Matrix softness regulates plasticity of tumour-repopulating cells via H3K9 demethylation and Sox2 expression

Abstract

Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of cancer cells critical in cancer progression. However, the underlying mechanisms of how TRCs maintain their self-renewing capability remain elusive. Here we show that relatively undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening, histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC self-renewal.

Full text

ARTICLE
Received 6 Jun 2014 |Accepted 8 Jul 2014 |Published 6 Aug 2014
Matrix softness regulates plasticity of
tumour-repopulating cells via H3K9 demethylation
and Sox2 expression
Youhua Tan1,2,*, Arash Tajik2,*, Junwei Chen1,*, Qiong Jia1, Farhan Chowdhury3, Lili Wang1, Junjian Chen1,
Shuang Zhang1, Ying Hong1, Haiying Yi1, Douglas C. Wu2, Yuejin Zhang1, Fuxiang Wei1, Yeh-Chuin Poh1,2,
Jihye Seong2,4,w, Rishi Singh2, Li-Jung Lin4, Sultan Dog
˘anay3,5, Yong Li6, Haibo Jia1, Taekjip Ha3,5,
Yingxiao Wang7, Bo Huang6,8 & Ning Wang1,2
Tumour-repopulating cells (TRCs) are a self-renewing, tumorigenic subpopulation of
cancer cells critical in cancer progression. However, the underlying mechanisms of how
TRCs maintain their self-renewing capability remain elusive. Here we show that relatively
undifferentiated melanoma TRCs exhibit plasticity in Cdc42-mediated mechanical stiffening,
histone 3 lysine residue 9 (H3K9) methylation, Sox2 expression and self-renewal capability. In
contrast to differentiated melanoma cells, TRCs have a low level of H3K9 methylation that is
unresponsive to matrix stiffness or applied forces. Silencing H3K9 methyltransferase G9a or
SUV39h1 elevates the self-renewal capability of differentiated melanoma cells in a Sox2-
dependent manner. Mechanistically, H3K9 methylation at the Sox2 promoter region inhibits
Sox2 expression that is essential in maintaining self-renewal and tumorigenicity of TRCs both
in vitro and in vivo. Taken together, our data suggest that 3D soft-fibrin-matrix-mediated cell
softening, H3K9 demethylation and Sox2 gene expression are essential in regulating TRC
self-renewal.
DOI: 10.1038/ncomms5619 OPEN
1Laboratory for Cell Biomechanics and Regenerative Medicine, Department of Biomedical Engineering, School of Life Science and Technology, Huazhong
University of Science and Technology, Wuhan, Hubei 430074, China. 2Department of Mechanical Science and Engineering, College of Engineering, University
of Illinois at Urbana–Champaign, Urbana, Illinois 61801, USA. 3Institute for Genomic Biology, University of Illinois at Urbana–Champaign, Urbana, Illinois
61801, USA. 4Department of Bioengineering, University of Illinois at Urbana–Champaign, Urbana, Illinois 61801, USA. 5Center for Biophysics and
Computational Biology and Center for the Physics of Living Cells, Department of Physics, University of Illinois at Urbana–Champaign, Howard Hughs Medical
Institute, Urbana, Illinois 61801, USA. 6Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, Hubei 430030, China. 7Department of Bioengineering and Institute of Engineering in Medicine, University of California–San Diego,
La Jolla, California 92093, USA. 8Department of Immunology, Institute of Basic Medical Sciences of Chinese Academy of Medical Sciences, Beijing 100005,
China. * These authors contributed equally to this work. wPresent address: Center for Neuro-Medicine, Brain Science Institute, Korea Institute of Science and
Technology, Hwarangno 14-gil 5, Seongbuk-gu, Seoul 136-791, South Korea. Correspondence and requests for materials should be addressed to N.W. (email:
nwangrw@illinois.edu).
NATURE COMMUNICATIONS | 5:4619 | DOI: 10.1038/ncomms5619 | www.nature.com/naturecommunications 1
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Tumour-initiating cells (TICs) are a self-renewing, highly
tumorigenic subpopulation of cancer cells. They play a
critical role in the initiation and progression of cancer1.
These tumorigenic cells exhibit high chemo-resistance to
conventional chemotherapeutic drug treatment and therefore
are speculated to be key players in cancer relapses after
chemotherapy2. However, the concept of TICs has been
controversial. Past reports show that a high percentage (425%)
of human melanoma cells can generate a tumour in a NOD-SCID
interleukin-2 receptor-gchain null (IL2rg/) mouse3,4,
suggesting that there is no hierarchy of clonal repopulation in
melanoma. We recently developed a mechanical method of
selecting TICs from cancer cell lines and primary cancer cells by
culturing single cancer cells in soft fibrin gels5. Remarkably, in
addition to being able to generate local primary tumours in wild-
type syngeneic mice, when injected in tail veins, as few as ten of
such cells can generate distant metastatic colonization and grow
tumours in the lungs of wild-type non-syngeneic mice. Therefore,
we functionally define these soft-fibrin-gel-selected melanoma
cells as tumour-repopulating cells (TRCs) based on their high
efficiency in repopulating tumours in wild-type syngeneic and
non-syngeneic mice. Soon after our report, three other groups
independently provide strong experimental evidence in mice that
TRCs do exist in brain6, skin7and intestinal8tumours. In vivo
imaging of unperturbed tumours further confirmed the existence
of TRCs9,10. However, the underlying mechanisms of how
TRCs maintain their self-renewing capability remain elusive.
In the current study, we demonstrate that melanoma TRCs
exhibit plasticity in mechanical stiffening, histone 3 lysine
residue 9 (H3K9) methylation, Sox2 expression and self-
renewal. Three-dimensional (3D) soft fibrin matrices promote
H3K9 demethylation and increase Sox2 expression and self-
renewal, whereas stiff ones exert opposite effects.
Results
Self-renewal plasticity of TRCs. It is known that soft substrates
can sustain self-renewal of mouse embryonic stem cells11 and
substrate rigidity can regulate the fate of mesenchymal stem
cells12, indicating that rigidity of extracellular matrix plays an
important role in the maintenance and regulation of stem cell
properties. As TRCs are selected from a population of melanoma
cells that are usually cultured on rigid plastic, we asked what
would happen if we plated these TRCs back to rigid substrates. To
determine the effect of substrate rigidity on the gene expression,
we cultured TRCs on rigid plastic for 1, 3, 5 and 7 days, and
quantified their Sox2 expression. TRCs gradually lost Sox2
expression in both mRNA and protein levels along with the
culture time on plastic (Fig. 1a,b and Supplementary Fig. 18c,d).
Sox2 expression of TRCs dramatically decreased after 1 day and
was as low as that of control cells after 3 days on plastic. Other
stem cell genes Bmi-1,C-kit,Nestin and Tert, which are
upregulated in TRCs5, were also downregulated after culture on
plastic (Supplementary Fig. 1).
To examine the functional consequences of the loss of Sox2 and
other stem cell genes, we re-plated those TRCs back into 90-Pa
soft fibrin gels after culture on rigid substrates for 1, 3, 5 and 7
days, respectively. The growth rate of spheroids in fibrin matrices
successively decreased with the culture time of TRCs on plastic
(Fig. 1c), which is not a result of the increased apoptosis rate
(Supplementary Fig. 2). Moreover, colony number was also
decreased (Supplementary Fig. 3). After 7-day culture on plastic,
Sox2
Control
TRC
1 day
3 days
5 days
7 days
Control
TRC
1 day
3 days
5 days
7 days
GAPDH
GAPDH
TRC
1 day
3 days
5 days
7 days
*
***
*
*
*
**
**
*
*
*
*
Control
800
700
600
500
Colony size (×103 μm3)
400
300
200
100
012
Culture time on plastic (day) Culture time of TRC on plastic (day)
Control75310
0
0.05
Cell stiffness (kPa)
0.1
0.15
0.2
0.25 0.15 kPa
0.6 kPa
8 kPa
Glass
345
Sox2
200
180 **Sox2
*
160
140
Relative expression
14
12
10
8
6
4
2
0
Figure 1 | Inhibition of Sox2 expression and self-renewal of TRCs on 2D rigid substrates. (a)Sox2 expression at both mRNA level (top panel) and
protein level (bottom panel). Control: B16-F1 cells cultured on plastic. TRC: Control B16-F1 cells were cultured in 90-Pa fibrin gels for 5 days. 1 day, 3 days,
5 days or 7 days: TRCs were seeded on 2D rigid dishes for 1, 3, 5 or 7 days. Images are representatives of three independent RT–PCRs and two independent
western blotting experiments. (b) Quantification of Sox2 expression by real time RT–PCR. Mean±s.e.m.; n¼3 independent experiments. *Po0.05.
