Attenuation of Cell Mechanosensitivity in Colon Cancer Cells during In Vitro Metastasis
et al. (2012) Attenuation of Cell Mechanosensitivity in Colon Cancer Cells during In
Vitro Metastasis. PLoS ONE 7(11): e50443. doi:10.1371/journal.pone.0050443
Attenuation of Cell Mechanosensitivity in Colon Cancer Cells during In Vitro Metastasis
Xin Tang 0
Qi Wen 0
Theresa B. Kuhlenschmidt 0
Mark S. Kuhlenschmidt 0
Paul A. Janmey 0
Taher A. Saif 0
Fan Yuan, Duke University, United States of America
0 1 Department of Mechanical Science and Engineering, College of Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America, 2 Departments of Physiology , Physics, and Bioengineering , Institute for Medicine and Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America, 3 Department of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America , 4 Micro and Nanotechnology Laboratory (MNTL) , University of Illinois at Urbana-Champaign , Urbana, Illinois , United States of America
Human colon carcinoma (HCT-8) cells show a stable transition from low to high metastatic state when cultured on appropriately soft substrates (21 kPa). Initially epithelial (E) in nature, the HCT-8 cells become rounded (R) after seven days of culture on soft substrate. R cells show a number of metastatic hallmarks . Here, we use gradient stiffness substrates, a bio-MEMS force sensor, and Coulter counter assays to study mechanosensitivity and adhesion of E and R cells. We find that HCT-8 cells lose mechanosensitivity as they undergo E-to-R transition. HCT-8 R cells' stiffness, spread area, proliferation and migration become insensitive to substrate stiffness in contrast to their epithelial counterpart. They are softer, proliferative and migratory on all substrates. R cells show negligible cell-cell homotypic adhesion, as well as non-specific cell-substrate adhesion. Consequently they show the same spread area on all substrates in contrast to E cells. Taken together, these results indicate that R cells acquire autonomy and anchorage independence, and are thus potentially more invasive than E cells. To the best of our knowledge, this is the first report of quantitative data relating changes in cancer cell adhesion and stiffness during the expression of an in vitro metastasis-like phenotype.
Funding: The work is financially supported by National Science Foundation (NSF) grants No. ECCS 07-25831, 10-02165 and NIH RO1 083272-03. XT was funded
by NSF Grant 0965918 IGERT: Training the Next Generation of Researchers in Cellular and Molecular Mechanics and BioNanotechnology. The funders had no role
in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Most cancer deaths are caused by metastasis and not by the
primary parent tumor [2,3,4,5,6]. During metastasis, malignant
cancer cells escape from the tumor by detaching from one another
or from other cells and the extracellular matrix (ECM) [2,3,6,7].
The escaped cells actively express proteinases and alter their
adhesion ligands to degrade and modify their surrounding ECM
[3,4,5,8,9]. Simultaneously, they up-regulate their motility and
resistance to apoptosis for successful vascular spread and invasion
of distant healthy organs [6,7,10]. Concurrently, these cells lower
their stiffness [11,12,13,14], i.e., increase their compliance to flow
through small capillaries [4,15,16]. A quantitative study of the
mechanical properties of cancer cells during the early phases of
metastasis; however, is lacking [17,18,19,20], largely because of
the challenges in detecting the onset of metastasis in vivo and the
heterogeneity in biochemical and cellular properties of individual
tumor cells [3,17,21,22].
We recently discovered  that human colon carcinoma cells
(HCT-8) can consistently display an in vitro metastasis-like
phenotype (MLP) when cultured on soft hydrogel substrates with
appropriate mechanical stiffness (polyacrylamide gels with Youngs
modulus: 21,47 kPa [1,23]). HCT-8 cells are epithelial (E) in
nature. When cultured on soft substrates, they first form distinct
epithelial clusters or islands. After 7 days, the cells dissociate from
the islands, and assume a rounded shape (R cells). These R cells
are highly proliferative, migratory and they significantly
downregulate E-cadherin expression - typical hallmarks of metastasis
[1,24]. Furthermore, E to R transition is repeatable and
irreversible [1,24]. On hard substrates (3 GPa polystyrene
substrates), this E to R transition does not occur.
