycles of the cyclic compression stage. At the end of this sequence, the
externally applied pressure was sequentially increased first to 370 mbar, then to
400 mbar and finally 640 mbar, resulting in the respective piston contact pressures
of 23.8 kPa (Intermediate 1), 37.8 kPa (Intermediate 2) and 140 kPa (Severe). During
this stage, cells were compressed for up to 2 min at each pressure (Fig 1(d)) and
actin-GFP signals of the compressed cells were captured (Fig 3(b-c)). Throughout the
application of sequential cyclic compression, cells were allowed to adjust to each
next higher applied pressure. As shown in the pressure amount ladder in Fig 1(d),
pressure was applied gradually in 30 s among rising steps of the ladder until the
piston reached the focal plane of the cells and started cell deformation. After observing
that cells were distinctly deformed at a mild compression level but not to a disruption
or lysis level, compression was set to be in cyclic mode. Although the pressure was
changing suddenly along the cycles as per the settings of the selected cyclic mode
in the pressure controller, cells were not damaged when compressed as they resulted
in being resistant at this physiological value. Next, pressure application was continued
with gradual increase from 350 to 370 mbar, from 370 to 400 mbar and from 400 to 640
mbar in 30 s for each, as shown in Fig 1(d). Thus, cells could have the time to adjust
during the pressure increase and show their intrinsic response to the pressurized
stage of each compression level.”
Further explanation and discussion after the CTCF calculations, have been also added
in the next two paragraphs in text following the paragraph above in section “Live
cell actin profiles of cancer cells during sequential cyclic compression”. Overall,
the revised/added discussions (highlighted in the revised manuscript) include that
the lower actin-GFP signal and disruption of actin cytoskeleton in these experiments
indicates for cell plasticity during a cyclic compression in ovarian cancer when varying
higher pressures occur in the microenvironment.
The following sentence has been also added to Conclusions: “While actin deformation
at mild pressures (e.g. 15.6 kPa) in cyclic mode indicates for cell elasticity, the
resultant actin disruption indicates for cell plasticity that can emerge when ovarian
cancer cells are exposed to varying and higher pressures in a microenvironment.”
Although we think that a mechanical compression process is dynamic inside the body
and it can develop in a cumulative way (with increasing pressures over time), and
thus our sequential cyclic compression can mimic such process, we also think that
the reviewer is right on that actin profile investigation could be also done straight
on a particular higher pressure in a discrete way, should such sudden high pressures
emerge in the body.
For cell viability investigation in Onal et al 2021 Frontiers in Physics, we extensively
tried on multiple devices applying compression at 640 mbar (Severe pressure), and
checked for viability at initial time of the compression and after 1 hour of continuous
application at 640 mbar and then on other samples we tried sequential cyclic compression
up to 640 mbar with the stage of 640 mbar itself being for 2 min. At all applications,
cell viability result was similar for samples exposed to 640 mbar indicating that
cell response was due to the mechanical compression at this high pressure rather than
its independent experiment or sequential cyclic compression (that developed with varying
pressures up to 640 mbar similar to profiles that were applied in live cell actin
investigation here) or the durations of the applications.
Thus, similar to those compression assays and cell viability response in Onal et al
2021 Frontiers in Physics, for high pressures we expect that actin disruptions still
happens when the applications at each of Intermediate 2 or Severe level pressure ranges
are done independently and actin-GFP signal is measured, in similar way as it happened
in sequential cyclic compression shown in the current work.
12. Page 9, Line 304-306: Were these “resistant” cells in contact with the piston?
Characterization of the interface between the piston and the cell substrate AND/OR
other metrics (ie., nucleus shape/area) is needed to rule out artifact.
The resistant cells remained on the glass surface.
The relevant sentence has been slightly revised to prevent confusion: “Interestingly,
a few cells on the glass surface usually remained resistant to the increasing pressure
ranges, as evidenced by their retained fluorescent signal while compressed under the
micro-piston.”
The following clarification has been also added: “During the entire process, wide-field
imaging was used and the focus was kept on one focal plane on the glass surface where
cells were initially adhered to. At compressed stages, glass and PDMS piston surfaces
were in contact and at the same focal plane. At the rest stages when pressure is released
and piston is retracted back, to be able to gain the signal from piston surface there
would be need to bring the focal plane to the suspended piston level that is on average
108 �m above the glass surface. In the region under the micro-piston, there seem to be
variations in gained actin signal between the compressed and rest stages of each cycle
(Fig 4), but these were not statistically significant (p>0.05, see S1 File). Thus,
no significant amount of cell artifacts was attached to piston surface.”
In terms of background (light artifacts), this was extracted from the images as shown
in the equation in section “Imaging and data analysis”. Furthermore, the calculated
actin signals were compared between the region under piston and control region around
the piston. Actin signal in the control regions were also compared to each other across
the cycles and remained statistically insignificant (S1 File) as it was indicated
in the manuscript. The following discussion has been added to the text in the same
paragraph as above in Results and discussion: “Thus, the GFP-tagged actin signal in
control regions was robust during the entire process, as expected [27]. This can be
reference to that fluorescence changes in the region under micro-piston (i.e. decrease
in actin signal) was due to the impact of mechanical compression.”
