Introduction 1 Based on assay development & validation, a pilot screen was

conducted from two biased priority compound libraries at 10

uM, (0.1% DMSO, 72h) to identify EMT modulators.

Figure-4: Image Analysis of Single cell versus Organoid ROI Comparison of single cell and organoid ROI level image analysis measurements

with compound AA-18-163 used during assay development to calculate the EC50

for each fluorescent bioprobe to determine which method suitable for screening.

Maximum intensity projection from a 11 image z-stack was captured with 10x

objective lens. EC50 intensity values of each probe were calculated and plotted

within Harmony software.

Epithelial-mesenchymal transition (EMT) is linked

to the pathology of cancer with overwhelming

evidence from literature associating EMT as a

driving force for tumor progression and metastasis.

Here, we describe the assay development and

validation of a live-cell 3D multicellular tumor

spheroid model for high-content screening (HCS)

using a dual reporter EMT biosensor probe. The

outcome of this dual reporter expression is

engineered to identify compounds that inhibit,

modulate, or reverse EMT in real-time.

Key Features

• Phenotypic-based assay design of live-cell 3D

colorectal organoid cell model to screen drugs or

compounds that inhibit, modulate, or reverse EMT

• Assay validated for primary HCS on Operetta CLS™

high-content imager by capturing z-stack images

• Screen pilot focused libraries of 2,550 compounds

Cell Model: Colorectal cancer cell line SW620 was transduced

with lentiviral vector reporter pCDH1-vimentin promoter-EGFP

and E-cadherin promoter-mCherry-EFI-puro and selected with

puromycin. Subpopulations of vimentin positive, E-cadherin

positive, and double positive cells were purified by FACS

sorting. Selected subpopulation for vimentin positive (EGFP+)

cells were used in this study.

Figure-3: Images of Reference Control Compound Challenge

Organoids following treatment with 0.1% DMSO, 5uM, and 20uM reference

compound AA-18-207. Images were captured using 11 image planes at 10

micron intervals with a 10x objective lens using confocal on the Operetta CLS;

image is displayed as maximum intensity projection..

Figure-7: DMSO Tolerance & Replicates

(A) DMSO concentrations up to 4% was determined for both E-cadherin-

mCherry and Vimentin-EGFP expression after 72 hour exposure to mimic

drug treatment. A DMSO of 0.1% was selected for compound screening.

(B) Replicate EC50 dose response of E-cadherin-mCherry intensity from

organoid ROI measurement using reference control compound AA-18-207

after 72 hour treatment. Experiment-1 EC50 was 13 µM and Experiment-2

EC50 was 11 µM.

Figure-10: Screening Results from 30 microplates uploaded into

SpotFire HCP for visualization (left) and predication of number of

hits that cluster with reference compounds Neo and AA-18-207 or

no response (DMSO) from multivariate PCA (right).

Screening and Results

Summary • EMT is a complex phenomenon in cancer that is linked as a

major driving force in tumor progression and metastasis.

Here we show a novel 3D phenotypic dual reporter tumor

organoid model of EMT that is suitable for HCS probe and

drug discovery.

• We demonstrate our model’s capacity to identify and validate

novel small molecules that modulate or reverse EMT in real

time over 3 days using 3D high-content analysis. Lead

compounds can now be used to better understand EMT

driven disease but also develop targeted therapies against

EMT.

• We established that the Operetta CLS imager equipped with

PreciScan improved the screening logistics and efficiency of

identifying organoids based on different xy coordinates using

a fully automated process of pre-scan and re-scan.

• We determined that single cell analysis was not required for

primary HCS but provided insights for confirming hits.

• The HCS assay development & validation results met the

recommended guidelines for screening compounds (1).

• SpotFire® High-Content Profile module provided tools to

easily navigate and visual screening hits; additional the

ability for higher level statistical analysis including

multivariate PCA of all well-level data was measured to rank

order HCS features.