In this study, we first present a detailed investigation of
mechanosensitivity of both pre- and post-metastasis-like HCT-8
cells using a gradient stiffness substrate. The study reveals the loss
of mechanosensitivity of HCT-8 R cells in contrast to both the E
cells and normal fibroblasts. The stiffness of the R cells, measured
by AFM, becomes independent of substrate stiffness. In contrast,
the stiffness of E cells is correlated with the substrate stiffness.
Coulter counter and Bio-MEMS assays reveal that R cells have
low homotypic cell-cell adhesion and negligible non-specific
adhesion compared to E cells.
1. Weak adhesion between HCT-8 R cells and substrate
To explore how HCT-8 R cells respond to different
physiologically-relevant substrates of varying stiffness, HCT-8 R cells were
harvested from soft PA gels, expanded as described in Materials
and Methods and then cultured on fresh stiffness-gradient PA gel
substrates with stiffness varying continuously from 1 to 20 kPa
(Fig. 1a, left to right). The stiffness-gradient substrate is coated with
a uniform fibronectin concentration to allow cell attachment to the
substrate [25,26,27]. For comparison, both HCT-8 E cells and
normal Monkey Kidney Fibroblast (MKF) cells, without any prior
exposure to PA gels, were plated on the same stiffness gradient
substrates and surface functionalization (Fig. 1b and 1c). The
normal MKF cells were chosen as control because they are known
to be mechanosensitive to substrate stiffness . We found, in
contrast to HCT-8 E cells and normal MKF cells, HCT-8 R cells
constitutively showed very limited substrate contact areas
regardless of substrate stiffness. The R cells contact area with the
substrate is about 4060% of their apparent projected area. As
measured by 3D confocal microscopic imaging, the R cell contact
area with substrate is only 49.5620.9 mm2 (n = 34), which is
3.860.3 fold smaller than E cells (n = 47), suggesting that R cells
have weaker adhesion with the substrate than E cells. The weak
adhesion of R cells with substrate is also consistent with the
observation that R cells show a smaller projected area, than E cells
on the same stiffness substrate (Fig. 1d). The projected area of
isolated cells without any neighboring cell contact, of is 1.9 0.6
fold smaller for R cells (n = 68) than E cells (n = 61).
HCT-8 R cells also show a remarkable insensitivity to changing
the mechanical-stiffness of their culture substrate. They retain a
rounded phenotype and limited adhesion area to substrates
regardless of the substrates stiffness (Fig. 1a, indicated by white
arrows; Fig. 1d, 1e and 1g). When the substrate stiffness varied
over a 20-fold range, the spread area of single R cells increased
only about 27%, (from 156.2642.1 mm2 on a 1 kPa region
(n = 62) to 197.9683.6 mm2 (n = 56) on a 20 kPa region) (Fig. 1d).
Across the stiffness tested, the increase in R cells spread area is not
as dramatic as that of E and MKF cells. On 5 kPa, 10 kPa and
15 kPa regions, their spread areas are 158.2640.3 mm2 (n = 56),
182.3632.2 mm2 (n = 63), and 190.9682.5 mm2 (n = 57),
respectively (Fig. 1d). Also, the R cell shape factor changed only 7% from
0.960.2 on a 1 kPa region to 0.860.2 on a 20 kPa region (Fig. 1g;
The shape factor, S = 4*pA/P2, where A is the area of the cell and
P is the perimeter. S = 1 for perfect circular shape and 0 for
irregular shape), indicating constitutive rounded shape
independent of the substrate stiffness. On 5 kPa, 10 kPa and 15 kPa
regions, the shape factors of single R cells are 0.860.1, 0.860.2,
and 0.960.2, respectively (Fig. 1g). After prolonged culture (60
days), R cells did not show any reversal toward an epithelial
morphology on all substrates, regardless of stiffness, even very rigid
polystyrene (3 GPa). In addition, daily recording via video
microscopy indicates that R cells show no sign of impairment of
proliferative activity even after several months in culture. In
contrast, both HCT-8 E cells and MKF cells cultured on the same
type of stiffness gradient substrates show obvious sensitivity to the
mechanical stiffness of their culture substrate. The individual
isolated E cells spread area increases 2.5 fold over 20-fold substrate
stiffness change, from 239.66191.9 mm2 on the 1 kPa region to
578.16429.8 mm2 on the 20 kPa region (Fig. 1b, indicated by
white arrows). As substrates become rigid, the HCT-8 E cells
display a greater spread area, with their spread areas
270.8 201.7 mm2 (n = 51), 276.06104.8 mm2 (n = 62), and
442.76367.7 mm2 (n = 55) on 5 kPa, 10 kPa and 15 kPa regions,
respectively (Fig. 1b). Their shape factor decreased from 0.960.2
on the 1 kPa region to 0.660.2 on the 20 kPa region (Fig. 1g).
