poster: new and improved cellular health evaluation of 2d and 3d … · 2020. 8. 12. · new and...
TRANSCRIPT
Thermo Fisher Scientific • 29851 Willow Creek Road• Eugene, OR 97402 • thermofisher.comFor Research Use Only
0 .1 1 1 0 1 0 0
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1
2
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S p h e ro id
G A tre a tm e n t 26 h o u rs
[ 1 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
so
rb
an
ce
XT
T (
45
0-6
60
nm
)
IC50
4 hour XTT
38.41
22hr
36.49
27hr 47hr 51hr
27.54
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
1 1 0 1 0 0 1 0 0 0
0
2
4
6
S p h e ro id
G A tre a tm e n t 26 h o u rs
[ 2 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
so
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an
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XT
T (
45
0-6
60
nm
)
IC50
4 hour XTT
38.85
22hr
35.53
27hr 47hr 51hr
27.25
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
RESULTSSpheroids in Cancer Drug DiscoveryRelevant pharmacology & better tumor models
ABSTRACT
High throughput screening (HTS) is an effective method for
identifying putative active compounds for therapeutics. Assays that
evaluate changes in cellular functions are essential for
characterizing test compounds and their potential role as
pharmaceuticals.
Researchers in drug discovery and cancer research rely on
reproducible biological assays to guide medicinal chemistry
programs. A current goal in the cancer research field is to explore
and expand the use of microplate readers and assay systems into
the realm of 3D cellular models for drug discovery in anticancer
research, specifically aimed at modelling solid tumors to screen
compounds and confirm their activity. In this study, we screened
multiple therapeutic drugs using different HTS assays to establish
drug dose response curves and to understand the diversity in cell
health assays. Despite the lack of guidelines and enabling
technologies to study 3D cellular models, we establish the different
impact therapeutics have on 2D versus 3D cellular models using
existing cellular assays.
INTRODUCTION
The ability to use diverse cell health assays allows
researchers to monitor different cell health read-outs and
target specific cellular responses. The differences in
potency and drug incubation time between 2D and 3D
models indicate that drug effectiveness is dependent on
cellular microenvironment. The ability to use existing
microplate tools that are established for 2D cell models in
3D cell models provides a time and cost efficient basis for
understanding therapeutic effects on 3D cellular models,
allowing for rapid therapeutic characterization in drug
discovery with minimal investment.
MATERIALS AND METHODS
When comparing existing cell health assays, we evaluated
the differences between PrestoBlueTM HS and PrestoBlue.
The PrestoBlue HS assay has a larger dynamic range,
minimal background signal, and allows the removal of
hotspots (false-positives). The PrestoBlue HS assay has
the lowest detection threshold among the resazurin-based
fluorescent assays available for detection of cell viability.
With this advantage, the limit of quantification can be
studied. Additionally, as an improved reduction potential
assay, PrestoBlue HS can be used to interrogate the
differences between 2D and 3D cellular models.
We demonstrate that existing microplate assays, such as
PrestoBlue High Sensitivity (HS), CyQUANT® XTT, and
CyQUANT® Direct, can be used to quantify functional
differences between 2D and 3D cellular models. Using all
three cell health assays, we found that a longer incubation
time with drug is required to have an effect on the health of
3D models compared to 2D models. These results suggest
that drug treatment is less potent in 3D models than 2D
models, as demonstrated by a shift in the apparent IC50
values. Additionally, the dense compact structure of 3D
models is preserved at low to no concentrations of drug,
indicating that higher concentrations of drug are required to
kill cells grown in 3D culture compared to 2D monolayers.
Leticia A. Montoya, Bhaskar S. Mandavilli, and Madison Javitz,
Thermo Fisher Scientific, 29851 Willow Creek Road, Eugene, Oregon, USA, 97405
New and improved cellular health evaluation of 2D and 3D cellular models
using microplate reader assays
Working in 3D involves
formation of spheroids.
Spheroids are aggregates
that can either be grown in
suspension, encapsulated,
or grown on the top of a
3D matrix. CellEventTM Caspase 3/7 detection reagent measures cleaved caspase 3, hall mark of apoptosis cells
The spheroid size measurements is a phenotypic readout to measure drug effects.
