2016 incucyte™ live-cell analysis webcasts smart … · revealing the interplay between cancer...

Revealing the interplay between cancer and immune cells

Tim O’Callaghan

Product Manager

Smart cell assays for immuno-oncology2016 INCUCYTE™ LIVE-CELL ANALYSIS WEBCASTS

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 contents





✔ Case studies


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WEBINAR: Smart Cell Assays for Immuno-Oncology

The promise

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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Cancer immunotherapy - the promise and need


Immuno-oncology drug development

Challenges and complexities

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





✔ Case studies


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Commonly used cell assays for immuno-oncology

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Flow Cytometry

ELISpot

51Chromium Release LDH

SEAP Reporter Assay

MTT

Boyden chamber

Typically:• End-point• Non image based• Indirect read outs• Cell processing

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✔ Real-time analysis

✔ Living non-perturbed cells

✔ Non-exhaustive

✔ Long term as required

✔ Applicable across workflows

Capture full response kinetics

Environmental control is key

Cells are not depleted

Hours, days, weeks

Culture, manipulation, assay

✔ Image-based measurements Visualize cell interactions, verify metrics

Continuous live-cell imaging and analysis Image-based continuous (or semi-continuous) cellular measurements


Continuous live-cell analysisComparison verses end-point workflow

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Endpoint

Single time point

Continuous

e.g. Cell Health,

Morphology, Growth

e.g. Transfection, Plating,

Pre-treatments

Kinetic assay

Culture Manipulate Assay


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Automated real-time analysis

Fits into existing workflows

Image based measurements

Flexibility

Environmental control

Capacity, throughput

• Critical for longer term studies• e.g. within incubator

• Culture, manipulations & assays• Cells are not exhausted

• Run experiments in parallel • Multi-vessel, multi-user

• Measure full kinetic response• No sample prep, no end point

• Optics, readouts, cells, vessels • Multiplex assay readouts

• Characterise cell interactions• Validate measurements

Key features for live-cell analysis systems


Non-perturbing to cells

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Research flexibility Protocols for live-cell assays

Viable cell analysis 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

Reagents for live-cell analysis


✔ Multiplex reagents for additional insight

Webinar contents





✔ Case studies


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Continuous live-cell analysis for I-O research

✔ Continuously monitor & analyze key stages of immune response to cancer

Immune-cellkilling

Proliferation

Cell migration& invasion

Clustering & Proliferation

ChemotaxisTEM

Phagocytosis

Tumor cellsGrowth & metastasis

Immune cellsActivation & homing

Co-culturesKilling & Clearance


0 24 48 7220

40

60

80

100

Time (h)

Ph

ase o

bje

ct

co

nfl

uen

ce (%

)

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Assay principle Treatment 2

Control

Control

Treatment 2

Images and data generated with the IncuCyte ZOOM® live cell analysis system

Tumor cell growth and proliferation

✔ Label-free measures of growth/growth inhibition

✔ Rapidly profile multiple donor cells, isogenics cell lines etc

Treatment 1


Cell

co

nfl

uen

ce (

%)

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Assay principle


Tumor cell growth and proliferation

✔ Real-time viable cell counting in mono-/co-cultures

✔ Investigate the effect of micro-environment on tumor cell viability

SK-BR-3 Co-culture + Lapatinib

SK-BR-3 Monoculture + Lapatinib

NucLight™ Red labeled tumor cells


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Assay principle

Tumor cell migration and invasion

✔ Create scratch wounds in seconds in 96-well plates

✔ No labels required, amenable to wide range of cells

SEED CELLS

CREATE WOUNDS

ECM OVERLAY

LIVE CELL ANALYSIS

WoundMaker™96-pin wound making tool

ImageLock™ 96-well PlatesFor precision repeat imaging


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Assay principle


Tumor cell migration and invasion

✔ Compare migration and invasion profiles in one 96-well plate

✔ Investigate how treatments alter metastatic potential

SEED CELLS

CREATE WOUNDS

ECM OVERLAY

LIVE CELL ANALYSIS

Migration

Invasion (Matrigel)


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Immune-cellkilling

Proliferation



ChemotaxisTEM

Phagocytosis





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Assay principle


Immune cell activation: clustering and proliferation

✔ Monitor proliferation and clustering in real-time

✔ Label free, kinetic measurements

Non-Activated


Activated: Anti-CD3/IL2

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Assay principle

Chemotaxis and transendothelial migration

✔ Visualize and quantify migration toward chemoattractants

✔ No labels required, low cell usage

ClearView™96-well chemotaxis plate

COAT MEMBRANE

ADD CELLS & TREATMENT

ADD CHEMOATTRACTANT


-1 0 -9 -8 -7 -6

0

2 5

5 0

7 5

1 0 0

lo g [S D F -1 ] (M )

% M

igra

tio

n (

6 h

r)

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Assay principle

Chemotaxis and transedothelial migration

✔ Automatically monitor entire time course of chemotaxis

✔ Investigate migration across ECM or through an endothelial monolayer

COAT MEMBRANE

ADD CELLS & TREATMENT

ADD CHEMOATTRACTANT

Neutrophil Chemotaxis to IL-8 T Cell TEM to SDF-1α

[IL-8]