Assay Development

5

6

3

3D Live Cell Organoid HCS Assay Development & Validation

O. Joseph Trask1, Qiong Zhou2, Linfeng Li2, Adedoyin D. Abraham2,

Kevin Quick1 and Daniel V. LaBarbera2*

1 PerkinElmer, Hopkinton, MA; 2University of Colorado AMC, Denver, CO

PerkinElmer, Inc., 940 Winter Street, Waltham, MA USA (800) 762-4000 or (+1) 203 925-4602 www.perkinelmer.com

VWR International, 1310 Goshen Parkway, West Chester, PA 19380 USA (800) 932-5000 www.vwr.com

Automation Image

Acquisition

Image

Analysis

Data

Analysis Cell Model

Microplate

Selection

CellCarrier Ultra Opera

Operetta™ Harmony® High-Content

Profiler™ Janus®

PreciScan: Re-Scan (10x) Re-positions ROI of object

based on XY coordinates

Sin

gle

Cel

l Hoechst EGFP mCherry DRAQ7

Org

an

oid

DMSO% vs Fluorescence Signals

0.00

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Mean EGFP Intensity

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Mean

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herry

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Figure-2: PreciScan™ Intelligent Acquisition.

LEFT: Pre-scan transmission light images captured with 5x

objective lens. Upper panel shows xy position in well; (A) 3 z-

slice images over 200µ ; (B) maximum intensity projection

collapsed images from 3 slices; (C) ROI mask overlay.

RIGHT: Re-Scan fluorescent images from 4 channels (Hoechst,

EGFP, mCherry, and DRAQ7) of organoids captured with 10x

objective lens. Images are pseudocolored.

(D) 11 z-slice image stack at 10µ intervals, totaling 100µ;

(E) pseudocolored maximum projection intensity image; (F) ROI

image analysis overlay mask

2 Experimental Design &

Procedure

Figure – 1: 3D HCS Work Flow Process

A B C D E F

z3

z2

z1

z5

z1

z11

PreciScan: Pre-Scan (5x)

Identify XY coordinates for

every organoid ROI

L o g A A -1 8 -2 0 7 (M )No

rma

liz

ed

mC

he

rry

In

ten

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(%,

no

rma

liz

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to

DM

SO

)

- 3 -2 -1 0 1 2

0

2 0 0

4 0 0

6 0 0

8 0 0 E x p # 1 : E C 5 0 = 1 3 M

E x p # 2 : E C 5 0 = 1 1 M

A B

Assay Validation 4

Figure-8: Z’-factor determination

Following a 72 hour treatment with respective reference control compounds

(A) Neo for Vimentin-EGFP, the Z’-factor was 0.52 and (B) AA-18-207 for E-

cadherin-mCherry was 0.62. Note, a ratiometric measurement of mCherry /

EGFP intensity provided a Z-factor of 0.55, graph not shown.

Figure-9: Example of Compound Selectivity / Specificity

(A) The size of the organoid ROI area (µm2) decreased when E-cadherin-

mCherry expression increased using a known selective a compound that targets

E-cadherin. (B) Compounds that did not alter E-cadherin expression showed

no evidence in changes to organoid ROI area. Plotted in Harmony.

0

1000

2000

3000

4000

Mean E

GF

P Inte

nsity average Z' from 30 plates = 0.53

DMSO Neo

0

5000

10000

15000

Mean m

Cherr

y Inte

nsity average Z' from 30 plates = 0.62

DMSO AA-18-207

A B

The following conditions were determine for screening:

• DMSO tolerance

• Dose response replicates

• Z-factor for selected HCI features

• Selective / Specificity of test compounds

• Pilot screen to determine screening conditions & outcome

Cell Seeding & Compound Treatment

• Pre-coat microplates (CellCarrier Ultra) with 0.75% agarose,

incubate at RT.

• Seed 20,000 cells/well in 96w or 5,000 cells/well in 384w

• Incubate microplates for 3 days with 2% Matrigel overlay.

