1 1 © 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
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