Across other stiffness tested, the single E cells shape factors are
0.860.2 (on 5 kPa region), 0.860.1 (on 10 kPa region), and
0.760.3 (on 15 kPa region), respectively. The mechanosensitivity
of MKF is even more pronounced as compared to HCT-8 cancer
cells (Fig. 1c). The spread area of individual isolated MKF cells
(Fig. 1c; indicated by white arrows) increases 5 fold across the
gradient substrate, from 286.4686.2 mm2 (n = 46) on the 1 kPa
region to 1421.76845.7 mm2 (n = 31) on the 20 kPa region
(Fig. 1d). As the substrate stiffness increases, their spread area
increases dramatically, and are 578.16373.1 mm2 (n = 62),
749.96355.5 mm2 (n = 63), and 1218.66773.5 mm2 (n = 59) on
5 kPa, 10 kPa and 15 kPa regions, respectively. Concurrently with
increasing substrate stiffness, single MKF cells spread to a more
irregular morphology, with their shape factor decreasing from
0.960.1 on the 1 kPa to 0.560.2 on the 20 kPa regions,
respectively (Fig. 1g). On the intermediate stiffness regions, i.e. 5
kPa, 10 kPa and 15 kPa regions, the shape factors of single MKF
cells are 0.760.3, 0.660.3 and 0.560.3, respectively. The weak
adhesion between HCT-8 R cells and the substrate, as well as the
independence of R cell morphology from substrate stiffness,
strongly suggest that R cells lose anchorage-dependence and
communication with their mechanical microenvironment. This
anchorage-independence can potentially promote R cells survival
in suspension, which is an essential hallmark of in vivo metastasis of
cancer cells [2,3,4,16,22].
2. HCT-8 R cells show weak cell-cell adhesion
On stiffness-gradient substrates, both HCT-8 E cells and MKF
cells show cell colony formation, especially on stiffer regions
(indicated by yellow arrows in Fig. 1b and 1c). The colony size is
positively correlated with the substrate stiffness. On substrate
stiffness 1 kPa, 5 kPa, 10 kPa, 15 kPa and 20 kPa gels the cell
colony sizes of HCT-8 E cells are 2962.261000.5 mm2,
3662.161105.3 mm2, 4249.56919.5 mm2, 9736.564032.7 mm2
and 11748.762144.9 mm2, respectively (Fig. 1f). For HCT-8 R
cells on the same stiffness substrates, the colony sizes are markedly
smaller than their E counterparts even when R cells are in contact
with neighboring cells for 3 days (Fig. 1a). On substrate stiffnesses of
1 kPa, 5 kPa, 10 kPa, 15 kPa and 20 kPa, the R cell colony sizes are,
1087.46338.3 mm2, 1449.86343.4 mm2, 3062.261326.9 mm2,
3849.66919.1 mm2 and 3912.161183.8 mm2, respectively (Fig. 1f).
We also observed that inside R cell colonies, the cell-cell contact
area is not as extensive as in E cell colonies. R cells appear to be just
touching each other at point-contacts (Fig. 1a). These results suggest
R cell-cell adhesion is not sufficient for them to form cohesive
colonies or cell islands as do E and MKF cells.
Furthermore, it is interesting to note that as HCT-8 E cells or
MKF cells undergo homotypic cell-cell adhesion, their individual
cell areas and cell shape factor become remarkably less substrate
stiffness-dependent (Fig. 1b and 1c, indicated by yellow arrows).
Individual cell areas and shape factors of single HCT-8 E cells
inside cell islands on 1 kPa gels are 785.66299.4 mm2 and
0.760.1, respectively, which is similar to those on 20 kPa gels,
892.86322.1 mm2 and 0.660.1 (Fig. 1e and 1 h). Same
characteristics are observed on intermediate stiffness, the cell area and
shape factor of individual HCT-8 E inside islands are
526.76187.0 mm2 and 0.860.1 on 5 kPa gels,
633.96421.4 mm2 and 0.660.2 on 10 kPa gels, and
723.16515.2 mm2 and 0.660.2 on 15 kPa gels. For individual
MKF cells inside islands, their cell area and shape factor are
928.56374.0 mm2 and 0.560.3 on 1 kPa gels, 892.86415.7 mm2
and 0.560.3 on 5 kPa gels, 1098.16564.6 mm2 and 0.560.2 on
10 kPa gels, 1008.86223.7 mm2 and 0.360.2 on 15 kPa gels, and
1160.66429.7 mm2 and 0.460.1 on 20 kPa gels (Fig. 1e and 1 h).
