2
Lack of efficacy and toxicity are still
the main reason’s of high attrition rates
Kola I. and Landis J.; 2004; Nature review drug discovery
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
3
'Why 3D?' Such sessions are a thing of the past now
because there is an accumulating body of evidence -
joined by the recent article from Leslie and colleagues -
demonstrating the importance and utility of 3D culture
systems to discover and model biological process with in
vivo relevance.
Editorial “3D culture reveals a signaling
network”
Senthil K Muthuswamy
Breast Cancer Research, 2011
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
4
Better in vitro Biology
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Drug-Target
interactions
Drug – Cell
response
Drug – Tissue
response
Drug – Organism
response
High failure rate
Reducing failure rate
5
Advancing cell-based assays
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Resembling native-like cell functionality to
improve predictive power of cell-based assays.
6
3D Microtissue culture in
hanging drops
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Seeding
Medium
After 1 hour
Cells
After 2-4 days Tumor tissue
Medium/air interface
8
Size Reproducibility
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
HCT116-colon
cancer MTs
9
Microtissue analysis in specific
spheroid assay plates (GravityTRAPTM)
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
10
Microtissue Models:
From Discovery to Safety
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Tumor
Kidney
Liver Brain
Heart
Islets Vasculature
Bone
Skin
Cartilage
Epithelial
Barrier
Ganglia
12
Spheroid cell heterogenity
Adapted from Friedrich J. Int J Radiat Biol. 2007 Nov-Dec;83(11-12):849-71.
secondary
necrosis
Spheroid model Monolayer
Homogenous cell population
No diffusion of substrates required
Low to now production of ECM
Heterogeneous cell population
Diffusion required
High levels of ECM
perfused
capillary
secondary
necrosis
Native tumor
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
13
Tumor spheroid expression profile
closer to in vivo
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
N. A. Cody et. Al., BMC Med Genomics. 2008
•
•
•
•
•
L LSC
LIP
TSC
S
TIP
OV90
14
A 3D Filter can significantly reduce
the number of candidates Screening for apoptosis inducer
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Fayad et al. 2011
10’000 cpds
382 cpds
2D screen 25 uM, 72h
40 cpds
3D screen 25 uM, 72h
11 cpds
3D screen 12.5 uM, 72h
HCT116, colon cancer
15 www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Example of a 3D filter Screening for an apoptosis inducer
Fayad et al. 2011
16
Cell Viability
CellTiter-Glo*
Cell Death
CytoTox-ONE*
Apoptosis
Caspase-3/7 Glo*
Redox State
GSH/GSSG-Glo*
Metabolism
P450-Glo Luciferin-IPA*
Biochemical assays evaluated with
InSphero’s microtissues:
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Lytic Assays Non-Lytic
Assays
Cell Death/Growth
NanoLuc
17
The holy grail of lytic assays for
microtissues: Tissue Disruption
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Buffer 1 Buffer 2 Buffer 3 Buffer 4 Buffer 5 PBS
Flu
ore
sce
nce
act
ivit
y (
RF
U)
human liver MTs
rat liver MTs
18
Cell number assessment in
microtissues
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
0
2000
4000
6000
8000
10000
12000
1000 2000 4000 6000 8000
Ce
ll N
um
be
r
Size MT (cells)
Theoretical Cell Number
Cell number determined by picogreen
0
5000
10000
15000
0 4000 8000 12000
RF
U
Cell number single cells
Calibration curve with single cell
supspension (input cell
population)
y = 1.6265x R² = 0.9686
Assay: PicoGreen
Model: NIH3T3
19
Cell viability: ATP
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Model: SNB-19 human glioblastoma cells
Monolayer
• Varied cell number at inoculation
• Day 4 MT transferred for study
CellTiter-Glo protocol optimization
• 3X pipet mix at CTG Reagent add’n
• 20 min incubation prior to read
Spheroid
20
Caspase 3/7: Critical Time Window
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
6 hrs 24 hrs 72 hrs
IC50: 0.31uM
21
Apoptosis: Caspase 3/7 acitivty
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
4h 24h 48h
RL
U/m
icro
tiss
ue
Staurosporin treatment
Caspase 3/7 activity
Control
10 microM
Model: 3D Insight rat liver microtissue
DISCRIMINATING DRUG EFFECTS IN MULTI-
CELL TYPE MICROTISSUES
Drug discovery
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
In collaboration with:
Pomega Ltd., Madison US and Dübendorf CH
Sirion Biotech GmbH, Munich
Tecan Austria GmbH, Grödig
23
Colon cancer co-culture
NIH3T3-tGFP : HCT116-NanoLuc
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
HCT-116:
NIH3T3
HCT-116
HE EGFR
A B C
D E F
24
Monitoring distinct cell populations
in a co-culture model
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
0
100
200
300
400
500
0
250
500
750
0 5 10 15
RF
P-s
ign
al [
RL
U]
Dia
mte
r [µ
m]
Time [days]
NIH3T3RFP
Diamter [µm]
RFP signal
0
20
40
60
80
100
0
250
500
750
0 2 4 6 8
Lu
cife
rase
[R
LU
x10
5 ]
Dia
mte
r [µ
m]
Time [days]
HCT116Luc
Diamter [µm]]
Luciferase
0
10
20
30
40
50
0
250
500
750
0 2 4 6 8
Lu
cife
rase
[R
LU
x10
5 ]
Dia
mte
r [µ
m]
Time [days]
NIH3T3RFP:HCT116Luc
Diamter [µm]
Luciferase
0
100
200
300
400
500
0
250
500
750
0 2 4 6 8
RF
P-s
ign
al [
RL
U]
Dia
mte
r [µ
m]
Time [days]
NIH3T3RFP:HCT116Luc
Diamter [µm]
RFP-signal
25
IC50 determination in co-culture
systems
Com
poun
d
ATP
[µM]
nano
Luc
[µM]
RFP
[µM]
STS 1,25 1,23 n.d.