13. Page 10-11: the authors should consider discussing impact of the observed disruption
of actin /no recovery due to cyclic compression in ovarian cancer and the relevance
to cell plasticity
The following discussion has been added in section “Live cell actin profiles of cancer
cells during sequential cyclic compression”:
“The observed disruption of actin cytoskeleton in these experiments indicates for
cell plasticity during a cyclic compression in ovarian cancer when varying higher
pressures occur in the microenvironment. There is need for more studies in literature
to compare our results on cell plasticity after cyclic or dynamic compression applied
sequentially up to high pressures. For cell actin deformation at milder pressures,
however, a distinct change in actin cytoskeleton morphology was observed by Asem et
al. for mesothelial cells, interacting with ovarian cancer cells, under compression
that is statically applied at ∼3 kPa for 24 h in a bulk compression system. As a result,
the interaction between ovarian cancer cells and mesothelial cells by enhanced formation
of actin-based tunneling nanotubes (TNTs), and hence metastatic progression in ovarian
cancer, was promoted under compression [7].”
14. Page 13, figure 5 b-d needs to include details of significance tests conducted
and the significance levels detected.
Intended comparison is shown on the figures. Figure caption is revised.
“Fig 5. ……. Student’s t-test was used to determine the statistical significance of
nuclear deformations in compressed and recovered cells after compression.”
Rest of all the significance test results (p-values) are in S2_File excel file, newly
added supplementary file.
Same revisions have been applied for Figure 6 in caption and S2_File.
15. Page 12, line 352: it is not clear what specific heterogeneity the authors mean
here
Heterogeneity word was clarified to prevent confusion by revising the relevant sentence
into “This divergence might emerge from the variations in decrease of cell volume
under compression and nuclear envelope wrinkling and ability of each cell to recover
these changes after mild compression [13,28].”
16. Page 12, line 391: minute differences (?)
“Minute” word in “……minute differences in cell behaviour…..” has been removed from
line 391 and from the Abstract, to prevent any confusion. It was meant that the pressure
change was minute (e.g. ~5 kPa difference between Fig 5 and Fig 6) but there were
changes in cell response.
It was also removed from the sentence in the section “Actin cytoskeleton and nuclei
profiles of cancer cells after compression and recovery”: “The phenotypical response
of compressed cells during recovery after compression at 20.8 kPa differed observably
from that for 15.6 kPa, demonstrating the capability of the flexible microdevice to
capture minute differences in cell behaviour after being compressed at various pressures
in a physiologically relevant manner.”
17. Page 13: in Fig 5(a) 15.6 kPa (o time), actin signal looks higher in compressed
area compared to cells in control area? The authors should also explain the unevenness
of brightness in phase contrast images
The distinct actin deformation by the increased fluorescence signals at the edges
of the cells were shown on Fig 5(a) by representative arrows as it was noted on figure
caption:
“Representative arrows (white) show the distinct actin deformation by the increased
fluorescence signals at the edges of the cells in the compressed groups under the
micro-piston at zero time after compression.”
An explanation has been added in text: “Fig 5(a) shows the distinct actin deformation
by the increased fluorescence signals at the edges of the cells in the compressed
groups under the micro-piston at zero time after compression, compared to the control
(non-compressed) groups around the micro-piston. Thus, membrane bulges formed during
cell compression at 15.6 kPa as observed in S1 Movie and fixed directly after compression
appeared as increased actin signal at cell edges. Conversely, at 24 h-recovery after
compression, cell actin deformation seems recovered in the group under the micro-piston
as appeared similar to the control group.”
A note about the unevenness of brightness in phase contrast images has been added
to the caption of Fig 5(a): “The unevenness of brightness in phase contrast images
is due to that imaging focal plane was on cells cultured on glass while the micro-piston,
brought back to static state after compression, was suspended on average 108 �m above the glass.”
18. Fig. 5 & 6 quantification of nucleus metrics: how many independent experiments
were conducted?
Figure captions are revised.
“Fig 5. …… (mean ± s.e.m. n = 588, 230, 358, 174 cell nuclei from at least two or
three independent experiments per each group).”
“Fig 6. …… (mean ± s.e.m. n = 406, 162, 342, 143 cell nuclei from at least two or
three independent experiments per each group).”
19. Page 12, line 380: authors should explain why no significant changes in nucleus
area were observed, as it would be expected given the high stress cells were experiencing.
The authors should also explain the visible difference in two “0 (CR)” groups in Figure
6 (b)
All the statistical significance values for nuclear deformations in compressed and
recovered cells after compression are noted in S2_File, newly added as supplementary
file.
S2 File caption “Statistical significance levels among all groups, obtained with Student's
t-test run for nuclear deformations in compressed and recovered cells after compression
presented in Fig 5 and Fig 6.” has been added. Fig 5 and Fig 6 captions have been
also updated.
We added the following result and discussion for the difference between the two "0
(CR)" values: “There is statistically significant change in nuclei area between the
control groups of zero-time and of 24 h-recovery after compression (see S2 File).