Drug Potency on Monolayer (2D) and Spheroid (3D) Cellular Models: (A) As incubation time increases with
gambogic Acid, the drug becomes more potent to 2D monolayer cells. Gambogic Acid is less potent to 3D spheroids
than to 2D monolayer cells; shift in IC50 values. (B) High concentrations of Gambogic Acid drug are required to have
an effect on spheroid health. Long incubation time (greater than 24 hours) is required with Gambogic Acid to effect
Spheroid health.
0 .1 1 1 0 1 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
M o n o la y e rT r e a t e d w i t h 1 0 u L P B fo r 2 1 h r s
T r e a t m e n t w i th d r u g 1 9 h r s a f te r p la t in g
G a m b o g ic A c id (u M )
RF
U (
56
0/
59
0)
+/
- S
D
2 -3 h o u rs
1 9 h o u rs
EC50
2-3 hours
8.588
19 hours
3.359
in c u b a t io n t im e w i t h G ATreatment time with GA
Optimization of viability reagents for 3D spheroid analysis
1 1 0 1 0 0 1 0 0 0
0
1 0 0
2 0 0
3 0 0
S p h e r o idT r e a t e d w i t h 1 0 u L PB
T r e a t e d w i th d r u g 1 9 h o u r s a f te r p la t in g
G a m b o g ic A c id (u M )
Flu
ore
sc
en
ce
56
0/5
90
nm 2 h o u rs G A
2 4 h o u rs G A
4 8 h o u rs
7 2 h o u rs
EC50
2 hours GA
156.2
24 hours GA
53.37
48 hours
28.44
72 hours
25.95
Treatment time with GA
Measuring Cellular Health on Monolayer (2D) and Spheroid (3D) Cellular Models
First Step is determining concentration of reagent. CyQUANT XTT gives a better signal/noise ratio and same
IC50 value when used at twice the concentration on Spheroids when compared to one times the concentration on
monolayer cellular models.
Second step is determining incubation time with reagent. The incubation time with reagents is extended
when working with spheroids when compared to monolayer. Highly treated spheroids presents a very low
signal turn-on, further confirming the presence of dead cells.
Measuring Cellular Health on microplate reader VarioskanTM LUX versus High Content Analysis System CX7
0 .1 1 1 0 1 0 0
0
2 0 0
4 0 0
6 0 0
8 0 0
M o n o la y e rT r e a t e d w i t h 1 0 u L P B fo r 2 1 h r s
T r e a t m e n t w i th d r u g 1 9 h r s a f te r p la t in g
G a m b o g ic A c id (u M )
RF
U (
56
0/
59
0)
+/
- S
D
2 -3 h o u rs
1 9 h o u rs
EC50
2-3 hours
8.588
19 hours
3.359
in c u b a t io n t im e w i t h G ATreatment time with GA
1 1 0 1 0 0 1 0 0 0
0
5
1 0
1 5
2 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
S p h e r o idT r e a t e d w i t h 1 x C Q D fo r 3 h r s
T r e a tm e n t w i t h d r u g f o r 4 8 h r s
G a m b o g ic A c id (u M )
Va
rio
sk
an
LU
X
Flo
ure
sc
en
ce
50
0/5
30
Hig
h C
on
ten
t CX
7
Flo
ure
sc
en
ce
50
0/5
30
L U X
C X 7
IC50
LUX
~ 16.75
CX7
19.76
LUX
IC50 16.75
CX7
IC50 19.76
Enhanced Assay Performance
High Sensitivity Resazurin-based Reagents: An innovative purification process was developed that removes
contaminants from the original PrestoBlue and alamarBlue products to make the High Sensitivity versions (HS)
of both reagents.
