11 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer

Quantitative Analysis of 3D Microtissue Growth & Biomarker Intensity

Jacop G. Tesdorpf, Ph.D.

Director of High Content Instruments,

PerkinElmer

22

Strategic Goal

COST OF TESTING

DA

TA Q

UA

LIT

Y

Small Animal

Cell

DNA, RNA, Protein

Human

Tissue

Revolutionary Platforms Building The In Vitro To In Vivo To Human Bridge

Maestro, Vectra systems

IVIS & FMT systems

LabChip technology

Zephyr & JANUS

Automation

EnVision multilabel

detection

Opera, & Operetta HCS systems

EnSpire multimode detection

Automation, cell plate

Instruments

Reagents

& Services

NGS

Fluorescent agents

SeeGene

GPCRs

Labeled Cells & Markers

Rads, Alpha, LANCE, DELFIA Assays

Labels & Markers,

Fluorescence, Bioluminescence

AlphaLISA, Rads, LANCE, DELFIA reagents

Labels & Markers,

TSA reagents,

Tissue services

InformaticsColumbus data storage & analysis

Volocity 3D image analysis

TrueQuant fluorescent imaging

Geospiza LIMS sample tracking

Cambridgesoft eNotebooks

Pre

clin

ical

Clin

ical

In vitro In vivo

33

Strategic Goal

COST OF TESTING

DA

TA Q

UA

LIT

Y

Moving Assays Towards Higher Translational Relevance…

Pre

clin

ical

Clin

ical

In vitro In vivo

Human

DNA, RNA, Protein

Cell

Small

Animal

Tissue

More relevant disease models• Co-cultures

• 3D cultures & tissue

• Stem derived cell lines• Primary cells, personalized screens

• Toxicity testing

More relevant readouts• Phenotypic information

• Dynamic behaviour

• Spatial information• Network relations

3D Cell Culture,

Microtissues

...Requires Live Cells & 3D

44

Integration of in vitro & in vivo agents

Integrin αvβ3/αvβ5: IntegriSense 645

Furthering biomarker & pathway studies

Fluorescent Agents for Disease Imaging

In vivo & In vitro agents

Translational fluorescence markers

Biological specificity

Activatable

Targeted

Vascular

Red & NIR wavelengths

Quantitative imaging

A. Fluorescence microscopy showing 4T-1 tumor cells labeled with IntegriSense 645

B. Reflectance imaging of murine breast adenocarcinoma4T-1 tumors implanted in nude mice

C. Tomographic imaging of murine breast adenocarcinoma4T-1 tumors implanted in nude mice

A B C

55

Arthritis

Integrin αvβ3 Inflammation

Renin

Oncology

Bone Biology

Hypertension

Cardiovascular

Gastric emptying

Cathepsin K

Hydroxyapatite

Vascular

Neutrophil Elastase

Physiologic

Infectious disease Cathepsins B, L, S

MMPs 2, 7, 9, 13

Fluorescent Agents for Disease Imaging

Furthering Biomarker & Pathway Studies

Hypoxia (Carbonic Anhydrase IX )

Bacteria

Folate

Her2/Neu

Annexin

66

450 500 550 600 650 700 750 800 850 900

Ab

sorb

ed li

gh

t

Wavelength [nm]

HB

HBO2

Tissue

Versatile in vivo dyes with high quality in vitro imaging capabilities

Fluorescent Near Infrared (NIR) Agents for Translational Research

Low absorption/scattering of NIR light in tissue enables in vivo imaging

NIR agents allow translational use from biochemistry to cellular assays to in vivo testing

Comparable biologic interactions by applying in vivo agents in vitro

VivoTag 680, …

GFP Alexa 633 ®

Tamra

Ovalbumin-induced Asthma Model; Imaged with FMT

2500 + MMPSense 680 ®

In Vivo ImagingMicroscopy

77

Fluorescent Near Infrared Agents: Robust Biomarker Readouts

Agent Categories

Vascular Targeted ActivatableD

etec

tio

n Physiology

Biological

Structure/Expression

Biological

Function

Increasing value of functional information

Val

ue

Mec

han

ism

• Vascularity, blood

pooling or leakage

• Protein distribution or

expression levels

• FL upon protease

specific cleavage

88

Arthritis

Integrin αvβ3 Inflammation

Renin

Oncology

Bone Biology

Hypertension

Cardiovascular

Gastric emptying

Cathepsin K

Hydroxyapatite

Vascular

Neutrophil Elastase

Physiologic

Infectious disease Cathepsins B, L, S

MMPs 2, 7, 9, 13

Fluorescent Agents for Disease Imaging

Furthering biomarker & pathway studies

Hypoxia (Carbonic Anhydrase IX )