Although these are not exactly the same control groups in the same conditions and
we intend to compare the test group (e.g. zero-time or 24 h-recovery after compression)
with their own control groups, we consider that the observed significant difference
could be due to cell-cell signaling between the cells in compressed and control groups
during that 24 h-recovery period after compression. In the groups of zero-time after
compression, we did not allow control and compressed cells enough time to further
interact with each other as they were fixed right after the compression.”
We further added the following discussion for the absence of statistical change in
nucleus area between the control and compressed groups at zero-time after compression,
in the associated paragraph: “While we expect a statistically significant change between
the compressed and its control group at zero-time after compression at 20.8 kPa similar
to the results observed in our previous report [5], we attribute the absence of such
a significant change to cells being cultured on Matrigel-coating here as opposed to
PLL-coating used in the previous experiments. Since Matrigel is expected to better
represent the cancer cell microenvironment, it thus aided in cell growth into denser
monolayers and formation of supporting extracellular matrix layer, that can alter
sensing of the applied stress for the compressed cells.”
Reviewer #3: Onal and colleagues present a previously published method to dynamically
compress a monolayer of cells. In this article, they use this device to briefly study
how compression can impact actin expression, through a live reporter.
The study is technically sound. However, there are some points that need to be addressed
before I can reach a decision:
1/ The claim of novelty of the device needs to be taken away. The device does not
seem different from the one published by the authors in 2021 in Frontiers in Physics,
and is inspired by the one published by Mishra et al (PNAS 2017), which should be
acknowledged. In their 2021 paper, the authors notably already perform cyclic compression.
Can the authors comment on what is really new, and if nothing is, can they transform
the abstract and parts of introduction / conclusion to have it more focused on the
study on actin?
Multiple parts in the manuscript have been revised and novelty section has been improved
as in the last paragraphs of the Introduction, as highlighted in the revised manuscript.
Abstract and Conclusion have been also revised.
We thank the reviewer for letting us know about Mishra et al (PNAS 2017) which we
were not aware of. We have gone through the details of the microfluidic device presented
in the supplementary of the paper. The device includes 2 layers of PDMS with micro
patch pad array of 15-30 �m height which can be obtained in a single master. Design and applications are more
adapted to yeast-size cells. Thus, the two platforms developed by Onal et al 2021
Frontiers in Physics and Mishra et al 2017 PNAS are different in dimensions and fabrication
methods. Our microfluidic platform is composed of 3 layers of PDMS with microchannels
and micro-pistons of 200 – 300 �m height and we provide dynamic cell compression at mammalian cell-culture suited
dimensions. The 3 layers must be fabricated separately as UV lithography of a single
master with varying structures at 200-300 �m (i.e. a micro-channel of 300 mm with a suspended micro-piston of 200 �m height and also attached to a membrane of ~200 �m thick at the same time) for a fluidic layer is not feasible to pattern as in the
method of Mishra et al. to obtain small structures in a patterned single fluidic layer.
One of the advantages of our platform is that dimensions of any layer can be changed
easily as the layer dimensions are not depending on each other by a single master.
The spin coating method to obtain such large structures as monolithically attached
to PDMS membrane in our platform has been also very novel to obtain structures of
200 �m height and 300 �m diameter/length micro-pistons as attached to varying membrane thicknesses (varied
from 100 �m up to 345 �m) at high resolution and high fidelity on a 4-inch Si/photoresist master. Cell loading
methods are also quite different between the two papers. We use piston-retracted loading,
which is a dynamic method and pistons are retracted up towards control microchannel
while loading cells into the bottom channel to ensure homogenous distribution of cells
under and around micro-piston. About the compression values applied, Mishra et al.
used 7 psi (~48 kPa) widely in their experiments and they mention that the pressure
level they use is physiologically relevant, as yeast cells can experience up to about
100 psi (~690 kPa) when grown in confined geometries. These values, especially up
to 690 kPa, used in yeast applications are quite high pressures compared to physiology
of cancer cell compression microenvironments. Additionally, it is not clear whether
7 psi used Mishra et al. in their device is externally applied pressure or contact
pressure. Since no contact pressures are mentioned in their paper (including for supplementary
as well), we consider that the mentioned value is externally applied pressure and
there would be need to predict what were the internal/contact pressures via more device
characterization methods. In our studies we typically characterize both externally
applied pressures by a pressure pump and internal/contact pressures experienced inside
the microdevice as PDMS is a hyperelastic material.
2/ I was surprised that the authors present their method as a great strategy to apply
cyclic compressive stresses, but did not really present results on this part (playing
with frequency for instance, etc). It is discussed on p.15, but no experiments are
presented. Have the authors played with this parameter? If not, maybe it would be
better to put less emphasis on this in the manuscript. Also, can the authors provide
a rationale for the frequency of the oscillations used in this study?
S1 Movie and S2 Movie, and Fig 1(d) representing how the sequential cyclic compression
application was done for S2 Movie and Fig 3, show temporal evolution of the dynamic
cell compression and a clear control of the process.
