Leyna Zhao Ph D ACEA Biosciences Inc San Diego California USA

Impedance‐Based
Using Impedance
Based Approaches for Measuring Cell
Using
Impedance
Based Approaches for Measuring Cell‐Mediated
Cell Mediated Mediated
Cytotoxicity and Antibody
Antibody‐Dependent
Cell‐Mediated
Cytotoxicity and Antibody
Dependent Cell
Mediated Cytotoxicity (ADCC)
Dependent Cell
Mediated Cytotoxicity (ADCC)
Leyna Zhao, Ph.D. ACEA Biosciences Inc. San Diego, California. USA
Leyna
Zhao,
Zhao Ph.D.
Ph D ACEA Biosciences Inc.
Inc San Diego,
Diego California.
California USA
INTRODUCTION
The most common method for measuring cell
mediated cytotoxicity is the release assay based on
h lloss off target
b
h
g cellll membrane
g y Within
the
integrity.
up to 4 hours following effector cell addition
resulting in target cell lysis,
lysis either the radioactive
release from target
g
cells p
pre‐labeled with
Chromium 51 or Indium‐111
Chromium‐51
Indium 111 is measured,
measured or the
release of naturally occurring substances,
substances such as
lactate dehydrogenase ((LDH),
H), into the culture
medium
di
i assayed.
is
d Release
R l
off these
th
substances
bt
thus serves as an indirect measure of the extent of
cell damage due to effector cell
cell‐mediated
mediated target cell
l i
lysis.
Alt
Alternative
ti endpoint
d i t methods
th d also
l include
i l d
flow cytometry,
cytometry enzyme‐linked immunosorbent
assay‐based
assay
based
granzyme
measurement,
and
morphometric
h
t i analyses
l
b microscopy.
by
i
Here we describe an impedance
impedance‐based
based real
real‐time
time
l b lf
label‐free
method
th d that
th t can automated
t
t d capture
pt
th
the
kinetics of the cell mediated or antibody‐dependent
antibody dependent
cell mediated cytolysis (ADCC) of the target cancer
cells.
ll
T determine
To
d t
i
if cell‐mediated
ll
di t d cytotoxicity,
yt t i ity
and specifically ADCC,
ADCC can be investigated using an
impedance
based
xCELLigence
system,
the
impedance‐based
response
off tumor
cells
p
t
ll ((as target
t g t cells)
ll ) to
t natural
t l
cells) in the
killer (NK) cell activity (as effector cells),
presence or absence of immunoglobulin G isotype
isotype‐
specific
p ifi antibody,
ib dy, was measured.
d Importantly,
I p
ly, it
i is
i
shown that the addition of NK cells in suspension,
suspension
over a monolayer of adherent tumor cells,
cells does not
produce
p
d
i p d
impedance
changes,
h g , because
b
the
h NK cells
ll
do not come in contact with the electronic sensor.
sensor
However the secretion of perforins and granzymes
However,
by these
by
h
non‐adherent
dh
NK cells
ll does
d
activate
i
caspases
inducing
tumor
cell
apoptosis
apoptosis.
Dysfunctional and dying tumor cells detach from
the
h sensor electrode,
l
d , reducing
d
g the
h number
b off viable
bl
and adhering cells on the electrode surface.
surface
Overall,
ll, our results
l show
h
that
h the
h impedance‐base
p
b
technology is a label‐free
label free alternative to the
traditional end‐point assays.
The automated
assays
readouts p
provide direct,, sensitive and specific
p
meas rement of target cell changes both short‐
short
measurement
(days) It allows the easy
term (hrs) and long‐term (days).
quantification of the cell‐mediated
cell mediated cytotoxicity and
evaluation
l ti off the
th potencies
t i for
f specific
ifi antibodies.
tib di
RESULTS
1. Dynamic Monitoring of NK Cell‐mediated Cytolysis
1
Dy
i M it i g f NK C ll
di t d Cyt ly i
A
B
C D
B
C
D
Fi 1 (A) Dynamic monitoring of NK cell‐mediated cytolysis of NIH 3T3 cells. Mnk
Fig 1. (A) D
i
it i
f NK ll
di t d t l i f NIH 3T3 ll M k cells or YAC ll
YAC
( g
(negative control) cells were added to each well in triplicate. (B) Time‐dependent cytolytic )
p
( )
p
y y
activity of mNK cells. The cytolytic activity at a given time point was calculated and presented activity of mNK
cells The cytolytic activity at a given time point was calculated and presented
as the percentage of cytolysis (C) The morphological examination of cytolysis by mNK
th
t
f t l i (C) Th
h l i l
i ti
f t l i b NK cells. (D) ll (D)
The xCELLigence RTCA SP system in a conventional tissue culture incubator.
g
y
3. Real
3.
