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Next-Generation Safety
Assessment Tool for Advancing
In Vivo to In Vitro Translation
Commercial Lead in Immunology
ImmuONE
Louis Scott, PhD
Victoria Hutter, PhD
Chief Scientific Officer
ImmuONE
© 2 0 2 4 IMMUONE
INNOVATIVE SOLUTIONS FOR
INHALATION SAFETY
In vitro specialists for immune responses
ImmuONE webinar
Wednesday 28th February 2024
Prof Victoria Hutter
Chief Scientific Officer, ImmuONE
© 2 0 2 4 IMMUONE
• Overview of in vitro inhalation toxicity assessment
• The models
• The exposure
• The assessment
• The interpretation
• Past, present and future – next generation tools and assessments
• How in vitro respiratory models complement and replace in vivo models
title
Overview
© 2 0 2 4 IMMUONE
title
In vitro inhalation toxicity assessment
The
model
The
exposure
The assessment
methodology
The
interpretation
• Specialized functions
• Upper & lower respiratory tract
• Conducting & pulmonary airways
• Inhalation toxicity typically has airway
epithelial focus
The respiratory tract
© 2 0 2 4 IMMUONE
The cellular composition of the airway epithelia differs
significantly
(a) In the upper airways, ciliated cells are
interspersed with secretory cells
(b) At lower levels they are interspersed with
club cells
(c) 2 cell types in the alveolar regions-squamous
cell Types I and II
The respiratory tract
Biological barriers: cells
© 2 0 2 4 IMMUONE
• Lung lining fluid
- Total lung volume 15-70 mL
- Ions but low protein content
• Conducting airways: mucus
- Mucopolysaccharide
- Influence on diffusion and absorption
• Respiratory airways: alveolar lining fluid
- Contains phospholipid surfactant and protein
- Monolayer coverage
- Also pools of excess lining fluid
Biological barriers: lining fluid
© 2 0 2 4 IMMUONE
Increasing complexity
Epithelial
2D cell lines
Epithelial cell lines
cultured in 3D at the
air-liquid interface
(ALI)
Primary human
cells 3D ALI
Commercial primary
human tissues 3D ALI
Spheroids and
organoids
Lung on a chip
Co-culture cell
line/primary models
cultured at the ALI
Models
© 2 0 2 4 IMMUONE
• Homo- or heterogenous populations
• Continuous cell lines has acquired the ability to proliferate indefinitely, either through genetic or
artificial modifications
• Lung specific cell models
• Bronchial epithelial – Calu-3, 16HBE14o-, BEAS-2B,
NuLi,
• Alveolar epithelial – A549, H441, hAeLVi
• Fibroblast – MRC5, CCD19-Lu
• Endothelial
• Alveolar macrophages – THP1, U937
Cell lines
© 2 0 2 4 IMMUONE
• Epithelial cells cultures at the air-liquid interface (ALI) generate a model
more morphologically representative of the airway epithelium than
cells cultured under submerged conditions
• Columnar morphology
• Increased cilia-like structures
• Increased glycoprotein secretions
• Reduced TEER/ZO1 staining
Submerged or LLI ALI Calu-3 cells at the ALI
Air-liquid interface culture
© 2 0 2 4 IMMUONE
• Primary cells are isolated directly from humans using
enzymatic or mechanical methods
• Biologically relevant
• Physiologically similar
• Short life
• Patient variability
• Commercial airways tissues
• Combine multiple donors
NHBE primary cells at ALI
Primary cell culture models
© 2 0 2 4 IMMUONE
Variety of co-culture models in literature
• epithelial, endothelial, fibroblast, immune
(alveolar macrophage)
Cells selected depending on response/mechanism
of interest
• irritation/viability – epithelial
• inflammation – macrophage (epithelial
limited)
• sensitisation – epithelial, endothelial,
macrophage, dendritic cells
ImmuLUNG
Co-culture of human alveolar macrophage-
like cells with alveolar epithelial cells
✓ 24 Transwell® format
✓ Immortalised cells from lung
origin
✓ Air-liquid interface culture
✓ Up to 14 days in culture (in house)
Co-culture models
© 2 0 2 4 IMMUONE
3D structures composed of multiple cells
Organoids
• complex clusters of organ-specific cells
• made from stem cells or progenitor cells
• self-assemble when given a scaffolding
• disease modelling, drug discovery, regenerative medicine,
cancer research and tumour pathophysiology
Spheroids
• simple clusters of broad-ranging cells
• stick together without the need for a scaffold
• can’t self-assemble or regenerate
• understanding tumour microenvironments
Spheroids and organoids
© 2 0 2 4 IMMUONE
• Simulate human physiology within the system
• Mechanical forces - breathing
• Physiological flow - microfluidics
• Closer human physiological representation of the cells/tissues
Organ-on-a-chip models
© 2 0 2 4 IMMUONE
What is physiological relevance
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• Contain all lung cell types present in
tissue (at the time of slicing)
• Can be maintained for 3-4 weeks
• Acute and chronic
Precision cut lung slices
© 2 0 2 4 IMMUONE
Right cell types for
the desired
response/function
Which model?
Increasing
complexity not
always needed
- establish clear
benefit of adding
more cell types
Optimal for
exposure/test item
(media, system)
Optimal for the
right assessment
techniques
Compatible with
the assessment
endpoints
Skill of the end user
and time to
optimise
Model selection
© 2 0 2 4 IMMUONE
• Human lung physiology (vs rat)
• Particle characteristics
• Deposition
• Interaction with lining fluid
• Cellular exposure
Lung physiology
© 2 0 2 4 IMMUONE
Exposure
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Regional deposition
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• Deposition modelling
• Broad range that covers toxic to non-toxic
• mg/area
• Lung area 100-130m2
• In vitro area (24 well plate – 0.33cm2)
Dosing in vitro
© 2 0 2 4 IMMUONE
• Solution
• Suspension
• Aerosol
In vitro exposure
© 2 0 2 4 IMMUONE
• Exposure is reasonably certain
• If aqueous solubility is acceptable
• Non-specific binding
• Potential for cellular metabolism
• Potential interaction with cell culture
medium
• Less physiologically relevant
• Flooding the airways with liquid
Dosing in solution
© 2 0 2 4 IMMUONE
• Liquid aerosols
• 15-300 µL nebulised volume
• 3-6 min exposure time
Dosing using aerosols
© 2 0 2 4 IMMUONE
Dry powder
• Powder placed in loading system
• Expansion chamber sealed
• Powder aerosolised under high pressure
breaks up agglomerates)
• Powder settles on samples (gravitational
sedimentation)
Dosing using aerosols
© 2 0 2 4 IMMUONE
Continuous flow exposure
• Gases, complex mixtures, particles
• 6-24h
• Grams of material
Continuous dosing
© 2 0 2 4 IMMUONE
Does it kill cells?