Further explanation of the profiles in Fig 1(d) has been added at the end of 1st paragraph
of the section “Live cell actin profiles of cancer cells during sequential cyclic
compression”: “Throughout the application of sequential cyclic compression, cells
were allowed to adjust to each next higher applied pressure. As shown in the pressure
amount ladder in Fig 1(d), pressure was applied gradually in 30 s among rising steps
of the ladder until the piston reached the focal plane of the cells and started cell
deformation. After observing that cells were distinctly deformed at a mild compression
level but not to a disruption or lysis level, compression was set to be in cyclic
mode. Although the pressure was changing suddenly along the cycles as per the settings
of the selected cyclic mode in the pressure controller, cells were not damaged when
compressed as they resulted in being resistant at this physiological value. Next,
pressure application was continued with gradual increase from 350 to 370 mbar, from
370 to 400 mbar and from 400 to 640 mbar in 30 s for each, as shown in Fig 1(d). Thus,
cells could have the time to adjust during the pressure increase and show their intrinsic
response to the pressurized stage of each compression level.”
The application rates and durations of the process in S1 Movie was already shown on
the moving graph in the movie itself and was also clearly noted on its caption: “Temporal
evolution of the dynamic cell compression within Matrigel-coated micro-piston devices.
Time-lapse live cell imaging with 100 ms per frame was recorded for ladder pressure
increase from 0 kPa up to 15.6 kPa and a short cycle at 15.6 kPa with gradual increase
(from 0 kPa to 15.6 kPa) and decrease (from 15.6 kPa to 0 kPa) in 30 s for each. These
steps were sequentially followed by 1 h-long cyclic compression between 0 kPa and
15.6 kPa (piston contact pressure from simulation). Cells were compressed in the temporal
evolution of the dynamic pressure control within Matrigel-coated micro-piston devices
and dynamic changes in cells during the cyclic compression were captured in detail
at a rate of 100 ms per frame.”
During the mechanical characterizations of the microdevice itself, multiple profiles
that the platform could apply was shown in Onal et al. 2021 Frontiers in Physics.
Thus, it was discussed and noted in this manuscript, for the frequency of the oscillations
as well in the Results and discussion, section “Comparison of the experimental and
computational results in micro-piston device”: “Although the platform is capable of
temporal control of cell compression (e.g. to decrease, increase or remove the pressure
at any certain time interval), duration of each stage in a cycle was set to 5 minutes,
corresponding to a 5-min compressed stage followed by 5-min rest stage in each cycle.
This was chosen to allow cells enough time during each stage to complete bulge formation
or recovery, processes which were considered important for the investigation of cell
deformation and recovery after compression. Sensor readings in S1 Movie showed that
the micro-piston actuated according to externally applied pressures with reliable
control and could be operated in short durations gradually or suddenly when needed.”
This was further discussed with a next paragraph added following the paragraph above
in the same section: “Cyclic compression in the study of Ho et al. was applied only
for 6 minutes in total [2], significantly shorter than the entire duration of a cyclic
compression in this work, and results presented here suggest that cell deformation
at compression stage and recovery at rest stage depends on the temporal length of
the compression on cells. Particularly observable in S1 Movie, cell membrane bulge
formation slowly developed during the compression stage while cells were responding
to contact pressure at the interface with the PDMS piston. This was despite the fact
that the piston was actuated onto the cells rapidly during cyclic compression as directed
by the external pressure controller. As shown by the pressure sensor readings in the
same movie, PDMS membrane and piston were consistently responding to amount, duration
and mode of the applied pressure. Thus, the gradual deformation of cells and bulge
formation during compression stages must originate in the response type of the cells
to mechanical load when in solid contact with the PDMS piston. During rest stages,
where the compression was lifted, cell bulges did not fully recover right after compression
was removed, suggesting that the cells require a certain amount of time to recover
compressed cellular parts (S1 Movie). Based on these observations of temporal evolution
of the cell compression application, and comparison of application durations in this
study with the study by Ho et al. [2], we hypothesize that this recovery also depends
on the duration and number of the cycles.”
And supported with the paragraph revised and moved to Introduction, while introducing
the literature frequencies and this work:
“To date, dynamic compression experiments in literature include frequencies of 0.1
– 30 Hz with an applied stress of 5.1, 9.3, 12.9 and 18.7 kPa on breast cancer cells
over short durations (30 – 300 s), as used by Takao et al., who observed a mixed mode
of apoptosis and necrosis-dominant cell death in mechanically compressed monolayers
in a bulk compression platform [18]. In contrast, work by Novak et al. used a frequency
of 0.05 Hz for cyclic compression applied at 3.9 – 6.5 kPa for 24 hours [3]. They
observed an increase in proliferation capacity and decrease in apoptosis for OVCAR3
and OVSAHO ovarian cancer cells cultured in 3D hydrogel components in a bioreactor
at millimeter scales. In another example, Ho et al. applied cyclic compression by
alternating between external pressures of 10 and 15 psi (68.9 and 103.4 kPa) at 0.5
Hz for 6 minutes [2]. Using a single cell microfluidic compression platform, their
application resulted in full recovery and thus no permanent plastic deformation in
MCF10A normal breast epithelial cells after compression [2]. In our work we add to
these examples by using the capabilities of our compression platform to apply cyclic
compression with a low frequency in microfluidic settings. In particular, we utilize
this functionality to provide a first investigation of the effects of cell compression,
deformation and recovery after compression on SKOV-3 ovarian cancer cells. As part
of this we provide evidence that the amount of the pressure and duration of the application
clearly affects morphology during cell compression and recovery. While compression
frequencies in an in vivo tumor microenvironment are not yet fully known, cyclic compression
is important for maintenance of cell culture during mechanical compressions in an
in vitro experimental setup, in particular as it enhances media flow underneath the
micro-pistons.”
