Download Smart cell assays for immuno-oncology

2016 INCUCYTE™ LIVE-CELL ANALYSIS WEBCASTS
Smart cell assays for immuno-oncology
Revealing the interplay between cancer and immune cells
Tim O’Callaghan
Product Manager
Webinar contents
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Cancer immunotherapy - the promise, the need & challenges
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Common immune cell assays
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Continuous live-cell imaging & analysis
✔
New assays & reagents for immuno-oncology
✔
Case studies
✔
Summary & perspective
2
Webinar contents
✔
Cancer immunotherapy - the promise, the need & challenges
✔
Common immune cell assays
✔
Continuous live-cell imaging & analysis
✔
New assays & reagents for immuno-oncology
✔
Case studies
✔
Summary & perspective
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Cancer immunotherapy - the promise and need
The promise
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Harnessing the immune system to fight cancer
Immune specificity and memory
Potential for safer, more effective treatments
Recent preclinical and clinical success
The need
 I-O drugs have shown success in the clinic
 However many patients need next gen treatments
 Non-responders
 Relapse
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Immuno-oncology drug development
Challenges and complexities
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Tumor heterogeneity and evolution
Variable and complex response kinetics
Off target activity
Combination therapies
Opportunity for in vitro cell based assays
 Ready access to relevant immune cell types
 Valuable in vitro & ex vivo studies
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Webinar Contents
✔
Cancer immunotherapy - the promise, the need & challenges
✔
Common immune cell assays
✔
Continuous live-cell imaging & analysis
✔
New assays & reagents for immuno-oncology
✔
Case studies
✔
Summary & perspective
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Commonly used cell assays for immuno-oncology
Boyden chamber
SEAP Reporter Assay
ELISpot
51Chromium
Flow Cytometry
Release
LDH
MTT
Typically:
• End-point
• Non image based
• Indirect read outs
• Cell processing
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Continuous live-cell imaging and analysis
Image-based continuous (or semi-continuous) cellular measurements
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Real-time analysis
Capture full response kinetics
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Image-based measurements
Visualize cell interactions, verify metrics
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Living non-perturbed cells
Environmental control is key
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Non-exhaustive
Cells are not depleted
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Long term as required
Hours, days, weeks
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Applicable across workflows
Culture, manipulation, assay
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Continuous live-cell analysis
Comparison verses end-point workflow
Culture
Manipulate
Assay
Endpoint
Single time point
Continuous
e.g. Cell Health,
Morphology, Growth
e.g. Transfection, Plating,
Pre-treatments
Kinetic assay
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Key features for live-cell analysis systems
Capacity, throughput
Automated real-time analysis
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•
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Run experiments in parallel
Multi-vessel, multi-user
Measure full kinetic response
No sample prep, no end point
Environmental control
Image based measurements
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•
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Critical for longer term studies
e.g. within incubator
Fits into existing workflows
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Culture, manipulations & assays
Cells are not exhausted
Characterise cell interactions
Validate measurements
Flexibility
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Optics, readouts, cells, vessels
Multiplex assay readouts
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Reagents for live-cell analysis
Non-perturbing to cells
Viable cell analysis
Research flexibility
Protocols for live-cell assays
Dead cell analysis
Cell clearance
Cell counting
Cytolysis
Apoptosis
Phagocytosis
Genetically encoded fluorescent proteins
Membrane impermeable dyes
Enzyme activated/PS probes
pH sensitive membrane dye
NucLight™ BacMam 3.0
Cytotox Reagents
Caspase-3/7 Reagent
pHrodo® Cell Labeling Kit
NucLight™ Lenti
Annexin V Reagents
✔ Multiplex reagents for additional insight
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Webinar contents
✔
Cancer immunotherapy - the promise, the need & challenges
✔
Common immune cell assays
✔
Continuous live-cell imaging & analysis
✔
New assays & reagents for immuno-oncology
✔
Case studies
✔
Summary & perspective
12
WEBINAR: Smart Cell Assays for Immuno-Oncology
Continuous live-cell analysis for I-O research
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Tumor cells
Growth & metastasis
Immune cells
Activation & homing
Co-cultures
Killing & Clearance
Proliferation
Clustering &
Proliferation
Immune-cell
killing
Cell migration
& invasion
Chemotaxis
TEM
Phagocytosis
Continuously monitor & analyze key stages of immune response to cancer