(c) 2D rigid substrates inhibit TRC growth. Significant differences between Control and TRC, or 1 or 3 days from Day 1 to 5 in the 90-Pa fibrin gels
(all Po0.05, except TRC at day 2, where P¼0.068, 1 day at day 2, where P¼0.52, and 3 days at Day 2 and Day 3, where P40.15). No differences
between Control and 5 or 7 days from Day 1–5 (all P40.06, except 5 days at Day 1 and Day 3, where Po0.04). Each data point was averaged from
at least 30 colonies. *Po0.05 between TRC and all other groups. (d) 2D rigid substrates promote stiffening of TRCs with substrate rigidity. B16-F1 cells
cultured on plastic were used as a control. All were compared with ‘0.15 kPa’ in each group. Mean±s.e.m.; *Po0.01. Each data point was averaged from
4150 cells from two independent experiments. All the statistics were conducted using Student’s t-test throughout the manuscript unless otherwise
specified.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5619
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TRC colonies proliferated at a similar rate as control cells
(harvested from rigid dishes). The lower colony size and number
suggest that rigid substrates significantly reduce the self-renewal
capability of TRCs, likely due to the loss of Sox2 and other stem
cell gene expressions.
Besides the differences in gene expression patterns, TRCs also
differ from control melanoma cells in their non-stiffening
response to the increase in substrate rigidity5. To explore
whether switching to rigid plastic may also change biophysical
properties of TRCs, we first seeded them on two-dimensional
(2D) rigid plastic for 1–7 days, and then quantified their stiffness
on polyacrylamide (PA) gels of different rigidities. TRCs started
to stiffen with substrate rigidity after 1-day culture on plastic
(Fig. 1d). The stiffening response was more evident after 3 and 5
days. Interestingly, after 7-day culture on plastic, the stiffness and
the stiffening response of TRCs were essentially the same as those
of control melanoma cells (Fig. 1d).
Regulation of H3K9 methylation by matrix and soluble factors.
Our data have shown major differences in self-renewal gene
expression and biophysical properties between TRCs and control
melanoma cells. As these two types of cells are genetically iden-
tical, we hypothesized that epigenetics may play a role in it. A
large body of work has shown aberrant changes in epigenetic
features during tumour initiation and progression13,14, including
histone modification. Among those aberrations, H3K9 is
hypermethylated in several types of tumours15,16. We asked
what role H3K9 methylation plays in TRCs. To explore H3K9
methylation of TRCs, we developed a CFP-YPet H3K9
fluorescence resonance energy transfer (FRET) biosensor (fused
with a nuclear localization signal (NLS)) modified from a
previously reported CFP–YFP H3K9 biosensor17 to quantify
H3K9 di- and trimethylation (me2 and me3) in the nucleus of
single cells (Supplementary Fig. 4a). This FRET probe uniquely
measures the spatial distribution of H3K9 methylation level in the
nucleus and revealed that H3K9 me2 and me3 mostly occurred at
the nuclear periphery (Supplementary Fig. 4b). Culturing control
cells in 90-Pa fibrin gels for merely 3 h resulted in H3K9
demethylation (Fig. 2a). TRCs did not elevate their H3K9
methylation (within 15 h of plating) with substrate stiffness on
PA gels coated with either collagen-1 or fibrinogen (Fig. 2b,d); in
contrast, control cells increased their H3K9 methylation with
substrate stiffness on PA gels coated with collagen-1 but not with
fibrinogen (Fig. 2b,c). Interestingly, high level of H3K9
methylation in control cells was associated with high cellular
tractions on collagen-1-coated substrates and inhibition of the
tractions decreased H3K9 methylation (Supplementary Fig. 5),
suggesting that high endogenous forces transmitted via a
1
/a
2
b
1
(collagen-1 receptor) rather than via a
v
b
3
(fibrinogen receptor)
might be responsible for the stiff substrate-mediated H3K9
methylation, consistent with a report that activation of b
1
integrin
but not b
3
integrin elevates cell traction forces18.
To further explore the role of physical forces in H3K9
methylation, we applied exogenous forces via RGD (Arg-Gly-
Asp)-coated magnetic beads to single cells using magnetic
twisting cytometry19 (Fig. 3a,b). On fibrinogen-coated stiff
substrates (Glass), the applied forces induced significant
increases in H3K9 methylation at the nuclear periphery within
60 min only in control cells, but not in TRCs (Fig. 3a–c). The
force-induced H3K9 methylation was dependent on force
magnitude and specifically mediated via b
1
integrin, as low
forces, forces applied via beads coated with poly-L-lysine, or
forces applied after blocking b
1
integrin with monoclonal
1.45
1.4
1.35
H3K9-me (YPet/CFP)
H3K9-me (YPet/CFP)
1.3
1.25
1.2
1.15
1.1
1
0.9
1.1
1.2
1.3
1.4
1.5
1.6 Control on Fib
TRC on Fib TRC on Col-I
*
*
*
Control on Col-I
1.7
0.8 0.15 kPa 0.6 kPa 2 kPa
Substrate stiffness
8 kPa
8 kPa2 kPa0.6 kPa0.15 kPa
CollagenFibrinogen
2D 3 h 19 h 48 h
3D fibrin
Control TRC
8 kPa
2
1
0
2
1
0
2 kPa0.6 kPa0.15 kPa
CollagenFibrinogen
Plastic
*
Figure 2 | Matrix rigidity mediated H3K9 methylation in control cells but not in TRCs. (a) H3K9 methylation is lower in 90-Pa fibrin gels than
on rigid plastic. Control B16-F1 cells were transfected with the H3K9 FRET methylation biosensor and then plated either on gelatin-coated rigid plastic
(2D plastic) or in the 90-Pa soft 3D fibrin gel (3D Fibrin). H3K9 methylation was measured at the indicated times after cell plating. Mean±s.e.m.;
n475 cells for each value (*Po0.001). (b) H3K9 methylation in control B16-F1 cells (Control) but not in TRCs increases on stiff PA substrates coated with
collagen I (Col-1) but not fibrinogen (Fib). PA, polyacrylamide. H3K9 methylation was measured 15h after cell plating. Mean±s.e.m., n440 cells
for each value (*Po0.005).Representative FRET images of a cell nucleus for control cells (c) and TRCs (d) on PA gels coated with collagen I (top panel)) or
fibrinogen (bottom panel). Scale bars, 5 mm.
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antibody, was not able to induce H3K9 methylation (Fig. 3d). In
addition, silencing H3K9 methyltransferase G9a or SUV39h1
(knockdown efficiency was B50%; Fig. 4a) not only reduced the
baseline level of H3K9 methylation in control melanoma cells
(Fig. 4c), but also completely blocked the force-induced H3K9
methylation (Fig. 3d), consistent with a report that these two
enzymes are critical to H3K9 methylation20. In contrast, silencing
or inhibiting H3K9 demethylase KDM7 with small interfering
RNA (siRNA) or with Daminozide in cells in 3D soft fibrin gels
increased H3K9 methylation levels (Fig. 4b). These data suggest
that H3K9 methylation levels in these melanoma cells are
controlled by both methyltransferases and demethylases. H3K9
methylation also depended on intactness of the cytoskeleton and
cellular tension, as disrupting actin microfilaments with
Latrunculin A or microtubules with colchicine and inhibiting
myosin light chain kinase with ML7 significantly lowered H3K9
methylation levels (Fig. 4d). Although TRCs did not increase
H3K9 methylation with substrate stiffness within 15 h (Fig. 2b,d),
they started to increase H3K9 methylation on collagen-1-coated
plastic after 48 h (Fig. 3e). In contrast, TRCs treated with retinoic
acid (a nonspecific soluble differentiation factor) for 60 min
(similar in duration for external force-induced H3K9me; see
Fig. 3c) increased H3K9 methylation levels (Fig. 3f), suggesting
H3K9 methylation may be associated with the differentiation
of TRCs.