Once these cells establish cell-cell contacts, the E and MKF cells
show cell spreading on very soft 1 kPa gels, suggesting the cell-cell
signals overwhelm the cell-substrate signals (the left region in
Fig. 1b and 1c, indicated by yellow arrows). The majority of
8 R cells; however, remain rounded, with same apparent cell area
and shape factor as those of isolated R cells, even when in contact
with neighboring cells (Fig. 1a, indicated by yellow arrows). This R
cell phenotype results in generally smaller R cell colony area
compared to E cell islands consisting of similar cell numbers
(Fig. 1f). The individual cell areas and shape factors of single R
cells inside R cell colonies on 1 kPa gels are 151.8633.4 mm2 and
1.060.1, respectively, and is similar to those on 20 kPa gels
(169.6630.5 mm2 and 0.960.2), respectively, as well as those of
single R cells displaying no cell-cell contacts (Fig. 1e and 1 h). On
5 kPa, 10 kPa and 15 kPa gels, the cell area and shape factor of
individual HCT-8 R cells inside islands are 156.2652.3 mm2 and
0.860.1, 142.8647.2 mm2 and 0.960.0, and 160.7633.4 mm2
and 0.860.2, respectively. This unique phenotype persists even
after R cells are cultured on the very stiff polystyrene substrates (3
GPa) for prolonged culture times (months); again suggesting weak
cell-cell adhesion among R cells. Taken together, these results
suggest that during or after E-to-R transition, R cells acquire cell
autonomy that is characterized by markedly reduced cell-cell and
cell-substrate adhesive contacts.
3. R cells have reduced homotypic cell-cell adhesive activity
Besides estimating the cell-cell adhesion qualitatively based on
their contact morphologies, we further used the coulter counter
assay to quantitatively study the functional loss of HCT-8 cell-cell
adhesion following E-to-R transition. The coulter counter
measures the rate and degree of cell adhesion by quantifying the
reduction in the number of single cells in suspension as cell
aggregates form as a function of time [1,29,30]. The kinetics of
specific homotypic cell-cell adhesion for cancerous epithelial
HCT-8 E and R cells were measured and compared. Normal
(non cancerous) Ma104 epithelial cells were used as a control. We
found that disassociated HCT-8 R cells (harvested from 21 kPa
PA substrates) displayed a markedly lower rate and extent of
cellcell adhesion as compared to the original HCT-8 E cells cultured
on hard polystyrene substrates (Fig. 2). Previous studies have
shown that after 120 minutes of incubation, 84.864.0% of the
HCT-8 R cells remained as single cells, in contrast to 37.666.1%
of original HCT-8 E cells and 5.260.7% of normal Ma104 cells
. This remarkable result strongly indicates that the cell-cell
adhesive activity of HCT-8 R cells is almost completely lost after
they disassociate from E cell islands. This result is consistent with
our finding of reduced E-Cadherin expression on R cells [1,24].
The reduction in cell surface adhesiveness was also seen when
non-specific adhesion forces between HCT-8 surfaces and
SiO2coated Bio-MEMS probes were measured.
