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PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com
Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model
Jen Carlstrom, Jeanine Hinterneder, Lindsay Nelson, and Stephen Hurt
#5793
Introduction 1 E-cadherin and Fibronectin modulated
by TGF-β measured with AlphaLISA
in monolayer cultures
To assess the changes in IL-6 secretion over time, 4000 cells were plated in
multiple wells in 2D and 3D. At each time point, supernatants were removed and
cells lysed and aliquots were frozen at -20°C. AlphaLISA was used to measure
IL-6 levels in the samples and GAPDH was used to normalize to potential
differences in cell number due to differences in growth rates. The dramatic
difference in IL-6 secretion over time in 2D compared to 3D cannot simply be
explained by differences in cell numbers.
GAPDH levels may be used to
correlate cell number differences
between 2D and 3D over time
AlphaLISA assay
E-cadherin and fibronectin quantification
using AlphaLISA. A) AlphaLISA signal
detecting E-cadherin and fibronectin in cell
lysates for various cell numbers before and
after TGF-β treatment in monolayer cultures.
B) Concentrations of E-cadherin and
fibronectin interpolated from standard curves
(C) generated in lysis buffer and EMEM.
DU 145 cells were plated in 2D and 3D cultures
at 5 concentrations and GAPDH levels were
assessed in lysates using the AlphaScreen
SureFire GAPDH kit (cat# TGRGDS500). As
shown here, the amount of GAPDH is ~4-fold
lower in 3D cultures.
A) AlphaLISA data showing increases
in IL-6 secretion over time between
2D and 3D cultures. B) GAPDH levels
in lysates from 2D and 3D cultures. C)
IL-6 levels normalized to GAPDH
levels over time.
Changes in IL-6 levels over time
between 2D and 3D
Summary
• We confirmed that treatment of DU 145 cells with TGF-β is
sufficient for inducing changes in both EMT biomarker
expression and cellular morphology in monolayer cultures.
• AlphaLISA and LANCE (TR-FRET) biomarker assays can be used
to measure ECM-associated protein modulation caused by
human transforming growth factor-beta (TGF-β) induction of
EMT in a 3D Spheroid model of human prostate carcinoma.
• E-Cadherin is downregulated by TGF-β in both 2D and 3D
cultures, wherease Fibronectin is increased significantly only in
2D monolayer cultures.
• Monolayer cultures proliferate considerably more than cells in
spheroid cultures.
• PMA and TGF-β treatment induced significant differences in IL-6
secretion levels between 2D and 3D cultures.
• TGF-β affects proliferation or viability of cells in 2D cultures
differently than in 3D cultures.
• These data illustrate the differences in protein expression levels
and in cellular tolerance for treatment between a human prostate
cell line grown in monolayers versus 3D spheroids.
Modulation of E-cadherin & Fibronectin
Levels in 2D and 3D cultures
3
11
12
10
4
Epithelial-mesenchymal transition (EMT) is characterized by
rearrangement of the extracellular matrix (ECM) and differential
regulation of ECM proteins. We induced EMT in DU 145 cells using
TGF-β and phorbol-12-myristate-13-acetate (PMA) and compared
expression levels of specific biomarkers, including E-cadherin,
fibronectin, and IL-6, using AlphaLISA® and LANCE® (TR-FRET)
assay technologies. We confirmed that treatment with TGF-β is
sufficient for inducing changes in both EMT biomarker expression
and in promoting development of characteristic mesenchymal
stromal cell morphology in monolayer cultures. However, in 3D
spheroid cultures, we only observed a partial EMT response to the
same TGFβ treatment as demonstrated by changes in the expected
biomarker expression pattern. Cellular proliferation, growth and
vitality were assessed using ATPlite luminescence assays and
confocal microscopy of live-stained cells with a high content
imaging system. Though we observe increased proliferation in
monolayer cultures compared to 3D spheroids, the changes
observed in protein expression patterns cannot be sufficiently
explained by differences in cell number or viability. These data
illustrate the differences in protein expression levels and in cellular
tolerance for compound treatment between a human prostate
cancer cell line grown in monolayers and those same cells grown in
3D spheroids.