H3K9 demethylation-induced Sox2 expression is critical to
TRC self-renewal. As H3K9 methylation has been associated
with inactivation of certain genes13–15, we postulated that
H3K9 demethylation might be related to Sox2 expression. Our
western blotting analysis showed that control cells expressed
higher levels of H3K9 me2 and H3K9 me3 compared with
TRCs (Supplementary Fig. 6a). Importantly, silencing H3K9
methyltransferase G9a or SUV39h1 in control cells significantly
increased their Sox2 expression (Fig. 5a and Supplementary
Fig. 7a), suggesting that H3K9 demethylation can increase Sox2
expression. To further investigate how H3K9 demethylation
promotes Sox2 expression, we performed chromatin immuno-
precipitation (ChIP) assay to quantify H3K9 me2 and me3 levels
+ Stress
10 min0 min
Control
30 min 60 min
1.15
1.10
1.05
1.00
0.95 0 10203040506070
+24.5 Pa stress
*
Control
TRC
Normalized YPet/CFP
1.15
1.10
1.05
1.00
0.90
0.95
0 10203040
TIme (min)
TIme (min)
50 60 70
SUV39h1 siRNA
G9a siRNA
PLL
RGD+β1mAb
RGD 12.3 Pa
RGD 17.5 Pa
+stress
Normalized YPet/CFP
10 min0 min 30 min 60 min
1.8
0
1.8
0
+ Stress
10 min0 min
TRC
30 min 60 min
10 min
1.3
1.25 *TRC
1.2
1.15
1.1
1
1.05
H3K9-me (YPet/CFP)
0.95
0.9 19 24 48
Culture time on glass (h) Culture time on 8 kPa PA gels (min)
0
0.9
0.95
1
1.05
1.1
1.2 Control 10 μM RA
*
H3K9-me (YPet/CFP)
1.15
10 30 6072 96 120 144 168
0 min 30 min 60 min
Figure 3 | Exogenous forces increase H3K9 methylation level via b1 integrin. Exogenous forces increase H3K9 methylation in control B16-F1 cells (a) but
not in TRCs (b). Top: bright-field and fluorescence overlay image of the cells; the magnetic bead is shown as a black dot. Bottom: representative
FRET images showing the spatial distribution of H3K9 methylation in the nucleus during loading. Cells were plated on 8 kPa PA gels coated with fibrinogen
and loaded at 24.5 Pa, 0.3 Hz with RGD-coated ferromagnetic beads. H3K9 methylation clearly increases in aat the nuclear periphery 30–60 min after
loading. Scale bars, 10 mm (top), 5mm (bottom). (c) Quantification of H3K9 methylation in aand bduring loading. (d) No force-induced elevation
of H3K9 methylation was observed when the externally applied stress magnitude was equal or o17.5 Pa, when b
1
integrin was blocked by a monoclonal
antibody, when the force was applied via a poly-L-lysine coated bead, or when siRNA was transfected to knockdown G9a and SUV39h1, which are
methyltransferases responsible for dimethylation and trimethylation of H3K9, respectively. Knockdown of G9a and SUV39h1 was confirmed by western
blottings that these proteins reduced by B50% when compared with control. (e) H3K9 methylation in TRCs increases after 2-day culture on glass
dishes. TRCs were cultured on collagen I-coated glass-bottomed dishes and H3K9 methylation was measured at indicated times. Mean±s.e.m., n440
cells for each time point, *Po0.05. (f) Short time treatment with retinoic acid (RA) increases H3K9 methylation in TRCs. TRCs were cultured on
collagen I-coated 8-kPa PA gels for 15 h. Cells were then treated with or without (Control) 10 mM RA. Note that all the values were normalized to the value
at 0 min (15 h after plating). Mean±s.e.m., n440 cells for each value, *Po0.05.
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in the promoter region of Sox2. H3K9 me2 and H3K9 me3 levels
were B50% higher in the promoter region of Sox2 in control cells
than in TRCs (Fig. 5b and Supplementary Fig. 6b). Silencing G9a
or SUV39h1 in control cells greatly decreased their H3K9-me2 or
H3K9-me3 levels in the promoter region of Sox2 (Fig. 5b). Plating
TRCs on plastic for 7 days elevated H3K9 me2 and me3 to the
same levels as those in control cells (Fig. 5b and Supplementary
Fig. 6b). These results suggest that G9a and SUV39h1 methylated
H3K9 at the Sox2 promoter site blocks Sox2 expression. In
contrast, TRCs did not express G9a and SUV39h1 proteins and
silencing Sox2 in TRCs did not increase the expression of G9a or
SUV39h1 (Supplementary Fig. 8), suggesting that G9a and
SUV39h1 may be independent of Sox2 expression.
As knocking down G9a or SUV39h1 can induce Sox2
expression in control cells, we further explored whether H3K9
demethylation can promote their self-renewal capability. Silen-
cing G9a or SUV39h1 in control cells significantly increased the
colony growth in soft fibrin gels to a level similar to that of TRCs
(Fig. 5c and Supplementary Fig. 7b), but the colony number was
still twofold less than that of TRCs (Supplementary Fig. 7c,d).
However, simultaneously silencing G9a (or SUV39h1) and Sox2
completely abrogated the colony growth but not the colony
number (Supplementary Fig. 9), suggesting that H3K9 demethy-
lation promotes self-renewal capability of melanoma cells in a
Sox2-dependent manner. H3K9 methylation is regulated by both
methyltransferases and demethylases. Silencing KDM7, an
important demethylase for H3K9 (ref. 21), significantly elevated
H3K9 methylation (Fig. 4b) and suppressed colony growth and
number (Fig. 6a,b), probably through inhibition of Sox2
expression (Fig. 6c). Treatment with two separate inhibitors of
KDM4 (ref. 22) (another demethylase for H3K9) led to dose-
dependent inhibition of colony size and colony number
(Supplementary Fig. 10). Taken together, these data suggest that
H3K9 demethylation by deactivating methyltransferases or
activating demethylases increases Sox2 expression and thus
promotes self-renewal capability.
We further investigated the functional role of Sox2 in
maintaining self-renewal of TRCs in vitro and in vivo. Silencing
Sox2 greatly decreased the expressions of Sox2,Bmi-1 and C-kit in
TRCs (Supplementary Fig. 11). Colony growth was suppressed by
490% in Sox2-knocked down TRCs in comparison with that of
control TRCs (Fig. 5d), suggesting that Sox2 is essential in
maintaining self-renewal of TRCs in vitro.Sox2 silencing had no
effects on control melanoma cells (these cells express little Sox2)
that were grown on plastic and were then embedded in 3D stiff
fibrin gels, suggesting that there were little off-target effects of
Sox2 silencing (Supplementary Fig. 12). Overexpressing Sox2 in
control melanoma cells increased the colony number but not
colony growth (Supplementary Fig. 13), indicating that this
intervention increases the number of self-renewing cells but does
not alter cell cycle duration of a self-renewing cell. Importantly,
subcutaneously injecting 100 of TRCs treated with Sox2 short
hairpin RNA (shRNA) into wild-type syngeneic C57BL/6 mice
did not generate any tumours by day 19. In contrast, palpable
tumours were observed for untreated TRCs by day 13 and treated
with scrambled control shRNA by day 15. By day 28, 2 out of 16
mice for TRCs treated with Sox2 shRNA had small tumours
(Supplementary Table 1), whereas for untreated TRCs 9 out of 16
mice formed large tumours; for TRCs treated with scrambled
shRNA, 3 out of 8 formed tumours. The formation of tumours in
TRCs treated with Sox2 shRNA appearing at late stages probably
reflects the fact that the knockdown of Sox2 by shRNA was
transient such that Sox2 expression came back in these cells after
shRNA effect was depleted. As expected from previous findings5,
injecting 100 of control melanoma cells (harvested from rigid
plastic) did not form any tumours (0/8; Supplementary Table 1).
These data demonstrate that Sox2 expression is necessary to
sustain self-renewal capability of TRCs both in vitro and in vivo.
