Technologies & Innovation
for Phenotypic Screening
Simple Readouts for Complex Biology
Christian N. Parker
Svenja Zietzling, Heinz Ruffner and Olaf Galuba
Overview – 25 minutes
 A new grammar for drug discovery.
 Description of crypt biology
 Example of using this as a phenotypic readout
• Mucositis
 Challenges to screening
 Future perspectives
A New Grammar of Drug Discovery
By identifying key nodes in
biological systems it allows
1) Better description of the
disease.
2) Allows common nodes to
be identified allowing
treatments to be targeted to
the mechanism
3) A rational means to apply
compounds targeting a single
node to multiple diseases.
Cell based screening:
Identifying the syntax of biology
Cyclopia
Cyclopamine
Vismodegib
Birth defects found
in cattle feeding
on wild corn lily,
first reported in
1957
Identified from wild
corn lily after an
11 year
investigation by
USDA
Approved 30th Jan
2012 for treatment
of basal-cell
carcinoma
http://en.wikipedia.org/wiki/Cyclopamine
Epithelial Intestinal Regeneration
The intestinal stem cell niche
5
Small Intestinal Organoid System
Primary cells, stem-niche visualization, growth over-time
LGR5-GFP cell
Stem cell
Gut lumen
Organoid
lumen
Niche
Paneth cell
Villus domain
Crypt domain
Villus
•
•
•
•
6
Matrigel, EGF, Noggin, RSPO1
Primary cell culture, indefinite growth
All cell types present
Physiology maintained
Tracking growth of a single cell
Shows regeneration over 27 days
day2
day15
day3
day5
day17
day7
day20
day9
day13
day22
day27
7
Tracking growth of a single cell
Shows how failure to close crypt leads to death
day 2
day 17
day 5
day 7
day 20
day 9
day 13
day 22
day 27
8
Summary of crypt cultures
Biological features
Primary cells
Untransformed
Not immortalized
Continuous culture
Requires correct structure to continue growth
9
Radiation-Sensitized Organoid Proliferation Assay
Identification of protective/toxic agents
10
Regenerative Process
Damage response in small intestine following radiation
Correction
& Remodel
• Drug discovery
Differentiated
cells
Repair complete
Proliferation
Overshoot
• Understand biology
Initial death
Intestinal stem cell number
Small intestine crypt stem
cell damage response
Recapitulate small intestine
damage response in a
physiological ex vivo setting
Damage response (hours)
Stem cells
11
Correlation to in vivo effect
Rspo and been show to have positive effects
12
Epithelial Regeneration: Drug Discovery
RSPO is efficacious in preclinical mouse models of mucositis
Radiation-induced mucositis
Chemotherapy-induced mucositis
Day -3 -2
-1
0
1
2
3
4
Palifermin
5-FU
Day
-3
-2
-1
0
1
2
3
RSPO1
4
Dissections
60
Ileum (# BrdU crypts/section)
50
40
30
20
10
Vehicle
RSPO1 / Vehicle treatment i.v.
Lab Hintzen
13
RSPO1
0
5-FU
RSPO1
Palifermin
-
+
-
+
+
-
+
-
+
+
Lab Smith
Intestinal Mucositis – RSpondin effect
 Treatment: recombinant hu RSpo 1, 10mg/kg i.v.
 Irradiation: 9Gy, whole body
Vehicle i.v.
R Spondin1 i.v.
 The effect is strong, visible and measurable
 Irradiation and timing are optimized for intestinal damage
Lab Hintzen
14
Screening with crypt
Practical aspects : Isolation and preparation
15
Summary of crypt cultures
Biological features : in vitro steps
16 | Presentation Title | Presenter Name | Date | Subject | Business Use Only
Handling crypt cultures
Automation challenges remain
17 | Presentation Title | Presenter Name | Date | Subject | Business Use Only
Expected results
Fluorescence
simple readout for complex biology
• Translation model
• Develop medium throughput
assay
• Assess effect of compounds
on organoid growth under
patho-physiological conditions
Viability
Fluorescence
• Robust assay in 384-well
format
Active cpd
Negative cpd
Cpd concentration
18
Screening results
Plate to plate variability
Activity-Sample Normalized
(DMSO =100%) Log Scale
Controls
Compounds-arrayed by number
19
Organoid Proliferation/Viability
Assessment of toxic agents in organoid cultures
 Cpd library screens
• Medium throughput
Proliferation (early passage organoids)
• Single concentration, 1 time-point measurement
Increase in viability
Decrease in viability
20
Proliferation (late passage organoids)
Complex culture compared to standard 2D culture
Differences in sensitivity to GR agonists
Proliferation (early passage organoids)
DMSO
Growth inhibition Cell line
1
2
3
Compound 1
-5
-6
-1
Compound 2
9
-5
0.5
Compound 3
2
0
1
DMSO
21 | Eurotox 2013 | Heinz Ruffner | Sept 2, 2013 | Stem cells: Discovery, Toxicology | Business Use Only
Proliferation (late passage organoids)
Dose response of hit from screen
Differences in sensitivity to GR agonists
Normalized Activity
Repeat 1
Repeat 2
Compound Concentration
22
Perspectives
 Improvements to plating consistency
 Improvements to activity normalization
 Additional readouts
• Morphology
• Activity
 Marked Stem cells
 Markers of differentiation
23
Organoid Morphology
Different treatments generate different crypt morphology
Ruffner, 2012
24 | Eurotox 2013 | Heinz Ruffner | Sept 2, 2013 | Stem cells: Discovery, Toxicology | Business Use Only
Assay concept
Correlation between Readouts
Assay concept
4500
R² = 0.8336
4000
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0
-100000
-500
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25 | Presentation Title | Presenter Name | Date | Subject | Business Use Only
1. Treat crypts
2. Monitor Alamar blue
(reports on # of cells)
3. Monitor Luciferase
(reports on # of SC)
4. Look for:
a. Increase in cells
b. Increase ratio of
SCs
Mouse Small Intestine Crypt Organoid Cultures
A physiological complex system: Target ID & validation, toxicology
26
Acknowledgements
Fred Bassilana
Tewis Bouwmeester
Feng Cong
Sumon Datta
Lucy Kirchhofer
Susan Kirkland
Juliet Leighton-Davies
Gabriele Hintzen
Bernd Kinzel
27
Monika Pikiolek
Adrian Salathe
Joëlle Sprunger
Jan Tchorz
Elizabeth Wiellette
Natasa Zamurovic
LGR5-IRES-Luc Readouts
average AlamarBlue
500000
400000
300000
200000
100000
mockKGFCGP060106 ABG424
BBD175
BBK291
CHX298
LJI197
LKW002
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
250ng/ml
-100000
-
0
LMZ740
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mockKGFCGP060106
ABG424
BBD175
BBK291
CHX298
LJI197
LKW002
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
10µM
5µM
1µM
250ng/ml
4500
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0
-500
-
average SteadyGlo
LMZ740