Materials and Methods 2
Cell culture and treatment: DU 145 cells (ATCC® HTB-81™) were seeded (100 μL/well) into
PerkinElmer 96-well CellCarrier™ (6005550) or CellCarrier Spheroid ULA 96-well microplate
(3D) (6055330) and grown for at least 18 hours (see spheroid growth illustration below). Cells
were serum starved for 24 hours prior to treatment with recombinant human TGF-β1 (BioLegend,
580702) or PMA (Sigma cat# P1585) to induce EMT. In some experiments, cells were treated
with 2.5 μM of TGF-β inhibitor SD 208 (Sigma, S7071) for two hours followed by 48 hours with
TGF-β (5 ng/mL) in a total volume of 100 μL.
Biomarker quantification assays: For biomarker detection assays, 50 μL of media was
removed (for testing supernatants). Cells were then lysed with 50 μL of 2X Alpha SureFire® Ultra
lysis buffer (ALSU-LB-10mL) for 10 minutes. Lysates and supernatants were frozen at -80°C
and later thawed for testing in AlphaLISA and LANCE assays (see models for each assay type
below). For AlphaLISA assays detecting E-cadherin (AL370C), fibronectin (AL351C), and
human IL-6 (AL223C), 5 μL of each lysate or supernatant sample was added to a 384-well white
OptiPlate (6007290) and assays were performed according to the manual. For Fibronectin
LANCE Ultra TR-FRET assay (TRF1351C), 15 μl of lysate was added to a 384-well white
OptiPlate and assays were performed according to the manual. AlphaLISA and LANCE assays
were measured on a standard EnVision® multilabel plate reader using standard Alpha Settings
and reading TR-FRET with the Laser excitation.
ATPliteTM 1step and ATPliteTM 1step 3D assays: Cellular proliferation, growth & vitality were
measured by assessing the concentration of ATP using ATPlite 1step and ATPlite 1step 3D
luminescence-based assays following kit protocols. Luminescence was measured using the
EnSightTM multimode plate reader.
Cellular imaging: For cellular imaging, cultures were first labeled with Hoechst 33342 (Life
Technologies, #H3570), Tetramethylrhodamine (TMRM; Life Technologies, #T-668) and CellTox
Green (Promega, #G8742). Monolayer cultures were imaged using the cellular imaging module
of the EnSight plate reader using Brightfield and UV fluorescence filters and cell numbers were
quantified using the Count Nuclei function. 3D Spheroid cultures were imaged with the 10X long
WD objectives on the Operetta® and Opera PhenixTM High Content Imaging systems using
Brightfield and appropriate fluorescence optics. Cross-sectional spheroid area was measured
with Harmony software using an intensity cutoff in the UV channel (Hoechst).
