Topic:

Application Note

Cancer,Immuno-oncology
Cells:
A549, lung adenocarcinoma cell line

Immune cell-mediated killing of A549
cancer target cells in real-time

Tools:AtlaZ

Nanion Technologies GmbH, Munich.
TUM, Munich

Summary
Cancer remains one of the leading causes of death, with,

T- lymphocytes expressing CD8 (cluster of differentiation 8), aaccording to the World Health Organisation (WHO), aroundco-receptor for the T-cell receptor (TCR). Those effector cells

10 million people dying due to the disease in 2020 (1). Chemo-exhibit an antigen-induced cell-mediated cytotoxicity byand radio-therapy are still the dominant treatment types, but

triggering intracellular pathways leading to apoptosis of target

advancing therapies such as immuno-therapy have emerged

A549 cells.

as tools to fight against the disease.
The impedance of planar gold-film electrodes that are used
In general, identifying T cells that kill cancer cells in vivo is

as growth substrate for adherent cells reveals changes incritical to the development of successful cell therapies. Theelectrode coverage or cell behavior (Figure 1B). Real-timelabel-free AtlaZ immune cell killing assay can be used toimpedance data provide insights in various cell phenotypes,

measure rate of killing at Effector : Target (E:T) ratios to predict

such as cell morphology, proliferation, lateral migration orin vivo activity. In order to gain a deeper understanding ofcytotoxicity even over prolonged periods of time.

cancer cells, real-time and continuous monitoring is necessary
to access kinetic and phenotypic information.

A crucial advantage over standard assays, e.g. filter basedmethods using labels and either an optical or radiometric

The platform used here, AtlaZ, is a quantitative live-cell analysis

detection technique, is the continuity of cell monitoring.

system and allows for cellular research on cell adhesion
and proliferation, cytotoxicity, GPCR, morphology andEndpoint assays using dyes or labels quantify the potency

barrier function, label-free and in real-time. Recordings canof effector cells to kill target cells by either measuring target

be performed in up to six 96-well plates simultaneously orcell viabiliy (i.e. MTT or flow cytometry) or membrane integrity

independently. Electrical impedance spectroscopy (2,3) as(i.e. LDH). Those assays are labor-intensive and lack kinetic

the methodology behind the AtlaZ system, in combination withthe throughput of 6 x 96-wells allows for a so far unmet quantity
and richness of information which can be gained from cells.

AHere we used the human A549 cell line (Figure 1A) which
is a widely utilized epithelial lung adenocarcinoma cell linethat was derived from a primary lung tumor. The effector
cells co-cultured in our assay were purified human cytotoxic

B
compound-induced effectcell detachment

Cell Signal

Figure 1: A Cancer cells A549 one day aftercell confluency

loss of tight junctions
loss of membrane integrity

cell proliferation
cell adhesion

plating on Nanion sensor plates (left), after twodays (middle) and after four days (right) B The Cell
Signal value offers information on cell adherence,
proliferation or cell death.

Electrode

Electric current

Time (h)Suspended cell

Adherent cellinformation of the recorded effects.

growth of A549 cells was observed in wells with only A549 cells(Figure 2, grey line).

Unique culture plates with integrated electrodes as used in
the AtlaZ system enable long-term measurements over several

The Kill Time 50 values were calculated to investigate atweeks. Thus real-time data on cell adhesion, proliferation andwhat timepoint and which ratios of effector cells killed thecompound effects can be aquired.

cancer cells to 50%. Fig. 3 B shows that 50% of A549 cells werekilled after approx. 13 h (E:T = 1:1) or 6 h (E:T = 2:1 and 3:1).

Results

These results demonstrate the capability of CD8 T cells to kill
Here we used the human A549 cancer cell line derived from aA549 cancer cells.

primary lung tumor. Next to the proliferating A549 cells we usedeffector cells expressing CD8, which is is predominantly present

In summary, AtlaZ allows for label-free and real-time cellularon the surface of cytotoxic T-cells, but can also be found on

research on cell adhesion, proliferation and cytolysis.

e.g. natural killer cells.

Measurements can be performed in up to 6 x 96-well plates
simultaneously or independently. The AtlaZ system provides

Figure 1 shows the lung adeno-carcinoma cell line A549 asa versatile tool for in vitro cell monitoring addressing theadhered on the cell culture flasks on days 1, 2 and 4 post-

demands for versatility, physiological relevance andplating.

throughput.