Does it affect cell/tissue
function/change cell behaviour?
No
Can they recover?
Yes
Yes
No
Yes
( )
No
Mechanistic
assessment
• how does it kill cells?
• how is cell/tissue
function affected?
• what is the recovery
time?
• Adverse Outcome
Pathways (AOP)
In vitro assessment
© 2 0 2 4 IMMUONE
• Viability
- PI, LDH
- mitochondrial activity
- ATP
• Barrier
- TEER
- paracellular marker
• Histology
Untreated
Standard assessments: epithelial
© 2 0 2 4 IMMUONE
• Cilia beating frequency • Mucociliary clearance
Other epithelial assessments
© 2 0 2 4 IMMUONE
• Inflammation
• Cytokines
• Cell surface markers
• Markers for cell activation
• Growth factors
• Prostaglandins
• Acute phase proteins (CRP)
• Oxidative stress (ROS, RNS)
• Individual cell
characterisation and
population characterisation
Other stress/inflammatory assessments
© 2 0 2 4 IMMUONE
• What is the mechanism of death?
• What is the mechanism of inflammation?
• sensitisation, irritant
• Is there the potential for longer term
toxicology/airway remodelling?
• fibrosis
• Not all cells in a model/tissue will be have
the same
• average population characteristics
• individual cell characteristics
Genomics (DNA)
Transcriptomics (RNA)
Proteomics (Protein)
Mechanistic driven assessment
© 2 0 2 4 IMMUONE
Multi-endpoint analysis for mechanism-driven risk
assessment
Exposure: Solution vs
suspension vs aerosol
Multiple endpoint assessment
Multi-endpoint analysis for mechanism-driven risk
assessment
Exposure: Solution vs
suspension vs aerosol
Barrier properties
Epithelial-immune
interactions
Morphology
Surfactant
production
Viability
Multiple endpoint assessment
Multi-endpoint analysis for mechanism-driven risk
assessment
Cell size
Vacuolation profile
Cell shape
Cytotoxicity/cell
membrane integrity
Metabolic activity
Cell number
Exposure: Solution vs
suspension vs aerosol
Barrier properties
Epithelial-immune
interactions
Morphology
Surfactant
production
Viability
Multiple endpoint assessment
© 2 0 2 4 IMMUONE
Multi-endpoint analysis for mechanism-driven risk
assessment
Cell size
Vacuolation profile
Cell shape
Polarisation (CD
markers)
Phagocytosis
Cytokines
Cell lipid profiles
Genetic analysis
Cytotoxicity/cell
membrane integrity
Metabolic activity
Cell number
Exposure: Solution vs
suspension vs aerosol
Barrier properties
Epithelial-immune
interactions
Morphology
Surfactant
production
Viability
Proteomic analysis
Multiple endpoint assessment
© 2 0 2 4 IMMUONE
Multi-endpoint analysis for mechanism-driven risk
assessment
Cell size
Vacuolation profile
Cell shape
Polarisation (CD
markers)
Phagocytosis
Cytokines
Cell lipid profiles
Genetic analysis
Cytotoxicity/cell
membrane integrity
Metabolic activity
Cell number
Exposure: Solution vs
suspension vs aerosol
Barrier properties
Epithelial-immune
interactions
Morphology
Surfactant
production
Viability
Proteomic analysis
Cell migration Cell velocity
Multiple endpoint assessment
© 2 0 2 4 IMMUONE
High content analysis (cell painting)
© 2 0 2 4 IMMUONE
4 h
24 h
48 h 48 h
4 h
24 h
48 h 48 h
4 h
24 h
48 h 48 h
Control Known toxicant Test item A
Phenotype fingerprinting for safety profiling
© 2 0 2 4 IMMUONE
4 h
24 h
48 h 48 h
4 h
24 h
48 h 48 h
4 h
24 h
48 h 48 h
Control Known toxicant Test item A
Phenotype fingerprinting for safety profiling
© 2 0 2 4 IMMUONE
air
control
known
toxicant
Phenotype can indicate toxicity mechanism
© 2 0 2 4 IMMUONE
air
control
known
toxicant
Phenotype can indicate toxicity mechanism
© 2 0 2 4 IMMUONE
MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
CELL A REA ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
NUMBER OF VA CUO LES ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑
A REA O F VACUO LA TIO N ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
Compound C (low)
Compound C (high)
Compound D (low)
Compound D (high)
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
Compound C (low)
Compound C (high)
Compound D (low)
Compound D (high)
Nuclei
Membrane
Permeability
Mitochondrial
Activity Cytoplasm Merged
Control
Compound C
(low)
Compound C
(high)
04
Test item (low conc)
Test item (high conc)
Phenotype indicative of toxicity mechanism
© 2 0 2 4 IMMUONE
MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
CELL A REA ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
NUMBER OF VA CUO LES ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑
A REA O F VACUO LA TIO N ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
Compound C (low)
Compound C (high)
Compound D (low)
Compound D (high)
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑
Control
PBS
Compound A (low)
Compound A (high)
Compound B (low)
Compound B (high)
Compound C (low)
Compound C (high)
Compound D (low)
Compound D (high)
Nuclei
Membrane
Permeability
Mitochondrial
Activity Cytoplasm Merged
Control
Compound C
(low)
Compound C
(high)
04
Test item (low conc)
Test item (high conc)
Phenotype indicative of toxicity mechanism
© 2 0 2 4 IMMUONE
• What is the purpose of the in vitro test
• Differentiate response
• Benchmark response
• Correlate response
• In vitro-in vivo correlation
• Appropriate endpoints for direct correlation (morphology, blood markers)
• Appropriate compound set with sufficient in vivo characterisation
• Mechanism-driven risk assessment
• Adverse Outcome Pathways (AOP)
• Integrated Approaches to Testing and Assessment (IATA)
• IATA NAMS for refining assessment of chlorothalonil
Interpretation and context
© 2 0 2 4 IMMUONE
Human in vitro
Rat in vivo
(lung pathology)
Rat ex vivo
(BAL)
Rat in vitro
Rat in vivo
Interspecies comparisons
© 2 0 2 4 IMMUONE
• Wide range of in vitro models, dosing systems and assessments available
• Defining the question and purpose of the in vitro assessment is key
• Does it kill cells?