Neg Ctr Neg Ctr
siRNA #1 siRNA #1
G9a
GAPDH
GAPDH
SUV39h1
170kDa
40kDa
40kDa
40kDa
G9a
GAPDH
GAPDH
SUV39h1
H3K9-me (YPet/CFP)
1.55
1.5
1.45
1.4
1.35
1.3
1.25
1.2
Control
2 μM daminozide
KDM7 siRNA #1
KDM7 siRNA #2
***
31948
Culture time (h)
Control G9a
siRNA
SUV39h1
siRNA
**
**
0.7
0.9
1.1
1.3
1.5
H3K9-me (YPet/CFP)
H3K9-me (YPet/CFP)
Control
0.7
0.9
1.1
1.3
1.5 ** ** **
*
*
Colch ML7 LatA
a
cd
b
Figure 4 | H3K9 methylation levels depend on methyltransferases and demethylases and the tensed cytoskeleton. (a) Representative images of
western blotting of G9a or SUV39h1 expression after siRNA knockdown. Control melanoma cells were transfected with two sets of G9a/SUV39h1
siRNAs (1 and 2), respectively, and western blotting assays were performed to confirm the knockdown efficiencies to be 48% (1 siRNA) and 45%
(2 siRNA) for G9a, and 58% (1 siRNA) and 43% (2 siRNA), respectively. Three independent experiments showed similar results. (b) Silencing or inhibiting
demethylase KDM7 increases H3K9 methylation of control melanoma cells in soft fibrin gels. Control cells were transfected with H3K9 FRET
biosensors and KDM7 siRNAs (1 and 2) or negative control siRNA, or treated with KDM7 inhibitor daminozide. The cells were seeded into 3D soft fibrin
gels and H3K9 methylation levels were measured after 3, 19 or 48 h. Mean±s.e.m.; n440 cells for each data point; *Po0.05. (c) Knockdown of G9a or
SUV39h1 decreases the H3K9 methylation in control B16-F1 cells. (d) Inhibiting microtubule, myosin light chain kinase (MLCK) or actin polymerization
decreases H3K9 methylation. Control B16-F1 cells were incubated with Colchicine (Colch, 10 mM for 30 min) to inhibit microtubule polymerization,
ML7 (25mM for 30 min) to inhibit myosin light chain kinase, or Latrunculin A (LatA, 1 mM for 30 min) to disrupt actin filaments. Mean±s.e.m; n440 cells
for each data point; *Po0.05; **Po0.001.
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Because of the importance of Sox2 and H3K9 demethylation in
TRCs, we asked what roles Sox2 expression and H3K9
methylation might play in the selection and propagation of TRCs
by soft fibrin gels. When control melanoma cells were transfected
with Sox2 shRNA and cultured in 90-Pa fibrin gels, both growth
and number of spheroids were inhibited (Supplementary
Fig. 14a,b). Moreover, treatment with Daminozide, a highly
selective inhibitor of KDM7, significantly inhibited TRC spheroid
growth number (Supplementary Fig. 14c,d). Therefore, these data
suggest that H3K9 demethylation-dependent regulation of Sox2
expression is important in TRC selection and propagation.
Mechanism of cell stiffening with substrate stiffness. To elu-
cidate the mechanisms of the stiffening response plasticity, we
examined the potential role of Cdc42 in cell stiffening because of
its known role in inducing filopodia formation and cell spreading
in B16-F1 melanoma cells23. TRCs expressed B50% less Cdc42
than control cells; plating TRCs on plastic for 5 and 7 days
increased their Cdc42 expression by B30% and B100%,
respectively (Fig. 7a). Overexpression of Cdc42 in TRCs rescued
the cell stiffening response with substrate rigidity (Fig. 7b),
whereas silencing Cdc42 in control melanoma cells or in TRCs on
plastic for 5 days completely abolished the stiffening response
(Fig. 7c). Silencing Cdc42 in melanoma cells on rigid plastic
downregulated H3K9 me2 but not H3K9 me3 (Fig. 7d and
Supplementary Fig. 15a,b) and had no effects on Sox2 expression
(Supplementary Fig. 15c), suggesting that Cdc42, cell stiffening
and H3K9 methylation might be linked via tension-dependent
cytoskeletal mechanotransduction, contributing to the biological
plasticity of TRCs.
Inhibition of TRC self-renewal by 3D stiff fibrin matrix. When
the cells were cultured from 2D rigid dish to 3D soft matrix, both
matrix dimensionality and stiffness were altered. To further
determine whether it is the 3D matrix stiffness or the effect of
switching from 2D to 3D matrix that impacts on the self-renewal
capability of TRCs, we cultured control B16-F1 cells in 3D soft
(90-Pa) or 3D stiff (1,050-Pa) fibrin gels for 5 days. These cells
were then seeded into either soft or stiff fibrin gels. After re-
seeding, the cells grew better in soft fibrin gels no matter whether
they were derived from soft or stiff fibrin gels; in contrast, once
re-seeded in stiff fibrin gels, colony growth and number were
inhibited (Fig. 8a,b), suggesting that switching from a 3D soft
matrix to a 3D stiff matrix inhibits self-renewal and promotes
quiescence of the TRCs. When culturing control melanoma cells
in soft and stiff fibrin gels, a 10-fold increase in substrate stiffness
leads to a B1.25-fold increase in H3K9 me2 and a 50% increase
in H3K9 me3 on the protein level, and a B90% decrease in Sox2
expression (Fig. 9). Importantly, switching from a stiff matrix
(1,050-Pa) to a soft matrix (90-Pa) significantly decreased H3K9
methylation and elevated Sox2 expression by B5-fold (Fig. 8c,d).
Taken together, these data suggest that 3D stiff fibrin matrix can
inhibit TRC self-renewal via H3K9 methylation-mediated Sox2
suppression.
TRC
Neg Ctr
G9a
SUV39h1
Sox2
GAPDH
200 *
*
Sox2
Sox2 promoter
H3K9 me2
1.6
1.4
1.2
0.8
0.6
0.4
Fold change in H3K9me
0.2
0
TRC
TRC+
7 days
Neg Ctr
siRNA
1
H3K9 me3
*
*
180
160
140
6
4
2
0TRC Neg Ctr SUV39h1G9a
Control cells with siRNA #1
Negative control
350
300
250
200
150
100
50
012
Culture time (day)
3**
*
45
G9a siRNA #1
SUV39h1 siRNA #1
TRC
Colony size (×103 μm3)
Negative control
600
500
400
300
200
100
012
Culture time (day)
3
*
*
*
*
45
Scrambled control
Sox2 shRNA #1
Sox2 shRNA #2
Sox2 shRNA #3
Sox2 shRNA #4
Colony size (x103 μm3)
Relative expression
Figure 5 | H3K9 demethylation-mediated Sox2 expression is essential for in vitro tumour growth. (a) Silencing G9a or SUV39h1 promotes Sox2
expression in control melanoma cells, quantified by real-time RT–PCR. Neg Ctr: negative control. *Po0.05. (b) H3K9 di- (me2) and trimethylation
(me3) levels in the promoter region of Sox2 quantified by the ChIP assay. TRC þ7 days: TRCs were cultured on plastic for 7 days; Neg Ctr, siRNA: control
melanoma cells were treated with negative control siRNA, G9a (for H3K9 me2) or SUV39h1 (for H3K9 me3) siRNA #1, respectively. *Po0.05.
(c) H3K9 demethylation in control melanoma cells promotes their self-renewal capability. Control cells were transfected with G9a or SUV39h1 siRNA and
then re-plated in soft fibrin gels. (d) Silencing Sox2 greatly inhibits growth of TRC spheroids. TRCs were transfected with Sox2 shRNAs or scrambled
shRNA. These cells were then re-plated into 90-Pa fibrin gels. Each data point was averaged from at least 40 colonies. In c, significant differences between
Negative Control and G9a siRNA 1 or SUV39h1 siRNA 1 or TRC from day 2 to day 5 (*Po0.05, except G9a siRNA 1 at day 2, where P¼0.09).
In d, significant differences between Negative control or Scrambled control and Sox2 shRNA 1 to 4 from day 2 to day 5. (*Po0.05). Mean±s.e.m.; n¼3
independent experiments for all subfigures.
