Application Note

Topic:CAR T,
Immuno-oncology
Cells:
A549, SKOV3CAR T cell-mediated killingof cancer cells

Tools:AtlaZ

Nanion Technologies GmbH, Munich.
TUM, Munich.

Summary
The US Food and Drug Administration has approved a number

the methodology behind the AtlaZ system, in combination withof chimeric antigen receptor (CAR) T-cell therapies. Due

the throughput of 6 x 96-wells allows for a so far unmet quantity

to the nature of CAR T cells as “living drugs”, they display

and richness of information which can be gained from cells.

a unique toxicity profile. As CAR T-cell therapy is extending
towards multiple diseases and being broadly employed inhematology and oncology, being able to reliably predict
treatment efficacy and a quantification of responses are ofhigh relevance. Furthermore, for continued breakthroughs,
novel CAR designs are needed. This includes different antigenbinding domains such as antigen-ligand binding partners
and variable lymphocyte receptors (1). Now, after amazing
advances for treating blood cancers, CAR T cell therapy is
showing promise for solid tumors.
In general, identifying T cells that kill cancer cells in vivo is
critical to the development of successful cell therapies. The
label-free AtlaZ immune cell killing assay can be used tomeasure rate of killing at Effector:Target (E:T) ratios to predictin vivo activity. In order to gain a deeper understanding ofcancer cells, real-time and continuous monitoring is necessary
to access kinetic and phenotypic information. Such monitoring

We investigated A549, adenocarcinomic human alveolar
basal epithelial cells, and SKOV3 cells which are a humanovarian carcinoma cell line. In principle, addition of effector
T-lymphocyte cells are co-cultured with the cancer cells,
and cytolysis of the target cells can be measured. In ourassay, were used CAR T-cells directed to the receptor
EGFR on the target cells. Those CAR T-cells exhibit anantigen-induced cell-mediated cytotoxicity by triggering
intracellular pathways leading to apoptosis of target A549
or SKOV3 cells. We observed a time- and ratio-dependent
decrease of the Cell Signal, representing apoptosis ofcarcinoma cells.
The impedance of planar gold-film electrodes that are used
as growth substrate for adherent cells reveals changes inelectrode coverage or cell behavior, e.g. upon treatment
with CAR T cells (Figure 1). Real-time impedance data provide

captures also unique toxicity profiles of CAR T cells.
The platform used here, AtlaZ, is a quantitative live-cell analysis

ACancer cellsystem and allows for cellular research on cell adhesion
and proliferation, cytotoxicity, GPCR, morphology andCAR T cellbarrier function, label-free and in real-time. Recordings canbe performed in up to six 96-well plates simultaneously orindependently. Electrical impedance spectroscopy (2, 3) as

B

Quantitative testing of CAR T cells

CAR T or compound-induced effect:cell detachment

Cell Signal

Figure 1: A CAR T cells are genetically altered T

cell confluency

loss of tight junctions
loss of membrane integrity

cell proliferation
cell adhesion

cells which can locate and destroy cancer cells
effectively. B The Cell Signal value offers information
on cell adherence, proliferation or cell death ofcancer cells upon treatment with CAR T cells.

Time (h)Electrode

Electric current

Suspended cell

Adherent cell

insights in various cell phenotypes, such as cell morphology,

completely removed the medium in which target cells haveproliferation, lateral migration or cytotoxicity even overbeen seeded). The impedance signal of the cell monolayer

prolonged periods of time.

was recorded every 2 minutes, and is plotted using the unitlessparameter Cell Signal (Figure 2A). The data revealed that

A crucial advantage over standard assays, e.g. filter based

increasing E:T (Effector:Target) cell ratios induced a ratio-methods using labels and either an optical or radiometricdependent apoptosis of A549 cells as well as of SKOV3 cells

detection technique, is the continuity of cell monitoring.

seen as a decrease of the Cell Signal. The lowest ratio ofadded CAR T cells, 1:3, had a larger killing effect on SKOV3

Endpoint assays using dyes or labels quantify the potency

cells compared to A549 cells. Uninterrupted attachment ofof effector cells to kill target cells by either measuring target

cells was observed in wells with only untransduced control cells

cell viabiliy (i.e. MTT or flow cytometry) or membrane integrity

or only medium (Figure 2A, grey lines).