• What is the long-term effect?
• What is the mechanism of toxicity/cell interaction?
• Examples of how in vitro respiratory models complement and potentially replace in vivo
models
Summary
© 2 0 2 4 IMMUONE
© 2 0 2 4 IMMUONE
Profiling alveolar
macrophage responses to
inhaled compounds
Dr. Louis Scott
Commercial Lead in Immunology
© 2 0 2 4 IMMUONE
• Concerns over safety and initiation of immune
responses in the distal airways in preclinical
studies is one of the reasons over a third of
candidate inhaled compounds fail.
• Highly vacuolated or "foamy" alveolar
macrophages (FM) observed in pre-clinical rat
studies poses safety concerns, despite not
knowing the correlation to hindering progress of
promising candidates into humans
title
Background: “foamy” macrophages
in vivo
in vitro
Forbes et al., 2014
© 2 0 2 4 IMMUONE
• HCIA combines alveolar
macrophage morphology/ lipid
endpoints as an in vitro tool for
inhaled safety assessment.
• Morphometric endpoints that
directly correlate with in vivo
observation.
title
Purpose and methodology
in vivo in vitro
© 2 0 2 4 IMMUONE
title
HCIA screens
HCA screens overview
A - Cell Health and Morphology Assay
• Nuclear staining (Hoechst 33342)
• Mitochondrial membrane potential (MitoTracker Red)
• Membrane permeability (Image-It Dead Green)
• Cytoplasm staining (Cell Mask)
B - Lipid content and Phagocytosis Assay
• Nuclear staining (Hoechst 33342)
• Phospholipidosis detection (Lipidtox Red)
• Neutral lipids detection (Neutral lipid green)
• Phagocytosis activity (Crimson beads)
Lipid content staining
10 µM Imipramine, NR8383
Cell health staining,
Air-treated, BAL
© 2 0 2 4 IMMUONE
title
Median morphology parameters of untreated and amiodarone-
treated rat and human macrophage cell models.
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Number of vacuoles (human)
Number
of
vacuoles
per
cell
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10
15
Number of vaculoes (rat)
Number
of
vacuoles
per
cell
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Numbe
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0
100
200
300
400
500
Cell area (human)
Cell
area
[µm
2
]
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0
Numb
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100
120
140
160
180
200
220
Cell area (rat)
Cell
area
[µm
2
]
Vacuolation Cell area
Hoffman et al., 2017
© 2 0 2 4 IMMUONE
title
Median lipid parameters of untreated and amiodarone-treated
rat and human macrophage cell models.
Neutral lipids Phospholipids
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0.05
0.10
Relative
fluo
Phos
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0.10
0.15
0.20
Neutral Lipids (human)
Relative
fluorescence
intensity
Neutral
lipid
Green
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0.1
Relative
fluor
Phosp
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0.1
0.2
0.3
0.4
0.5
Neutral Lipids (rat)
Relative
fluorescence
intensity
Neutral
lipid
Green
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0.25
Phospholipids (human)
Relative
fluorescence
intensity
Phospholipid
Red
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Phospholipids (rat)
Relative
fluorescence
intensity
Phospholipid
Red
Hoffman et al., 2017
© 2 0 2 4 IMMUONE
title
Macrophage Responses to Amiodarone: ‘elevated’
responses
• No significant difference (p > 0.05) was observed
between untreated cells and those exposed to 10 μM
amiodarone (known inducer of phospholipidosis) when
comparing median values, despite observable
differences in histogram profiles.
• Average population statistical analysis was not a suitably
sensitive descriptor to depict the morphology and lipid
changes observed in cells exposed to amiodarone which
impacted less than 20% of the total cell population.
• Therefore, an alternative analytical approach which
included only the cell population with elevated
characteristics in comparison with the untreated cells
was considered.
Hoffman et al., 2017
Untreated Amiodarone
© 2 0 2 4 IMMUONE
title
Macrophage Responses to Amiodarone: ‘elevated’
responses
HCA screens overview
Hoffman et al., 2017
Parameter Inclusion criteria
Elevated mitochondrial
activity
% cells > mean mitochondrial activity + 2nd SD of the untreated cell
population
Elevated membrane
permeability
% cells > mean membrane permeability + 2nd SD of the untreated cell
population
Elevated cellular area % cells > mean cell area + 2nd SD of the untreated cell population
Elevated vacuole number
per cell
% cells > mean vacuole number per cell + 2nd SD of the untreated cell
population
Elevated vacuole area per
cell
% cells > mean vacuole area per cell + 2nd SD of the untreated cell
population
Elevated phospholipid
content
% cells > mean phospholipid content per cell + 2nd SD of the untreated cell
population
Elevated neutral lipid
content
% cells > mean neutral lipid content per cell + 2nd SD of the untreated cell
population
**Summary of diagnostic criteria employed in foamy macrophage investigation.
E Median value untreated
F Median value amiodarone
G Population of cells elevated number of vacuoles per cell
© 2 0 2 4 IMMUONE
title
Cell health, morphology and lipid multi-parameter profiles after
amiodarone exposure to rat macrophages
24h
Hoffman et al., 2017
48h • In contrast to median value
analysis, multi-parameter
profile analysis of the
elevated population
characteristics revealed an
impact on elevated
phospholipid content and
cell health markers as
expected for the
phospholipidosis inducer,
amiodarone.
title
In vivo to in vitro translation
© 2 0 2 4 IMMUONE
• Rat alveolar macrophages were more
sensitive to amiodarone exposure than
human cells.
• All alveolar lung macrophage cell
models require analysis of cell sub-
populations rather than average
population data.
• HCIA provides a method for profiling
human and rat alveolar macrophage
responses in vitro.
© 2 0 2 4 IMMUONE
• 12 compounds, categorized as FM- and
non-FM-inducers.
• In vitro tests deemed all compounds
suitable for animal studies.
• In vivo studies revealed that some
compounds led to macrophage
accumulation and FM responses.