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Discussion
Increasing evidence has demonstrated that TRCs truly exist as a
highly tumorigenic subpopulation that is critical to tumour
initiation and progression. TRCs also exhibit high resistance to
conventional chemotherapeutic drug treatment, which has been
speculated to drive cancer relapse after chemotherapy. Therefore,
TRCs may be one of the pivotal targets to achieve complete
tumour eradication24. In the present study, we investigated how
TRCs maintain their self-renewal capability. As TRCs appear to
be less differentiated, we set out to determine their specific
lineage. Our data show that TRCs exhibited Sox2highMitflow
phenotype and control melanoma cells exhibited Sox2 Mitf þ
phenotype (Supplementary Fig. 16a,b), consistent with published
results25–27 and suggesting that TRCs resemble melanoblasts that
are Sox2lowMitflow and control melanoma cells resemble
melanocytes that are Sox2 Mitf þin these two gene expression
patterns27. However, expression of Mitf upstream regulators,
Pax3 (ref. 28) and Sox10 (ref. 27), and melanocyte-specific genes,
tyrosinase-related protein 1 (Tyrp1)28, dopachrome tautomerase
(DCT)28 and glycoprotein non-metastatic melanoma protein B
(Gpnmb)29, was similar in TRCs as in control cells (Supplemen-
tary Fig. 16c,d). These findings suggest that melanoma TRCs may
be progenitor cells of control B16-F1 cells but more differentiated
than melanoblasts. Moreover, knocking down Sox2 in TRCs
increased Mitf expression (Supplementary Fig. 17a), while
silencing Mitf (knockdown efficiency B80%; Supplementary
Fig. 17b) in control melanoma cells did not affect Sox2 expression
(Supplementary Fig. 17c), suggesting that self-renewal gene Sox2
is independent of Mitf. Importantly, when TRCs are plated on the
rigid dish, expression of melanocyte-related gene Mitf gradually
increased (Supplementary Fig. 18), demonstrating Mitf plasticity
of TRCs when substrate rigidity was elevated, opposite in
direction as the plasticity of Sox2 expression (see Fig. 1a,b).
Sox2 is known for its critical role in the maintenance of
pluripotency25 and fate determination30 of stem cells. Sox2 also
sustains self-renewal capability of TICs26. Inhibition or silencing
of Sox2 leads to loss of tumorigenicity26,31.Sox2 dysfunction in
cancer32,33 is known, but it is not clear what triggers Sox2
expression in cancer. Our current study reveals a critical
role of the mechanics of 3D matrix in Sox2 expression in a
subpopulation of tumour cells. Importantly, silencing Sox2 greatly
inhibits spheroid growth in vitro and tumour generation in vivo.
TICs are heterogeneous in terms of their transient, long-term, or
delayed-contributing effects on tumour growth34. Although we
cannot exclude the possibility that TRCs isolated from soft fibrin
matrices are also heterogeneous, our previous finding that as few
as ten of these TRCs are sufficient to generate local and distant
tumours5suggests that these TRCs are distinct from those TICs.
Cancer cells exhibit aberrant epigenetic features, including
global changes in DNA methylation and altered histone
modification35. On the other hand, stem cells have an open
chromatin structure and abnormal epigenetic regulation may be
prone to malignant transformation36. Here we show that H3K9
demethylation by silencing G9a or SUV39h1 can greatly promote
the self-renewal capability of control melanoma cells in a Sox2-
dependent manner, whereas silencing H3K9 demethylases
inhibits self-renewal. It should be noted that in contrast to the
increase in TRC colony number and growth observed following
culture in soft substrates, silencing H3K9 methyltransferases has
significant functional effects on colony growth but not colony
number and the levels of Sox2 remain 30- to 40-fold lower than
those found in TRCs. The exact mechanism is not clear at this
350
300
250
200
150
Colony size (×103 μm3)
100
KDM7 siRNA #1
KDM7 siRNA #2
Neg Ctr
50
01
*
***
#
#
2
Culture time (day)
1.2
Relative expression
0.8
0.6
0.4
0.2
0
Ne
g
Ctr KDM7 siRNA #1 KDM7 siRNA #2
Sox2
*
1
345
350
300
250
200 *
****
#
#
#
150
Colony number
100
KDM7 siRNA #1
KDM7 siRNA #2
Neg Ctr
50
012
Culture time (day)
345
Figure 6 | Silencing H3K9 demethylase KDM7 greatly inhibits colony size and number through suppression of Sox2 expression. Knocking down
KDM7 using two different siRNAs significantly inhibits colony size (a) and number (b). Control melanoma cells were transfected with KDM7 siRNA 1 or 2,
and then cultured in 90-Pa fibrin gels. Colony number was counted and colony size was monitored until day 5 (n¼2). There is a significant difference
between Neg Ctr and KDM7 siRNA 1 in afrom day 2 through day 5 (all Po0.00001) and in bfrom day 1 through day 5 (all Po0.01), and between Neg Ctr
and KDM7 siRNA 2 in (a) at day 4 and day 5 (both Po0.005) and in bfrom day 3 through day 5 (all Po0.05). ‘*’ and ‘#’ indicates significant differences
between Neg Ctr and KDM7 siRNA 1 or 2, respectively. (c) Silencing KDM7 significantly suppresses Sox2 expression. Control melanoma cells were
transfected with KDM7 siRNA 1 or 2, or negative control siRNA and then cultured in 90-Pa fibrin gels. After 5 days, Sox2 expression was quantified by real
time RT–PCR. *Po0.05; n¼3 independent experiments.
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time, but it may be partly due to the fact that only 50% of G9a/
SUV39h1 is knocked down, whereas there are no detectable levels
of G9a/SUV39h1 in TRCs (Supplementary Fig. 8) and/or other
self-renewal promoting effects of silencing G9a/SUV39h1 are at
work. In addition, it is not clear why silencing Sox2,
overexpressing Sox2, or inhibiting KDM4, has different effects
from silencing methyltransferases on colony number and size.
These issues need to be investigated in the future by dissecting out
specific signalling pathways. Nevertheless, our data suggest that
H3K9 demethylation and methylation, modulated by force-
induced chromatin remodelling mediated by the cytoskeleton,
can regulate Sox2 expression and play important roles in self-
renewal and differentiation of TRCs. Our results show that stiff
matrix induced H3K9 methylation occurs at the nuclear
periphery just under the nuclear envelope. The applied stress
(via the magnetic bead) on the cell surface via integrins also
induces H3K9 methylation at the periphery of the nucleus,
suggesting that stiff matrix-induced H3K9 methylation may be (at
least) in part due to the elevation of endogenous stresses in the
cell. These findings are in line with a recent report that force-
induced stiffening of isolated nuclei depends on an intact LINC
(linker of nucleoskeleton and cytoskeleton) pathway37.Cdc42
silencing decreases H3K9me2 by B20%, similar to that after
disruption of cytoskeletal filaments with drugs (Fig. 4d),
suggesting that the effect of silencing Cdc42 might be important
in altering cell functions. However, silencing Cdc42 has no effect
on H3K9me3 or Sox2, suggesting that some molecule(s) upstream
of Cdc42 may be important in controlling H3K9me3 and Sox2.
Furthermore, our current findings suggest that increasing matrix
rigidity affect Cdc42-mediated cell stiffening, H3K9 methylation
and Sox2 suppression, but whether these are independent or
linked pathways in controlling TRC function remains unclear and
requires additional experiments in the future. Published reports
suggest that G9a/SUV39h1-mediated H3K9 methylation
contributes to cancer initiation and/or progression13,14,16,38,39,
different from our findings that G9a or SUV39h1 knockdown
leads to Sox2 expression. This discrepancy may lie in the fact that
in those previous studies differentiated cancer cells are assayed,
whereas in our present study TRCs are analysed. Although there
are reports on Sox2 expression in several types of undifferentiated
tumour cells26,31, there is, to the best of our knowledge, no report
of H3K9 methylation or Sox2 expression in TRCs when
mechanical microenvironment is perturbed. Therefore, our
findings on low force-mediated H3K9 demethylation and thus
Sox2 gene expression are novel. Furthermore, we find that H3K9
methylation can directly inhibit Sox2 expression. Nevertheless,
the potential roles of other histone modifications, such as H3K4
and/or H3K27 methylation, acetylation and phosphorylation, and
DNA methylation, remain to be elucidated. In addition, the
mechanisms of in vivo Sox2 expression/suppression with the link
to H3K9 demethylation/methylation and matrix compliance/cell
stiffening need to be investigated in the future.
We show that a uniform stiff fibrin gel functions as a physical
barrier to prevent the formation and growth of TRC spheroids
through elevated H3K9 methylation-induced Sox2 suppression,
supporting a recent postulate on breast cancer progression40.