(i.e. LDH). Those assays are labor-intensive and lack kinetic
information of the recorded effects.

The Kill Time 50 values were calculated to investigate at

Unique culture plates with integrated electrodes as used inwhat timepoint and which ratios of effector cells killed thethe AtlaZ system enable long-term measurements over several

cancer cells to 50%. Figure 2B shows that 50% of A549 cellsweeks. Thus real-time data on cell adhesion, proliferation andwere killed after approx. 50 h (E:T = 1:1) or 9 h (E:T = 3:1). SKOV3

compound effects can be acquired.

cells were killed to 50% after approx. 46 h (E:T = 1:1) or 8 h (E:T= 3:1).

Results

The AtlaZ Control software calculates and displays Cytolysis in

In our assay, were used CAR T-cells directed to the receptor% (Figure 3). The data were retrieved at t = 73 h and showed

EGFR on the target cells A549 and SKOV3. Those CAR T-cells

that increasing E:T cell ratios induced a ratio-dependent

exhibit an antigen-induced cell-mediated cytotoxicity

apoptosis. The lowest amount (1:3) of CAR T cells that wereby triggering intracellular pathways leading to apoptosis

added to A549 or SKOV3 cells did not induce a 50% cell deathof target cells. As a control, we used untransduced cells(Figure 2B), but they induced a cytolysis of only 10% (A549) or

added to the target cells in the same ratio. We observed

20% (SKOV3) (Figure 3). Figure 3 shows as well that ratios 1:1

a time- and ratio-dependent decrease of the Cell Signal,and 3:1 did induce a 84% and 100% cytolysis of A549 cells, andrepresenting apoptosis of carcinoma cells.

a 75% and 100% cytolysis in SKOV3 cells, respectively.

Figure 1 shows the lung adeno-carcinoma cell line A549 asadhered on the 96-well AtlaZ sensor plates on days 1, 2 and 4
post-plating.

These results demonstrate the capability of CAR T cells to kill
A549 and SKOV3 cancer cells in a ratio- and time-dependentway.

The A549 cell seeding density was 40 k cells/well and 80 k cells/
well for SKOV3. The cells then adhered and proliferated for

In summary, AtlaZ allows for label-free and real-time cellular

the next 24 h. Next, at t = 24 h, CAR T cells were added at an

research on cell adhesion, proliferation and cytolysis.

effector T-cell to target A549 cell ratio of 1:1, 1:3 and 3:1. The

Measurements can be performed in up to 6 x 96-well plates

added volume of CD8 T effector cells was 200 µl (after havingsimultaneously or independently. The AtlaZ system provides
a versatile tool for in vitro cell monitoring addressing theAA549 cells

BA549 cells

throughput.

SKOV3 cells

CAR T (1:1)
n=2

CAR T (1:1)
n=2

CAR T (1:3)
n=2demands for versatility, physiological relevance andSKOV3 cells

CAR T (3:1)
n=2

CAR T (1:3)
n=2

CAR T (3:1)
n=2

Figure 2: A CAR T cells added in different ratios
to A549 (left) or SKOV3 (right) cells at t = 24 hafter plating. B Kill Time 50 values calculated frommeasurements as shown in A (+/- SD, n = 2 wells
each. UTD = untransduced control cells).

Figure 3: A Cytolysis in percent (%) as a timecourse
and as calculated from the timepoint t = 73 hA

A549 cells

SKOV3 cells

A549 cells

SKOV3 cells(B). The ratios 1:1 and 3:1 did induce a 84% and100% cytolysis of A549 cells, and a 75% and 100%cytolysis in SKOV3 cells, respectively. The ratio 1:3
induced a 10% (A549) or 20% (SKOV3) cytolysis.