• Compounds were discontinued from
further development due to potential
human safety issues.
title
Profiling of alveolar macrophage responses to
inhaled modalities
© 2 0 2 4 IMMUONE
title
CrackIT: profiling of alveolar macrophage
responses to inhaled modalities
Non-FM-inducers FM-inducers
salbutamol
hemisulphate
β2-agonist amiodarone
hydrochloride
cationic amphiphilic drug
formoterol
fumarate
β2-agonist imipramine
hydrochloride
cationic amphiphilic drug
dihydrate,
salmeterol
xinafoate
β2-agonist chlorpromazine
hydrochloride
cationic amphiphilic drug
ipratropium
bromide
muscarinic receptor
antagonist
dibucaine
hydrochloride
cationic amphiphilic drug
beclomethasone
dipropionate
steroidal antiflammatory drug staurosporine Pro-apoptotic
fluticasone
propionate
steroidal antiflammatory drug etoposide Pro-apoptotic
budesonide steroidal antiflammatory drug campthotecin Pro-apoptotic
© 2 0 2 4 IMMUONE
title
Multiparameter analysis of rat alveolar
macrophages
B C
Rat macrophages treated in vitro with Amiodarone in various concentrations for 24 hours
(A) 0µM
(B) 10µM
(C) 50µM
InCell Analyser images, magnification 40x
A
© 2 0 2 4 IMMUONE
title
Multiparameter analysis of rat alveolar
macrophages
0
50
100
150
24h
0
50
100
150
48h
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
t
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
24h
Parameter
24h
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
48h
Parameter
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
0
50
100
150
48h
%
Cell
Population
Untreated
Amiodarone
Imipramine
Dibucaine
Chlorpromazine
Staurosporine
Etoposide
Campthotecin
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
24h
Parameter
%
Cell
Population
0
50
100
150
24h
%
Cell
Population
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
48h
Parameter
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
t
h
a
a
t
t
a
c
c
c
s
s
0
50
100
150
48h
%
Cell
Population
Untreated
Amiodarone
Imipramine
Dibucaine
Chlorpromazine
Staurosporine
Etoposide
Campthotecin
Non-FM-inducers FM-inducers
© 2 0 2 4 IMMUONE
title
Multiparameter analysis of human alveolar
macrophages
Human macrophages treated in vitro with amiodarone in various concentrations for 24 hours
(A) 0µM
(B) 1µM
(C) 10µM
(D) 50µM
InCell Analyser images, magnification 40x
A
B C
A B C D
© 2 0 2 4 IMMUONE
title
Multiparameter analysis of human alveolar
macrophages
Non-FM-inducers FM-inducers
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
24h
Parameter
%
Cell
Population
0
50
100
150
24h
%
Cell
Population
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
48h
Parameter
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
t
t
t
0
50
100
150
48h
%
Cell
Population
Untreated
Amiodarone
Imipramine
Dibucaine
Chlorpromazine
Staurosporine
Etoposide
Campthotecin
e
a
e
a
I
n
t
I
n
t
e
a
a
c
a
c
a
c
d
s
d
s
24h
n
t
l
t
h
e
a
e
a
I
n
t
I
n
t
e
a
a
c
a
c
a
c
d
s
d
s
0
50
100
150
48h
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
24h
Parameter
24h
C
e
l
l
C
o
u
n
t
C
e
l
l
H
e
a
l
t
h
N
u
c
A
r
e
a
N
u
c
/
C
e
l
l
A
r
e
a
M
i
t
o
I
n
t
P
e
r
m
I
n
t
C
e
l
l
A
r
e
a
#
V
a
c
%
V
a
c
A
r
e
a
/
#
V
a
c
P
h
o
s
p
h
o
l
i
p
i
d
s
N
e
u
t
r
a
l
L
i
p
i
d
s
0
50
100
150
48h
Parameter
%
Cell
Population
Untreated
Salbutamol
Formoterol
Salmeterol
Ipratropium
Beclomethasone
Fluticasone
Budesonide
t
h
a
a
t
t
a
c
c
c
s
s
0
50
100
150
48h
%
Cell
Population
Untreated
Amiodarone
Imipramine
Dibucaine
Chlorpromazine
Staurosporine
Etoposide
Campthotecin
Summary
© 2 0 2 4 IMMUONE
• All alveolar lung macrophage cell models responded to challenge with amiodarone, but detection of
these required analysis of cell sub-populations rather than average population data.
• Human alveolar macrophages profiles showed that cells were less sensitive to stimuli.
• HCIA-derived profiles differentiated between safe and unsafe responses in macrophages.
• Multi-parameter methodology has the potential to offer an early non-clinical screening tool to predict
the safety of candidate inhaled medicines.
• The comparison of in vitro cell responses from a variety of rat ex vivo and human and rat in vitro
models allows for a direct comparisons to be made.
IN VITRO INNOVATION SERVICES FOR
EMPOWERED DECISION MAKING
© 2 0 2 4 IMMUONE
ImmuONE provides innovative respiratory in vitro cell
culture products and contract research services for
inhalation safety testing. Our focus is supporting
companies and researchers to understand the
interaction and safety implications of inhaled products
with immune cells in the airways.
© 2 0 2 4 IMMUONE
Text box
Delivering in vitro solutions
In vitro services for inhalation
testing
In vitro alveolar macrophage
assays
Immune-competent in vitro
human airways models
© 2 0 2 4 IMMUONE
Contact us!
