Poster: New and improved cellular health evaluation of 2D...

Thermo Fisher Scientific • 29851 Willow Creek Road• Eugene, OR 97402 • thermofisher.comFor Research Use Only

0 .1 1 1 0 1 0 0

1 0 0 0

1 5 0 0

2 0 0 0

N ic lo s a m id e (u M )

1 1 0 1 0 0 1 0 0 0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

G a m b o g ic A c id (u M )

1 1 0 1 0 0 1 0 0 0

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

4 hour XTT

27hr 47hr 51hr

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

S p h e ro id

4 hour XTT

27hr 47hr 51hr

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

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

2 -3 h o u rs

1 9 h o u rs

2-3 hours

19 hours

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

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

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

2 hours GA

24 hours GA

48 hours

72 hours

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

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

2 -3 h o u rs

1 9 h o u rs

2-3 hours

19 hours

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

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

~ 16.75

IC50 16.75

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

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 )

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

PrestoBlue HS

PrestoBlue

alamarBlue HS

alamarBlue

1 1 0 1 0 0 1 0 0 0

P re s to B lu e

a la m a rB lu e

PrestoBlue HS

PrestoBlue

alamarBlue HS

alamarBlue

Resazurin-based Reagents

T im e

P re s to B lu e 1 .0

C e llT ite r -B lu e

10 60 120

Time (minutes)

T im e

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

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 )

1 1 0 1 0 0 1 0 0 0

P re s to B lu e

a la m a rB lu e

PrestoBlue HS

PrestoBlue

alamarBlue HS

alamarBlue

CyQUANT XTT

incubation time

Concentration

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

S p h e ro id

4 hour XTT

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

S p h e ro id

4 hour XTT

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

S p h e ro id

4 hour XTT

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

S p h e ro id

4 hour XTT

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

5 0 0 0

1 0 0 0 0

1 5 0 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 )

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

5 0 0 0

1 0 0 0 0

1 5 0 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 )

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

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

~ 16.75

1 1 0 1 0 0 1 0 0 0

5 0 0 0

1 0 0 0 0

1 5 0 0 0

2 0 0 0 0

~ 16.75

MitoTrackerTM Orange

0 .0 1 0 .1 1 1 0 1 0 0 1 0 0 0

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 )

G a m b o g ic A c id A m id e (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

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 )

G a m b o g ic A c id A m id e (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

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 )

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

O x y d a t iv e S tre s s & D N A c o n te n t

0 1 2 3 4 5 6

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 )

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

1 0 0 0

1 5 0 0

2 0 0 0

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 )

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

IC50 4.03

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

IC50 22.08

IC50 18.57

Varioskan LUX analysis

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Scientific and its subsidiaries unless otherwise specified

Poster: New and improved cellular health evaluation of 2D...

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