Real‐time,
time, label
label‐free
free monitoring of NK92 cell
monitoring of NK92 cell‐mediated
mediated cytolysis of cytolysis of
DU145 ll
DU145 cells
A
Fig 2. (A) Quantitative measurement of cytolytic activity of mNK
Fig 2. (A) Quantitative measurement of cytolytic activity of mNK cells on NIH 3T3 target cells. (B) Time
cells on NIH 3T3 target cells. (B) Time‐
dependent cytolytic activity of mNK
dependent
cytolytic activity of mNK cells at different E/T ratios. (C) Quantitative measurement of cytolytic cells at different E/T ratios (C) Quantitative measurement of cytolytic
activity of NK92 cells on MCF7 target cells. (D) Time‐dependent cytolytic activity of NK92 cells at different E/T i i
f 92 ll
C
ll ( ) i
d
d
l i
i i
f 92 ll
diff
/
ratios.
f
5. Label‐free
5. Label
free assessment of NK cell
assessment off NK cell‐mediated
mediated cytolysis using a variety of target cell cytolysis using a variety off target cell
lines
li
A
B
B
Fig 5 (A) The NK92
Fig 5. (A) The NK92‐mediated
mediated cytolysis of 7 different cancer lines. The percentage of cytolysis indicated for each cytolysis of 7 different cancer lines The percentage of cytolysis indicated for each
liline is calculated based on the Cell Index value of individual wells 8 hours after NK92 cells were added; cytolysis i
l l t db d
th C ll I d
l
f i di id l ll 8 h
ft NK92 ll
dd d t l i
reached a maximum at that time. (B) The mNK‐mediated cytolysis of 9 different cell lines. The percentage of ( )
y y
p
g
cytolysis indicated for each line is calculated based on the Cell Index values of individual wells 12 hours after
cytolysis indicated for each line is calculated based on the Cell Index values of individual wells 12 hours after mNK
NK cells were added; cytolysis reached a maximum at that time.
ll
dd d t l i
h d
i
t th t ti
Fig 3. (A) This plot shows data normalized to the last time point before NK cell addition. (B) (A) This plot shows data normalized to the last time point before NK cell addition. (B)
Fig 3. Same data as (A), but with non‐treated control cells (red) defined as baseline. Plots were Same
data as (A) but with non treated control cells (red) defined as baseline Plots were
generated using RTCA Software 1.1.
d
f
4
Effect of anti‐IGF‐1R Ab on
NK cell‐mediated cytolysis of DU145
4. Effect of anti‐IGF‐1R Ab
on NK cell‐mediated cytolysis of DU145 cells (E:T
2.75:1)
cells (E:T=2.75:1)
METHODS
SUMMARY
The present study shows the feasibility for using the xCELLigence System to monitor both
cell‐mediated
cell‐mediated
NK cell
mediated tumor cell cytolysis and antibody dependent cell
mediated cytotoxicity
non‐invasive
findings
by Gl
Glamann
(ADCC) iin a llabel‐free,
b lf
i
i manner, corroborating
b ti earlier
li fi
di
b
and Hansen (2006)
(2006). It was possible to show both the quantitative effect of adding
different amounts of NK92 cells, as well as the additive cytolytic effect of introducing an
NK92‐dependent
NK92 d
d t anti‐IGF‐1R
ti IGF 1R monoclonal
l
l antibody.
tib d
The xCELLigence System is thus ideal for directly monitoring ADCC without the need for
labeling the target cells or using a chemical reporter. Importantly, the entire cytolytic
process can now be
p
b measured
d using
i g impedance
i p d
electronic
l t i sensing
i g to
t detect
d t t both
b th
expected and unexpected cellular responses,
responses an impossible task for previously described
conventional end
end‐point
point assay formats.
Adherent Adherent
Target Cells
Target Cells
B
Add Effector
Cells
dd ff
ll
C
The xCELLigence Real Time Cell Analyzer
The xCELLigence Real Time Cell Analyzer
Dead Target D
dT
Cells
C ll
A
B
A
Microelectrode
2. Label‐free and quantitative measure of cytolytic activity of mNK
2
L b lf
dq
tit ti
f yt lyti
ti ity f NK cells and NK92 ll
d NK92
cells
C
Cytolysis
t l i
E Plate 96
E‐Plate 96
Fig 4.
g ((A) Plot of normalized CI values of the entire 88 hours of the experiment. Data are )
p
normalized to the last time point before NK cell addition and curves were plotted with control
normalized to the last time point before NK cell addition and curves were plotted with control wells (DU145 cells only) set as baseline. (B) Calculation of IC50 after 60 hours. (C) Calculation of ll ( U14
ll
l )
b li
( )C l l i
f IC 0 f 60 h
(C) C l l i
f
time‐dependent IC50 from 9 hours after NK cell addition to the end of the experiment at 88 hours. p
p
All plots were generated using the RTCA Software
All plots were generated using the RTCA Software.
SELECTED PUBLICATIONS
SELECTED
PUBLICATIONS
1 Determining optimal cytotoxic activity of human Her2neu specific CD8 T cells by comparing
1.
the
h Cr51
C 51 release
l
assayy to the
h xCELLigence
CELLig
system.
y
E ki CL,
Erskine
CL, Henle
H l AM,
AM, Knutson
K
KL J Vis
KL.J
Vi
Exp 2012 Aug 8;(66):e3683
Exp.
2. Real‐time p
profilingg of NK cell killingg of human astrocytes
y
usingg xCELLigence
g
technology.
gy
K Angel CE,
CE Glass M,
M Graham ES.
ES J Neurosci Methods.
Methods 2011 Sep 15;200(2):173‐80
15;200(2):173 80
Moodley K,
3. Unique functional status of natural killer cells in metastatic stage IV melanoma patients and
3
its
Fregni
it modulation
d l ti by
b chemotherapy.
h
th
F
i G,
G Perier
P i A,
A Pittari
Pitt i G,
G Jacobelli
J b lli S,
S Sastre
S t X,
X Gervois
G
i N,
N
Allard M,
M Bercovici N,
N Avril MF,
MF Caignard A.
A Clin Cancer Res.
Res 2011; 17(9); 2628
2628–37
37.