0 2 4 6

0 .0

0 .4

0 .8

1 .2

1 1 2 0 n M

3 7 3 .3 3 n M

1 2 4 .4 n M

4 1 .5 n M

1 3 .8 n M

4 .6 n M

1 .5 n M

C o n tro l

T im e (h )

No

rm

ali

se

d T

op

Su

rfa

ce

Co

nfl

ue

nc

eC

ell A

rea

(T

op

Su

rfa

ce

) N

orm

alize

d t

0h

EC50 17.5 nM


% T

EM

(6

h)

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Immune-cellkilling

Proliferation



ChemotaxisTEM

Phagocytosis





0 3 0 6 0 9 0 1 2 0

0

5 0 0

1 0 0 0

1 5 0 0

2 0 0 0

2 5 0 0

T im e (h )

Via

ble

tu

mo

r c

ell

co

un

t (r

ed

nu

cle

i p

er m

m2)

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Assay principle

Images and data generated with the IncuCyte ZOOM® live cell analysis system (SK-OV-3 cells)

Immune cell killing - T cell killing, ADCC

✔ Direct measurements of tumor cell death and viability

✔ Highly flexible format – use your choice of effector/target cells

Activated: Anti-CD3/IL-2

Non-Activated

SEED TARGET CELLS

ADD TREATMENTS

ADD IMMUNE CELLS

Caspase-3/7 ReagentNucLight™ Red Reagent

0 3 0 6 0 9 0 1 2 0

0

2 5

5 0

7 5

1 0 0

1 2 5

1 5 0

1 7 5

T im e (h )

Ca

sp

as

e-3

/7 o

bje

ct

co

un

t (p

er m

m2)

Non-Activated

Activated: Anti-CD3/IL-2


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Assay principle

Images and data generated with the IncuCyte ZOOM® live cell analysis system (Jurkats)

Cell clearance - efferocytosis

✔ Quantify & validate phagocytosis using time-lapse imaging

✔ Simple labeling of your choice of target cells

Apoptotic Target Cells

Non-Apoptotic Target Cells

0 6 1 2 1 8 2 4

0 .0

0 .5

1 .0

1 .5

2 .0

T im e (h )

To

tal

Re

d O

bje

ct

Are

a x

10

4 (

µm

²/Im

ag

e)

Non-Apoptotic Target Cells

Apoptotic Target Cells

IncuCyte™ pHrodo® Red Cell Labeling Kit


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Assay principle

Bone marrow derived macrophagesCCRF-CEM T lymphoblasts

Cell clearance - antibody mediated cell phagocytosis

✔ Visualize and measure antibody mediated phagocytosis in real time

✔ Anti-CD47 and antibody dependent cellular phagocytosis (ADCP)

IncuCyte™ pHrodo® Red Cell Labeling Kit

CD47 blockade - uncloaking tumor cells



Webinar contents





✔ Case studies


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Zumwalde, N. et al. J. Immunol. 191, 3681–93 (2013).

Visual analysis of cluster formation

• 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

Automated analysis of T cell clustering kineticsSingle cell clustering Cluster aggregation

Case study 1:

Probing the mechanisms of T cell activation

Zumwalde, N. A. PhD Dissertation, University of Minnesota (2013)



http://www.ncbi.nlm.nih.gov/pubmed/23997225

http://conservancy.umn.edu/handle/11299/151553

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McCormack, E. et al. Cancer Immunol. Immunother. 62, 773–85 (2013).

Donor T cell variability influences killing kinetics

• 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

Bispecific T cell engagers induce dose dependent & selective killing

Case study 2:

Tumor cell killing by redirected T cells

Adams, K. PhD Dissertation, Cardiff University (2013)


Images and data generated with the IncuCyte ZOOM® live cell analysis system Images and data generated with the IncuCyte ZOOM® live cell analysis system

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3624013&tool=pmcentrez&rendertype=abstract

http://orca.cf.ac.uk/47578/

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Kinetic monitoring of T cell exhaustionLive-cell analysis correlates with flow cytometry

Case study 3:

Tumor cell killing by CAR T cells

No modulation of CAR T cell ‘serial killing’



Ligand modulation of CAR T cell ‘serial killing’

No ligand

With ligand

Data provided by an immunotherapy biopharma company Data provided by an academic IncuCyte® customer

CTRL

TD1

TD2

TD3

TD4

No T cells

CTRL

TD1

TD2

TD3

TD4

No T cells

PRODUCTIVITY

Value of continuous live-cell imaging and analysis

Real time observation and measurement

Full time-course – capture response kinetics

Visualise cell interactions -make direct measurements

Multiplex readouts & cell morphology build confidence

Images & movies verify biology

Workflow enhancements

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

BIOLOGICAL INSIGHT

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Wrap up and perspective

✔ I-O drug development requires an understanding of immune and tumor cell response kinetics

✔ Common cell based assays lack temporal resolution of cellular events & visualization of cell activity

✔ 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

✔ 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

✔ Follow up e-mail:

Webcast Recording

Copy of Webinar Slides (pdf)

✔ Learn more:

essenbioscience.com/communications/immuno-oncology/

✔ Live cell imaging & analysis publications

essenbioscience.com/publications

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Questions and comments?

✔Contact us at: [email protected]

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2016 incucyte™ live-cell analysis webcasts smart … · revealing the interplay between cancer...

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