Bacteria

Folate

Her2/Neu

Annexin

ProSense

99 The First Step To Identifying A Truly Translational Agent Platform

Biochemical Cathepsin Activity Assays

0

200

400

600

800

1000

1200

1400

Cat

hep

sin

BC

ath

epsi

nL

Cat

hep

sin

SC

ath

epsi

nK

Leg

um

ain

Cat

hep

sin

D MM

P-2

MM

P-9

MM

P-1

2

MM

P-1

3

Flu

ore

scen

ce S

ign

al (

RF

U)

ProSense 680

ProSense 750

0

200

400

600

800

1000

1200

1400

1600

1800

0 6 12 18 24

Flu

ore

scen

ce S

ign

al (

RF

U)

Time (h)

Cathepsin BCathepsin LCathepsin SCathepsin K

In vitro biochemical assays for disease associated proteases shows ProSense is an

pan-cathepsin activatible agent

Inhibitor studies possible as well

Cysteine Cathepsins

1010

Operetta High Content Analysis System

HCA for everylab

High image quality and confocal option

Large field of view

Automated workflow for unbiased results

Ideal for multi-user and multi-department

environment

Designed for flexibility and ease for all

applications

Standard

Operetta

1111

Harmony workflow oriented software

1212

HT-29 colon adenocarcinoma cells

Cell toxicity assay using 5-FU treatment on the Operetta

5-FU induces apoptosis & cell death

Monitoring Viable & Non-viable Cells

Hoechst labels all cells equally

Yo-Pro is specific to apoptotic cells

Bobo labels dead cells only

Moving towards more relevant, complex disease models...

Cathepsin Biomarker Visualization In Cancer Cells

40 µM

20 µm

80 µM 160 µM 5-FU

Hoechst, Yo-Pro-1, Bobo-3Hoechst, Yo-Pro-1, Bobo-3 Hoechst, Yo-Pro-1, Bobo-3

No 5-FU

ProSense 680 visualizes cathepsin activity in cellular lysosomes

20 µm Operetta, 40x LWD

ProSense 680HT-29 colon carcinoma cells

1313

Cathepsin Biomarker Visualization In Cancer Cells

ProSense Provides Information On Cancer Biomarker Expression & The Cell Metabolism

20 µm

ProSense 680 is activated in living & early apoptotic cancer cells

Cathepsin activity declines in 5-FU induced late apoptosis

ProSense allows discrimination between early & late apoptotic cells

72 h of 5-FU treatment [80 µM]

Viable[Hoechst positive +

ProSense positive]

Early apoptotic[Yo-Pro-1 positive +ProSense positive]

Late apoptotic[Yo-Pro-1 positive +

ProSense negative]

Dead[Bobo-3 positive + ProSense negative]

1414 Microtissues Become Available For High Throughput Applications

Towards More Relevant Disease Models: 3D Microtissues

Scaffold-free spheroid production in hanging drops

Cells produce endogenous extracellular matrix

Cell-cell & cell-ECM interactions provide native environment

More physiologic conditions compared to 2D cell culture

Seeding MT formation 2-4 days

Transfer Assay ready

Data courtesy insphero

1515

Live HT-29 cancer microtissues were treated with 5-Fluorouracil (5-FU) or Staurosporine

Microtissue growth was analyzed 72 h after treatment

Dose-dependent inhibition of microtissue growth

Microtissues As 3D Cell Model For Drug Efficacy Testing

Response Of 3D Microtissues To Drug Treatment

100 µm

A

B

C

A

B

C

Operetta, non confocal, Brightfield, 10x hNA

1616

Arthritis

Integrin αvβ3 Inflammation

Renin

Oncology

Bone Biology

Hypertension

Cardiovascular

Gastric emptying

Cathepsin K

Hydroxyapatite

Vascular

Neutrophil Elastase

Physiologic

Infectious disease Cathepsins B, L, S

MMPs 2, 7, 9, 13

Agent Portfolio: Rapidly Expanding Across Key Biomarkers

Hypoxia (Carbonic Anhydrase IX )

