3d live cell organoid hcs assay development & validation · 2018-03-19 · 3d live cell...
TRANSCRIPT
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
0.25
0.50
1.00
2.00
4.00
0
1000
2000
3000
0
200
400
600
800
1000
Mean
EG
FP
In
ten
sit
y
(backg
rou
nd
su
btr
acte
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DMSO (%)
Mean EGFP Intensity
Mean mCherry Intensity
Mean
mC
herry
Inte
nsity
(b
ackg
rou
nd
su
btra
cte
d)
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
sit
y
(%,
no
rma
liz
ed
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 : [email protected]
*
DMSO% vs Fluorescence Signals
0.00
0.25
0.50
1.00
2.00
4.00
0
1000
2000
3000
0
200
400
600
800
1000
Mean
EG
FP
In
ten
sit
y
(backg
rou
nd
su
btr
acte
d)
DMSO (%)
Mean EGFP Intensity
Mean mCherry Intensity
Mean
mC
herry
Inte
nsity
(b
ackg
rou
nd
su
btra
cte
d)
DMSO% vs Fluorescence Signals
0.00
0.25
0.50
1.00
2.00
4.00
0
1000
2000
3000
0
200
400
600
800
1000
Mean
EG
FP
In
ten
sit
y
(backg
rou
nd
su
btr
acte
d)
DMSO (%)
Mean EGFP Intensity
Mean mCherry Intensity
Mean
mC
herry
Inte
nsity
(b
ackg
rou
nd
su
btra
cte
d)
EC50 =46.6
EC50=42.2
EC50=9.2
EC50=9.9
EC50=7.5 EC50=6.0
EC50=5.4 EC50=5.0
For research use only. Not for use in diagnostic procedures.