Excitation
680 nm
Emission
615 nm
Streptavidin coated
Donor Bead Anti-Analyte Antibody 2
AlphaLISA Acceptor Bead
Biotinylated
Anti-Analyte
Antibody 1
Analyte
T re a tm e n ts (2 D , 5 0 0 0 c e lls s e e d e d /w e ll)
E-c
ad
he
rin
(g
/mL
)
N o T re a tm e n t + T G F - S D 2 0 8 + T G F - S D 2 0 8 o n ly
0
2 .01 0 - 9
4 .01 0 - 9
6 .01 0 - 9
S u p e rn a ta n tsL y s a te s
T re a tm e n ts (2 D , 5 0 0 0 c e lls s e e d e d /w e ll)
Fib
ro
ne
cti
n (
g/m
L)
N o T re a tm e n t + T G F - S D 2 0 8 + T G F - S D 2 0 8 o n ly
0
5 .01 0 - 9
1 .01 0 - 8
1 .51 0 - 8
2 .01 0 - 8
S u p e rn a ta n tsL y s a te s
T re a tm e n ts (3 D , 5 0 0 0 c e lls s e e d e d /w e ll)
Fib
ro
ne
cti
n (
g/m
L)
N o T re a tm e n t + T G F - S D 2 0 8 + T G F - S D 2 0 8 o n ly
0
1 .01 0 - 9
2 .01 0 - 9
3 .01 0 - 9
L y s a te s S u p e rn a ta n ts
T re a tm e n ts (3 D , 5 0 0 0 c e lls s e e d e d /w e ll)
E-c
ad
he
rin
(g
/mL
)
N o T re a tm e n t + T G F - S D 2 0 8 + T G F - S D 2 0 8 o n ly
0
5 .01 0 - 1 0
1 .01 0 - 0 9
1 .51 0 - 0 9
2 .01 0 - 0 9L y s a te s S u p e rn a ta n ts
Log [protein] (g/ml)
Alp
haL
ISA
Sig
nal
103
104
105
106
107
-12 -11 -10 -9 -8 -7 -6
Fibronectin
E-Cadherin
N u m b e r o f c e lls p la te d
Alp
ha
LIS
A S
ign
al
5 0 0 0 2 5 0 0 6 2 5 0 5 0 0 0 2 5 0 0 6 2 5 0
0
2 .51 0 4
5 .01 0 4
7 .51 0 4
1 .01 0 5
1 .51 0 5
2 .01 0 5
2 .51 0 5
3 .01 0 5T G F b ( + ) ( f ib r o n e c t in )
T G F b ( - ) ( f ib r o n e c t in )
T G F b ( + ) ( E -c a d h e r in )
T G F b ( - ) ( E - c a d h e r in )
c e lls p la te d
Pro
tein
(g
/ml)
5 0 0 0 2 5 0 0 6 2 5 5 0 0 0 2 5 0 0 6 2 5
0
1 .01 0 -9
2 .01 0 -9
3 .01 0 -9
4 .01 0 -9
5 .01 0 -9
6 .01 0 -9
T G F b ( + ) ( F ib r o n e c t in )
T G F b ( - ) ( F ib r o n e c t in )
T G F b ( + ) ( E -c a d h e r in )
T G F b ( - ) ( E - c a d h e r in )
E-cadherin (A, C) and fibronectin (B, D) levels in supernatants and lysates from 2D (A, B) and
3D (C, D) cultures before and after treatment with TGF-β and/or TGF-β inhibitor SD 208.
Examining TGF-β effects on Fibronectin
expression with LANCE TR-FRET 7
C e ll N u m b e r P la te d
LA
NC
E (
66
5/6
15
* 1
0K
)
2 0 0 0 (2 D ) 8 0 0 0 (3 D )
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
2 5 0 0 N o T G F -
5 n g /m l
1 0 n g /m l
2 5 n g /m l
5 0 n g /m l
IL -6 (s e c re te d )
Alp
ha
Sig
na
l
2 D 3 D
0
2 0 ,0 0 0
4 0 ,0 0 0
6 0 ,0 0 0
8 0 ,0 0 0
1 0 0 ,0 0 0 3 .5 h r
2 0 h r
2 6 h r
4 5 h r
5 0 h r
G A P D H (in ly s a te s )
Alp
ha
Sig
na
l
2 D 3 D
0
1 0 ,0 0 0
2 0 ,0 0 0
3 0 ,0 0 0
4 0 ,0 0 0
5 0 ,0 0 02 0 h r
2 6 h r
4 5 h r
5 0 h r
IL -6 (n o rm a liz e d to G A P D H )
Alp
ha
Sig
na
l (n
orm
ali
ze
d t
o G
AP
DH
)
2 D 3 D
0
1 0 ,0 0 0
2 0 ,0 0 0
3 0 ,0 0 0
4 0 ,0 0 0
5 0 ,0 0 0
6 0 ,0 0 0
7 0 ,0 0 0
8 0 ,0 0 0
9 0 ,0 0 0
2 0 h r
2 6 h r
4 5 h r
5 0 h r
PMA -induced changes in E-cadherin
and IL-6 expression
PMA has been shown to induce EMT also. Here, we show E-cadherin levels are
decreased in 2D and less so in 3D after treatment with 150 nM PMA.