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0Control TRC 5 days 7 days
TRC on plastic
Relative expression
Cdc42
*
*
Cell stiffness (% baseline)
0
50
100
150
200
250
300
350
TRC+empty
vector
Glass
8 kPa
0.6 kPa
0.15 kPa
***
TRC+Cdc42
overexpression
500
450
400
350
300
250
200
Cell stiffness (% baseline)
150
100
50
0
Glass
8 kPa
0.6 kPa
0.15 kPa *
*
*
*
*
*
*
TRC on
plastic
+scrambled
siRNA
TRC on
plastic
+Cdc 42
siRNA
Control
+scrambled
siRNA
Control
+Cdc 42
siRNA
Relative methylation
1.4
1.2
0.8
0.6
0.4
0.2
0
1
H3K9me2 H3K9me3
Neg Ctr
Cdc42 siRNA
a
cd
b
Figure 7 | Cdc42 expression regulates stiffening response and H3K9 me2 in melanoma cells. (a) TRCs express lower level of Cdc42 compared with
control melanoma cells and TRCs after culture on rigid plastic for 5 or 7 days. n¼6; *Po0.05; two independent experiments. (b) TRCs regain cell stiffening
responses after overexpression of Cdc42. Cells were plated on Collagen-I-coated PA gels with different stiffnesses or on glass-bottomed dishes.
Experiments were carried out 6 h after plating. Relative changes in cell stiffness were represented with respect to the cell stiffness on 0.15 kPa PA gel as the
baseline. TRC þempty vector: TRCs were transfected with pEGFP-N1 vector; TRC þCdc42 overexpression: TRCs were transfected with Cdc42-EGFP
plasmid. n4150 cells; *Po0.001. (c) Cdc42 knockdown inhibits cell stiffening in control cells and TRCs plated on plastic for 5 days. Relative changes in cell
stiffness were represented with respect to the cell stiffness on 0.15 kPa PA gel as the baseline. n4100 cells; *Po0.001. Control: B16-F1 cultured on rigid
plastic; TRC on plastic: TRCs were cultured on plastic for 5 days; Control þCdc42 siRNA: control melanoma cells were transfected with Cdc42 siRNA;
Control þScrambled siRNA: control cells were transfected with scrambled siRNA; TRC on plastic þCdc42 siRNA: TRCs were cultured on plastic
for 5 days and then transfected with Cdc42 siRNA; TRC on plastic þScrambled siRNA: TRCs were cultured on plastic for 5 days and then transfected with
scrambled siRNA. Mean±s.e.m.; ‘*’ indicates significant difference compared with ‘0.15 kPa’. (d) Quantification analysis of western blotting of H3K9me2
and me3 after Cdc42 knockdown in control melanoma cells. Mean±s.e.m.; n¼3 independent experiments; *Po0.05.
ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms5619
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Melanoma cells in the same soft fibrin gels can form round
colonies only at z-heights far away from the rigid plastic bottom
(Supplementary Fig. 19); as these cells experience the same
porosity but different elasticity, these data demonstrate that it is
matrix stiffness but not porosity that dictates round colony
generation and self-renewal. Furthermore, addition of soluble
fibrinogen ligands to the 90-Pa fibrin matrix (total ligand amount
is equivalent to that of 1,050-Pa fibrin gels) did not alter colony
growth (Supplementary Fig. 20), suggesting that the difference in
colony growth between 90-Pa and 1,050-Pa fibrin gels may not be
due to ligand density availability. It is noteworthy that the H3K9
methylation results and the corresponding traction data (and
their inhibition by myosin light chain kinase inhibitor ML7)
suggest that different integrin subsets (that is, collagen-1-
mediated b1 integrin binding versus fibrinogen-mediated b3
integrin binding) can trigger different responses in H3K9
methylation even for the same substrate stiffness.
Our data suggest that TRCs may have different cell fates in
matrices of various mechanical and biochemical microenviron-
ments (Fig. 10). In a soft matrix, TRCs can proliferate well and
grow into a local tumour. In the presence of soluble differentia-
tion factors, TRCs cannot proliferate even in soft matrix. In
contrast, a stiff matrix restricts TRC growth and makes them
quiescent. However, on matrix degradation and remodelling, the
stiff matrix may become locally softened, reactivating TRCs to
proliferate, while some areas of local stiffening may promote
TRCs to differentiate. Locally switching from 3D stiff matrices to
3D soft matrices and vice versa may be able to regulate TRC self-
renewal, as suggested by our current results from in vitro culture.
This scenario may partially explain why tumour relapses several
years after surgery and therapies. These data indicate that stiff
matrix may be used as an effective strategy to suppress self-
renewal of TRCs.
Published reports have shown that tension-dependent matrix
stiffening is critical in breast cancer progression41–43. At a first
look, it appears that there is a discrepancy in our findings and the
results of these reports. A close examination reveals that our
findings and theirs may be referring to different cancer cells at
different stages during cancer progression. It is known that tissue
stiffening alone cannot drive mammary epithelial transformation
without ErbB2 activation43. It is possible that integrin-mediated,
tension-dependent matrix stiffening facilitates differentiation and
invasion of those TRCs only in the presence of matrix
degradation by various proteases42 that generate more porous
matrices of heterogeneous rigidity. This explanation is supported
by the experimental finding that matrix metalloproteinease 14-
mediated local softening of the invasive front of mammary
epithelial cells is necessary to overcome the stiff elastic resistance
of the collagen matrix44. As tens of thousands of cancer cells
differentiate on the stiff matrix, invade and then intravasate, few
undifferentiated or partially differentiated cancer cells such as
TRCs may follow these differentiated cancer cells and disseminate
into the blood vessel. It is possible that only these soft TRCs can
survive the distant organs to form metastatic colonization, the
major rate-limiting step in clinically detectable macroscopic
metastasis45. This interpretation may explain why metastatic
600
500
400
90 Pa to 90 Pa
90 Pa to 1,050 Pa
1,050 Pa to 90 Pa
1,050 Pa to 1,050 Pa
1,050 Pa to 1,050 Pa
1,050 Pa to 90 Pa
90 Pa to 90 Pa
90 Pa to 1,050 Pa
1,050 Pa to 90 Pa
1,050 Pa to 1,050 Pa
*****
#
#
#
#
Colony size (x103 μm3)
300
200
100
0
600
800
1,000
400
Colony number
200
0
12345
Culture time (day)
Culture time in fibrin
g
els (h)
1,050 Pa to 1,050 Pa
0
2
4
6
8
10
Sox2 *
Relative expression
1,050 Pa to 90 Pa
24 48
*
*
1.7
1.6
1.5
H3K9-me (YPet/CFP)
1.4
1.3
1.2
12345
Culture time (day)
***
*
*
#
#
Figure 8 | Stiff matrix inhibits self-renewal of TRCs. Stiff fibrin gels inhibit colony growth (a) and number (b). ‘90-Pa to 90-Pa’ and ‘90-Pa to
1,050-Pa’: control B16-F1 cells were cultured in 90-Pa fibrin gels for 5 days and these cells were then re-plated into 90-Pa and 1,050-Pa fibrin gels,
respectively. ‘1,050-Pa to 90-Pa’ and ‘1,050-Pa to 1050-Pa’: control B16-F1 cells were cultured in 1050-Pa fibrin gels for 5 days and these cells were
then re-plated into 90-Pa or 1,050-Pa fibrin gels. Spheroid size and number were measured from Day 1 through Day 5. In aand b, significant differences
between ‘90-Pa to 90-Pa’ and ‘90-Pa to 1,050-Pa’ from Day 1 through Day 5 (all Po0.01); significant differences between ‘1,050-to 90-Pa’ and ‘1,050-Pa to
1050-Pa’ at day 4 and day 5 in a(both Po0.02) and from day 1 to day 4 in b(all Po0.05). Mean±s.e.m.; n¼2 independent experiments. Each data
point was averaged from 430 colonies. ‘*’ and ‘#’ indicates significant differences between ‘90-Pa to 90-Pa’ and ‘90-Pa to 1,050-Pa’ and between ‘1,050-to
90-Pa’ and ‘1,050-Pa to 1050-Pa’, respectively. (c) Switching to 3D soft matrix decreases H3K9 methylation. Mean±s.e.m.; n450 cells from two
independent experiments; *Po0.05. (d) Switching to 3D soft matrix increases Sox2 expression. Mean±s.e.m.; n¼3 independent experiments; *Po0.05.
In cand d, control cells were cultured in 1,050-Pa fibrin gels for 5 days and then re-plated into 90-Pa or 1,050-Pa gels. After 24 or 48 h in cand 5 days in d,
their H3K9 methylation was measured by FRET and Sox2 expression was quantified by real time RT–PCR.