info@immuone.com
www.immuone.com
ImmuONE
in

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Next-Generation Safety Assessment Tools for Advancing In Vivo to In Vitro Translation

  • 1. Next-Generation Safety Assessment Tool for Advancing In Vivo to In Vitro Translation Commercial Lead in Immunology ImmuONE Louis Scott, PhD Victoria Hutter, PhD Chief Scientific Officer ImmuONE
  • 2. © 2 0 2 4 IMMUONE INNOVATIVE SOLUTIONS FOR INHALATION SAFETY In vitro specialists for immune responses ImmuONE webinar Wednesday 28th February 2024 Prof Victoria Hutter Chief Scientific Officer, ImmuONE
  • 3. © 2 0 2 4 IMMUONE • Overview of in vitro inhalation toxicity assessment • The models • The exposure • The assessment • The interpretation • Past, present and future – next generation tools and assessments • How in vitro respiratory models complement and replace in vivo models title Overview
  • 4. © 2 0 2 4 IMMUONE title In vitro inhalation toxicity assessment The model The exposure The assessment methodology The interpretation
  • 5. • Specialized functions • Upper & lower respiratory tract • Conducting & pulmonary airways • Inhalation toxicity typically has airway epithelial focus The respiratory tract © 2 0 2 4 IMMUONE
  • 6. The cellular composition of the airway epithelia differs significantly (a) In the upper airways, ciliated cells are interspersed with secretory cells (b) At lower levels they are interspersed with club cells (c) 2 cell types in the alveolar regions-squamous cell Types I and II The respiratory tract Biological barriers: cells © 2 0 2 4 IMMUONE
  • 7. • Lung lining fluid - Total lung volume 15-70 mL - Ions but low protein content • Conducting airways: mucus - Mucopolysaccharide - Influence on diffusion and absorption • Respiratory airways: alveolar lining fluid - Contains phospholipid surfactant and protein - Monolayer coverage - Also pools of excess lining fluid Biological barriers: lining fluid © 2 0 2 4 IMMUONE
  • 8. Increasing complexity Epithelial 2D cell lines Epithelial cell lines cultured in 3D at the air-liquid interface (ALI) Primary human cells 3D ALI Commercial primary human tissues 3D ALI Spheroids and organoids Lung on a chip Co-culture cell line/primary models cultured at the ALI Models © 2 0 2 4 IMMUONE
  • 9. • Homo- or heterogenous populations • Continuous cell lines has acquired the ability to proliferate indefinitely, either through genetic or artificial modifications • Lung specific cell models • Bronchial epithelial – Calu-3, 16HBE14o-, BEAS-2B, NuLi, • Alveolar epithelial – A549, H441, hAeLVi • Fibroblast – MRC5, CCD19-Lu • Endothelial • Alveolar macrophages – THP1, U937 Cell lines © 2 0 2 4 IMMUONE
  • 10. • Epithelial cells cultures at the air-liquid interface (ALI) generate a model more morphologically representative of the airway epithelium than cells cultured under submerged conditions • Columnar morphology • Increased cilia-like structures • Increased glycoprotein secretions • Reduced TEER/ZO1 staining Submerged or LLI ALI Calu-3 cells at the ALI Air-liquid interface culture © 2 0 2 4 IMMUONE
  • 11. • Primary cells are isolated directly from humans using enzymatic or mechanical methods • Biologically relevant • Physiologically similar • Short life • Patient variability • Commercial airways tissues • Combine multiple donors NHBE primary cells at ALI Primary cell culture models © 2 0 2 4 IMMUONE
  • 12. Variety of co-culture models in literature • epithelial, endothelial, fibroblast, immune (alveolar macrophage) Cells selected depending on response/mechanism of interest • irritation/viability – epithelial • inflammation – macrophage (epithelial limited) • sensitisation – epithelial, endothelial, macrophage, dendritic cells ImmuLUNG Co-culture of human alveolar macrophage- like cells with alveolar epithelial cells ✓ 24 Transwell® format ✓ Immortalised cells from lung origin ✓ Air-liquid interface culture ✓ Up to 14 days in culture (in house) Co-culture models © 2 0 2 4 IMMUONE
  • 13. 3D structures composed of multiple cells Organoids • complex clusters of organ-specific cells • made from stem cells or progenitor cells • self-assemble when given a scaffolding • disease modelling, drug discovery, regenerative medicine, cancer research and tumour pathophysiology Spheroids • simple clusters of broad-ranging cells • stick together without the need for a scaffold • can’t self-assemble or regenerate • understanding tumour microenvironments Spheroids and organoids © 2 0 2 4 IMMUONE
  • 14. • Simulate human physiology within the system • Mechanical forces - breathing • Physiological flow - microfluidics • Closer human physiological representation of the cells/tissues Organ-on-a-chip models © 2 0 2 4 IMMUONE
  • 15. What is physiological relevance © 2 0 2 4 IMMUONE
  • 16. • Contain all lung cell types present in tissue (at the time of slicing) • Can be maintained for 3-4 weeks • Acute and chronic Precision cut lung slices © 2 0 2 4 IMMUONE
  • 17. Right cell types for the desired response/function Which model? Increasing complexity not always needed - establish clear benefit of adding more cell types Optimal for exposure/test item (media, system) Optimal for the right assessment techniques Compatible with the assessment endpoints Skill of the end user and time to optimise Model selection © 2 0 2 4 IMMUONE
  • 18. • Human lung physiology (vs rat) • Particle characteristics • Deposition • Interaction with lining fluid • Cellular exposure Lung physiology © 2 0 2 4 IMMUONE
  • 19. Exposure © 2 0 2 4 IMMUONE
  • 20. Regional deposition © 2 0 2 4 IMMUONE
  • 21. • Deposition modelling • Broad range that covers toxic to non-toxic • mg/area • Lung area 100-130m2 • In vitro area (24 well plate – 0.33cm2) Dosing in vitro © 2 0 2 4 IMMUONE
  • 22. • Solution • Suspension • Aerosol In vitro exposure © 2 0 2 4 IMMUONE
  • 23. • Exposure is reasonably certain • If aqueous solubility is acceptable • Non-specific binding • Potential for cellular metabolism • Potential interaction with cell culture medium • Less physiologically relevant • Flooding the airways with liquid Dosing in solution © 2 0 2 4 IMMUONE
  • 24. • Liquid aerosols • 15-300 µL nebulised volume • 3-6 min exposure time Dosing using aerosols © 2 0 2 4 IMMUONE
  • 25. Dry powder • Powder placed in loading system • Expansion chamber sealed • Powder aerosolised under high pressure breaks up agglomerates) • Powder settles on samples (gravitational sedimentation) Dosing using aerosols © 2 0 2 4 IMMUONE