1 1 0 1 0 0 1 0 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
T ra n s fo rm o f 3 D s p h e ro id s -o n c e lls -P B H S , P B , a B H S , a B -d ru g d o s e -6 0 m in in c u b a t
G a m b o g ic A c id (µ M )
Flu
ore
sc
en
ce
56
0/5
90
nm
P re s to B lu e H S
P re s to B lu e
a la m a rB lu e H S
a la m a rB lu e
IC50
PrestoBlue HS
17.64
PrestoBlue
21.86
alamarBlue HS
20.00
alamarBlue
17.05
1 1 0 1 0 0 1 0 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
T ra n s fo rm o f 3 D s p h e ro id s -o n c e lls -P B H S , P B , a B H S , a B -d ru g d o s e -6 0 m in in c u b a t
G a m b o g ic A c id (µ M )
Flu
ore
sc
en
ce
56
0/5
90
nm
P re s to B lu e H S
P re s to B lu e
a la m a rB lu e H S
a la m a rB lu e
IC50
PrestoBlue HS
17.64
PrestoBlue
21.86
alamarBlue HS
20.00
alamarBlue
17.05
Resazurin-based Reagents
10 m
inu
tes
1 h
ou
r
2 h
ou
rs
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
T im e
sig
na
l to
ba
ck
gro
un
d
P re s to B lu e H S
P re s to B lu e 1 .0
C e llT ite r -B lu e
Sig
nal/B
ackg
rou
nd
10 60 120
Time (minutes)
10 m
inu
tes
1 h
ou
r
2 h
ou
rs
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
T im e
RF
U (±
SE
M)
(56
0/
59
0)
P re s to B lu e H S
P re s to B lu e 1 .0
C e llT ite r -B lu e
PrestoBlue (Thermo Fisher Scientific)
PrestoBlue HS (Thermo Fisher Scientific)
CellTiter-Blue® (Promega)
Performance Comparison of resazurin-based reagents: (A) High Sensitivity reagents have a 100% increase
in the signal-to-background ratio and greater dynamic range when compared to other resazurin-based
reagents. (B) Cellular reduction kinetics on A549 spheroids (C) A549 spheroids were treated with Gambogic
Acid for 24 hours and cell viability was measured with resazurin-based reagents (fluorescence response was
measured at 60 minute).
0 2 4 6 8
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
3 D s p h e ro id s -o n c e lls -v ia b ility - t im e c o u rs e
T im e (h o u rs )
Flu
ore
sc
en
ce
56
0/5
90
nm
1 1 0 1 0 0 1 0 0 0
0
1 0 0
2 0 0
3 0 0
4 0 0
5 0 0
T ra n s fo rm o f 3 D s p h e ro id s -o n c e lls -P B H S , P B , a B H S , a B -d ru g d o s e -6 0 m in in c u b a t
G a m b o g ic A c id (µ M )
Flu
ore
sc
en
ce
56
0/5
90
nm
P re s to B lu e H S
P re s to B lu e
a la m a rB lu e H S
a la m a rB lu e
IC50
PrestoBlue HS
17.64
PrestoBlue
21.86
alamarBlue HS
20.00
alamarBlue
17.05
CyQUANT XTT
incubation time
IC50s
(µM)
Concentration
(µM)
1X 2X
4 hours 38.4 38.9
22 hours 36.5 35.5
51 hours 27.5 27.3
1 1 0 1 0 0 1 0 0 0
0
1
2
3
4
5
6
S p h e ro id
G A tre a tm e n t 26 h o u rs
[ 1 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
so
rb
an
ce
XT
T (
45
0-6
60
nm
)
IC50
4 hour XTT
38.41
22hr
36.49
27hr
37.24
47hr
29.67
51hr
27.54
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
1 1 0 1 0 0 1 0 0 0
0
1
2
3
4
5
6
S p h e ro id
G A tre a tm e n t 26 h o u rs
[ 1 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
so
rb
an
ce
XT
T (
45
0-6
60
nm
)
IC50
4 hour XTT
38.41
22hr
36.49
27hr
37.24
47hr
29.67
51hr
27.54
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
4 hours
51 hours
22 hours
1 1 0 1 0 0 1 0 0 0
0
1
2
3
4
5
6
S p h e ro id
G A tre a tm e n t 26 h o u rs
[ 1 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
so
rb
an
ce
XT
T (
45
0-6
60
nm
)
IC50
4 hour XTT
38.41
22hr
36.49
27hr
37.24
47hr
29.67
51hr
27.54
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
1 1 0 1 0 0 1 0 0 0
0
1
2
3
4
5
6
S p h e ro id
G A tre a tm e n t 26 h o u rs
[ 1 X ] C y Q U A N T X T T
G a m b o g ic A c id (u M )
Ab
so
rb
an
ce
XT
T (
45
0-6
60
nm
)
IC50
4 hour XTT
38.41
22hr
36.49
27hr
37.24
47hr
29.67
51hr
27.54
4 h o u r X T T
2 2 h r
2 7 h r
4 7 h r
5 1 h r
4 hours
51 hours
22 hours
0 .1 1 1 0 1 0 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
N ic lo s a m id e
A 5 4 9 c e lls
N ic o ls a m id e (M )
Ce
ll E
ve
nt
Gre
en
(5
03
/53
0 n
m)
Mito
Tra
ck
er O
ra
ng
e
C e ll E v e n t G re e n
M ito T ra c k e r O ra n g e
0 .1 1 1 0 1 0 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
N ic lo s a m id e
A 5 4 9 c e lls
N ic o ls a m id e (M )
Ce
ll E
ve
nt
Gre
en
(5
03
/53
0 n
m)
Mito
Tra
ck
er O
ra
ng
e
C e ll E v e n t G re e n
M ito T ra c k e r O ra n g e
High-Content Screening Platforms provide special and temporal
resolution.