CIS >500 >500 n.d.
TAX 1,08 0,50 n.d.
DMSO 0,01 uM 0,2 uM 2,5 uM
TAX
CIS
DMSO 15,6 uM 62,5 uM 250 uM
DMSO 0,16 uM 0,63 uM 2,5 uM
STS
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
LIVER MICROTISSUES
Pre-clinical safety
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
27
3D InSightTM rat liver microtissues
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
In collaboration with Jan
Hengstler and Seddik Hammad
28
3D InSightTM human liver microtissues
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Cryopreserved
Hepatocytes
Cryopreserved
NPCs
Production in
GravityPLUS
Assaying in
GTRAP
29
Acetominophen-induced liver
microtissue toxicity
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
30
Metabolic activity of human liver
microtissues
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
0
5000
10000
15000
20000
25000
30000
35000
40000
hLiMT - NPC hLiMT + NPC
RL
U
CYP3A4-induction DMSO control
20 uM Rif, 48h
31
Correct Prediction of Toxicity
Where Classical Methods Fail
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Endpoint: ATP content
INFLAMMATION-MEDIATED LIVER TOXICITY
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Pre-clinical safety
Kupffer-Macrophages are
incorporated in rLiMTs
Nuclei :405 Dapi
Kupffer macrophages: rat CD68
Bile canaliculie: DPPIV
In collaboration with Marianne Uteng, Pierre Moulin
and Francois Pognan, Novartis Pharma Basel
0.00
50.00
100.00
150.00
200.00
250.00
300.00
IL-6
(p
g/m
l)
no LPS
10 ug/ml LPS
Pure
Heps
Heps + Kupffer
Cells
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
34
Morphological characterization of
human liver microtissues
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
CK8
CD68
50 µm
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
48h LPS-treatment (10ug/ml)
IL-6
(p
g/w
ell
)
hLiMT - NPC
hLiMT + NPC
IL-6 secretion
35 www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Why did we develop InSphero’s 3D models?
Example: Trovan (Pfizer)
Introduction in 1997
2.5 million prescriptions
4 patients needed liver transplantation
6 patients died
Withdrawn 1998
Estimated loss for Pfizer: US$ 8.5 billion
Trovan toxicity was not detected in-vitro
36 www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
0
200
400
600
800
1000
1200
control 9 uM 28 uM 86 uM 260 uM
Hep61, 72h Trovafloxacin
LD
H in
su
pe
rna
tan
t
-LPS
+LPS
0
200
400
600
800
1000
1200
control 9 uM 28 uM 86 uM 260 uM 780 uM
Hep61, 72h Levofloxacin
LH
D in
su
pe
rna
tan
t
-LPS
+LPS
Inflammation-mediated idiosyncratic
trovafloxacin toxicity rLiMT
Trovafloxacin/Levofloxacin treatment
Readout: Released LDH (Cytotox-Glo, Promega)
Trovafloxacin [µM] Levofloxacin [µM]
37 www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
0
50
100
150
200
250
300
350
400
450
500
control 9 uM 28 uM 86 uM 260 uM
LZ9, HEP/NHDF/NPC, 72h Trovafloxacin
RF
U L
HD
in s
up
ern
ata
nt
-LPS
+LPS
0
50
100
150
200
250
300
350
400
450
500
control 9 uM 28 uM 86 uM 260 uM 780 uM
LZ9, HEP/NHDF/NPC 72h Levofloxacin
RF
U L
HD
in s
up
ern
ata
nt
-LPS
+LPS
Inflammation-mediated idiosyncratic
trovafloxacin toxicity hLiMT
Trovafloxacin/Levofloxacin treatment
Readout: Released LDH (Cytotox-Glo, Promega)
Trovafloxacin [µM] Levofloxacin [µM]
38
Acknowledgements
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013
Dr. Terry Riss
Joanna Stevenson
Kevin Kopish
Dr. Dan Lazar
Dr. Christian Thirion
Dr. Michael
Salomon
Dr. Jacob Tesdorpf
Dr. Karin Boettcher
Stefan Letzsch
Frauke Hänel
Dr. Claudio Thoma
Prof. Dr. Wilhelm Krek
Christian Oberdanner Helen Gill
Paul Walker
39
Questions Welcome
www.insphero.com | Tour of the Prom’s - Lausanne | 13.03.2013