4. Cell stiffness changes reflect the mechanosensitivity
In addition to substrate stiffness-dependent cell morphology
changes, HCT-8 E cells also showed varied cell stiffness dependent
on culture substrate rigidity. Using atomic force microscopy
(AFM), the cell stiffness of HCT-8 E cells cultured on
stiffnessgradient substrates is determined by indentation using
siliconnitride cantilevers with a spring constant of 148.14 pN/nm (with
consistent cell indentation speed 0.1 mm/sec). Hertz theory (see
Materials and Methods) was used to extract the elastic modulus of
the indented cells. To facilitate the comparison between different
cells on same substrate stiffness, we designated 5 equal-space
regions across the entire stiffness range, with region 1 spanning a
stiffness of 1-4 kPa, regions 5 with stiffness 58 kPa, 912 kPa, 13
16 kPa, 1720 kPa respectively (Fig. 3a). Using AFM, we found
HCT-8 E cells increase their cell stiffness as the substrates become
more rigid. From region 1 to region 5, HCT-8 E cells stiffness
progressively increased from 1.460.9 kPa to 1.960.8 kPa, to
2.161.4 kPa, to 2.261.3 kPa, and to 3.862.0 kPa, respectively
(n = 6,10 for each region; Fig. 3b). In particular, it is worth noting
that the gradient of cell stiffness increase (Fig. 3a) seems to match
the gradient of gel substrate stiffness increase. These results are
consistent with those previously reported , and suggests that
HCT-8 E cells are highly responsive to the delicate variation of
their microenvironmental mechanical signals. The stiffness of
HCT-8 R cells; however, on all the different stiffness substrates,
appears invariant at 0.560.4 kPa, indicating that R cells have a
very limited or no interaction with their mechanical
microenvironment. The AFM study also indicated that R cells are
mechanically softer than E cells, which potentially may enhance
their malleability to allow more efficient invasion of target tissues
following in vivo metastasis.
5. E cell islands show high non-specific adhesion compared to R cells
The surface non-specific adhesions of HCT-8 E cells (4th day of
culture on PA gel) and R cells were measured using a
microfabricated bio-MEMS force sensor (Fig. 4 and Materials and
Methods) [1,30,31]. The sensor consists of a microcantilever beam
with calibrated force-displacement relation (see Materials and
Methods). There is a flat probe (width 15 mm and depth 5 mm)
attached to the beam, which forms adhesive contact with the cells
(Fig. 4a). The sensor is made from single crystal silicon, and is
coated with a thin layer of native silicon oxide (SiO2). The probe
and the sensor are not functionalized. The sensor is manipulated
with an x-y-z piezo stage. The flat probe is brought in contact with
E-cell islands lateral convex surface at the boundary. Each E-cell
island consists of 100 s of cells with multiple cells stacking at the
island periphery (Fig. 4b). After a 2-minute contact, the force
sensor is pulled away horizontally from the cell island at a constant
speed of 2.160.4 mm/s (Fig. 4c). The contact time was chosen as
2 minutes, since prolonged contact duration might result in
cellular deposition of ECM on probe and complicate the analysis.
Due to the cell-probe adhesion, the sensor beam deforms during
retraction, i.e., cells apply a restoring force against detachment.
The short contact duration between the cell and the probe
prevents the activation of cell integrins and the formation of any
focal adhesion on the probe (takes .30 minutes to form [32,33]).
Therefore, only non-specific adhesive interactions can be formed
between the cell surface and the SiO2-coated probe.
Figure 3. Stiffness and morphology of HCT-8 E cells correlate with substrate rigidity. Using Atomic Force Microscopy, the stiffness of
8 E cells cultured on the gradient substrate is determined. The HCT-8 E cells increase their cell stiffness as the substrates become more rigid. To
facilitate the comparison between different cells on same substrate stiffness, five equal-spaced regions across the entire stiffness range are
designated: region 1 covers 14 kPa, region 2 covers 58 kPa, region 3 covers 912 kPa, region 4 covers 1316 kPa, and region 5 covers 1720 kPa.
(a) From region 1 to region 5, the E cell stiffness progressively increases with values 1.4260.85 kPa to 1.9060.77 kPa, 2.0661.39 kPa, 2.1561.28 kPa,
and 3.8261.98 kPa, respectively. In contrast, on gel substrates with same stiffness gradient, the post-metastatic R cells show almost invariant cell
stiffness. (b) Phase-contrast pictures of HCT-8 E cells on gradient PA substrates.
We found that, during retraction of the bio-MEMS sensor,
Ecell islands stretch locally by 1520 mm resulting in a conical shape
(see both schematics in Fig. 4c and phase-contrast pictures in
Fig. 5). Note this stretch is different from that due solely to
membrane tether, which consists of stretching only the
phospholipid bilayer. During probe retraction, the cone is continuously
stretched with increasing contact angle h, while the cell contact
with the probe drops in a stepwise fashion (Fig. 5b-5d). The
increase of force between cell and probe is reflected in the
progressive increase of gap between a fixed reference and the
probe (from D0 to D1 and D2). Cell force is calculated from the
change of gap and force-deformation calibration of the sensor
spring. At a critical value of force, Fc, the cone suddenly detaches
from probe (Fig. 5d-e). For E-cells, Fc is the maximum force on the
force-displacement curves. We consider Fc as a measure of
cellprobe adhesion. We measured Fc for 12 such cell clusters and
obtained Fc = 256.3633.7 nN (n = 12). Similar experiments with
R cells show negligible cell-probe adhesion with Fc
= 1.1460.13 nN (n = 25; Fig. 6). Hence, R cells have negligible
non-specific adhesion compared to E cells and thus appear to be in
a lubricated state perhaps enabling them to be adapted to
passage through vascular capillary beds during in vivo metastasis.