High-Content Screening Platforms are equipped with wide field or
confocal optics to allow for ease of multiplexing.
Assays for microplate readers allow for rapid quantification of cell
health measurements and enzyme activity.
3D High Content Screening Platform: CellInsightTM CX7
untreated6 µM60 µM
Niclosamide treated 3D spheroids2D Monolayer 3D spheroids
Cellular
Activity
Cell to Cell Cell to ECM
Cellular
Interactions
Cellular Adhesion,
Proliferation, & modified genes
Proliferative Ring &
Apoptotic core
Micro-
environment
Immune Response therapy Gradients to Oxygen,
metabolites &
nutrients
MitoTrackerTM detection reagent measures
mitochondria health in live cells. The reagent is
dependent upon membrane integrity.
Mitochondria health is inversely proportional to
the apoptotic pathway.
Assessing Spheroid Health using High Content Analysis
untreated6 µM60 µM
Gambogic Acid treated 3D spheroids
Using microplater reader VarioskanTM LUX to drug screen before image analysis on HCA System
Existing solution- and fluorescence-based assays can be analyzed by microplate readers for initial drug
discovery questions. Similar IC50 values are determined by using existing assays on the microplate reader and
High Content System.
CONCLUSIONS
Monolayer and Spheroids can be monitored across multiple platforms with multiple different
reagents.
Existing microplate assays can be used to quantify functional differences between 2D and 3D
cellular models.
Pharmaceutical drugs can be analyzed on a microplate as initial studies before further
analysis on imaging platform
using existing cell functional
assays.
Increase in Cell Viability
CyQUANT Direct
High Throughput Drug Screening. The IC50 values of multiple
drugs were determined by multiplexing with CellEvent Green and
MitoTracker Orange and analyzing on the VarioskanTM LUX
microplate reader.