  • 26. Continuous flow exposure • Gases, complex mixtures, particles • 6-24h • Grams of material Continuous dosing © 2 0 2 4 IMMUONE
  • 27. Does it kill cells? Does it affect cell/tissue function/change cell behaviour? No Can they recover? Yes Yes No Yes ( ) No Mechanistic assessment • how does it kill cells? • how is cell/tissue function affected? • what is the recovery time? • Adverse Outcome Pathways (AOP) In vitro assessment © 2 0 2 4 IMMUONE
  • 28. • Viability - PI, LDH - mitochondrial activity - ATP • Barrier - TEER - paracellular marker • Histology Untreated Standard assessments: epithelial © 2 0 2 4 IMMUONE
  • 29. • Cilia beating frequency • Mucociliary clearance Other epithelial assessments © 2 0 2 4 IMMUONE
  • 30. • Inflammation • Cytokines • Cell surface markers • Markers for cell activation • Growth factors • Prostaglandins • Acute phase proteins (CRP) • Oxidative stress (ROS, RNS) • Individual cell characterisation and population characterisation Other stress/inflammatory assessments © 2 0 2 4 IMMUONE
  • 31. • What is the mechanism of death? • What is the mechanism of inflammation? • sensitisation, irritant • Is there the potential for longer term toxicology/airway remodelling? • fibrosis • Not all cells in a model/tissue will be have the same • average population characteristics • individual cell characteristics Genomics (DNA) Transcriptomics (RNA) Proteomics (Protein) Mechanistic driven assessment © 2 0 2 4 IMMUONE
  • 32. Multi-endpoint analysis for mechanism-driven risk assessment Exposure: Solution vs suspension vs aerosol Multiple endpoint assessment
  • 33. Multi-endpoint analysis for mechanism-driven risk assessment Exposure: Solution vs suspension vs aerosol Barrier properties Epithelial-immune interactions Morphology Surfactant production Viability Multiple endpoint assessment
  • 34. Multi-endpoint analysis for mechanism-driven risk assessment Cell size Vacuolation profile Cell shape Cytotoxicity/cell membrane integrity Metabolic activity Cell number Exposure: Solution vs suspension vs aerosol Barrier properties Epithelial-immune interactions Morphology Surfactant production Viability Multiple endpoint assessment © 2 0 2 4 IMMUONE
  • 35. Multi-endpoint analysis for mechanism-driven risk assessment Cell size Vacuolation profile Cell shape Polarisation (CD markers) Phagocytosis Cytokines Cell lipid profiles Genetic analysis Cytotoxicity/cell membrane integrity Metabolic activity Cell number Exposure: Solution vs suspension vs aerosol Barrier properties Epithelial-immune interactions Morphology Surfactant production Viability Proteomic analysis Multiple endpoint assessment © 2 0 2 4 IMMUONE
  • 36. Multi-endpoint analysis for mechanism-driven risk assessment Cell size Vacuolation profile Cell shape Polarisation (CD markers) Phagocytosis Cytokines Cell lipid profiles Genetic analysis Cytotoxicity/cell membrane integrity Metabolic activity Cell number Exposure: Solution vs suspension vs aerosol Barrier properties Epithelial-immune interactions Morphology Surfactant production Viability Proteomic analysis Cell migration Cell velocity Multiple endpoint assessment © 2 0 2 4 IMMUONE
  • 37. High content analysis (cell painting) © 2 0 2 4 IMMUONE
  • 38. 4 h 24 h 48 h 48 h 4 h 24 h 48 h 48 h 4 h 24 h 48 h 48 h Control Known toxicant Test item A Phenotype fingerprinting for safety profiling © 2 0 2 4 IMMUONE
  • 39. 4 h 24 h 48 h 48 h 4 h 24 h 48 h 48 h 4 h 24 h 48 h 48 h Control Known toxicant Test item A Phenotype fingerprinting for safety profiling © 2 0 2 4 IMMUONE
  • 40. air control known toxicant Phenotype can indicate toxicity mechanism © 2 0 2 4 IMMUONE
  • 41. air control known toxicant Phenotype can indicate toxicity mechanism © 2 0 2 4 IMMUONE
  • 42. MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ CELL A REA ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ NUMBER OF VA CUO LES ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ A REA O F VACUO LA TIO N ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ Control PBS Compound A (low) Compound A (high) Compound B (low) Compound B (high) Compound C (low) Compound C (high) Compound D (low) Compound D (high) Control PBS Compound A (low) Compound A (high) Compound B (low) Compound B (high) MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ Control PBS Compound A (low) Compound A (high) Compound B (low) Compound B (high) Compound C (low) Compound C (high) Compound D (low) Compound D (high) Nuclei Membrane Permeability Mitochondrial Activity Cytoplasm Merged Control Compound C (low) Compound C (high) 04 Test item (low conc) Test item (high conc) Phenotype indicative of toxicity mechanism © 2 0 2 4 IMMUONE
  • 43. MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ CELL A REA ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ NUMBER OF VA CUO LES ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ ↓ ↓ ↓ − − − ↑ ↑ ↑ A REA O F VACUO LA TIO N ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ ↓ − ↑ Control PBS Compound A (low) Compound A (high) Compound B (low) Compound B (high) Compound C (low) Compound C (high) Compound D (low) Compound D (high) Control PBS Compound A (low) Compound A (high) Compound B (low) Compound B (high) MITOCHO NDRIA L A CTIVITY ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ − − − − − − − − − − − − − − − − − − − − − − − − − − − ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ ↑ Control PBS Compound A (low) Compound A (high) Compound B (low) Compound B (high) Compound C (low) Compound C (high) Compound D (low) Compound D (high) Nuclei Membrane Permeability Mitochondrial Activity Cytoplasm Merged Control Compound C (low) Compound C (high) 04 Test item (low conc) Test item (high conc) Phenotype indicative of toxicity mechanism © 2 0 2 4 IMMUONE
  • 44. • What is the purpose of the in vitro test • Differentiate response • Benchmark response • Correlate response • In vitro-in vivo correlation • Appropriate endpoints for direct correlation (morphology, blood markers) • Appropriate compound set with sufficient in vivo characterisation • Mechanism-driven risk assessment • Adverse Outcome Pathways (AOP) • Integrated Approaches to Testing and Assessment (IATA) • IATA NAMS for refining assessment of chlorothalonil Interpretation and context © 2 0 2 4 IMMUONE