To our knowledge, the present study is the first to describe and
evaluate the change in mechanosensitivity in human colon cancer
cells during a metastasis-like transition produced by solely by
changing the mechanical microenvironment during in vitro culture.
In an earlier paper we reported HCT-8 cells execute an E-to-R
transition on 21,40 kPa stiffness substrates . The present study
effectively employs a combinatorial assay system approach using
stiffness-gradient substrates, Coulter counter assay, atomic force
microcopy (AFM) and Bio-MEMS force sensors to explore the
quantitative mechanosensitivity change of human colon
carcinoma HCT-8 epithelial E cells as they transit to rounded-shape R
cells. We found, triggered by the appropriate substrate rigidity
cues, that HCT-8 R cells lose their sensitivity to both the substrate
microenvironment as well as their interaction with neighboring R
and E cells. As a result, HCT-8 R cells acquire autonomy for
survival as anchorage-independent, mobile cells, which is an
essential feature of the early events of cancer cell metastasis
The physical properties of culture substrates are found to widely
affect the phenotypes and gene expression of a number of normal
and cancerous cells [1,17,18,35,36,37,38,39,40,41,42,43,44,45,
Figure 4. Surface non-specific adhesion of E cell islands measured using a micro-fabricated bio-MEMS force sensor. (a) The
nonfunctionalized micro-fabricated Si force sensor with a flat probe and with known force-deflection relation is manipulated by a high-resolution x-y-z
Piezo-stage to contact cell islands lateral convex surface (on x-y plane). (b) Confocal microscopy of cell islands show the height of islands is on the
order of 30,50 mm. The vertical height of bio-MEMS probe is 5,10 mm. (c) After a 2-minute contact, force sensor is horizontally pulled away at a
constant speed of 2.160.4 mm/s. While the cell adhesion between the probe and cell surface hinders retraction of the sensor, the sensor beams
deform by d, giving the force F. Note that the probe is not functionalized. The 2-minute contact between the probe and cells prevents the activation
of cell integrins and the formation of any cell focal adhesion, which takes .30 minutes to form.
46,47,48,49,50,51,52,53,54,55]. To respond to substrate stimuli,
cells adhere to and spread on the substrate followed by sensing and
processing both mechanical and chemical signals [26,37,44,
46,49,53,55,56,57,58,59,60,61]. As we have previously shown ,
after 7-day culture on soft substrates, HCT-8 cells undergo an E to R
transition characterized by R cells dissociating from the parent
epithelial cell layer or cell islands. These dissociated R cells show
remarkably diminished adhesion (both specific and non-specific
Figure 6. Surface non-specific adhesion of R cells measured by micro-fabricated bio-MEMS force sensor. (a) Adhesive force of R cells on
MEMS probe as the probe is moved away from the cells after 2 min contact (n = 25). (be). Phase-contrast images of R cells and MEMS probe when
non-specific adhesion between them is measured. The maximum detachment force measured is ,2.5 nN, while the cell deformation is barely
noticeable. Scale bar: 40 mm.
[1,24,29]) compared to their E cell counterparts. Unlike E cells, the
dissociated HCT-8 R cells show substrate-stiffness independent
cellsubstrate interactions. Their proliferation is not impaired by weak
anchorage with the culture substrate or to other cells (Fig. 1).
Anchorage independence is a distinguishing feature of metastatic
cells [7,21,36]. Indeed, our recent in vitro basement membrane cell
invasion assays indicate that HCT-8 R cells are significantly more
invasive than E cells .
