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Mechanical Stretch Kills Transformed Cancer Cells
Ajay Tijore1, Mingxi Yao1, Yu-Hsiu Wang1, Yasaman Nematbakhsh2, Anushya Hariharan1,
Chwee Teck Lim1,2,3, Michael Sheetz1,4*
1Mechanobiology Institute, National University of Singapore, Singapore 117411
2Department of Biomedical Engineering, National University of Singapore, Singapore, 117575
3Biomedical Institute for Global Health Research and Technology, National University of Singapore,
Singapore 117599
4Department of Biological Sciences, Columbia University, New York, NY10027
*Correspondence:
E-mail: ms2001@columbia.edu
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Abstract
Transformed cancer cells differ from normal cells in several important features like anchorage
independence, Warburg effect and mechanosensing. Consequently, transformed cancer cells
develop an anaplastic morphology and respond aberrantly to external mechanical forces.
Consistent with altered mechano-responsiveness, here we show that transformed cancer cells
from many different tissues have reduced growth and become apoptotic upon cyclic stretch as do
normal cells after the transformation. When matrix rigidity sensing is restored in transformed
cancer cells, they survive and grow faster on soft surface upon cyclic stretch like normal cells but
undergo anoikis without stretch by activation of death associated protein kinase1 (DAPK1). In
contrast, stretch-dependent apoptosis (mechanoptosis) of transformed cells is driven by stretch-
mediated calcium influx and calcium-dependent calpain 2 protease activation on both collagen
and fibronectin matrices. Further, mechanosensitive calcium channel, Piezo1 is needed for
mechanoptosis. Thus, cyclic stretching of transformed cells from different tissues activates
apoptosis, whereas similar stretching of normal cells stimulates growth.
Keywords: malignant transformation, cell stretching, apoptosis, mechanical force, cancer
treatment, piezo calcium channel, DAPK1, calpain.
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Introduction
The transformed phenotype was initially described in early studies of cancer cell growth,
since tumor cells would often grow on soft agar plates, while normal cells from the same tissue
required a rigid surface for growth (Hamburger and Salmon, 1977). Recent studies have shown
that transformed cancer cells from many different tissues lack rigidity sensors. Restoration of
rigidity sensing, through cytoskeletal protein expression in cancer cells, blocks transformed
growth (Wolfenson et al., 2016; Yang et al., 2018). Further, normal cells can become
transformed by depleting cytoskeletal proteins that are required for rigidity-sensing. This raises
many questions regarding the transformed phenotype, since altering cytoskeletal protein levels in
cancer cell lines restores rigidity-dependent growth and reciprocal alterations in normal cells
cause transformed growth in many different tissue backgrounds. As the morphology and
mechanical properties of the transformed cancer cells are dramatically different from the normal
cells, there may be substantial differences in the mechano-sensitivity between normal cells and
transformed cancer cells.
Several reports have indicated that cancer cell growth is vulnerable to mechanical forces.
Studies showing inhibition of tumor growth after stretching or exercise in mice model could be
explained through a mechanical force-dependent growth inhibition (Berrueta et al., 2018; Betof
et al., 2015). Fluid shear-induced killing of circulating tumor cells and adhesive cancer cells can
also be explained by increased sensitivity to mechanical forces (Lien et al., 2013; Regmi et al.,
2017). In addition, ultrasonic and shock-wave therapies have demonstrated that cancer cells are
particularly sensitive, although concerns were raised about increased metastasis and healthy
tissue damage (Lin et al., 2012; Marano et al., 2017; Nicolai et al., 1994; Zequi et al., 2018). We
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designed this study to investigate the effect of mechanical cyclic stretch on the behavior of
normal versus transformed cancer cells from a variety of different tissue backgrounds.
Because the presence or absence of a rigidity-sensing complex correlates strongly with the
normal (rigidity-dependent) and transformed growth (rigidity-independent) states, respectively, it
is relevant to understand the important elements of the rigidity sensors. They are transient
sensors that involve the assembly of sarcomeric units of ~2 µm in length with anti-parallel actin
filaments anchored to the matrix adhesion sites. Myosin IIA contraction pulls the adhesions to a
constant displacement of about 100 nm for 30 seconds irrespective of substrate rigidity
(Wolfenson et al., 2016). If the matrix is rigid, then the contractile force will exceed ~25 pN and
the adhesions will be reinforced. Adhesion disassembly and eventual cell apoptosis will occur if
the surface is soft. Many different tumor suppressors are part of the rigidity sensors including
TPM2.1 (formerly known as Tm1), myosin IIA, DAPK1, α-actinin 4 and receptor tyrosine
kinases like AXL and ROR2 (Helfman et al., 2008; Qin et al., 2018; Yang et al., 2016; Yang et
al., 2018). A common mechanism of the malignant transformation is the depletion of TPM2.1 or
the increased expression of Tpm3 (Raval et al., 2003; Stehn et al., 2013). Decreasing the ratio of
TPM2.1 to Tpm3 causes the loss of matrix rigidity sensing and enables transformed cell growth.
This process can occur even in the normal cells (Wolfenson et al., 2016; Yang et al., 2018). In
contrast, restoration of TPM2.1 level in many cancer cells re-establishes rigidity-dependent
growth and inhibits transformation in those cells (Yang et al., 2018). Other mechanosensory
cytoskeletal proteins (myosin IIA, α-actinin, filamin A, AXL, ROR) are part of the rigidity
sensing complex. Depletion of these sensory proteins and other proteins like scaffolding proteins
(caveolin1) promotes cell transformation, while their restoration in cancer cells will block
transformed cell growth (Lin et al., 2015; Wolfenson et al., 2016; Yang et al., 2016; Yang et al.,
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2018). Thus, the transformed state of cancer cells appears in many different tissue backgrounds
to result from the loss of rigidity sensing, i.e. transformed cells grow on soft surfaces because
they fail to sense the surface softness.
There are a number of common features of cancer cell mechanics that differ from normal
cells, including a decrease in the rigidity of the cortical cytoskeleton (Lin et al., 2015;
Swaminathan et al., 2011) and increased traction forces on matrices (Kraning-Rush et al., 2012).