  • 45. Human in vitro Rat in vivo (lung pathology) Rat ex vivo (BAL) Rat in vitro Rat in vivo Interspecies comparisons © 2 0 2 4 IMMUONE
  • 46. • Wide range of in vitro models, dosing systems and assessments available • Defining the question and purpose of the in vitro assessment is key • Does it kill cells? • What is the long-term effect? • What is the mechanism of toxicity/cell interaction? • Examples of how in vitro respiratory models complement and potentially replace in vivo models Summary © 2 0 2 4 IMMUONE
  • 47. © 2 0 2 4 IMMUONE Profiling alveolar macrophage responses to inhaled compounds Dr. Louis Scott Commercial Lead in Immunology
  • 48. © 2 0 2 4 IMMUONE • Concerns over safety and initiation of immune responses in the distal airways in preclinical studies is one of the reasons over a third of candidate inhaled compounds fail. • Highly vacuolated or "foamy" alveolar macrophages (FM) observed in pre-clinical rat studies poses safety concerns, despite not knowing the correlation to hindering progress of promising candidates into humans title Background: “foamy” macrophages in vivo in vitro Forbes et al., 2014
  • 49. © 2 0 2 4 IMMUONE • HCIA combines alveolar macrophage morphology/ lipid endpoints as an in vitro tool for inhaled safety assessment. • Morphometric endpoints that directly correlate with in vivo observation. title Purpose and methodology in vivo in vitro
  • 50. © 2 0 2 4 IMMUONE title HCIA screens HCA screens overview A - Cell Health and Morphology Assay • Nuclear staining (Hoechst 33342) • Mitochondrial membrane potential (MitoTracker Red) • Membrane permeability (Image-It Dead Green) • Cytoplasm staining (Cell Mask) B - Lipid content and Phagocytosis Assay • Nuclear staining (Hoechst 33342) • Phospholipidosis detection (Lipidtox Red) • Neutral lipids detection (Neutral lipid green) • Phagocytosis activity (Crimson beads) Lipid content staining 10 µM Imipramine, NR8383 Cell health staining, Air-treated, BAL
  • 51. © 2 0 2 4 IMMUONE title Median morphology parameters of untreated and amiodarone- treated rat and human macrophage cell models. U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0 5 10 15 20 Number of vacuoles (human) Number of vacuoles per cell U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0 5 10 15 Number of vaculoes (rat) Number of vacuoles per cell U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0 5 Numbe U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0 100 200 300 400 500 Cell area (human) Cell area [µm 2 ] U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0 Numb U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 100 120 140 160 180 200 220 Cell area (rat) Cell area [µm 2 ] Vacuolation Cell area Hoffman et al., 2017
  • 52. © 2 0 2 4 IMMUONE title Median lipid parameters of untreated and amiodarone-treated rat and human macrophage cell models. Neutral lipids Phospholipids U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0.00 0.05 0.10 Relative fluo Phos U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0.00 0.05 0.10 0.15 0.20 Neutral Lipids (human) Relative fluorescence intensity Neutral lipid Green U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0.0 0.1 Relative fluor Phosp U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0.0 0.1 0.2 0.3 0.4 0.5 Neutral Lipids (rat) Relative fluorescence intensity Neutral lipid Green U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0.00 0.05 0.10 0.15 0.20 0.25 Phospholipids (human) Relative fluorescence intensity Phospholipid Red U n t r e a t e d 2 4 h A m i o d a r o n e 2 4 h U n t r e a t e d 4 8 h A m i o d a r o n e 4 8 h 0.0 0.1 0.2 0.3 0.4 Phospholipids (rat) Relative fluorescence intensity Phospholipid Red Hoffman et al., 2017
  • 53. © 2 0 2 4 IMMUONE title Macrophage Responses to Amiodarone: ‘elevated’ responses • No significant difference (p > 0.05) was observed between untreated cells and those exposed to 10 μM amiodarone (known inducer of phospholipidosis) when comparing median values, despite observable differences in histogram profiles. • Average population statistical analysis was not a suitably sensitive descriptor to depict the morphology and lipid changes observed in cells exposed to amiodarone which impacted less than 20% of the total cell population. • Therefore, an alternative analytical approach which included only the cell population with elevated characteristics in comparison with the untreated cells was considered. Hoffman et al., 2017 Untreated Amiodarone
  • 54. © 2 0 2 4 IMMUONE title Macrophage Responses to Amiodarone: ‘elevated’ responses HCA screens overview Hoffman et al., 2017 Parameter Inclusion criteria Elevated mitochondrial activity % cells > mean mitochondrial activity + 2nd SD of the untreated cell population Elevated membrane permeability % cells > mean membrane permeability + 2nd SD of the untreated cell population Elevated cellular area % cells > mean cell area + 2nd SD of the untreated cell population Elevated vacuole number per cell % cells > mean vacuole number per cell + 2nd SD of the untreated cell population Elevated vacuole area per cell % cells > mean vacuole area per cell + 2nd SD of the untreated cell population Elevated phospholipid content % cells > mean phospholipid content per cell + 2nd SD of the untreated cell population Elevated neutral lipid content % cells > mean neutral lipid content per cell + 2nd SD of the untreated cell population **Summary of diagnostic criteria employed in foamy macrophage investigation. E Median value untreated F Median value amiodarone G Population of cells elevated number of vacuoles per cell
  • 55. © 2 0 2 4 IMMUONE title Cell health, morphology and lipid multi-parameter profiles after amiodarone exposure to rat macrophages 24h Hoffman et al., 2017 48h • In contrast to median value analysis, multi-parameter profile analysis of the elevated population characteristics revealed an impact on elevated phospholipid content and cell health markers as expected for the phospholipidosis inducer, amiodarone.
  • 56. title In vivo to in vitro translation © 2 0 2 4 IMMUONE • Rat alveolar macrophages were more sensitive to amiodarone exposure than human cells. • All alveolar lung macrophage cell models require analysis of cell sub- populations rather than average population data. • HCIA provides a method for profiling human and rat alveolar macrophage responses in vitro.