Our discovery of an E-to-R transition in HCT-8 colon
adenocarcimona cells suggests that appropriate substrate
mechanical softness may promote or aid in initiation of the early events in
cancer cell metastasis, and ironical loss of mechanosensitivity,
which could aid in vascular spread to distal tissue target sites. This
study reveals that colon cancer cells can attain this trait solely by
culture on the appropriately soft substrate. We are currently
evaluating whether R cells display enhanced metastatic behavior
in animal studies as compared to E cells. If E to R transition
correlates with acquisition of enhanced metastatic activity,
manipulation of the mechanical microenvironment may serve as
an attractive in vitro model for investigating the early events of
cancer cell metastasis as well as for screening of possible
antimetastatic therapeutic agents.
Materials and Methods
1. Cell culture, microscopy imaging and PA gels preparations
Human colon adenocarcinoma HCT-8 cells (ATCC No.:
CCL244) were cultured in RPMI 1640 (Gibco No.: 23400062)
supplemented with 2 grams of sodium bicarbonate per liter, giving
final concentrations of 10% horse serum (Gibco No.: 26050088),
16 antibiotic-antimycotic (Gibco No.: 15240062) and 1 mM of
sodium pyruvate (Gibco No.: 11360) . Ma104 cells (embryonic
African green monkey kidney) were obtained from M.A.
Bioproducts and cultured in MEM (Gibco No.: 41500018)
supplemented with 2 grams of HEPES per liter, 2.2 grams of
sodium bicarbonate per liter, 16 antibiotic-antimycotic as above,
and 5% fetal bovine serum (Gibco No.: 16140). The monkey
kidney fibroblast (MKF) cell line (CV-1, ATCC, Manassas, VA)
was cultured in a medium with 90% DMEM (ATCC, Manassas,
VA), 10% FBS (ATCC, Manassas, VA) and 16
antibioticantimycotic (Gibco No.: 15240062). The cell density before
plating was counted with standard hemocytometer. Standard cell
culture incubator was used to provide the culture condition with
sufficient humidity, 37uC temperature, and 5% CO2. An inverted
optical microscope (Olympus IX81, Olympus America) with an
objective 206 and a high-speed SPOT camera was used to record
cell phenotypes and deformation behavior [1,30,48,62,63,64].
Polyacrylamide (PA) gels were prepared following the protocols
described in the literature [1,64]. The PA gels of different rigidities
were fabricated with varying relative concentrations of acrylamide
(Bio-Rad) and N, N- methylene bis-acrylamide (Bio-Rad) to
obtain different cross-link extents. For 21 kPa PA gels, the mol./v
concentrations of acrylamide and N, N- methylene bis-acrylamide
are 8% and 0.13%, respectively. All gels were covalently coated
with 25 mg/mL fibronectin (BD).
2. Bio-MEMS force sensor calibration and experimental setup
We characterized the non-specific adhesion strength of the
HCT-8 cells using a novel Bio-MEMS force sensor [30,63]. Forces
were measured using two micromechanical beams with a spring
constant 3.48 nN/mm and calibrated using a tungsten
microneedle with known stiffness (0.091 N/m) [1,30,31,48,62,64,65,66,67].
The tungsten microneedle is 6 mm long and 22 mm in diameter.
The force vs. beam deflection characteristics of MEMS force
sensor were calibrated using a tungsten microneedle and best fitted
to (Eqn. 1):
where R-square = 0.9936. In Eqn. (1), F is the net force acting on
the probe along the force sensor backbone and D is the
displacement of the probe. Here D = D0 + d, where D0 is the
initial deflection of the sensor beam and d is the additional
deformation due to applied force F. Both F and D are in SI units,
Newton and meter, respectively. Before measuring cell adhesion,
the sensor was sterilized using Alcohol and DI water multiple
times. During the experiment, the T-shaped sensor probe was
allowed to contact the cell lateral membrane for 2 minutes and
was then moved away horizontally. Due to cell adhesion, the
sensor beams deform during retraction by d, giving the force
according to Eqn. (1). Note that the probe is non-functionalized by
any extracellular matrix proteins and only has a coating of SiO2 on
the surface. Therefore, non-specific adhesive interactions were
formed between the cell and the SiO2-coated probe. The entire
pulling process lasts 1030 seconds.