In addition, there are multiple reports demonstrating the altered expression and function of
calcium channels as well as increased calpain activity in cancer cells (Azimi et al., 2014; Storr et
al., 2011). In this study, we explore various aspects of the transformed cancer cells that cause
selective apoptosis of the transformed, but not of the normal cells. Surprisingly, we find that the
cyclic stretching of transformed cancer cells inhibits their growth and activates apoptosis.
Restoration of matrix rigidity sensing in transformed cancer cells by TPM2.1 expression results
in increased growth and survival upon cyclic stretch, particularly on soft surfaces. Transformed
cell death results from increased calcium entry upon stretch that activates calpain 2 mediated-
mitochondrial apoptosis.
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Results
Cancer Cells Elongate upon Cyclic Stretch: Magnitude, Frequency and Rigidity
Dependence
To understand the impact of cyclic stretch on cancer cell behavior, we used the MDA-MB-231
metastatic breast cancer cell line which is known for being highly aggressive. Cyclic stretching
of MDA-MB-231 cells on fibronectin-coated flat PDMS (2 MPa, rigid) for 6 hrs caused
prominent cell elongation (quantified by the cell aspect ratio (AR)) (Figures 1A and S2A). In the
case of MDA-MB-231 cells, the highest AR was at 5% cyclic strain (AR ~12) in contrast to 1%
(AR ~3) and non-stretched cells (AR ~2). In addition, a single stretch (5% for 6 hrs) did not alter
cell elongation (AR was the same as non-stretched cells). Next, to assess the effect of stretching
frequency on MDA-MB-231 cell morphology, cells were subjected to different frequencies (0.1
Hz to 1 Hz) for 6 hrs. Interestingly, cells displayed frequency-dependent cell elongation with
maximum elongation observed at 0.5 Hz with 5% cyclic strain (AR ~11) (Figure 1B). On the
other hand, cells showed less elongation at 0.1 Hz (AR ~4) and no significant difference in the
elongation for 1 Hz vs. 0.5 Hz was observed. Thus, the cancer cell elongation in response to
cyclic stretch optimally at a frequency of 0.5 Hz and a strain of 5% was chosen to minimize
damage to normal cells.
To investigate the effect of matrix rigidity on stretch-dependent cancer cell elongation,
MDA-MB-231 cells were cyclically stretched (5%, 0.5 Hz) on surfaces with different rigidity
(Figure 1C). On soft pillars (~8 kPa), cells failed to display elongation in response to cyclic
stretch and showed similar AR values on stretch and non stretch surfaces (Figure 1D). In fact,
cells were round on soft surfaces which resembled the morphology of non-stretched cells on soft
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surfaces. In contrast on rigid pillars (~55 kPa) and flat PDMS surfaces (2 MPa), cells had an
elongated morphology, while non-stretched cells had a non-polarized morphology on both
surfaces. In addition, we observed that cyclic stretching was responsible for a small increase in
the cell area irrespective of the matrix rigidity (Figure 1D).
To delineate the influence of extracellular matrix protein (ECM) on stretch-dependent
cancer cell morphology, similar stretching experiments were repeated with MDA-MB-231 cells
cultured on collagen-I coated surfaces (Figure S3A). Again, cells elongated after cyclic stretch
on flat PDMS surfaces (AR ~9) in comparison to non-stretched cells (AR ~3). In contrast,
stretching did not result in the cell elongation on soft surfaces (AR ~3). Further, the stretch-
dependent cell area increment was noticed on rigid surfaces only. In short, MDA-MB-231 cells
responded similarly on fibronectin and collagen-I coated surfaces by showing elongated and
round morphology on rigid and soft surfaces respectively upon cyclic stretch (Figure 1E).
Rigidity-Dependent Cancer Cells Spread like Normal Cells upon Cyclic Stretch
MDA-MB-231 cells lack the cytoskeletal protein TPM2.1 which is required for proper
rigidity sensing (Wolfenson et al., 2016). When TPM2.1 was restored to MDA-MB-231 cells
(rigidity-dependent cancer cells) and they were cyclically stretched (5%, 0.5 Hz) for 6 hrs,
spread area increased with no change in AR in stark contrast to the wild-type MDA-MB-231
(Figures 2A and S2B) (AR~3 for both non-stretched and stretched MDA-MB-231-TPM2.1
cells). Further, we tested whether cancer cells from another tissue, SKOV3 (human ovarian
adenocarcinoma which lacks endogenous TPM 2.1) would behave similarly to MDA-MB-231
cells. SKOV3 cells also demonstrated cyclic stretch-dependent cell elongation (AR~7) after 6 hrs
compared to the non-stretched SKOV3 cells (AR~3) (Figure 2B). No increase in the cell area
with stretch was observed for SKOV3 cells. However, when TPM2.1 was expressed in SKOV3
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cells, they spread more symmetrically (AR~3) and had a 2.5 fold increase in cell area upon
stretch when compared with wild-type SKOV3 (Figure 2C). Thus, rigidity-dependent MDA-MB-
231 and SKOV3 cells (with restored level of TPM2.1) behaved like wild-type mouse embryonic
fibroblasts (MEFs) and breast epithelial cells (MCF10A) upon cyclic stretch (Figure 2D and
S4A).
To determine if the loss of TPM 2.1, which caused transformation in MEFs and MCF10A,
would cause these cells to behave similarly to the transformed cancer cells, we knocked down
TPM2.1 in those cells (Figure 2E & S4B). Although wild-type-MEFs and -MCF10A showed a
significant increase in spread area with no increase in AR after 6 hrs of cyclic stretching (5%, 0.5
Hz), TPM2.1 KD-MEFs and –MCF10As were elongated upon stretch (Figure 2D and S4A).
Statistical analysis confirmed that there was no significant difference in AR values of wild-type
cells before and after stretch, whereas TPM 2.1 KD cells demonstrated a two-fold increase in AR
after stretch (Figure 2F and S4A). Moreover, there was a stretch-dependent increase in cell area
in both wild-type and TPM2.1 KD MEF cells similar to MDA-MB-231 cells after stretch. On the
other hand, in MCF10A, we only observed a stretch-dependent cell area increment in wild-type
cells. Overall, these results indicate that the cytoskeletal protein TPM2.1 is required for proper
sensing of the external mechanical forces that promoted cell spreading in response to the periodic
stretching (Figure 2G).
