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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)