3/ I have some difficulties with the motivations in the introduction for the choice
of ovarian cancer cells. In cancer, compressive stress is very slowly increasing,
and not cyclically changing. Dynamic stress, though, can be found in the heart or
during breathing, or also through the peristaltic motion during digestion. These examples,
which would be great to motivate the dynamic capabilities of the device, are not presented,
and instead, the focus is made on cancer. Moreover, I would not call 16 or 21 kPa
as “mild” compressive stresses: these are rather important. The authors may wish to
re-write their introduction if they want to introduce the device through its dynamic
capabilities. But in this case, ovarian cancer cells may not be the best model (although,
the authors can just say they use these cells as a model cell line for this test study).
The first two paragraphs have been revised to better reflect on the importance of
studying cyclic compression, and dynamic peritoneal environment of ovarian cancer
cells. 2nd paragraph give insights on the physiological values that have been in the
literature and further values that should be investigated:
“While some cells experience permanent plastic deformation after a repetitive mechanical
tensile loading and unloading, the impact of such repetitive compression on deformation
of cells remains largely unknown [1,2]. As such, the ability to apply cyclic compression
is crucial for any experimental setup aimed at the study of mechanical compression
taking place in cell and tissue microenvironments [3–5]. For instance, in ovarian
cancer, cells are exposed to chronic compressive stress from different sources such
as tumor growth, displacement within stromal tissue and hydrostatic pressure out of
ascitic fluid in peritoneal cavity [3,6]. Ascites-induced compression exists in the
peritoneal microenvironment due to the ascites volume that can reach >2L and increases
ovarian cancer cell adhesion to peritoneum, shown by Asem et al. using in vivo assay
[7]. This peritoneal microenvironment is continuously affected by the movements of
the surrounding organs, musculoskeletal dynamics, and gravity [6]. Thus, ovarian cancer
cells might experience cyclic compression profiles during chronic exposure to compression
from different sources and due to anatomical location of the ovary. Mimicking such
compression profiles and physiological pressure values in an in vitro dynamic compression
system is necessary to further study impact of compressive stress in cancer.
Compressive stress is estimated to reach 18.9 kPa for human tumors and can exceed
20 kPa based on the experimental data from murine tumors, while those values can be
even higher in situ with the impact of the surrounding extracellular matrix in tumor
microenvironment [3,8]. Compared to other cancer types such as breast and metastatic
bone niches, compression studies have been limited for ovarian cancer, including for
investigating the impact of various amounts of applied pressures [6]. Pressure values
existing in literature that are applied for ovarian cancer have been in a range from
∼3 kPa to 6.5 kPa [3,7,9]. Ovarian cancer cell response at higher applied pressures
that can occur in the body need further investigation.”
When it comes to categorization as Mild compression this was introduced and used so
in Onal et al, 2021, Frontiers in Physics while investigating cell viability response
to a wide range of pressures in 4 categories. In those previous experiments, we showed
SKOV-3 ovarian cancer cells were highly viable as on average 94% for Mild (15.6–15.9
kPa) and 77% for Intermediate 1 (23.8–26.8 kPa). Thus, we preferred to continue to
use the same categorization, as it was also used in this manuscript while applying
sequential cyclic compression to study dynamics of live cell actin, from Mild to Intermediate
1 to Intermediate 2 and Severe pressures.
Similarly, and actually beyond the mild and intermediate pressures we used in our
study for ovarian cancer cells, Hosmane et al. (2011, Lab on a Chip) showed application
of compression on single axons in the range of 0-250 kPa and categorized as mild (<55
kPa), moderate (55 - 95 kPa) and severe levels (>95 kPa).
