Document

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

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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

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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

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Summary of crypt cultures Biological features

Primary cells Untransformed Not immortalized Continuous culture Requires correct structure to continue growth

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Radiation-Sensitized Organoid Proliferation Assay Identification of protective/toxic agents

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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

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Epithelial Regeneration: Drug Discovery RSPO is efficacious in preclinical mouse models of mucositis Radiation-induced mucositis

Chemotherapy-induced mucositis Day -3 -2

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Palifermin 5-FU Day

-3

-2

-1

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1

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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

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+ -

+ + -

+ -

+ +

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

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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

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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 3500 3000 2500 2000 1500 1000 500 0 -100000

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0

100000

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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

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Acknowledgements

Fred Bassilana Tewis Bouwmeester Feng Cong Sumon Datta Lucy Kirchhofer Susan Kirkland Juliet Leighton-Davies Gabriele Hintzen Bernd Kinzel

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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 4000 3500 3000 2500 2000 1500 1000 500 0 -500

-

average SteadyGlo

LMZ740