Cyclic Stretch Reduces Cancer Cell Growth but Stimulates Normal Cell Growth
MDA-MB-231 cells are known for aggressive growth behavior. Therefore, we tested the
effect of cyclic stretch on MDA-MB-231, MDA-MB-231-TPM2.1 and normal cell growth
(Figure 3A). Unlike previous studies of cyclic stretching effects on fibroblast growth (Cui et al.,
2015), cyclic stretching (5%, 0.5 Hz) of MDA-MB-231 cells for 9 hours significantly reduced
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their proliferation on fibronectin coated rigid and soft surfaces (Figure 3B and 3C). Similarly,
cyclic stretch-dependent inhibition of proliferation was observed on the collagen-coated surfaces
(Figure S3B). However, a dramatic shift in the effect of stretch on proliferation was found when
MDA-MB-231-TPM2.1 cells were stretched. MDA-MB-231-TPM2.1 cells exhibited a higher
proliferation rate with cyclic stretch than without on both rigid and soft surfaces. When a similar
experiment was repeated using normal cells like MEFs and MCF10A as the control cells, these
cells also had a higher proliferation rate (similar to the MDA-MB-231-TPM2.1 cells) with cyclic
stretch than without on both surfaces (Figure 3B, 3C and S4C). After MEFs were transformed by
TPM2.1 KD, cyclic stretch significantly inhibited proliferation in comparison with the non-
stretched cells. Thus, transformation of MEFs by TPM2.1 KD caused a similar behavior to
transformed MDA-MB-231 cancer cells with cyclic stretch, indicating that cells in the
transformed state had increased sensitivity to stretch-induced growth inhibition. In contrast,
normal cells and TPM2.1 expressing MDA-MB-231 cells displayed elevated growth upon cyclic
stretch.
To further verify the importance of TPM2.1 in regulating cell proliferation, we analyzed
SKOV3 cells (no endogenous TPM2.1). Interestingly, cyclic stretch again caused a reduction in
the proliferation of SKOV3 cells on rigid surfaces, while the proliferation rate increased in
TPM2.1 expressing SKOV3 cells with cyclic stretch (Figure S5). Thus, transformed cell growth
was inhibited by cyclic stretch, whereas in cancer cells that were normalized by TPM2.1
expression, cyclic stretch activated growth.
HT1080 (human fibrosarcoma) cells exhibited normal levels of TPM2.1 but were
transformed because of high levels of Tpm3 that competed with TPM2.1 and blocked rigidity
sensing (Gateva et al., 2017; Yang et al., 2018). As predicted from other transformed cells
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results, cyclic stretch decreased the proliferation rate of HT1080 cells on rigid and soft surfaces
(Figure 3D). Thus for several different cell lines from different tissues, cyclic stretch inhibited
transformed cell growth and promoted normal cell growth (Figure 3E).
Cyclic Stretch Triggers Cancer Cell Apoptosis but Protects Normal Cells
To determine if inhibition of growth correlated with an increase in cell apoptosis, cells
were analyzed for apoptosis after 24 hrs of cyclic stretch by annexin-V immunostaining. As
shown in Figure 4A, cyclic stretch activated substantial apoptosis in MDA-MB-231 cells on rigid
and particularly on soft surfaces. Closer inspection of apoptotic cells confirmed that they
exhibited the characteristics of apoptosis such as membrane blebbing and cell rounding.
Interestingly, the apoptosis rate was much higher on the soft surface (34%) where cyclic stretch
did not cause cell elongation as compared to the rigid one (18%) (Figure 4B). We observed a
similar trend of apoptosis on collagen-coated rigid and soft surfaces (Figure S3C). Further, after
transformation of MEFs and MCF10A cells by TPM2.1 KD, cyclic stretching caused a dramatic
increase in apoptosis on rigid (20% for MEFs and 54% for MCF10A) and soft (35% for MEFs
and 84% for MCF10A) surfaces when compared with the non-stretched control (Figure 4A, 4B
and S4D). To strengthen the significance of cyclic stretch in promoting transformed cell
apoptosis, MDA-MB-231 cell apoptosis was compared after 24 and 48 hrs of cyclic stretch. We
found a notably higher extent of apoptosis (37-39%) after 48 hours on both rigid and soft
surfaces (Figure S6) that was likely an underestimate, since many of the cells that died early
were washed away. Thus, cyclic stretch caused apoptosis of transformed cells from several
different tissues (Figure 4C).
To determine if cyclic stretching conditions affected normal cells, the apoptosis assay was
performed on rigidity-dependent cells (including TPM2.1 restored cancer cells). When MDA-
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MB-231-TPM2.1 cells were stretched, there was negligible apoptosis on rigid (6%) or soft (10%)
surfaces (Figure 4A and 4B). However, a dramatic increase in apoptosis (21%) was observed in
non-stretched MDA-MB-231-TPM2.1 cells on soft surfaces, showing that TPM2.1 restored
cancer cells behaved as normal rigidity-dependent cells. To further reconfirm these results,
similar experiments were performed on normal cells (MEFs and MCF10As) as the control cells.
Both stretched and non-stretched normal cells revealed negligible apoptosis (2-7%) on rigid
surfaces (Figure 4A, 4B and S4D). However, on soft surfaces, non-stretched control cells
experienced a substantial increase in apoptosis (41-45%), whereas cyclic stretching of the soft
surface protected them from apoptosis (5-10%). Thus, cyclic stretching of rigidity-dependent
cells inhibited apoptosis, particularly on soft surfaces, whereas cyclic stretching of their
transformed counterparts increased apoptosis on soft surfaces.
