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

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

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

“Foamy” cells may not always be associated with phospholipids

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

CRACK IT Phase 2

NC3R CrackIT

Challenge

Academic Collaboration

Technology Providers

Sponsors

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

• 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)

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

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

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

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

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

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

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

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

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)

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

• 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)

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)

• 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

Experimental screening:

Compound selection and screening

Plus and

additional 12

blinded

compounds

from the

sponsor

companies

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