CyQUANTTM Direct Green CellEventTM Green
1 1 0 1 0 0 1 0 0 0
0
5
1 0
1 5
2 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
S p h e r o idT r e a t e d w i t h 1 x C Q D fo r 3 h r s
T r e a tm e n t w i t h d r u g f o r 4 8 h r s
G a m b o g ic A c id (u M )
Va
rio
sk
an
LU
X
Flo
ure
sc
en
ce
50
0/5
30
Hig
h C
on
ten
t CX
7
Flo
ure
sc
en
ce
50
0/5
30
L U X
C X 7
IC50
LUX
~ 16.75
CX7
19.76
1 1 0 1 0 0 1 0 0 0
0
5
1 0
1 5
2 0
0
5 0 0 0
1 0 0 0 0
1 5 0 0 0
2 0 0 0 0
S p h e r o idT r e a t e d w i t h 1 x C Q D fo r 3 h r s
T r e a tm e n t w i t h d r u g f o r 4 8 h r s
G a m b o g ic A c id (u M )
Va
rio
sk
an
LU
X
Flo
ure
sc
en
ce
50
0/5
30
Hig
h C
on
ten
t CX
7
Flo
ure
sc
en
ce
50
0/5
30
L U X
C X 7
IC50
LUX
~ 16.75
CX7
19.76
MitoTrackerTM Orange
0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0
0 .0
0 .2
0 .4
0 .6
0 .1 5
0 .2 0
0 .2 5
0 .3 0
0 .3 5
D r u g T r e a te d S p h e r o id s
C e ll E v e n t G re e n A n a ly s isT r e a t e d w i th d r u g 1 9 h o u r s a f te r p la t in g
D ru g (u M )
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
N ic lo s a m id e (u M )
G a m b o g ic A c id A m id e (u M )
G a m b o g ic A c id (u M )
N o c o d a z o le (u m )
0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0
4
5
6
7
8
0
2
4
6
8
D r u g T r e a te d S p h e r o id s
M ito T r a c k e r O r a n g eT r e a te d w i th d r u g 1 9 h o u r s a fte r p la t in g
D ru g (u M )
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
N ic lo s a m id e (u M )
G a m b o g ic A c id A m id e (u M )
G a m b o g ic A c id (u M )
N o c o d a z o le (u m )
0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0
4
5
6
7
8
0
2
4
6
8
D r u g T r e a te d S p h e r o id s
M ito T r a c k e r O r a n g eT r e a te d w i th d r u g 1 9 h o u r s a fte r p la t in g
D ru g (u M )
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
Va
rio
sk
an
LU
X
Flu
ore
sc
en
ce
50
0/5
30
nm
N ic lo s a m id e
G a m b o g ic A c id A m id e
G a m b o g ic A c id
N o c o d a z o le
1 1 0 1 0 0 1 0 0 0
0
2
4
6
0
2
4
6
8
1 0
O x y d a t iv e S tre s s & D N A c o n te n t
G a m b o g ic A c id (u M )
Ce
llR
OX
De
ep
Re
d (
64
4/6
65
nm
)
Ho
ec
hs
t (36
1/4
97
nm
)
0 1 2 3 4 5 6
0
5
1 0
1 5
2 0
0
1
2
3
4
5
F lu o r e s c e n c e B a s e d A s s a y s
5 k A 5 4 9 s p h e r o id s
T im e (h o u rs )
Cy
QU
AN
T D
ire
ct
Gre
en
(50
0/5
30
nm
)
Mit
oT
ra
ck
er O
ra
ng
e
(55
4/5
76
nm
)
2 6 0 G A u M -D ire c t
2 5 0 u M M ito O R
0 u M -M ito O R
0 G A u M -D ire c t
2 5 0 u M C e llR o x
0 u M C e llR O X
CyQUANT Direct Green + 250 µM GA
CyQUANT Direct Green (Untreated)
MitoTracker Orange + 250 µM GA
MitoTracker Orange (Untreated)
0 1 0 2 0 3 0 4 0 5 0 6 0
0
5 0 0
1 0 0 0
1 5 0 0
2 0 0 0
0
2
4
6
S o lu t io n B a s e d C e ll V ia b ility A s s a y s5 k A 5 4 9 s p h e r o id s
T im e (h o u rs )
Pre
sto
Blu
e
Flu
ore
sc
en
ce
56
0/5
90
nm
Cy
QU
AN
T X
TT
Ab
so
rb
an
ce
45
0-6
60
nm
PrestoBlue (Untreated)
PrestoBlue + 250 µM GA
CyQUANT XTT (Untreated)
CyQUANT XTT+ 250 µM GA
CyQUANTTM Direct Green detection reagent measures DNA content and
cytotoxicity of individual cells.
Solution-based cell viability assays provide information on entire cell
populations rather than tracking the behavior of individual cells.
After some initial optimization, both types of reagents (solution and individual
cell analysis) can be used to analyze cellular function and cellular viability on
a microplate reader.
CellInsightTM CX7VarioskanTM LUX
LUX
IC50 4.03
CX7
IC50 1.53
Multiple Modes of cellular function can
be monitored by microplate reader and
existing cell functional assays
CellROX
Hoescht
1X = monolayer working concentration
2X = two times monolayer working concentration
(A) (B) (C)
(A)(B) (C)
1324-E
LUX
IC50 22.08
CX7
IC50 18.57
Varioskan LUX analysis
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