Cyclic compression has been also applied to breast cancer cells, normal breast epithelial
cells, and other ovarian cancer cell lines, as revised to Introduction:
“To date, dynamic compression experiments in literature include frequencies of 0.1
– 30 Hz with an applied stress of 5.1, 9.3, 12.9 and 18.7 kPa on breast cancer cells
over short durations (30 – 300 s), as used by Takao et al., who observed a mixed mode
of apoptosis and necrosis-dominant cell death in mechanically compressed monolayers
in a bulk compression platform [18]. In contrast, work by Novak et al. used a frequency
of 0.05 Hz for cyclic compression applied at 3.9 – 6.5 kPa for 24 hours [3]. They
observed an increase in proliferation capacity and decrease in apoptosis for OVCAR3
and OVSAHO ovarian cancer cells cultured in 3D hydrogel components in a bioreactor
at millimeter scales. In another example, Ho et al. applied cyclic compression by
alternating between external pressures of 10 and 15 psi (68.9 and 103.4 kPa) at 0.5
Hz for 6 minutes [2]. Using a single cell microfluidic compression platform, their
application resulted in full recovery and thus no permanent deformation in MCF10A
normal breast epithelial cells after compression [2]. In our work we add to these
examples by using the capabilities of our compression platform to apply cyclic compression
with a low frequency in microfluidic settings. In particular, we utilize this functionality
to provide a first investigation of the effects of cell compression, deformation and
recovery after compression on SKOV-3 ovarian cancer cells. As part of this we provide
evidence that the amount of the pressure and duration of the application clearly affects
morphology during cell compression and recovery. While compression frequencies in
an in vivo tumor microenvironment are not yet fully known, cyclic compression is important
for maintenance of cell culture during mechanical compressions in an in vitro experimental
setup, in particular as it enhances media flow underneath the micro-pistons.”
Thus, different to bulk compression platform by Takao et al. (2019, Biology Open),
bioreactor compression platform at millimeter scales by Novak et al. (2020, Cancers)
and single cell microfluidic compression platform by Ho et al. (2018, Frontiers in
Bioeng and Biotech), in our platform with microchannels of 200 – 300 �m height we provide dynamic cell compression at mammalian cell-culture suited dimensions
that can be adapted to 2D (cell monolayers, individual cells) and 2.5 (dense cultures
with hydrogel coatings, cluster of cells) and in the future work 3D (spheroids and
cell-embedded hydrogels) cultures.
As the reviewer pointed out, this method can be applied to all other cell types, as
it was noted in Conclusions:
“As demonstrated here with controlled micro-scale mechanical cell compression and
recovery in a flexible microdevice, our comprehensive method will provide a more accurate
replication of cell-physiological mechanisms to study both short- and long-term effects
of compression in cellular microenvironments.”
4/ The actin intensity result seems correlated to survival, oddly. Can the authors
plot these two parameters alongside as a function of pressure on same plot? Can the
authors speculate on the decrease observed under pressure? Finally, have the authors
checked for bleaching, by changing the sequence of compression for instance?
Cell viability results for the applied pressure ranges were reminded from Onal et
al, 2021, Frontiers in Physics to give an idea at what viability states the cells
were at these pressure ranges while investigating live cell actin cytoskeleton under
dynamic compression: “……. the actin-GFP transduced cancer cells were dynamically stimulated
and compressed with various intermediate actuation pressures in a sequential cyclic
manner, namely following a sequence from Mild (15.6–15.9 kPa) to Intermediate 1 (23.8–26.8
kPa), Intermediate 2 (37.8–41.7 kPa) and Severe (127.8–140 kPa) piston contact pressures.
The first two ranges of this sequence fell within physiological and upper physiological
pressure values and cell viability was high (on average 94% for Mild and 77% for Intermediate
1), while Intermediate 2 and Severe levels constitute hyperphysiological values and
cell viability decreased (on average 43% for Intermediate 2 and 20% for Severe) [5].”
Thus, our previous report showed mechanical stress induced cell damage and death towards
higher pressures so categorized as Intermediate 2 and Severe pressures, providing
the degree of the damage as per the applied pressures via viability assays conducted
in detail in Onal et al 2021 Frontiers in Physics.
The calculated actin signals were compared between the region under piston and control
region around the piston. Actin signal in the control regions were also compared to
each other across the cycles and remained statistically insignificant (S1 File) as
it was indicated in the manuscript in Results and discussion in section “Live cell
actin profiles of cancer cells during sequential cyclic compression”: “The change
in actin signal for compressed cells under micro-piston was statistically different
compared to non-compressed cells in control regions around the micro-piston at all
applied compression levels (p <0.05, see S1 File). Actin signal changes in the control
regions on the other hand, remained statistically insignificant (p >0.05).”
The following discussion has been added in the same paragraph: “Thus, the GFP tagged
actin signal in control regions was robust during the entire process, as expected
[27]. This can be reference to that fluorescence changes in the region under micro-piston
(i.e. decrease in actin signal) was due to the impact of mechanical compression.”
Thus, given that there was no statistically significant difference between the control
groups throughout the application, we do not have signs indicating for photobleaching.
The following sentence was noted in the same paragraph”:
“At the higher pressures of Intermediate 2 and Severe, actin of most of the cells
was disrupted compared to in the initial stages of Mild compression (p <0.05, see
S1 File).”
The next sentence in the same paragraph has been slightly revised to: “Interestingly,
a few cells on the glass surface usually remained resistant to the increasing pressure
ranges, as evidenced by their retained fluorescent signal while compressed under the
micro-piston.”
The following discussion as relevant to the observation sentence above has been added:
“During the entire process, wide-field imaging was used and the focus was kept on
one focal plane on the glass surface where cells were initially adhered to. At compressed
stages, glass and PDMS piston surfaces were in contact and at the same focal plane.