Cyclic Stretch Mediated-Cancer Cell Apoptosis Is Not Driven by DAPK1 Activation
Recently, we determined that tumor suppressor Death Associated Protein Kinase1
(DAPK1) causes rigidity-dependent cell apoptosis (anoikis) on soft surfaces but accelerates
adhesion assembly on rigid surfaces (Qin et al., 2018). To determine if DAPK1 was involved in
stretch-dependent cancer cell apoptosis, we blocked DAPK1 activity with a DAPK1 inhibitor
(DAPK1 inhibitor, 100 nM). DAPK1 inhibition decreased MEF apoptosis (14%) on non-
stretched soft surfaces (Figure 4D and Figure S7A). As previously reported, we also observed
distinct colocalization of DAPK1 with peripheral adhesions not only in stretched and non-
stretched normal cells (HFFs) on rigid surface but also in the cells on soft surfaces after cyclic
stretch (Figure S7B). In contrast, DAPK1 was cytoplasmic in normal cells on non-stretched soft
surfaces. When DAPK1 inhibitor was added to MDA-MB-231 cells during cyclic stretch, there
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was no decline in apoptosis rate on rigid or soft surfaces indicating that a DAPK1-independent
apoptotic pathway was involved in cyclic stretch-induced transformed cancer cell apoptosis.
Calpain is the Major Downstream Effector of Stretch-Induced Cancer Cell Apoptosis
Since DAPK1 was not involved in cyclic stretch-induced cancer cell apoptosis, we looked
for another apoptotic mechanism and found that calpain proteases were implicated in inducing
apoptosis in cancer cells (Storr et al., 2011). Calpains were generally activated by a rise in
intracellular calcium level and then triggered apoptosis via caspase activity. To test the potential
role of calpains in cyclic stretch-induced apoptosis, ubiquitous calpain (calpain 1 & 2) activity
was inhibited using the calpain inhibitor (ALLN). Addition of ALLN inhibited apoptosis of
MDA-MB-231 cells after cyclic stretch compared to the non-treated counterpart (Figure 5A and
S8). Next, to assess the role of specific calpains in cancer cell apoptosis, calpain knock down
cells were developed using specific siRNAs. When cyclic stretch experiments were performed
on calpain 1 and calpain 2 KD MDA-MB-231 cells, both calpain 1 and 2 KD cells had lower
levels of apoptosis than control MDA-MB-231 cells (Figure 5A and S8). However, calpain 2 KD
cells showed significantly lower apoptosis, indicating that calpain 2 played the major role in
triggering cyclic stretch-induced cancer cell apoptosis (Figure 5B).
To understand the downstream effector of calpain cleavage, we focused on the pro-
apoptotic molecule BAX which can be cleaved by calpains to induce stress-mediated apoptosis
(Wood et al., 1998). Upon activation, BAX molecules translocated from cytoplasm to the
mitochondria to initiate the mitochondrial apoptotic pathway (Wolter et al., 1997). To check if
BAX was a downstream effector of calpain, MDA-MB-231 cells were stretched in the presence
of the BAX inhibitor peptide V5 (blocks mitochondrial translocation of BAX). Interestingly,
BAX inhibition reduced cell apoptosis (~10%) in stretched MDA-MB-231 cells (Figure 5C).
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Thus, it appeared that BAX acted downstream of the calpain protease to initiate the
mitochondrial apoptotic pathway.
Cyclic Stretch-Induced Calcium Influx is the Upstream Effector of Cancer Cell Apoptosis
If stretch-induced apoptosis of transformed cells was caused by calpain, then the cellular level of
calcium should increase upon cyclic stretch. Although normal cells have tightly regulated
calcium channel expression and regulation (Azimi et al., 2014), greater calcium entry in
transformed cells due to cellular stress could have caused cell death via apoptosis (Baig et al.,
2016). To determine first if calcium entry was important, we treated cells with the nonspecific
calcium channel blocker, Gadolinium, before the application of stretch (Bourne and Trifaro,
1982). Interestingly it blocked the apoptosis in stretched cancer cells (Figure 6A), indicating a
possible role of calcium influx as the upstream effector of apoptosis. To actually measure
calcium levels during stretch, different cell lines (MDA-MB-231, MDA-MB-231-TPM2.1 and
MCF10A) were transfected with a calcium indicator, GECO1 and subjected to the cyclic stretch.
In the case of MDA-MB-231 cells, GECO1 intensity increased threefold after 2 hrs of cyclic
stretching. (Figure 6B) However, there was no increase in GECO1 intensity in MDA-MB-231-
TPM2.1 and MCF10A cells after 2 hrs of cyclic stretching, indicating that stretch triggered-
calcium influx did not take place in rigidity dependent-cancer cells and normal cells. Thus, a
stretch-induced increase in calcium levels was only found in the transformed cancer cells.
Mechanosensitive Piezo1 Channels Promote Cyclic Stretch-Induced Cell Apoptosis
The linkage between stretch-induced apoptosis and calcium entry implied the involvement of
mechanosensitive calcium channels. Piezo channels were activated by mechanical perturbations
of cells and enabled calcium entry to trigger intracellular calcium dependent signaling in many
physiological processes (Coste et al., 2010). We therefore compared the level of apoptosis in
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Piezo1 expressing HEK 293 cells (transformed human embryonic kidney cells) and Piezo1
knock out HEK 293 cells (piezo KO HEK 293) after cyclic stretch on rigid surfaces for 6 hrs.
Cyclic stretch caused an increase in the apoptosis of piezo expressing HEK 293 cells, while
negligible apoptosis was observed in piezo KO HEK 293 cells (Figure 7A). Transformed HEK
293 cells lack endogenous TPM2.1. When the apoptosis assay was performed on TPM2.1
restored HEK 293 cells either with or without Piezo1, negligible apoptosis was noticed in piezo
KO HEK 293 or Piezo1 expressing HEK 293 cells with TPM2.1 restored (Figure 7B and Figure
S9). Thus, we suggest that stretch-induced transformed cell apoptosis involved Piezo1 channels
as well as the transformed state.
