AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016...
Transcript of AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016...
![Page 1: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/1.jpg)
AIT conference November 2016
Crack It: Development of high content analysis screening tools
to assess lung macrophage responses in vitro
Dr Victoria Hutter and Dr Lea Ann DaileyUniversity of Hertfordshire/ Martin Luther University of Halle
![Page 2: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/2.jpg)
Foamy Macrophage (FM)
• Term used to describe lung macrophages that have taken on a granular or vacuolated
cytoplasmic appearance when viewed by light microscopy
• Do FM constitute an adaptive or adverse response?
• Can we predict and/or monitor FM development?
Forbes et al., - Advanced Drug Delivery Reviews 71 (2014) 15-33
![Page 3: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/3.jpg)
Drug induced phospholipidosis
A widespread concern in drug development
• Appearance of membrane-bound cytosolic inclusions with a lamellar or crystalloid structure
• Inducing drug accumulates in association with the excess phospholipid but alterations are
generally reversible after discontinuation of the drug
• If appears in pre-clinical drug development then raises concerns regarding potential
occurrence in human clinical trials especially because of pathologically similar appearance
to inborn errors of metabolism (Niemann-Pick disease)
• If phospholipidosis observed then considered potentially adverse despite a lack of
information regarding the consequences of the presence of phospholipidosis in cells
• FDA working group report (presentation by Mark Reasnor, ACPS meeting, APRIL 14, 2010)
http://www.fda.gov/downloads/AdvisoryCommittees/CommitteesMeetingMaterials/Drugs/Ad
visoryCommitteeforPharmaceuticalScienceandClinicalPharmacology/UCM210798.pdf
![Page 4: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/4.jpg)
“Foamy” cells may not always be associated with phospholipids
![Page 5: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/5.jpg)
Crack It Challenge
Inhalation Translation
• Do foamy macrophages constitute an
adaptive or adverse response?
• Can we screen for foamy macrophage
development?
• Can we achieve 3R targets in inhaled
product development?
Non-invasive, longitudinal monitoring
Influx of mononuclear
cells in thelung
Define FM phenotype
Measure inflammatory
response
Define rodent response to inhaled
pharmaceuticals
Understand the FM
response timeframe
Understand drug-
induced FM biology
![Page 6: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/6.jpg)
CRACK IT Phase 2
NC3R CrackIT
Challenge
Academic Collaboration
Technology Providers
Sponsors
![Page 7: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/7.jpg)
WP1 – In vitro FM Toolkit
WP2 - Advanced understanding of FM
responses in vivo
WP3 - Non-invasive in vivo
methodology
WP4 – Translation, commercialisation
& engagement to
Addressing the
Challenge
Advanced
understanding
of FM biology
Achieving 3R
targets
Innovation and
engagement
Project Manager: B. Forbes (KCL)
WP1 Leads
V. Hutter (UH) · H. Collins (KCL)
WP2 Leads
J. Bunch (NPL) · I. Mudway (KCL)
WP3 Leads
L. Dailey (KCL) · D. Murnane (UH)
WP4 Leads
C. Page (KCL) · R. Booth (NPL)
CRACK IT Phase 2: Project overview
KHN Consortium Inhalation Translation Platform
![Page 8: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/8.jpg)
• Development of an in vitro screening tool to ascertain:
– Detection and quantification of different foamy macrophage phenotypes in vitro
– Gain a better understanding of the foamy macrophage response timeframe
– Gain a better understanding of foamy macrophage biology
• Developing methodologies to include:
– Interspecies differences – rat (NR8383) vs human (U937) macrophages
– Exposure time – 4-96h (24h and 48h tested)
– Concentration of test substance – 0.01µM – 100µM in half log concentrations
– Presentation of drug to cell – as a solubilised solution or as suspended particles
Workpackage 1: In vitro foamy macrophage tool kitOverview
WP1 – In vitro FM Toolkit
WP1 Leads
V. Hutter (UH) · H. Collins (KCL)
![Page 9: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/9.jpg)
High content analysis imaging
In cell analyser 6000
• High content analysis in vitro screen,
widely used in drug discovery
• Rapid, automated method
• Individual cell data
• Images and subsequent numerical
analysis to render quantified data of
outputs
Kumar et al., – AIT 2014
Hoffman et al., – Molecular Pharmaceutics 12 (2015) 2675-2687
![Page 10: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/10.jpg)
Positive control
Nuclei Image-It DeadCell Mask MitoTracker
Cell Morphology Stains
Nuclear staining (Hoechst 33342);
Cytoplasm staining (Cell Mask)
Cell Health Stains
Mitochondrial membrane potential (MitoTracker Red);
Membrane permeability (Image-It Dead Green).