Interestingly, the IL-6 secretion levels increase for 2D and decrease for 3D after
PMA treatment.
8
A) E-cadherin levels
before and after PMA
treatment for 2D and 3D.
B) IL-6 levels secreted
into supernatants. Of 2D
and 3D cultures
measured with
AlphaLISA.
IL -6 (s u p e rn a ta n ts )
c e ll n u m b e r p la te d
Alp
ha
Sig
na
l
4 0 0 0 2 0 0 0 1 0 0 0
0
2 ,0 0 0
4 ,0 0 0
6 ,0 0 0
8 ,0 0 0
1 0 0 ,0 0 0
2 0 0 ,0 0 0
3 0 0 ,0 0 0 P M A (2 D )
N o P M A (2 D )
P M A (3 D )
n o P M A (3 D )
G A P D H le v e ls
c e ll n u m b e r p la te d
Alp
ha
Sc
re
en
Sig
na
l
0 1 ,0 0 0 2 ,0 0 0 3 ,0 0 0 4 ,0 0 0
0
5 0 ,0 0 0
1 0 0 ,0 0 0
1 5 0 ,0 0 0
2 0 0 ,0 0 02 D c u ltu re s
3 D c u ltu re s
Affects of TGF-β on cell viability 9
Excitation 320 or
340 nm
Fluorescent
Emission 615 nm
FRET TR-FRET Emission
665 nm
Analyte
Anti-analyte
ULight conjugate
Anti-analyte
Eu chelate
conjugate
LANCE TR-FRET assay
To see if 3D spheroids and more cells require
more TGF-β and larger cell numbers to
illicit a response, 10, 25, and 50 ng/mL were
tested. Higher concentrations did not
increase fibronectin levels further in 2D and
did not affect levels at all in 3D. To account
for differences in proliferation over time
between 2D and 3D, we plated 4X higher
number of cells for 3D (8000/well) than for
2D (2000/well).
TGF-β effects on morphology 6
- TGF-β + TGF-β
Brightfield images of DU 145
cells 48 hours post-treatment
with or without TGF-β
captured on the EnSight.
TGF-β treatment induces
development of the
characteristic mesenchymal
stromal cell morphology
indicative of EMT.
Representative images for
6000 cells/well shown (right).
Brightfield images of spheroids DU 145 cells 48 hours post-treatment with
increasing concentrations of TGF-β on the Operetta High Content Imaging system.
E C a d h e rin (ly s a te s )
c e ll n u m b e r p la te d
Alp
ha
Sig
na
l
8 0 0 0 4 0 0 0 2 0 0 0
0
5 0 ,0 0 0
1 0 0 ,0 0 0
1 5 0 ,0 0 0
2 0 0 ,0 0 0 P M A (2 D )
N o P M A (2 D )
P M A (3 D )
N o P M A (3 D )
5 ng/mL 10 ng/mL 25 ng/mL 50 ng/mL TGF-β No TGF-β
Model of Spheroid culture growth.
t = Zero
(Seeding)
Less than
8 hours
24 hours 2 days 3+ days Flask
Culture
CellCarrierTM Spheroid
ULA microplate.