To determine if the rise in cellular calcium level relied upon Piezo1 channels, cyclic stretch
was applied on GECO1 transfected Piezo1- and Piezo1 KO-HEK 293 cells and TPM2.1 restored
Piezo1 HEK 293 cells. Of these three different cells, only piezo1 expressing HEK 293 cells
showed a twofold increase in GECO1 intensity within 30 min on rigid surface, while rigidity-
dependent or Piezo1 KO cells did not display any significant increase in the intensity (Figure
7C). All together, these findings confirmed that stretch-induced calcium entry and subsequent
apoptosis in transformed HEK 293 cells depended upon Piezo1 channels.
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Discussion
These results indicate that transformed cells exhibit cyclic stretch-induced apoptosis
regardless of the tissue origin, whereas rigidity-dependent normal cells show cyclic stretch-
induced growth and survival. For several different cell lines, the presence of TPM2.1 causes
rigidity-dependent growth, while depletion of TPM2.1 results in transformed growth despite
different tissue backgrounds. In transformed cells, cyclic stretch initially causes cell elongation
on rigid surfaces that is dependent on the stretch frequency as well as the magnitude of strain.
The frequency and magnitude of stretch were in a physiologically relevant range for exercise and
those parameters were used in all further studies. Longer period of cyclic stretch inhibits
transformed cell growth and increases apoptosis, particularly on soft surfaces. In contrast, cyclic
stretch promotes normal cell growth and inhibits apoptosis, particularly on soft surfaces. We find
that this relationship holds for cells from breast, ovaries, kidneys, connective tissue and skin.
Further, the stretch-induced apoptosis is calpain dependent (primarily calpain 2) and calpain acts
downstream of stretch-mediated calcium influx. The mechanosensitive Piezo1 calcium channels
are needed for calcium influx in the background of the transformed cell state.
Numerous studies regarding the transformed cell state imply that normal cells from a
variety of different tissues can be transformed. During the cell transformation, there is a shift
from an epithelial to mesenchymal phenotype in many cases; but the transformed phenotype is
strongly correlated with the loss of rigidity sensing and a change in the cortical actin
organization that makes cancer cell softer (Lin et al., 2015; Wolfenson et al., 2016). However,
the traction forces that most cancer cells exert on matrices are higher than their normal
counterparts and restoration of rigidity sensing in those cancer cells causes a decrease in traction
forces (Northcott et al., 2018; Wolfenson et al., 2016; Yang et al., 2018). Thus, it is logical to
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consider the ‘transformed state’ as a distinct cell state that can occur in cells from widely
different tissues with different expression patterns. Further, cells from different tissues can be
toggled between the transformed and the normal state by changing the level of expression of
several cytoskeletal proteins that enable or block the matrix rigidity sensing (Yang et al., 2018).
A relatively common feature of transformed cancer cells is that they have elevated levels
of calcium channels and calpain protease (Azimi et al., 2014; Storr et al., 2011). The higher level
of a mechanosensitive calcium channels and their altered functioning could explain the increased
susceptibility of transformed cancer cells to stretch-induced apoptosis. However, the
transformation of fibroblasts and epithelial cells with TPM2.1 KD will also sensitize those cells
to stretch-induced apoptosis. This indicates that transformed cells in general, whether from a
tumor or from a normal cell background, will undergo apoptosis in response to the appropriate
mechanical forces that can be mimicked by cyclic stretch.
There are previous reports of mechanical force-induced cancer cell death. Continuous
flow forces can cause the apoptosis of several different cancer cells attached to surfaces, whereas
oscillatory flow forces do not (Lien et al., 2013). The apoptotic pathway involves bone
morphogenetic protein receptor, Smad1/5, and p38 MAPK unlike our findings. Another study
shows that high shear forces on suspended circulating tumor cells initiates apoptosis possibly
through an oxidative stress-induced mitochondrial apoptotic pathway (Regmi et al., 2017).
Further, recent findings reveal that the stretching of mice for ten minutes a day for four weeks
suppresses the breast cancer growth by 50% compared to the non-stretched controls (Berrueta et
al., 2018). From these and other studies, there are strong indications that transformed cancer cells
may be mechanically vulnerable and thus sensitive to mechanical forces. Here we show that the
increased mechanical sensitivity is linked to the transformed cell state and not to a specific organ
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or a specific cell type. Thus, it seems that this feature could be exploited to damage many
different types of tumor cells, particularly in a metastatic state where cells are not protected by
tumor fibrosis.
In Figure 8, we present a working model of mechanical stretch-induced transformed
cancer cell death, which we are describing as ‘mechanoptosis’. Prolonged application of
mechanical stresses on the transformed cells triggers a rise in calcium levels through altered
calcium homeostatic mechanisms, most likely increased Piezo1 activity. The rise in intracellular
calcium level causes activation of calpain 2 protease which initiates a mitochondrial apoptotic
pathway through its downstream effector, the BAX molecule. In contrast, mechanical stretching
of normal cells does not cause a rise in calcium levels because of a more tightly regulated
calcium homeostatic mechanism. Thus, cyclic stretch promotes the normal cell growth and
survival especially on the soft surfaces.
Although most cancer cells are transformed, there are many other aspects that are
important for tumor growth (Hanahan and Weinberg, 2000; Hirashima et al., 2013; Nishida et
al., 2006). Nevertheless, the transformed cell state appears to be associated with changes in
important cell functions such as the loss of rigidity sensing. Many other factors are also altered
beyond the loss of rigidity sensing in transformed cells, making it difficult to link the increased
mechanical sensitivity to a single factor. Despite the fact that we do not fully understand why
transformed cells are more sensitive to calcium loading with cyclic stretch, that sensitivity can
provide new approaches for killing cancer cells while actually stimulating the normal cell
growth. Hence, much more work is needed to determine if this feature can be exploited in an in
vivo setting to damage transformed cancer cells.