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Tumor cell growth and proliferation
Treatment 2
100
Control
80
(%) (%)
confluence
Cell
Phase
object
confluence
Assay principle
Treatment 1
60
Control
40
Treatment 2
20
0
24
48
72
Time (h)
Images and data generated with the IncuCyte ZOOM® live cell analysis system
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Label-free measures of growth/growth inhibition
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Rapidly profile multiple donor cells, isogenics cell lines etc
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Tumor cell growth and proliferation
NucLight™ Red labeled tumor cells
Assay principle
SK-BR-3 Co-culture + Lapatinib
SK-BR-3 Monoculture + Lapatinib
Images and data generated with the IncuCyte ZOOM® live cell analysis system
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Real-time viable cell counting in mono-/co-cultures
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Investigate the effect of micro-environment on tumor cell viability
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Tumor cell migration and invasion
Assay principle
SEED CELLS
ImageLock™ 96-well Plates
For precision repeat imaging
CREATE WOUNDS
ECM OVERLAY
WoundMaker™
96-pin wound making tool
LIVE CELL ANALYSIS
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Create scratch wounds in seconds in 96-well plates
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No labels required, amenable to wide range of cells
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Tumor cell migration and invasion
Assay principle
Migration
SEED CELLS
CREATE WOUNDS
Invasion (Matrigel)
ECM OVERLAY
LIVE CELL ANALYSIS
Images and data generated with the IncuCyte ZOOM® live cell analysis system
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Compare migration and invasion profiles in one 96-well plate
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Investigate how treatments alter metastatic potential
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Continuous live-cell analysis for I-O research
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Tumor cells
Growth & metastasis
Immune cells
Activation & homing
Co-cultures
Killing & Clearance
Proliferation
Clustering &
Proliferation
Immune-cell
killing
Cell migration
& invasion
Chemotaxis
TEM
Phagocytosis
Continuously monitor & analyze key stages of immune response to cancer
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Immune cell activation: clustering and proliferation
Activated: Anti-CD3/IL2
Assay principle
Non-Activated
Images and data generated with the IncuCyte ZOOM® live cell analysis system
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Monitor proliferation and clustering in real-time
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Label free, kinetic measurements
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Chemotaxis and transendothelial migration
Assay principle
COAT MEMBRANE
ADD CELLS & TREATMENT
ADD CHEMOATTRACTANT
ClearView™
96-well chemotaxis plate
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Visualize and quantify migration toward chemoattractants
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No labels required, low cell usage
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Chemotaxis and transedothelial migration
Assay principle
T Cell TEM to SDF-1α
Neutrophil Chemotaxis to IL-8
COAT MEMBRANE
ADD CELLS & TREATMENT
100
C o n tr o l
1 .5 n M
4 .6 n M
1 3 .8 n M
4 1 .5 n M
1 2 4 .4 n M
0 .4
3 7 3 .3 3 n M
% TEM (6h)
[IL-8]
0 .8
% M ig ra tio n (6 h r)
ADD CHEMOATTRACTANT
ce
e C o n flu e nt0h
p S u r fa cNormalized
d T oSurface)
a lis e(Top
o r mArea
NCell
Images and data generated with the IncuCyte ZOOM® live cell analysis system
1 .2
75
50
25
EC50 17.5 nM
1120 nM
0 .0
0
2
4
T im e ( h )
6
0
-1 0
-9
-8
-7
-6
lo g [S D F -1  ] (M )
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Automatically monitor entire time course of chemotaxis
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Investigate migration across ECM or through an endothelial monolayer
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Continuous live-cell analysis for I-O research
✔
Tumor cells
Growth & metastasis
Immune cells
Activation & homing
Co-cultures
Killing & Clearance
Proliferation
Clustering &
Proliferation
Immune-cell
killing
Cell migration
& invasion
Chemotaxis
TEM
Phagocytosis
Continuously monitor & analyze key stages of immune response to cancer
22
WEBINAR: Smart Cell Assays for Immuno-Oncology
Immune cell killing - T cell killing, ADCC
Assay principle
Caspase-3/7 Reagent
NucLight™ Red Reagent
Activated: Anti-CD3/IL-2
175
2500
Activated: Anti-CD3/IL-2
ADD TREATMENTS
Non-Activated
2
e rmm2 )m )
leei rpm
ll coobujenct t(rceodu nnut c(p
V ia b le
s pmaosre c-3e/7
C atu
150
SEED TARGET CELLS
2000
125
1500
100
75
Non-Activated
1000
50
500
25
ADD IMMUNE CELLS
00
00
3
30
0
60
0
6
T im
im e
e (h
(h ))
T
99 00
11 22 00
Images and data generated with the IncuCyte ZOOM® live cell analysis system (SK-OV-3 cells)
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Direct measurements of tumor cell death and viability
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Highly flexible format – use your choice of effector/target cells
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Cell clearance - efferocytosis
IncuCyte™ pHrodo® Red Cell Labeling Kit