Cell Health Outputs
• Total number of cells
• Nuclear/cytoplasm area
• Mean intensity of MitoTracker dye per cell
• Mean intensity of Image-It Dead dye per cell
Morphometric Analysis Outputs
• Total number of cells
• Nuclear and cytoplasm area
• Number of vacuoles per cell
• % area occupied by vacuoles
Assay development
Cell health and morphology
![Page 11: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/11.jpg)
Positive control
LipidTox GreenNuclei PhospholipidTox Red
Lipid Content Assay
Nuclear staining (Hoechst 33342);
Neutral lipid dye (LipidTox Green);
Phospholipids dye (PhosphlipidTox Red)
Lipid Content Outputs
• Total number of cells
• Mean intensity of LipidTox Green dye per cell
• Mean intensity of PhospholipidTox Red dye
per cell
Assay development
Lipid content
![Page 12: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/12.jpg)
Histogram analysis
Cell health parameters
0.0
20.0
40.0
60.0
80.0
100.0
0 25 50 75 100 125 150 175 200
Per
cen
tage
po
pu
lati
on
Nuclear Area (um2)
0.0
20.0
40.0
60.0
80.0
100.0
0 200 400 600 800
Per
cen
tage
po
pu
lati
on
Cell Area (um2)
NUCLEAR AREA (µm²) CELL AREA (µm²)
0.0
20.0
40.0
60.0
80.0
100.0
0 500 1000 1500 2000 2500 3000
Per
cen
tage
po
pu
lati
on
MitoTracker Red fluorescence intensity (a.u.)
0.0
20.0
40.0
60.0
80.0
100.0
0 100 200 300 400 500 600 700 800Per
cen
tage
po
pu
lati
on
Image-It Green fluorescence intensity (a.u.)
MITOCHONDRIAL ACTIVITY MEMBRANE PERMEABILITY\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
NR8383
48h
![Page 13: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/13.jpg)
Histogram analysis
Morphology
0.0
20.0
40.0
60.0
80.0
100.0
0 10 20 30 40 50
Per
cen
tage
po
pu
lati
on
Number of vacuoles per cell
0.0
20.0
40.0
60.0
80.0
100.0
0 20 40 60 80 100
Per
cen
tage
po
pu
lati
on
% area vacuolesper cell (%)
NR8383
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
0.0
20.0
40.0
60.0
80.0
100.0
0 10 20 30 40 50
Per
cen
tage
po
pu
lati
on
Number of vacuoles per cell
0.0
20.0
40.0
60.0
80.0
100.0
0 20 40 60 80 100
Per
cen
tage
po
pu
lati
on
% area vacuolesper cell (%)
U937
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
Vacuole Number Vacuole % Area
![Page 14: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/14.jpg)
Histogram analysis
Lipid content
0.0
20.0
40.0
60.0
80.0
100.0
0 500 1000 1500 2000 2500 3000
Per
cen
tage
po
pu
lati
on
LipidTox Green dye intensity (a.u.)
0.0
20.0
40.0
60.0
80.0
100.0
0 500 1000 1500 2000 2500
Per
cen
tage
po
pu
lati
on
PhospholipidTox Red dye intensity (a.u.)
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
Amiodarone
Cyclosporine
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
Amiodarone
Cyclosporine
NR8383
24h
0.0
20.0
40.0
60.0
80.0
100.0
0 500 1000 1500 2000 2500 3000
Per
cen
tage
po
pu
lati
on
LipidTox Green fluorescence intensity (a.c.)
0.0
20.0
40.0
60.0
80.0
100.0
0 500 1000 1500 2000 2500
Per
cen
tage
po
pu
lati
on
PhospholipidTox Red fluorescence intensity (a.c.)
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
Amiodarone
Cyclosporine
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
Amiodarone
Cyclosporine
NR8383
48h
Neutral lipids Phospholipids
![Page 15: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/15.jpg)
Positive control
Cell Health Determination
Cell Health Purpose
Elimination of low viability cells from
morphometric analysis based on low
mitochondrial activity
“HEALTHY” CELLS > MEAN - 2ND ST DEV
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
0 500 1000 1500 2000 2500 3000
Per
cen
tage
po
pu
lati
on
MitoTracker Red fluorescence intensity (a.u.)