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Material and Methods:
Cell Culture
All cell lines MDA-MB-231(gift from Dr. Jay Groves, MBI, NUS), SKOV3 (gift from Dr. Ruby
Huang, CSI, NUS), HT1080 (ATCC), human foreskin fibroblasts (HFF, ATCC), transformed
human embryonic kidney cells (piezo expressing HEK 293 and piezo knock-out HEK 293, gift
from Boris Martinac, VCCRI Sydney and Ardem Patapoutian) and mouse embryonic fibroblasts
(MEF, ATCC) were cultured in high glucose DMEM (Thermo Fisher Scientific) supplemented
with 10% FBS and 1% penicillin and streptomycin at 37°C in 5% CO2 environment. MCF10A,
human breast epithelial cells (ATCC) were cultured in specialized growth medium as mentioned
before (Wolfenson et al., 2016). For cell experiments, high-glucose DMEM containing 10% FBS
and 1% penicillin and streptomycin was used. Cells were trypsinized using TrypLE (Thermo
Fisher Scientific) and cultured on human plasma fibronectin (10 μg/ml, Roche) or rat tail
collagen-I (20 μg/ml, Sigma) coated PDMS surface. Pharmacological inhibitors used are as
follows: DAPK1 inhibitor (100 nM, EMD Millipore), calpain inhibitor ALLN (100 μM, Sigma
Aldrich), non-specific calcium channel blocker gadolinium chloride (20 μM, Sigma Aldrich)
BAX inhibitor peptide V5 (200 μM, Merck). For inhibition assays, inhibitors were added to the
medium one hour post cell seeding and cyclic stretching was applied one to two hours after
addition of inhibitors. In general, cyclic stretching was applied 20 min after the cell seeding on
the cell stretching post (unless otherwise stated).
Plasmids and Transfection
For cell transfection using GFP tagged-TPM2.1 plasmid and RFP tagged-GECO1 calcium
indicator, Neon electroporation system (Invitrogen) was used according to the manufacturer’s
protocol. To perform TPM2.1 knockdown, cells were plated in a six well plate. TPM2.1 siRNA
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(Qiagen) transfection was performed on the next day using lipofectamine RNAiMAX
(Invitrogen) according to the manufacturer’s protocol. Calpain siRNAs were prepared by Protein
Cloning Expression Facility, MBI, NUS using following siRNA sequences adapted from
previous reports. The calpain siRNA primers were: Calpain 1 primer (5’-AAGAC
CUAUGGCAUCAAGUGG-3’; 5’-AAGAAGUUGGUGAAGGGCCAU-3; ’5’-AAGCUAGU
GUUCGUGCACUCU-3’; 5’-AAG AGGAGAUUGACGAGAACU-3’ (Upla et al., 2008; Wu et
al., 2006) and Calpain 2 primer (5’-AAGACUUCACCGGAGGCAUUG-3’; 5’-AAGAUG
GGCGAGGACAUGCAC-3’) (Ma et al., 2012; Xu et al., 2010). Calpain knockdown cells were
generated by cell transfection using lipofectamine RNAiMAX.
Pillar and Flat Surface Fabrication
Pillar substrates were prepared with PDMS (10:1 prepolymer to curing agent ratio, Sylgard 184;
Dow Corning) spin coating on plastic molds at 1800 rpm for 1 min and cured at 80°C for 3 hrs.
The PDMS membrane was then peeled off the mold to serve as the surface for cell culture as
shown in Figure S1A. Pillars were fashioned in a square grid with 0.5 μm diameter, 1.8 μm
(~8kPa, soft) or 0.8 μm (~55kPa, rigid) height and 1.5 μm centre-to-centre distance among
pillars (Lohner et al., 2018). The flat PDMS layer (2 MPa rigid, 60-70 μm thick) was prepared by
spin coating PDMS (10:1 prepolymer to curing agent ratio) on a plastic sheet at 1800 rpm for 1
min, cured at 80°C for 3 hrs and peeled off to treat as the loading surface for cell culture.
Stretching Device Fabrication and Operation
The scheme of stretching device fabrication was shown in Figure S1A. Standard lithography
technique was used to fabricate the PDMS stretching device. Briefly, PDMS solution (10:1
prepolymer to curing agent ratio) was spin coated on a silicon wafer containing microfeatures at
500 rpm for 30 sec and cured at 80°C for 3 hrs. PDMS layer embossed with microfeatures was
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peeled off, fixed to the glass bottom dish and subsequently covered with another thin PDMS
layer (60-70 μm thick). Glycerol (90%) was used as a lubricant to avoid sticking of thin PDMS
layer to the lower cell loading post. A PDMS cube with hole in middle was mounted on the top
of thin PDMS layer as shown in Figure S1A. Finally, the PDMS cube was connected to the
pump assembly. As depicted in Figure S1B & S1C, negative pressure was generated in the
hollow ring surrounding cell loading post by a pump which pulled the thin PDMS membrane
down to create membrane stretching. Different negative pressures were applied to measure the
magnitude of the strain generated at each pressure and the percent strain was then characterized
by measuring the changes in centre to centre distance between the pillars (Figure S1D & S1E).
To control the frequency of cyclic stretch, the Labview program was used.
Proliferation Assay
Cells were serum starved overnight to synchronize the cell cycle prior to cell seeding. EdU (5-
ethynyl-2’-deoxyuridine) reagent kit (Invitrogen) was used according to the manufacturer’s
instruction. The proliferation assay was performed after 9 hrs.
Apoptosis Assay
To assess cell apoptosis, Annexin V-Alexa Fluor 488 or Annexin V-Alexa Fluor 594 conjugates
(Thermofisher Scientific) were used according to the manufacturer’s protocol. Cells were
incubated with Annexin V solution after the specified time mentioned in the results section.
Immunocytochemistry and Fluorescence Microscopy
Cell samples were fixed with 4% paraformaldehyde (Thermofisher Scientific) in PBS for 10 min
and then permeabilized with 0.5% Triton X-100 for 5 min. Bovine Serum Albumin solution (2%)
(BSA) was used as a blocking buffer and samples were treated with BSA for 1 hr. Samples were
then incubated with primary antibodies of rabbit polyclonal anti DAPK1 (Abcam, 1:200), mouse
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21
monoclonal anti Paxillin (BD Bioscience, 1:400) and mouse monoclonal anti tropomyosin
(1:200, Merck) for 1 hr at 37°C followed by treatment with Alexa Fluor-488 and -594 secondary
antibodies (Invitrogen). For F-actin staining, cells were incubated with Alexa Fluor 594
phalloidin antibody (Invitrogen) for 1 hr. DAPI dye (Merck) was used to stain cell nucleus.