  • 57. © 2 0 2 4 IMMUONE • 12 compounds, categorized as FM- and non-FM-inducers. • In vitro tests deemed all compounds suitable for animal studies. • In vivo studies revealed that some compounds led to macrophage accumulation and FM responses. • Compounds were discontinued from further development due to potential human safety issues. title Profiling of alveolar macrophage responses to inhaled modalities
  • 58. © 2 0 2 4 IMMUONE title CrackIT: profiling of alveolar macrophage responses to inhaled modalities Non-FM-inducers FM-inducers salbutamol hemisulphate β2-agonist amiodarone hydrochloride cationic amphiphilic drug formoterol fumarate β2-agonist imipramine hydrochloride cationic amphiphilic drug dihydrate, salmeterol xinafoate β2-agonist chlorpromazine hydrochloride cationic amphiphilic drug ipratropium bromide muscarinic receptor antagonist dibucaine hydrochloride cationic amphiphilic drug beclomethasone dipropionate steroidal antiflammatory drug staurosporine Pro-apoptotic fluticasone propionate steroidal antiflammatory drug etoposide Pro-apoptotic budesonide steroidal antiflammatory drug campthotecin Pro-apoptotic
  • 59. © 2 0 2 4 IMMUONE title Multiparameter analysis of rat alveolar macrophages B C Rat macrophages treated in vitro with Amiodarone in various concentrations for 24 hours (A) 0µM (B) 10µM (C) 50µM InCell Analyser images, magnification 40x A
  • 60. © 2 0 2 4 IMMUONE title Multiparameter analysis of rat alveolar macrophages 0 50 100 150 24h 0 50 100 150 48h % Cell Population Untreated Salbutamol Formoterol Salmeterol Ipratropium Beclomethasone Fluticasone Budesonide t H e a l t h N u c A r e a N u c / C e l l A r e a M i t o I n t P e r m I n t C e l l A r e a # V a c % V a c A r e a / # V a c P h o s p h o l i p i d s N e u t r a l L i p i d s 24h Parameter 24h C e l l C o u n t C e l l H e a l t h N u c A r e a N u c / C e l l A r e a M i t o I n t P e r m I n t C e l l A r e a # V a c % V a c A r e a / # V a c P h o s p h o l i p i d s N e u t r a l L i p i d s 0 50 100 150 48h Parameter % Cell Population Untreated Salbutamol Formoterol Salmeterol Ipratropium Beclomethasone Fluticasone Budesonide 0 50 100 150 48h % Cell Population Untreated Amiodarone Imipramine Dibucaine Chlorpromazine Staurosporine Etoposide Campthotecin C e l l C o u n t C e l l H e a l t h N u c A r e a N u c / C e l l A r e a M i t o I n t P e r m I n t C e l l A r e a # V a c % V a c A r e a / # V a c P h o s p h o l i p i d s N e u t r a l L i p i d s 0 50 100 150 24h Parameter % Cell Population 0 50 100 150 24h % Cell Population C e l l C o u n t C e l l H e a l t h N u c A r e a N u c / C e l l A r e a M i t o I n t P e r m I n t C e l l A r e a # V a c % V a c A r e a / # V a c P h o s p h o l i p i d s N e u t r a l L i p i d s 0 50 100 150 48h Parameter % Cell Population Untreated Salbutamol Formoterol Salmeterol Ipratropium Beclomethasone Fluticasone Budesonide t h a a t t a c c c s s 0 50 100 150 48h % Cell Population Untreated Amiodarone Imipramine Dibucaine Chlorpromazine Staurosporine Etoposide Campthotecin Non-FM-inducers FM-inducers
  • 61. © 2 0 2 4 IMMUONE title Multiparameter analysis of human alveolar macrophages Human macrophages treated in vitro with amiodarone in various concentrations for 24 hours (A) 0µM (B) 1µM (C) 10µM (D) 50µM InCell Analyser images, magnification 40x A B C A B C D
  • 62. © 2 0 2 4 IMMUONE title Multiparameter analysis of human alveolar macrophages Non-FM-inducers FM-inducers C e l l C o u n t C e l l H e a l t h N u c A r e a N u c / C e l l A r e a M i t o I n t P e r m I n t C e l l A r e a # V a c % V a c A r e a / # V a c P h o s p h o l i p i d s N e u t r a l L i p i d s 0 50 100 150 24h Parameter % Cell Population 0 50 100 150 24h % Cell Population C e l l C o u n t C e l l H e a l t h N u c A r e a N u c / C e l l A r e a M i t o I n t P e r m I n t C e l l A r e a # V a c % V a c A r e a / # V a c P h o s p h o l i p i d s N e u t r a l L i p i d s 0 50 100 150 48h Parameter % Cell Population Untreated Salbutamol Formoterol Salmeterol Ipratropium Beclomethasone Fluticasone Budesonide t t t 0 50 100 150 48h % Cell Population Untreated Amiodarone Imipramine Dibucaine Chlorpromazine Staurosporine Etoposide Campthotecin e a e a I n t I n t e a a c a c a c d s d s 24h n t l t h e a e a I n t I n t e a a c a c a c d s d s 0 50 100 150 48h % Cell Population Untreated Salbutamol Formoterol Salmeterol Ipratropium Beclomethasone Fluticasone Budesonide I n t P e r m I n t C e l l A r e a # V a c % V a c A r e a / # V a c P h o s p h o l i p i d s N e u t r a l L i p i d s 24h Parameter 24h C e l l C o u n t C e l l H e a l t h N u c A r e a N u c / C e l l A r e a M i t o I n t P e r m I n t C e l l A r e a # V a c % V a c A r e a / # V a c P h o s p h o l i p i d s N e u t r a l L i p i d s 0 50 100 150 48h Parameter % Cell Population Untreated Salbutamol Formoterol Salmeterol Ipratropium Beclomethasone Fluticasone Budesonide t h a a t t a c c c s s 0 50 100 150 48h % Cell Population Untreated Amiodarone Imipramine Dibucaine Chlorpromazine Staurosporine Etoposide Campthotecin
  • 63. Summary © 2 0 2 4 IMMUONE • All alveolar lung macrophage cell models responded to challenge with amiodarone, but detection of these required analysis of cell sub-populations rather than average population data. • Human alveolar macrophages profiles showed that cells were less sensitive to stimuli. • HCIA-derived profiles differentiated between safe and unsafe responses in macrophages. • Multi-parameter methodology has the potential to offer an early non-clinical screening tool to predict the safety of candidate inhaled medicines. • The comparison of in vitro cell responses from a variety of rat ex vivo and human and rat in vitro models allows for a direct comparisons to be made.
  • 64. IN VITRO INNOVATION SERVICES FOR EMPOWERED DECISION MAKING © 2 0 2 4 IMMUONE ImmuONE provides innovative respiratory in vitro cell culture products and contract research services for inhalation safety testing. Our focus is supporting companies and researchers to understand the interaction and safety implications of inhaled products with immune cells in the airways.
  • 65. © 2 0 2 4 IMMUONE Text box Delivering in vitro solutions In vitro services for inhalation testing In vitro alveolar macrophage assays Immune-competent in vitro human airways models
  • 66. © 2 0 2 4 IMMUONE Contact us! info@immuone.com www.immuone.com ImmuONE in