Assay principle
Apoptotic Target Cells
1 .5
Apoptotic Target Cells
T o ta l R e d O b je c t A r e a x 1 0
4
(µ m ²/Im a g e )
2 .0
1 .0
Non-Apoptotic Target Cells
0 .5
Non-Apoptotic Target Cells
0 .0
0
6
12
18
24
T im e (h )
Images and data generated with the IncuCyte ZOOM® live cell analysis system (Jurkats)
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Quantify & validate phagocytosis using time-lapse imaging
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Simple labeling of your choice of target cells
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Cell clearance - antibody mediated cell phagocytosis
IncuCyte™ pHrodo® Red Cell Labeling Kit
Assay principle
CD47 blockade - uncloaking tumor cells
Images and data generated with the IncuCyte ZOOM® live cell analysis system
Bone marrow derived macrophages
CCRF-CEM T lymphoblasts
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Visualize and measure antibody mediated phagocytosis in real time
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Anti-CD47 and antibody dependent cellular phagocytosis (ADCP)
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Webinar contents
✔
Cancer immunotherapy - the promise, the need & challenges
✔
Common immune cell assays
✔
Continuous live-cell imaging & analysis
✔
New assays & reagents for immuno-oncology
✔
Case studies
✔
Summary & perspective
26
WEBINAR: Smart Cell Assays for Immuno-Oncology
Case study 1:
Probing the mechanisms of T cell activation
Automated analysis of T cell clustering kinetics
Visual analysis of cluster formation
Single cell clustering
Zumwalde, N. et al. J. Immunol. 191, 3681–93 (2013).
Cluster aggregation
Zumwalde, N. A. PhD Dissertation, University of Minnesota (2013)
Images and data generated with the IncuCyte ZOOM® live cell analysis system
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ICAM-1 KO and anti–LFA-1 integrin blockade inhibit the formation of T cell activation clusters
Long-term, automated, image-based analysis enable direct phenotypic measurements challenging by other technologies
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Case study 2:
Tumor cell killing by redirected T cells
Bispecific T cell engagers induce dose dependent & selective killing Donor T cell variability influences killing kinetics
McCormack, E. et al. Cancer Immunol. Immunother. 62, 773–85 (2013).
Images and data generated with the IncuCyte ZOOM® live cell analysis system
•
•
Adams, K. PhD Dissertation, Cardiff University (2013)
Images and data generated with the IncuCyte ZOOM® live cell analysis system
Selective analysis of target cell apoptosis to determine ImmTAC potency and specificity
Directed killing can be missed in short-term assays e.g. 51Cr- and extended LDH-release assays
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Case study 3:
Tumor cell killing by CAR T cells
Data provided by an immunotherapy biopharma company
Live-cell analysis correlates with flow cytometry
Data provided by an academic IncuCyte® customer
Kinetic monitoring of T cell exhaustion
No modulation of CAR T cell ‘serial killing’
CTRL
TD1
TD2
TD3
TD4
No T cells
No ligand
Ligand modulation of CAR T cell ‘serial killing’
CTRL
TD1
TD2
TD3
TD4
No T cells
With ligand
Images and data generated with the IncuCyte ZOOM® live cell analysis system
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WEBINAR: Smart Cell Assays for Immuno-Oncology
Value of continuous live-cell imaging and analysis
Real time observation and measurement
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Full time-course – capture response kinetics
Visualise cell interactions -make direct measurements
Multiplex readouts & cell morphology build confidence
Images & movies verify biology
BIOLOGICAL INSIGHT
Workflow enhancements
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Automated real time image gathering & analysis
96 & 384-well assays (> 1000 data points per hour)
Cells still viable for follow on studies e.g. Flow, ELISAs
Multi-vessel, multi-user
PRODUCTIVITY
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Wrap up and perspective
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I-O drug development requires an understanding of immune and tumor cell response kinetics
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Common cell based assays lack temporal resolution of cellular events & visualization of cell activity
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Continuous live-cell analysis enables real-time and direct cell based measurements providing
valuable biological insight and enhanced productivity for I-O drug researchers around the world
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Novel non-perturbing, live-cell reagents enable real-time multiplexed measures of immune cell
response to cancer
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Further information and next steps
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Follow up e-mail:
Webcast Recording
Copy of Webinar Slides (pdf)
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Learn more:
essenbioscience.com/communications/immuno-oncology/
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Live cell imaging & analysis publications
essenbioscience.com/publications
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Questions and comments?
✔Contact us at: [email protected]
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