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colonization is such a highly inefficient process, with only
B0.01% intravasated cancer cells being able to form metastatic
colonization45. The model that soft TRCs that are much softer
than differentiated cancer cells and that are the key players in
metastatic colonization is supported by a recent report that the
core of human breast tumour tissues is much softer than
peripheral tumour tissues and that metastatic tumour tissues in
mouse lungs are much softer than surrounding normal lung
tissues46. This soft cancer cell model is also supported by
numerous reports over the last two decades from several labs that
cancer cells, especially those metastatic cancer cells, are much
softer than normal cells or non-metastatic cancer cells47–52. The
long period of latency of disseminated carcinoma cells observed
clinically45 might be partially explained by the dormancy of the
TRCs inside the uniformly stiff matrix, which restrains them from
proliferating. This postulate needs to be tested in the future in
animal models.
Methods
Cell culture.Murine melanoma cell line B16-F1 was purchased from American
Type Culture Collection. Briefly, cells were cultured on rigid dishes with DMEM
cell culture medium supplemented with 10% fetal bovine serum (Invitrogen), 2 mM
L-glutamine (Invitrogen), 1 mM sodium pyruvate and 0.1 mM penicillin/strepto-
mycin at 37 °C with 5% CO
2
. Cells were passaged every 3–4 days using TrypLE
(Invitrogen). Glass or plastic culture dishes were coated with collagen-1
(0.2 mg ml 1) or sometimes with gelatin (0.1%) (denatured collagen) unless
otherwise specified.
3D fibrin gel preparation.Salmon fibrinogen and thrombin were purchased from
Reagent Proteins (CA, USA). Three-dimensional fibrin gels were prepared as
described previously5. In brief, fibrinogen was diluted into 2 mg ml 1with T7
buffer (pH 7.4, 50 mM Tris, 150 mM NaCl). Cells were detached from 2D rigid
dishes and cell density was adjusted to 104cells per ml. Fibrinogen and cell solution
mixture was made by mixing the same volume of fibrinogen solution and cell
solution, resulting in 1mg ml 1fibrinogen and 5,000 cells per ml in the mixture
(for 2, 4, 8 mg ml 1(180, 420 or 1,050 Pa, respectively) fibrin gels, the initial
concentration was increased to 4, 8 or 16 mg ml 1). Two hundred and fifty
microlitres of cell/fibrinogen mixture was seeded into each well of 24-well plate and
mixed well with pre-added 5 ml thrombin (100 U ml 1). The cell culture plate was
TRC
Soft matrix + soluble
differentiation factor
Local tumour
Matrix
degradation +
remodelling
TRC self-
renewal and/or
differentiation
Matrix
degradation
Remodelling
Invasion & metastasis
by TRCs
Dormancy
No growth
TRC=Tumour-
repopulating cell
DCC=Differentiated
cancer cell
Stromal cell
Soft matrix
Stiff matrix
Figure 10 | A working model of melanoma tumour growth and metastasis. TRCs proliferate in a soft matrix and eventually grow into a local tumour, while
a stiff matrix restricts their proliferation and makes TRCs dormant. TRCs in the soft matrix can invade and self-renew and/or differentiate when the matrix
is degraded and remodeled. When a soluble differentiation factor like retinoic acid is present, TRCs lose their proliferative property and stay quiescent even
in soft matrix. However, on matrix degradation, the dormant TRCs in the original stiff matrix start to self-renew in weakened and soft sites and to
differentiate in remodelled and stiffened sites, leading to invasion and eventual metastatic colonization by TRCs in very soft tissues/organs with rigidity of
B100 Pa, similar to those undifferentiated TRCs. It is known that majority of metastases from many types of solid tumours is found in very soft organs/
tissues such as the lung, brain, liver and bone marrow45, but the underlying mechanism of this metastatic tropism is not understood. As the bone marrow
stiffness is B100 Pa (ref. 54), brain stiffness is B200–1,000 Pa (ref. 54), liver stiffness is B400 Pa (ref. 55) and lung stiffness is 100–1,000 Pa (ref. 56), all
being very soft tissues, it is tempting to speculate that our model could be extended to metastases by other types of solid tumours, with implications in
providing possible partial explanation to the conundrum of metastatic tropism. Clearly, this working model needs to be tested rigorously in the future.
H3K9 me2
H3K9 me3
GAPDH
90-Pa
1,050-Pa
15kDa
15kDa
40kDa
3.5
3
2.5
Relative expression
2
1.5
1
0.5
0
90 Pa
1,050 Pa
H3K9 me2 H3K9 me3
1.4
1.2
1
0.8
Relative expression
0.6
0.4
0.2
090 Pa 180 Pa
Fibrin gels
420 Pa 1,050 Pa
*
*
*
** Sox2
a
c
b
Figure 9 | Stiff fibrin matrix elevates H3K9 methylation levels and
suppresses Sox2 expression. (a,b) Stiff fibrin matrix increases H3K9
methylation. Control cells were cultured in 90-Pa or 1,050-Pa fibrin gels for
5 days. Their H3K9 methylation level was analysed by western blotting assay
(a) and quantified in b.(c) Stiff fibrin matrix suppresses Sox2 expression.
Control B16-F1 cells were cultured in fibrin gels with the indicated stiffness for
5 days and then analysed for Sox2 expression by real-time RT-PCR.
Mean±s.e.m.; n¼3 independent experiments. *Po0.05; **Po0.01.
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then incubated in 37 °C cell culture incubator for 10 min. Finally, 1 ml of DMEM
medium containing 10% fetal bovine serum and antibiotics was added.
Reverse transcription-PCR and real-time RT–PCR analysis.Total mRNA was
isolated from the cells using the Trizol reagent according to the supplier’s
instruction (Invitrogen). Reverse transcription (RT) was performed using the
TransScript First-strand cDNA Synthesis SuperMix (TransGen), according to the
manufacturer’s protocol. RT–PCR was performed using a PCR kit (QIAGEN), and
real-time RT–PCR was performed using GoTaq qPCR Master Mix (Promega). The
data were normalized against mouse glyceraldehyde 3-phosphate dehydrogenase.
The sequences of all the primers for RT–PCR and real time RT–PCR were listed in
Supplementary Tables 2 and 3.
Chromatin immunoprecipitation assay.We performed ChIP assay following the
manufacturer’s instructions (ChIP Assay Kit, Millipore). Briefly, cells were sub-
jected to cross-linking with 1% formaldehyde in medium for 10 min at 37 °C and
then lysed in SDS buffer for 10 min on ice. Chromatin was sonicated to shear DNA
to an average length of 0.2–1.0 kb. Antibodies to di- and trimethyl histone H3
(Lys9) (Abcam) were used for immunoprecipitation. Normal mouse IgG (Santa
Cruz) was used as negative control. The immunoprecipitation was heated to
reverse the formaldehyde cross-linking and the DNA fragments in the precipitates
were purified by phenol/chloroform extraction and ethanol precipitation. The
immunoprecipitated DNA was quantified by real-time RT-PCR. Primers were sets
corresponding to Sox2 and glyceraldehyde 3-phosphate dehydrogenase (negative
control) promoter regions. The sequences of these promoter regions were listed in
Supplementary Table 4 and can be found in Transcriptional Regulatory Element
Database (Cold Spring Harbor Laboratory).
Western blotting assay.To quantify the expression levels of G9a, SUV39h1,
H3K9 di- and trimethylation, cells were lysed with 200 ml Laemmli sample buffer
(Beyotime). Twenty microlitres of each sample were separated by 8–15% SDS–
PAGE, blocked with 5% BSA overnight at 4 °C and incubated with primary anti-
bodies to G9a (Rabbit, 1:1,000, Cell Signalling, 3306), SUV39h1 (Rabbit, 1:1,000, Cell
Signalling, 8729), H3K9 di-methylation (Rabbit, 1:300, Millipore, 17–648) and tri-
methylation (Rabbit, 1:300, Millipore, 17–625) and GAPDH (Mouse, 1:1,000,
Abcam, ab8245) for 2 h at room temperature. Primary antibodies were detected with
goat anti-Rabbit IgG-HRP (1:2,000, Santa Cruz, sc-2004) or anti-Mouse IgG-HRP(,
1:2,000, Santa Cruz, sc-2005). The blots were developed using SuperSignal West Pico
chemiluminescent substrate (Millipore).