Fluorescence and bright field images were acquired using Delta Vision System (Applied
Precision) on an Olympus IX70 microscope and equipped with CoolSNAP HQ2 CCD camera
(Photometrics, Tucson, USA). Live cell imaging was done using same Delta Vision System
maintained at 37°C, 5% CO2 condition. Optical images were acquired with an EVOS digital
fluorescence microscope (Fisher Scientific).
Western Blot
The cells were harvested after at least 48 hrs with the respective siRNA’s, pelleted, washed with
PBS and then lysed with RIPA buffer (Sigma-Aldrich) supplemented with the 1X cOmplete
Protease Inhibitor cocktail tablet (Roche, Cat no: 4693116001). The lysed cell mixture was
centrifuged at 15000 rpm, 4°C for 30 mins. The supernatant was collected and mixed with 2X
loading dye (2X Lammeli Buffer, Bio-Rad, Cat no: 1610737) + Beta-Mercaptoethanol (Sigma-
Aldrich). The samples were then denatured for 10 mins at 95°C and run on a 4-20% Mini-
PROTEAN ® TGX precast protein gels (Bio-Rad, Cat no: 4561094). The gels were then
transferred onto membranes. The membranes were blocked with 5% BSA solution in 1X TBST
(Tris-Buffered Saline with Tween-20) for 1 hr and incubated with primary antibody overnight at
4°C. The membranes were washed three times in TBST (10 mins per wash) followed by
secondary antibodies in 1XTBST (Horse Radish Peroxidase –HRP) treatment for 1 hr. The
membranes were again washed three times in 1X TBST. The chemiluminescence of the
membranes was developed using Super Signal Femto Substrate Kit (Pierce) and developed using
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ChemiDoc Touch Imager (Bio-Rad). Primary antibodies and conditions used are as follows:
Calpain 1 antibody (Rabbit, 1:1000, Abcam ab39170), Calpain 2 antibody (Rabbit, 1:1000,
Abcam ab39165), TPM 2.1 (Mouse, 1:1000, Merck), GAPDH (Mouse, 1:3000, Abcam ab8245).
The secondary antibodies, HRP-conjugated goat anti-rabbit IgG (bio-Rad 170-6515) and goat
anti-mouse IgG (bio-Rad 170-6516) were used at half of the primary antibody concentration.
STATISTICAL ANALYSIS
Statistical difference was calculated using two-tailed Student t-test. P value <0.05 was
considered statistically significant. All data represented as mean ± standard deviation.
ACKNOWLEDGEMENTS
We would like to thank all group members of Sheetz lab. A.T and M. Y are supported by the
Singapore Ministry of Education Academic Research Fund Tier 3 (MOE grant No: 2016 T3-1-
002). M.S is supported by NIH grants, NUS grants and Mechanobiology Institute, National
University of Singapore.
AUTHOR CONTRIBUTION
M.S and A.T designed study; A.T performed cell experiments, device fabrication and data
analysis; A.T and M.Y performed calcium indicator experiments; Y.W fabricated molds; Y.N
and C.T.L engineered stretching device; A.H developed western blots; A.T and M.S wrote
manuscript. All authors read manuscript and commented on it.
CONFLICT OF INTEREST STATEMENT
The authors declare no competing financial interest.
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Figure Legends
Figure 1. Effects of Magnitude and Frequency of Cyclic Stretching on Cancer Cell
Morphology with Different Matrix Rigidities
(A) MDA-MB-231 cells displayed the highest elongation (AR ~12) at 5% cyclic strain on flat
PDMS surfaces (rigid) after 6 hrs. (B) Cancer cell elongation was also found to be dependent on
the frequency of stretching, demonstrating maximum elongation at 0.5Hz with 5% cyclic strain
(AR ~11). (C) Matrix rigidity influenced the cancer cell elongation. Representative images
illustrating cyclic stretch-dependent cancer cell elongation on rigid surfaces (rigid pillars and flat
PDMS) but not on the soft pillar surfaces. (D) Statistical data showing marked increases in the
cell AR on rigid surfaces only. However, a stretch-dependent cell area increase was observed on
both rigid and soft surfaces after cyclic stretch. (E) Schematic diagram illustrating matrix
rigidity-dependent cancer cell elongation upon cyclic stretch. n=100 cells for cell aspect ratio and
n=30 cells for cell area. Experiments were repeated at least three times. *P< 0.05, **P< 0.01,
***P< 0.001. Scale bar: 20 µm.
Figure 2. TPM2.1 Restored Cancer Cells Spread upon Cyclic Stretch
(A) TPM2.1 restored MDA-MB-231 cells exhibited less elongation (AR~3) and increase in
spread area after 6 hrs of cyclic stretch in contrast to wild-type MDA-MB-231, (n=30 cells). (B)
SKOV3 cells also showed an elongated morphology with 6 hrs of cyclic stretch (AR~7)
compared to non-stretched cells (AR~3). No significant increment in the cell area was noticed
after cyclic stretching, (n=30 cells). (C) TPM2.1 restored SKOV3 cells displayed less elongation
(AR~2) and increase in spread area after cyclic stretch, (n=30 cells). (D) Representative images
showing the morphology of wild-type- and TPM2.1 KD-MEFs on a rigid surface after 6 hrs with
and without cyclic stretch. (E) Western blots showing the knockdown of TPM2.1 in MEFs. (F)
Wild-type MEFs showed spread morphology (AR~2) in response to cyclic stretch after 6 hrs,
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while TPM2.1 KD MEFs were elongated (AR~4). The spread cell area increased for both wild-
type- and TPM 2.1KD-MEFs upon cyclic stretch, n=100 cells for aspect ratio and n=30 cells for
cell area measurement. (G) Schematic diagram showing the differential effect of cyclic stretch
on the morphology of transformed and normal cells. Experiments were repeated at least three
times. *P< 0.05, **P< 0.01, ***P< 0.001. Scale bar: 20 µm.