Serum starve & treatment
A)
C)
B)
C)
A)
D)
B)
A) B)
8 0 0 0 4 0 0 0 2 0 0 0 1 0 0 0
0
5 .01 0 4
1 .01 0 5
1 .51 0 5
2 .01 0 5
C e lls p la te d
Lu
min
es
ce
nc
e
+ T G F - (3 D )
N o T G F - (3 D )
2 0 0 0 1 0 0 0 5 0 0 2 5 0
0
5 .01 0 5
1 .01 0 6
1 .51 0 6
2 .01 0 6
2 .51 0 6
C e lls p la te d
Lu
min
es
ce
nc
e
+ T G F - (2 D )
N o T G F - (2 D )
8 0 0 0 4 0 0 0 2 0 0 0 1 0 0 0
0
5 .01 0 4
1 .01 0 5
1 .51 0 5
C e lls p la te d
Sp
he
ro
id S
ize
(
m2
)
T G F - (3 D )
N o T G F - (3 D )
Assessing cell proliferation in 2D & 3D 5
2 5 0 5 0 0 1 0 0 0 2 0 0 0 3 0 0 0 4 0 0 0 5 0 0 0
0
5 .01 0 5
1 .01 0 6
1 .51 0 6
2 .01 0 6
2 .51 0 6
3 .01 0 6
3 .51 0 6
A T P lite 1 s te p s ig n a ls
# c e lls p la te d
AT
Pli
te 1
ste
p
Lu
min
es
ce
nc
e
3 D s p h e ro id s
2 D c u ltu re s
0 1 ,0 0 0 2 ,0 0 0 3 ,0 0 0 4 ,0 0 0 5 ,0 0 0
0
2 5 ,0 0 0
5 0 ,0 0 0
7 5 ,0 0 0
1 0 0 ,0 0 0
1 2 5 ,0 0 0
S ig n a ls c o r re la te to th e c e ll n u m b e r p la te d
n u m b e r o f c e lls p la te d
AT
Pli
te 1
ste
p 3
D l
um
ine
sc
en
ce
R square 0.9647
0 2 0 ,0 0 0 4 0 ,0 0 0 6 0 ,0 0 0 8 0 ,0 0 0 1 0 0 ,0 0 0
0
2 5 ,0 0 0
5 0 ,0 0 0
7 5 ,0 0 0
1 0 0 ,0 0 0
1 2 5 ,0 0 0
S ig n a ls c o r re la te to s p h e ro id s iz e
s q u a re m ic ro n s
AT
Pli
te 1
ste
p 3
D l
um
ine
sc
en
ce
R square 0.9318
0 1 ,0 0 0 2 ,0 0 0 3 ,0 0 0 4 ,0 0 0 5 ,0 0 0
0
2 .01 0 4
4 .01 0 4
6 .01 0 4
8 .01 0 4
1 .01 0 5
S p h e ro id s iz e s c o r re la te to n u m b e r p la te d
# c e lls p la te d
sq
ua
re
mic
ro
ns
R square 0.9704
Hoechst
Monolayer (2D) cultures proliferate much more than spheroid (3D) cultures. 2D and 3D cultures
were plated at 7 concentrations, grown for 4 days and assessed with ATPlite 1step and ATPlite
1step 3D assays (with the final step being to transfer 50 μL of the reaction to a gray OptiPlate for
reading luminescence on the EnSight (n=3). 3D cultures were labeled with Hoechst, spheres
were imaged on the Operetta (C), and cross-sectional spheroid area measured using an intensity
cutoff (D). Spheroid area correlates to number plated (E) and luminescence signals (F).
Area measured
4000 seeded
C)
A)
D)
B)
E) F)
ATPlite 1step signals before and after
TGF-β treatment show an effect on
either proliferation or viability in
denser 2D cultures (A), but not 3D
spheroids (B). Spheroid area was not
affected by TGF-β treatment (C). 3D
cultures labeled with CellTox Green,
Hoechst, and TMRM (orange) show
no significant effect of TGF-β
treatment on cellular viability within
a spheroid (D).
TMRM Hoechst CellTox overlay
No TGF-β
+ TGF-β
A) B) C)
D)
A) B)
C)
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