At the rest stages when pressure is released and piston is retracted back, to be able
to gain the signal from piston surface there would be need to bring the focal plane
to the suspended piston level that is on average 108 �m above the glass surface. In the region under the micro-piston, there seem to be
variations in gained actin signal between the compressed and rest stages of each cycle
(Fig 4), but these were not statistically significant (p>0.05, see S1 File). Thus,
no significant amount of cell artifacts was attached to piston surface.”
Overall, for analysis of actin-GFP signal we had done CTCF calculations and have now
improved the graph to show the statical significances in Fig 4 in the revised manuscript.
When there was statistically significant loss in actin-GFP signal and as seen on S2
Movie, actin disruption happens and that indicates for plastic response of the cells
at those higher pressures. We added a discussion in the last paragraph of section
“Live cell actin profiles of cancer cells during sequential cyclic compression”: “The
observed disruption of actin cytoskeleton in these experiments indicates for cell
plasticity during a cyclic compression in ovarian cancer when varying higher pressures
occur in the microenvironment. There is need for more studies in literature to compare
our results on cell plasticity after cyclic or dynamic compression applied sequentially
up to high pressures. For cell actin deformation at milder pressures, however, a distinct
change in actin cytoskeleton morphology was observed by Asem et al. for mesothelial
cells, interacting with ovarian cancer cells, under compression that is statically
applied at ∼3 kPa for 24 h in a bulk compression system. As a result, the interaction
between ovarian cancer cells and mesothelial cells by enhanced formation of actin-based
tunneling nanotubes (TNTs), and hence metastatic progression in ovarian cancer, was
promoted under compression [7].”
In the section Conclusions it has been also added that “While actin deformation at
mild pressures (e.g. 15.6 kPa) in cyclic mode indicates for cell elasticity, the resultant
actin disruption indicates for cell plasticity that can emerge when ovarian cancer
cells are exposed to varying and higher pressures in a microenvironment.”
Minor points:
1/The paper is too affirmative at parts (for instance: p.9, “the results illustrate
how the actin cytoskeleton […] changes in response to compressive stress” is overstated
as the authors show a correlation, and do not provide a clear mechanism). The authors
need to be more careful on their claims.
We have gone through the entire manuscript and have done revisions and additions in
multiple parts, as highlighted in the revised manuscript.
We have revised Fig 4 and better referenced the associated Fig 1(d) and S1 File in
text. We have further explained the associated graphs and significance values and
interpreted and discussed the results. Thus, we have extensively revised the associated
section “Live cell actin profiles of cancer cells during sequential cyclic compression”
as highlighted in the revised manuscript.
2/ Could the authors put all pressure values in the same unit? We sometimes read kPa,
mbar or psi (which is by its very name not a pressure unit and not in the international
system of units), and a harmonization would help the reader.
We used mbar to indicate the externally applied pressures as per the settings and
calibrations on the pressure pump, pressure sensor and sensor readers used while running
this work. This would help towards repeatability of the work and the reader to choose
the appropriate pump, sensor, reader and their settings, should they like to use similar
methods. We used kPa to indicate for the piston contact pressures as conversion from
pascal by N/m2 computed on the mechanical modelling. Our piston contact pressures
given in kPa were compared to the literature values on contact pressures which are
also given in kPa in the associated references.
mbar and kPa are the two units that seem to be commonly used in the publications in
cell compression field.
A note was added in the methods section “Device operation for sequential cyclic compression”
to guide the reader on how it is a better way to read the units:
“Pressure values in this work are given in mbar for externally applied pressures,
as per calibrated settings of the pressure pump, pressure sensor and sensor reader,
while kPa is used for the piston contact pressures, which are based on conversion
from values computed using mechanical modelling.” The following sentence has been
also added to the end of this section for a better guidance: “Our piston contact pressures
given in kPa were compared to the literature values on contact pressures which are
also given in kPa in the associated references.”
As for psi, typically, we do not use psi in our work. psi values mentioned in our
manuscript belongs to Ho et al. (2021, Frontiers in Bioeng and Biotech) as externally
applied pressures and we also added their conversion to kPa in parentheses. We generally
intend to not change the units described in our references from the literature.
3/ Part at the end of page 8 read more like introduction and the end of page 14 /
beginning of page 15 read more like a discussion. Maybe they would be better placed
in these specific sections to avoid confusion.