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
\\\
Medium only
FCCP
Triton X-100
EXCLUSION
CRITERIA
MITOCHONDRIAL ACTIVITY
“HEALTHY” POPULATION
“Healthy” cells
Used to calculate:
• Nuclear area
• Cell area
• Mitochondrial Activity
• Cell membrane permeability
• Vacuole number and area
Setting up inclusion and exclusion thresholds
Cell health determination
![Page 16: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/16.jpg)
Positive control
PARAMETERS DESCRIPTION
CEL
LH
EALT
H
“HEALTHY” POPULATION Percentage of cells with mitochondrial activity > threshold
% VIABLE = # healthy per well / # total per well x 100%
NUCLEAR AREA Nuclear area stained by Hoechst Mean – 2nd StDev < % OF CELLS> mean+ 2nd StDev
NUCLEAR/CELL AREA Nuclear / Cell area ratio % OF CELLS > mean + 2nd StDev
ELEVATED MITCHONDRIAL
ACTIVITY
Fluorescence intensity of Mito Tracker stain
% OF CELLS > mean + 2nd StDev
ELEVATED CELL PERM Fluorescence intensity of ImageITDeadstain
% OF CELLS > mean + 2nd StDev
MO
RP
HO
LOG
Y ELEVATED CELL AREA Cellular area from cell mask stain % OF CELLS > mean + 2nd StDev
ELEVATED # VACUOLES Number of vacuoles per cell % OF CELLS > mean + 2nd StDev
ELEVATED % VACUOLES % Area vacuoles per cell % OF CELLS > mean + 2nd StDev
LIP
ID
CO
NTE
NT ELEVATED PHOSPHOLIPIDS Phospholipids accumulation % OF CELLS > mean + 2nd StDev
ELEVATED NEUTRAL LIPIDS Neutral lipids accumulation % OF CELLS > mean + 2nd StDev
Assay parameters
![Page 17: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/17.jpg)
Positive controlHE
ALT
HY
PO
PU
LA
TIO
N
NU
CL
EA
R
AR
EA
NU
CL
EA
R
/CE
LL
AR
EA
MIT
OA
CT
IVIT
Y
PE
RM
EA
BIL
ITY
CE
LL
AR
EA
# V
AC
% A
RE
AV
AC
PH
OS
PH
OL
IPID
S
NE
UT
RA
L
LIP
IDS
Cell Health Morphology Lipids
\\\\48 h
24 h
%
CE
LL
PO
PU
LA
TIO
N
Untreated cells
NR8383 (rat)
![Page 18: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/18.jpg)
Positive control
Cell Health Morphology Lipids
\\\\\\\\\\\48 h
24 h
%
CE
LL
PO
PU
LA
TIO
N
100
80
60
40
20
0
Untreated cells
U937 (human)
HE
ALT
HY
PO
PU
LA
TIO
N
NU
CL
EA
R
AR
EA
NU
CL
EA
R
/CE
LL
AR
EA
MIT
OA
CT
IVIT
Y
PE
RM
EA
BIL
ITY
CE
LL
AR
EA
# V
AC
% A
RE
AV
AC
PH
OS
PH
OL
IPID
S
NE
UT
RA
L
LIP
IDS
![Page 19: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/19.jpg)
• Reproducibility demonstrated via
heat map
• Untreated cells tested at three
different passages n=6 per
passage
Cell Health Morphology Lipids
MIN MAX
48H
24H
Experimental reproducibility
NR8383 (rat)
![Page 20: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/20.jpg)
Positive control
Cell Health Morphology LipidsCell Health Morphology Lipids
%
CE
LL
PO
PU
LA
TIO
N
24H EXPOSURE 48H EXPOSURE
0.1 µM
1 µM
10 µM
31 µM
Control
1. HEALTHY POPULATION 2. NUC AREA 3. NUC/CELL AREA 4.MITO ACT 5.PERM
6. CELL AREA 7. # VAC 8. % AREA VAC
9.PHOSPHOLIPIDS 10.NEUTRAL LIPIDS
0.1 µM
1 µM
10 µM
31 µM
Control
Amiodarone exposure
NR8383 (rat)
![Page 21: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/21.jpg)
• Optimised assay conditions
• Conducted a screen on blinded
compounds in development
• Scoping further options with analysis
approach
–Combining readouts for enhanced understanding of cell responses
• Considering other biomarkers (e.g.
M1/M2 detection, phagocytosis, etc)
• Expand biomarker and compound
screening with optimised assays
Current work
Non-invasive, longitudinal monitoring
Influx of mononuclear
cells in thelung
Define FM phenotype
Measure inflammatory
response
Define rodent response to inhaled
pharmaceuticals
Understand the FM
response timeframe
Understand drug-
induced FM biology
![Page 22: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/22.jpg)
Experimental screening:
Compound selection and screening
Plus and
additional 12
blinded
compounds
from the
sponsor
companies
![Page 23: AIT conference November 2016 Crack It: Development of high ... · AIT conference November 2016 Crack It: Development of high content analysis screening tools to assess lung macrophage](https://reader034.fdocuments.us/reader034/viewer/2022042911/5f43427f70d4b80c6f0895d5/html5/thumbnails/23.jpg)
Acknowledgments
• Aateka Patel
• Abhinav Kumar
• Anna Morgan
• Anthony Holmes
• Ben Forbes
• Clive Page
• Chris Walton
• David Hassall
• David Jones
• Darragh Murnane
• Doug Ball
• Helen Collins
• Ian Mudway
• Jan Klapwijk
• Jo Ann Rhodes
• Josephine Bunch
• Jo Taylor
• Lea Ann Dailey
• Martin Bootman
• Richard Booth
• Rory Stevens
• Simon Moore
• Val Millar
• Victoria Hutter
Funding body: NC3R NC/C013203/1