BMethods
AtlaZ platformThe AtlaZ platform (Nanion Technologies) provides
quantitative live-cell analytics by measuring the impedance(Ohm, W) of adherent cells as grown on 96-well plates withembedded planar gold-film electrodes (Nanion Technologies).
Real-time impedance data provide insights in various cell
phenotypes, such as cell morphology, proliferation, lateralmigration or cytotoxicity even over prolonged periods of time.
A crucial advantage over standard assays is the continuityof cell monitoring. Continuous measurements reveal thekinetics of cell behavior and allow an in-depth mechanisticWorkflow of AtlaZ immune cell-mediated killing assay
Target cells are seeded in AtlaZ sensor plates and they adhereon the surface with embedded gold electrodes (Figure4, 1).
Each well of the 96-well plate contains 1 center gold electrodewith 0.6 mm in diameter and one reference electrode. Afterthe target cells being adhered, the population starts toproliferate. When the cells reach confluency, non-adherent
effector cells (e.g. CAR T cells) are added (Figure 4, 2).

understanding without the need for time- and labor-intensive

Derived from the Cell Signal, the software automaticallyendpoint assays. For example, one of the crucial advantages

calculates and plots Cytolysis (%) over time and as a barof data derived from the AtlaZ platform is the possibility tograph at a specific time of interest. Furthermore, KT50 values

analyse dose-responses at any time during the experiment.

are plotted automatically.

Advanced information content is obtained by using multifrequency impedance readouts, which is possible with theAtlaZ Control software. With impedance readouts at different
frequencies (here a spectrum of 0.1 kHz – 100 kHz) it is possible
to further dissect physiological response into deeper levels,and thus zooming in on changes in membrane topography,
cell-cell or cell-matrix junctions. G-protein-coupled receptor(GPCRs) activation is one example here.
Immune-cell mediated killing assay
The Cell Signal values that are displayed were calculated
by subtracting the impedance in empty wells with medium,measured at the beginning of the experiment just before
cell addition. Data are presented in mean ± SD.
The AtlaZ assay is sensitive to adherent target cells, but not
to the presence of non-adherent immune effector cells. This
allows to specifically investigate target cell cytotoxicity andis an advantage over other assays where the presence of asecond cell type like effector cells could induce backgroundsignals.

Calculation of cytolysis and Kill Time 50
Percent (%) cytolysis was used to quantify cell death and is
calculated by AtlaZ Control software as follows:
Cytolysis (t, %) =D( ___k,t )DG no-effectorcells

D k,t( ___)
DG target +. 100

effectorcells

where Δk,t=Zt-Z0 and ΔG = Z0-Zmin. Zt is the impedancevalue after the addition of the treatment and Z0 is the last
impedance value before treatment addition. Zmin is thesmallest impedance value in the region before treatment
addition. This means that Cytolysis (t, %) is the effect of theeffector cells on the target cells at a certain time t, displayedin percent. Δk,t (k=killing; t= time) contains information on

the time-dependent cell death based on the impedancechange before and after treatment and ΔG considers the cellproliferation and growth (g = growth) post-seeding.

Next to Cytolysis (%) AtlaZ Control software also displays Kill
Time 50 values which are as well determined through theimpedance measurements.

Figure 4: Workflow of CAR T cell-mediated killing

2

Cell Signal

Goldelectrodeadhere and proliferate (1). When they reachconfluency, non-adherent effector (CAR T) cells
Proliferation

are added (2). The cell death of target cells isTime (h)measured and can be monitored as Cell Signal+ Effector cells

Non-adherent
effector cellover time.

Cell Signal

1assay. Target cells seeded on AtlaZ sensor plates,Adherent
target cellTime (h)Effector : Target ratio
neg. cntrl
1:2Cell Signal

3

1:1

Time (h)3:1pos. cntrl

References
1. Boucher JC, Davila ML. Chimeric Antigen Receptor Design
Today and Tomorrow. Cancer J. 2021 Mar-Apr 01;27(2):92-97.
doi: 10.1097/PPO.0000000000000514. PMID: 33750067.
2. Reiß, B. and Wegener, J. (2015) Impedance Analysis ofdifferent cell monolayers grown on gold-film electrodes.
IEEE Eng. Med. Biol. 7079-7082. org/10.1002/9780470015902.
a0025710
3. Lukic, S.; Wegener, J. (2015) Impedimetric monitoring of cellbased assays. Encyclopedia of Life Sciences, Wiley & Co.
https://doi.

Key findings
1. Our results demonstrate the capability of CAR T
cells to kill A549 and SKOV8 cancer cells.
2. AtlaZ allows for cellular research on cell adhesion,proliferation and cytotoxicity, label-free and in realtime.
3. Recordings can be performed in up to six 96-wellplates simultaneously or independently.

Nanion Technologies GmbH I Ganghoferstraße 70a, 80339 Munich, Germany+49 89 2190 95-0 I info@nanion.de I www.nanion.de