The following part has been moved to ‘Introduction’ from ‘Results and discussion’:
“To date, dynamic compression experiments in literature include frequencies of 0.1
– 30 Hz with an applied stress of 5.1, 9.3, 12.9 and 18.7 kPa on breast cancer cells
over short durations (30 – 300 s), as used by Takao et al., who observed a mixed mode
of apoptosis and necrosis-dominant cell death in mechanically compressed monolayers
in a bulk compression platform [15]. In contrast, work by Novak et al. used a frequency
of 0.05 Hz for cyclic compression applied at 3.9 – 6.5 kPa for 24 hours [3]. They
observed an increase in proliferation capacity and decrease in apoptosis for OVCAR3
and OVSAHO ovarian cancer cells cultured in 3D hydrogel components in a bioreactor
at millimeter scales. In another example, Ho et al. applied cyclic compression by
alternating between external pressures of 10 and 15 psi (68.9 and 103.4 kPa) at 0.5
Hz for 6 minutes [2]. Using a single cell microfluidic compression platform, their
application resulted in full recovery and thus no permanent plastic deformation in
MCF10A normal breast epithelial cells after compression [2]. In the following we add
to these examples by using the capabilities of our micro-piston platform to apply
cyclic compression with a low frequency in microfluidic settings. In particular, we
utilize this functionality to provide a first investigation of the effects of cell
compression, deformation and recovery after compression on SKOV-3 ovarian cancer cells.
As part of this we provide evidence that the amount of the pressure, duration of the
application and number of the cycles clearly affects morphology and compression in
this cell type. While compression frequencies in an in vivo tumor microenvironment
are not yet fully known, cyclic compression is important for maintenance of cell culture
during mechanical compressions in an in vitro experimental setup, in particular as
it enhances media flow underneath the micro-pistons.”
Some sentences are revised to
“…….. Using a single cell microfluidic compression platform, their application resulted
in full recovery and thus no permanent deformation in MCF10A normal breast epithelial
cells after compression [2]. In our work we add to these examples by using the capabilities
of our compression platform to apply cyclic compression with a low frequency in microfluidic
settings. ……. As part of this we provide evidence that the amount of the pressure
and duration of the application clearly affects morphology during cell compression
and recovery.”
The following part has been moved to discussion part of the section ‘Comparison of
the experimental and computational results in micro-piston device’ from the end of
‘Results and discussion’ (the end of page 14 / beginning of page 15):
“Cyclic compression in the study of Ho et al. was applied only for 6 minutes in total,
significantly shorter than the entire duration of a cyclic compression in this work,
and results presented here suggest that cell recovery after compression depends on
the temporal length of the compression on cells. The overall extent of recovery of
compressed cells represented by cell membrane bulges, the actin cytoskeleton and measurements
of shape descriptors of cell nuclei can yield further insight into the plasticity
of the cancer cells. Particularly observable in S1 Movie, cell plasticity depends
on the amount of pressure applied, as well as the timing. Cell bulge formation slowly
developed during the compression stage while cells were responding to contact pressure
at the interface with the PDMS piston. This was despite the fact that the piston was
actuated onto the cells rapidly as directed by the external pressure controller. As
shown by the pressure sensor readings in the same movie, PDMS membrane and piston
were consistently responding to amount, duration and mode of the applied pressure.
Thus, the gradual deformation of cells and bulge formation during compression stages
must originate in the response type of the cells to mechanical load when in solid
contact with the PDMS piston. During rest stages, where the compression was lifted,
cell bulges did not fully recover right after compression was removed, suggesting
that the cells require a certain amount of time to recover compressed cellular parts
(S1 Movie). Based on these observations of temporal evolution of the cell compression
application, and comparison of application durations in this study with the study
by Ho et al. [2], we hypothesize that this recovery also depends on the duration and
number of the cycles.”
Revised to
“Cyclic compression in the study of Ho et al. was applied only for 6 minutes in total
[2], significantly shorter than the entire duration of a cyclic compression in this
work, and results presented here suggest that cell deformation at compression stage
and recovery at rest stage depends on the temporal length of the compression on cells.
Particularly observable in S1 Movie, cell membrane bulge formation slowly developed
during the compression stage while cells were responding to contact pressure at the
interface with the PDMS piston. This was despite the fact that the piston was actuated
onto the cells rapidly during cyclic compression as directed by the external pressure
controller. As shown by the pressure sensor readings in the same movie, PDMS membrane
and piston were consistently responding to amount, duration and mode of the applied
pressure. Thus, the gradual deformation of cells and bulge formation during compression
stages must originate in the response type of the cells to mechanical load when in
solid contact with the PDMS piston. During rest stages, where the compression was
lifted, cell bulges did not fully recover right after compression was removed, suggesting
that the cells require a certain amount of time to recover compressed cellular parts
(S1 Movie). Based on these observations of temporal evolution of the cell compression
application, and comparison of application durations in this study with the study
by Ho et al. [2], we hypothesize that this recovery also depends on the duration and
number of the cycles.”
The following sentence that was at the end of the paragraph above has been slightly
revised and moved to last paragraph of “Results and discussion”:
“Based on this we conclude that the presented sequential cyclic compression and cell
recovery after compression method should be employed in the future to study the effects
of compression and decompression in intratumor vessels or endothelial cells to help
understand the structure and functionality of decompressed vessels, importance of
which was shown in a study by Padera et al. [25].”
Revised to
“For instance, we propose that the presented sequential cyclic compression and cell
recovery after compression method should be employed in the future to study the effects
of compression and decompression in intratumor vessels or endothelial cells to help
understand the structure and functionality of decompressed vessels, importance of
which was shown in a study by Padera et al. [34].”
- Attachments
- Attachment
Submitted filename: Response to Reviewers.pdf