FRET probe gene construction and plasmids.The plasmid of histone H3K9
methylation reporter (ID 22866) is purchased from Addgene (Cambridge, MA). In
brief, it includes a central fragment consisting of the methyl-lysine binding domain
of HP1 protein and a histone peptide, which was further fused in between an
amino-terminal CFP and a carboxy-terminal YFP17. This plasmid was digested
with BamHI and SacI, and fused to predigested pRsetB (Invitrogen) containing
C-terminal YPet and NLS. The reporter containing NLS was further digested and
inserted into pcDNA3 (Invitrogen) to create modified histone methylation reporter
for mammalian cell expression. The insert was sequenced (W. M. Keck Center for
Functional and Comparative Genomics, University of Illinois at Urbana–
Champaign) to verify the integrity of the coding sequence. FRET reporter was
transfected into the cells using Lipofectamine 2000 (Invitrogen) according to
manufacturer’s protocols.
Magnetic twisting cytometry.Magnetic twisting cytometry has been described
elsewhere19,53. RGD-coated or poly-L-lysin-coated ferromagnetic beads (Fe
3
O
4
,
4mm in diameter) were added to cells and then incubated for 30 min to allow for
integrin binding and clustering. Beads were then magnetized in horizontal
direction by applying a strong magnetic pulse (1,000 Gauss for 100 ms). A
sinusoidal oscillatory waveform was applied at 0.3 Hz for 1 h to the apical surface of
the cell. The amplitude of the magnetic field was varied at 35, 50 and 70 Gauss
corresponding to 11, 15.9 and 22.2 Pa applied stress. The apparent applied stress is
defined as the ratio of the applied torque to six times the bead volume and equals
the bead constant times the applied twisting field. Cells were plated on collagen-
coated (0.2 mg ml 1) or fibrinogen-coated (0.2 mg ml 1) PA gels with different
rigidities or glass/plastic. Stiffness was quantified 6 h later. Cells were maintained in
CO
2
-independent medium supplemented with 10% serum (Invitrogen) at 37 °C
during experiments.
A Leica inverted microscope integrated with the magnetic twisting cytometry
device and Dual-View system (Optical Insight) was used to simultaneously apply
stress and capture CFP and YFP (YPet) emission images. For emission ratio
imaging, the Dual-View MicroImager (Optical Insights) was used. CFP/YFP Dual
EX/EM (FRET) (OI-04-SEX2) has the following filter sets: CFP: excitation, S430/
25, emission S470/30; YFP: excitation, S500/20, emission S535/30. The emission
filter set uses a 515-nm dichroic mirror to split the two emission images. Cells were
illuminated with a 100-W Hg lamp. For FRET imaging, each CFP (1,344
pixels 512 pixels) and each YFP image (1,344 pixels 512 pixels) were
simultaneously captured on the same screen by using a charge-coupled device
camera (C4742–95-12ERG; Hamamatsu) and a 40, 0.55 numerical aperture air-
immersion objective. A customized Matlab (Mathworks) programme was used to
analyse CFP and YFP images and to obtain YFP/CFP emission ratio.
RNA interference.Cells were transfected with siRNA or shRNA, or com-
plementary DNA using Lipofectamine 2000 (Invitrogen) following the manu-
facturer’s protocol. Silencer Negative Control No. 1 siRNA (Invitrogen, AM4611)
was used a control in RNAi experiment. The construct sequence is 50-GGUGAU
CCUUAUGCUGUU-30Att for G9a siRNA 1 (Invitrogen, 90322), 50-GGUGAC
UUCAGAUGUGGCCtt-30for G9a siRNA 2 (Invitrogen, 90133), 50-GGUCCUU
UGUCUAUAUCAAtt-30for SUV39h1 1 (Invitrogen, 69566), 50-GGUGUACAAC
GUAUUCAUAtt-30for SUV39h1 2 (Invitrogen, 151927) and 50-AGUACUGCU
UACGAUACGGtt-30for negative control siRNA. KDM7 siRNA 1 (Santa Cruz,
sc-146320) is a pool of three different siRNA duplexes, sc-146320A, sc-146320B
and sc-146320C, the sequence of which is 50-GUAGUAUACCGCAGCUUAAtt-30,
50-GUGAAUGGUUAGCAAUACAtt-30and 50-GAGAAUGUCUCGCCUUU
CAtt-30, respectively. The sequence of KDM7 siRNA 2 is 50-GAAAUAACAUCAC
ACUUUAtt-30(Invitrogen, 503916). Mitf siRNA was purchased from Invitrogen
(155366) and the construct sequence is 50-GGUAUGAACACGCACUCUCtt-30.
Cdc42 siRNA was acquired from Invitrogen (catalogue number 66023) and the
construct sequence is 50-GGGCAAGAGGAUUAUGACAtt-30.Sox2 shRNAs were
obtained from Origene (TG515613). The construct sequence is 50-GCACTACCAG
AGCTAACTCAGATAGTACT-30for scrambled shRNA, 50-GTATAACATGAT
GGAGACGGAGCTGAAGC-30for shRNA 1, 50-AGACGCTCATGAAGAAGGAT
AAGTACACG-30for shRNA 2, 50-AGCTACGCGCACATGAACGGCTGGAG
CAA-30for shRNA 3, and 50-AACATGATGGAGACGGAGCTGAAGCCGCC-30
for shRNA 4. The Sox2 cDNA was obtained from Origene (MG204615). The
Cdc42-EGFP plasmid (ID 12975) was purchased from Addgene. pEGFP-N1 vector
(Clotech) was used as control.
Traction measurement..We followed the protocols of cell-traction quantification
published previously11. In brief, cells were plated on PA gels with red fluorescent
beads (0.2 mm in diameter; Molecular Probes, Invitrogen) embedded on the top
surface. Before and after cell trypsinization, fluorescent images were taken to
compute the displacement field of the beads using digital image correlation (DIC)in
a homebuilt MATLAB program. Cell tractions were then calculated from the
displacement field using inverse Boussinesq mathematical model.
Mice experiments.Four-week-old female C57BL/6 mice were purchased from
Wuhan University Center for Animal Experiment. All animals had received
humane care in compliance with the Principles of Laboratory Animal Care For-
mulated by the National Society of Medical Research and the guide for the National
Institutes of Health of USA. The protocol was approved by the Animal Care and
Use Committee of Wuhan University. B16-F1 cell spheroids were selected from 3D
90-Pa fibrin gels and pipetted to single cells. These TRCs were then transfected
with Sox2 shRNA or scrambled control via Lipofectamine 2000 (Invitrogen) fol-
lowing the manufacturer’s protocol. These cells were harvested and the cell number
was counted under microscopy. The cells were then suspended in a 0.9% NaCl
solution with appropriate cell density. One hundred of TRCs transfected with Sox2
shRNA or scrambled control were subcutaneously inoculated or intravenously
injected into the tail vein of wild-type C57BL/6 mice. The tumour growth of
injected mice was carefully monitored every day.
Colony number assay.By changing the focal planes along the zaxis (the direction
of gel depth), the colony number was counted view by view. At least three wells of
colonies were counted per condition per day.
Statistical analysis.All statistics (except the Fisher’s exact test for analyses of
mice experiments) were performed using a two-tailed Student’s t-test.
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Acknowledgements
We thank Wenting Yao for help. We thank Dr Alice Ting for help with the H3K9 FRET
biosensor. We thank Dr Fei Wang and his lab at UIUC for assistance. This work was
supported by the funds from Huazhong University of Science and Technology and US
NIH grant GM 072744 (to N.W.) and National Science Foundation through the Physics
Frontiers Center Program (0822613) (to T.H.). T.H. is an investigator with the Howard
Hughes Medical Institute. A.T. acknowledges partial financial support from Natural
Sciences and Engineering Research Council (NSERC) of Canada through PGS Doctoral
Scholarship. F.C. acknowledges support from IGB fellowship at UIUC.
Author contributions
N.W., Y.T. and A.T. conceived the project; N.W., Y.T., A.T., J.W.C., Q.J., H.J., F.C., T.H.
and B.H. designed the experiments. Y.T., A.T., J.W.C., Q.J., F.C., L.W., J.J.C., S.Z., Y.H.,
Y.Y., D.W., Y.Z., F.W., Y.C.P., J.S., R.S., S.D., Y.L. and L.J.L. carried out the experiments
and analysed the data. N.W., Y.T., A.T., J.W.C., J.S., F.C. and Y.W. wrote the manuscript.
Additional information
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How to cite this article: Tan, Y. et al. Matrix softness regulates plasticity of
tumour-repopulating cells via H3K9 demethylation and Sox2 expression. Nat. Commun.
5:4619 doi: 10.1038/ncomms5619 (2014).
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