Bacteria

Folate

Her2/Neu

Annexin

Tumor signaling & growth

Tumor migration & Metastasis

1717 3D Microtissues Resemble In Vivo Solid Tumors

Cancer Biomarker Visualization In 3D Microtissues

Characteristic labeling phenotypes observed in in vivo solid tumors

ProSense 680 shows a homogeneous staining comparable to mouse model

MMPSense 680 is mainly activated in isolated cells that may have escaped tumor

HypoxiSense 680 signal shows strongest hypoxic conditions in the core region of microtissues

100µ m

ProSense 680 MMPSense 680 HypoxiSense 680

Fluorescence +

brightfield

image

Fluorescence

image

Operetta, confocal, 10x hNA

1818

Hypoxisense 680 Intensity Is Size-dependant

100µm

Max core intensity: 3955 Max core intensity: 6348

Microtissues were grown using different cell seeding densities

HypoxiSense 680 intensity is size dependent

Quantification of HypoxiSense staining suggests small hypoxic areas in the core regions of microtissues

1919

Cathepsin Biomarker Visualization In Vivo

0

20

40

60

80

100

120

140

160

180

200

0 2 4 6 8 10 12 14 16

Calc

ula

ted

Tu

mo

r V

olu

me

(u

l)

Time (days)

Vernier Caliper Assessment

Control

5-FU treated

(47% decrease)

0

50

100

150

200

250

300

350

400

450

500

Control 5-FU

Pro

Se

ns

e F

luo

res

ce

nc

e V

olu

me

(m

m3)

Fluorescence Volume

*(46% inhibition)

0

10

20

30

40

50

60

70

80

Control 5-FU

Pro

Se

ns

e F

luo

res

ce

nce

(p

mo

l)

Total Fluorescence

#(53% inhibition)

Control 5-FU Control 5-FU

Mice bearing HT-29 tumor xenografts

ProSense 680 allows to monitor tumor progression

5-FU treatment significantly impacts tumor size & cathepsin biomarker activity

Epifluorescence FMT 2500 TomographyProSense680

2020

Biochemical protease assays

…Enabled By PerkinElmer Technology

Assays Of Increasing Biological Complexity & Relevance…

0

200

400

600

800

1000

1200

1400

Cat

he

…

Cat

he

…

Cat

he

…

Cat

he

…

Le

gu

…

Cat

he

…

MM

P-2

MM

P-9

MM

P-1

2

MM

P-1

3

Flu

ore

sc

en

ce

Sig

na

l (R

FU

)

ProSense…ProSense…

2D Cellular Assays

Max core intensity: 6348

3D Cellular Assays

In vivo Assays

FMT® in vivo Imaging

System

Operetta® High

Content Imaging

System

Volocity 3D Image

Analysis Software Microplates

Fluorescent

Reagents, in vitro

Operetta® High

Content Imaging

System

Fluorescent

Reagents, in vivo

Opera® High Content

Screening System

EnSpire® MultiMode

Plate Reader

UltraVIEW® VoX 3D

Live Cell Imaging System

2121

Thank You For Your Attention!

Thanks To:

Dr. Karin BoettcherThe Imaging Apps Team

Insphero AG

2222

Opera & Operetta: What‘s New?

2323

Harmony 3.1 Preview

STAR morphology properties Linear Classifier with more than 2

parameters

A more complete, statistically robust set of properties

for comprehensively describing cell morphology, distribution of intensity within regions, ...

For higher quality assays.

Symmetry

Treshold compactness

Axial

RadialProfile

Create statistically relevant cell classifications

by using more than 2 parameters.

2424

Opera 2.0 Upgrade

Digital Phase Contrast

� Brightfield LED

� Digital phase images

allow segmentation

� Label-free cell detection

New User Interface

� Columbus User Interface

� No scripting required

� Building blocks

� PhenoLOGIC machinelearning

4x Air Objective

� Low magnification

� Whole well imaging

� Rare events

� Rapid imaging

New Computing Hardware

� Better computingperformance

� Deal with larger amountof data

� Windows 7, 64 bit

� Acquire unlimited

number of fields