3. AFM calibration of cell island elastic modulus
Atomic force microscopy (Asylum) with silicon-nitride cantilever
having a spring constant k = 148.14 pN6nm21 (Veeco) was used
to characterize the stiffness of the HCT-8 cell monolayer. A
conical tip approximation (Eqn. 2) for the AFM tip was used to
extract the substrates Elastic modulus [1,48,68,69,70,71,72,73]:
where z and d are the cantilever base PZT displacement and the
cantilever tip deflection, respectively. z0 is the piezo-controllers
vertical position as the AFM tip touches the cell layers apical
surface, and d0 is the initial cantilever deflection prior to bending. v
is the Poissons ratio for cell layer (v = 0.3,0.5 in present study). a
= 35u is the half open-angle of cantilever tip. During experiments,
the curves of force versus sample indentation were obtained and
used to determine Elastic modulus distribution.
4. Coulter counter assay
The cells were harvested and individualized by trypsin/EDTA
treatment followed by restoration in complete culture medium
containing serum to neutralize residual trypsin. Since fibronectin
was used for cell adhesion on PA gel substrates and there was no
tissue present, trypsin/EDTA (not collagenase) were used to
remove cells from culture substrates into single cell state. The cell
suspensions were placed in 176100 mm capped polypropylene
tubes (Falcon No.: 352059) and were rotated end over end at 78
revolutions per minute in a conventional Labquake shaker
(Barnstead/Thermolyne Model No.: 41510) for 1 hour at 37uC
to allow recovery of any surface cell adhesion molecules (CAMs) or
other proteins. The recovery of CAMs following trypsinization was
guaranteed by identifying the increase in cell aggregate number as
incubation duration prolongs, as shown in Fig. 2. The
preincubation time was 1 hour because over-aggregation should be
avoided in adhesion-rate assay in order to differentiate the precise
adhesion rate kinetic effectively. Portions of the pre-incubated cells
(0.3 ml, ,56105 cells) were placed in flat bottom vials (Fisher
catalog No.: 0333926D) and rotated in a gyratory water bath
shaker (G-76, New Brunswick) at 12 rpm at 37uC for 5, 10, 20, 40,
60, 80, 100 and 120 minutes, respectively. At the end of each time
period, cells were diluted with 8 mL 0.9% saline and placed on ice
to stop further cell aggregation. The number of single cells present
at each time point was measured in the Coulter counter as
described in [1,29].
5. Immunofluorescent staining and confocal microscopy imaging
Cultures were fixed with 4% paraformaldehyde at 37uC for
30 minutes followed by the 15-minute permeabilization in 0.1%
Triton (6100) solution. Rhodamine phalloidin (520/650, red) was
used as fluorescent conjugate to stain specifically F-actin filaments.
Image-iTTM FX Signal enhancer (Invitrogen, Cat No.: I36933)
was used to block all non-specific binding and enhance the
imaging quality. The actin structures were imaged using
laserscanning confocal microscopy (Leica SP2, Heidelberg, Germany)
with appropriate fluorescent filters, and data were analyzed using
Andor IQ software (Andor technology Inc., USA). Multiple
images were combined using Amira (Advanced3DVisualization
and Volume Modeling) software (Fig. 4b).
6. Imaging processing and data analysis
Image stacks processing was performed using ImageJ (NIH) and
Photoshop CS3 (Adobe Inc.) software. Statistical data processing
and analysis were performed using Office Excel (Microsoft), Origin
Pro (OriginLab Corp.) and Matlab (the MathWorks) programs.
The bio-MEMS force sensor was fabricated with help from Dr. Yaguang
Lian, Mr. Marty Harris, Mr. Romans Hal and Mr. Edmond Chow at the
Micro and Nanotechnology Laboratory (MNTL), University of Illinois at
Urbana-Champaign (UIUC). AFM experiments were carried out with the
help from Mr. Scott MacLaren at the Materials Research Laboratory
(MRL), UIUC. Immuno-fluorescent staining and confocal microscopy
imaging were carried out at the Institute for Genomic Biology (IGB),
Digital Computer Laboratory (DCL), and Beckman Institute (BI-ITG),
UIUC with assistances from Dr. Duohai Pan, Dr. Jon Ekman, Mr. Darren
Matthew Stevenson, Dr. Mayandi Sivaguru, Dr. Glenn Fried and Mrs.
Joanne Manaster. We thank Mrs. Wen Yang (MSE, UIUC) for assistance
with data analysis.
Conceived and designed the experiments: XT QW PJ TS. Performed the
experiments: XT QW. Analyzed the data: XT. Contributed reagents/
materials/analysis tools: XT QW TK MK. Wrote the paper: XT TS.
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