Figure 3. Cyclic Stretch Inhibits Transformed Cell Proliferation
(A) Time line depicting the cell proliferation assay protocol. (B) Representative images showing
the effect of cyclic stretch on the growth of transformed and normal cells (growing cells were
labelled by the anti-EdU antibody). (C) Cyclic stretch inhibited proliferation of M-231 breast
cancer cells on rigid and soft surfaces, while in TPM2.1 restored M-231 cells cyclic stretch
increased proliferation. Similarly, cyclic stretching of MEFs (control cells) increased
proliferation, whereas cyclic stretch of TPM2.1 KD MEF inhibited cell proliferation, (n>1000
cells for M-231, MEFs, TPM2.1 KD MEFs and n>500 cells for TPM2.1 restored M-231). (D)
Cyclic stretching of HT1080 fibrosarcoma cells also inhibited proliferation on rigid and soft
surfaces, (n>1000 cells). (E) Illustration depicting the cyclic stretch-dependent decline in
transformed cell growth and increase in normal cell growth. Experiments were repeated at least
two times. *P< 0.05, **P< 0.01, ***P< 0.001. Scale bar: 100 µm.
Figure 4. Cyclic Stretch Potentiates Transformed Cell Apoptosis
(A) Representative images illustrating the effect of 24 hrs cyclic stretch on apoptosis in M-231,
TPM2.1 restored M-231, MEFs and TPM2.1 KD MEFs. Dotted white line indicates location of
MEFs. (B) Cyclic stretch promoted M-231 cell apoptosis on rigid and soft surfaces. However,
TPM2.1 restored M-231 cells experienced negligible apoptosis upon cyclic stretch. Likewise,
MEFs (control cells) also demonstrated insignificant apoptosis with cyclic stretch. However,
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non-stretched MEFs on soft surfaces displayed considerably high apoptosis similar to the non-
stretched TPM2.1 restored M-231 cells, indicating cell anoikis on the soft surface. In contrast,
TPM2.1 KD MEFs behaved like M-231 cells and experienced an increase in apoptosis after
cyclic stretch, (n>350 cells). (C) Diagram showing the effect of cyclic stretch on transformed
and normal cell apoptosis. (D) M-231 cells experienced notably high apoptosis upon stretch
even in the presence of DAPK1 inhibitor, suggesting the involvement of DAPK independent
apoptotic pathway. However, DAPK1 inhibition rescued the apoptosis in non-stretched MEFs on
soft surfaces showing that DAPK1 had a role in normal cell anoikis on soft surface, (n>300
cells). Data are representative of two independent experiments. *P< 0.05, **P< 0.01, ***P<
0.001. Scale bar: 50 µm.
Figure 5. Calpain Acts Downstream of Calcium to Initiate Cyclic Stretch-Induced
Apoptosis
(A) Cyclic stretch induced M-231 cell apoptosis decreased with Calpain inhibition by using
either a specific siRNA against Calpain-1 or -2, or a chemical inhibitor (ALLN). Specifically,
Calpain 2 KD caused greater inhibition of cell apoptosis compared to Calpain 1 KD, (n>1000
cells each for Calpain-1 and -2 knockdown assay and n>500 cell for ALLN inhibition assay and
control cells). (B) Schematic diagram highlighting the major role of Calpain 2 in promoting the
cyclic stretch-induced M-231 cell apoptosis. (C) M-231 cell apoptosis was further reduced by
BAX protein inhibition, implying that BAX protein acts downstream of the calpain protease,
(n>300 cells for BAX inhibition assay and n>600 cells for the control). Data are representative
of two independent experiments. **P< 0.01, ***P< 0.001. Scale bars: 50 µm.
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Figure 6. Cyclic Stretch-Mediated Calcium Influx is Upstream Activator of Transformed
Cell Apoptosis
(A) Cyclic stretch-dependent M-231 cell apoptosis was down regulated by non specific calcium
channel blocker, Gadolinium ion, (n>500 cells). Data are representative of two independent
experiments. ***P< 0.001. Scale bar: 50 µm. (B) Cells expressing GECO1 calcium indicator
were imaged on a cyclically stretched rigid surface. Representative time-lapse montages
displaying the progressive increase in GECO1 intensity in M-231 cells. In stark contrast, no such
increase in GECO1 intensity was observed in TPM2.1 restored M-231 cells and normal
MCF10A cells. After 2 hrs, the GECO1 intensity increased by three-fold in M-231 cells
compared to other groups, n>15 cells from three independent experiments were used. Scale bar:
50 µm.
Figure 7. Mechanosensitive Piezo1 Channels Needed for Cyclic Stretch-Induced Apoptosis
(A) Representative images illustrating elevated level of apoptosis in piezo expressing
transformed HEK293 cells upon cyclic stretch compared to piezo KO HEK 293 cells. (B) Piezo
expressing HEK293 cells exhibited significant apoptosis (~15%) upon cyclic stretch compared to
piezo KO HEK 293 and TPM2.1 restored HEK 293 either with or without Piezo1 (~4% in all
cases), (n>300 cells). Data are representative of two independent experiments. ***P< 0.001.
Scale bar: 50 µm. (C) Cells expressing GECO1 calcium indicator were monitored on a cyclically
stretched rigid surface. Representative time-lapse montages show a progressive increase in
GECO1 intensity in Piezo expressing HEK293 cells. No such increase in GECO1 intensity was
observed in TPM2.1 restored HEK293 and Piezo KO HEK293 cells. Two-fold increase in the
GECO1 intensity was observed in Piezo expressing HEK293 cells after 30 min compared to
other groups, n=16 cells from three independent experiments were used. Scale bar: 50 µm.
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Figure 8. Proposed Model of Transformed Cell Mechanoptosis
Prolonged cyclic stretching of transformed cells leads to intracellular calcium overloading,
activation of calpain protease and in turn initiation of the mitochondrial apoptotic pathway
through calpain downstream effector, BAX molecule. In contrast, mechanical stretching of
normal cells does not trigger calcium influx through their tightly regulated calcium channels. In
fact, it promotes the normal cell growth and survival through normal calcium homeostasis.
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Figure 6
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Figure 7
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Figure 8
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