• Using the JANUS to add 10µM of compound. Reference

controls compounds: AA-18-207 (upregulate E-cadherin) &

Neoamphimedine (down regulate Vimentin).

• Incubate compound for 72 hrs, 37oC, 5% CO2

• Label cells with 20 µM DRAQ7™ (BioStatus, LTD) and 27

µM of Hoechst 33342 for 1 hour, 37oC, 5% CO2.

• Wash with warmed media 1x, then fill wells with media.

Intelligent Image Acquisition: PreciScan™ (Harmony >v.4.5)

• Using the PerkinElmer Operetta CLS imager with

environmental control, pre-warm & stabilize to 37oC,5% CO2.

• Live organoids images were initially acquired with a 5x

objective lens from 3 z-slice image stacks (200 µ) using

transmission light.at 10% power, 10ms exposure times.

• Simultaneously, image analysis was performed to locate the

center of gravity XY coordinate positions of each organoid

detected and a region of interest (ROI) mask was generated to

segment organoids from debris or artifacts (see figure-2).

• PreciScan analysis of the organoid ROI was performed with

off-set XY coordinates to re-center the image re-scan for

fluorescent acquisition of 11 z-stack image slices at 20 µ

intervals, 100 ms exposure time. (see figure-2).

• Subsequent machine learning (Phenologics™) image analysis

was done on-the-fly using either the 5x, 10x, or 20x objective

lenses during development to determine size, shape, texture,

and intensity per cell object and per organoid ROI.

A

Figure-6: Pilot of PCA Multivariate Analysis of HCI Features More than 50 well level HCI features were processed in TIBO® SpotFire®

Analyst High-Content Profiler to determine the top HCS features for

multivariate analysis from all well-level features in preliminary study. First 3

PCA components were plotted.

Evaluate optimal settings for image acquisition (magnification,

confocality, number of z-stack image slices, exposure times); and

compare image analysis of single cell and whole organoid ROI

using known reference compounds to perturb EMT biosensor

reporter. Determine HCI feature list and if amenable for screening.

Magnification 1.25x

Widefield

5x

Widefield

10x Widefield

10x

Confocal

20x/W

Confocal

Field Number 1 1 1 1 4 - 6

3D Planes ND 11 11 11 41

Acquisition Time ND 31 min 24 min 30 min ND

Channels 1 4 4 4 5

Nuclei Detection - - -/+ ++ +++

*

* The 5x pre-scan time was ~2 minutes / 96w plate using transmission light. The 1.25x &

20x/W lenses were evaluated but not used for assay validation; the number of fields to

capture an organoid varied from 4-6 field frames / well with the 20x/W objective lens.

* *

*

References

Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK53196/

Acknowledgments This research was funded in part by grants from the CU Cancer Center, The State of Colorado, and the Department of Defense PRCRP (W81XWH-13-1-0344) awarded to Dr. LaBarbera.

Roy Edward, BioStatus Ltd for supplying DRAQ7™

Figure-5: Time Course Kinetics

Organoids (21 z-stack at 10x) acquired every 4 hours over 48 hours following

treatment with 4uM Neo, 10uM AA-18-207, or nothing. Vimentin-EGFP

intensity (left) and E-Cadherin-mCherry intensity (right).

DMSO

AA-18-207

E-Cad

Neo

Vimentin

Figure-11: Venn diagram of pilot screening results. 94 active

compound hits down-regulated Vimentin (V) expression; 39

active hits up-regulated E-cadherin (E) expression and 3

compounds reversed EMT (both down regulation of Vimentin

and up-regulation of E-cadherin.

B

V V E E 94 hits 39 hits 3 hits

* Correspondence : daniel.labarbera@ucdenver.edu

*

DMSO% vs Fluorescence Signals

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EC50 =46.6

EC50=42.2

EC50=9.2

EC50=9.9

EC50=7.5 EC50=6.0

EC50=5.4 EC50=5.0

joe.trask@perkinelmer.com

For research use only. Not for use in diagnostic procedures.