The A549 cell seeding density was 40 k cells/well, the cells
then proliferated and a plateau phase was reached approx.
after 14 h. Next, at t = 24 h, T-cells enriched from primary

Methods

PBMCs (Peripheral Blood Mononuclear Cells) were added atAtlaZ platform

an effector T-cell to target A549 cell ratio of 1:1 (50 k cells),

The AtlaZ platform (Nanion Technologies) provides

2:1 and 3:1. The impedance signal of the cell monolayer wasquantitative live-cell analytics by measuring the impedance

recorded every 15 minutes, and is plotted using the unitless(Ohm, W) of adherent cells as grown on 96-well plates withparameter Cell Signal (Figure 2). The data revealed thatembedded planar gold-film electrodes (Nanion Technologies).

increasing E:T (Effector:Target) cell ratios induced a ratiodependent reduction of the viability of A549 cells seen as aReal-time impedance data provide insights in various celldecrease of the Cell Signal.

phenotypes, such as cell morphology, proliferation, lateralmigration or cytotoxicity even over prolonged periods of time.

Based on the same data, the AtlaZ Control software also
calculates and displays Cytolysis in % (Figure 3A). The data
reveal that after approx. t = 27 h the cytolysis of A549 cells
gradually increases and reaches a maximum of 37%, 48%, 59%
and 57% in the presence of the effector to target cell ratio 1:2,1:1, 2:1 and 3:1, respectively. Uninterrupted attachment andA crucial advantage over standard assays is the continuity ofcell monitoring. Continuous measurements reveal the kinetics
of cell behavior and allow an in-depth mechanistic
understanding without the need for time- and labor-intensive
endpoint assays. For example, one of the crucial advantagesof data derived from the AtlaZ platform is the possibility toanalyse dose-responses at any time during the experiment.
Advanced information content is obtained by using multi-+ Effector cells

1000Cell Signal

frequency impedance readouts, which is possible with theAtlaZ Control software. With impedance readouts at different

untreated
1:2
1:1
2:1
3:1

frequencies (here a spectrum of 0.1 kHz – 100 kHz) it is possible
to further dissect physiological response into deeper levels,

500

001020Time (h)3040Figure 2: Effector cells added at t = 24 h after
plating of A549 cells. Increasing E:T cell ratios
induced a ratio-dependent reduction of the viabilityof A549 cells, represented as a reducing Cell Signal,data are shown +/- SEM.

Figure 3: A Cytolysis in percent (%) as calculated

Afrom the data seen in Fig. 2. B Kill time 50 shows

100Untreated
1:2

that 50% of A549 cells were killed after approx.

1:1

Cytolysis (%)

13 h (E:T = 1:1) or 6 hrs (E:T = 2:1 and 3:1). Dataare shown +/- SEM.

2:1

500B

3:1

253035Time (h)Kill Time 50% (h)15and thus zooming in on changes in membrane topography,

1050cell-cell or cell-matrix junctions is achievable. G-protein-N/A1:2

coupled receptor (GPCRs) activation is one example here.

1:1

2:1

3:1

Immune-cell mediated killing assay
To investigate PBMC-mediated cytotoxicity, A549 cells werecultured in DMEM medium (high glucose, 1% Pen/Strep, 10%

electrode. After the target cells being adhered, the population

FCS) for 4 days until they reached 80-90% confluency. With

starts to proliferate. When the cells reach confluency, non-a seeding density of 40 k cells/well they were then directly

adherent effector cells (e.g. immune cells) are added (Figure

plated on AtlaZ sensor plates without coating. The effector4, 2).

cells were obtained from the blood of healthy donors using aPercoll density gradient (PBMCs). CD8 T-cells were positively
selected and purified using magnetic beads. After purification,
the cells were incubated in T-cell medium (RPMI 1640, 10% FCS,1% P/S and 1% glutamine) and stimulated with the cytokine
IL-15 (10 ng/ml) for 5 days before being used for the co-culture

The cytolysis of the target cells is measured and displayed
as Cell Signal over time as seen in the figure, additionally theAtlaZ Control software calculates the data as Cytolysis (%) andcalculates and displays the data in a KT50 (Kill Time 50) plot (asshown in Figure 3)

experiments on AtlaZ. Effector cells were added to the AtlaZ
sensor plate at effector to target (E:T) ratios of 1:1, 2:1, andCalculation of cytolysis and Kill Time 50

3:1 24 hours after target cell seeding in a 100 μl volume at thePercent (%) cytolysis was used to quantify cell death and isdesired final cell concentration. The same volume was added

calculated by AtlaZ Control software as follows:

to negative control wells.
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 as mean ± SD.
The AtlaZ assay is sensitive to adherent target cells, but not
to the presence of non-adherent immune effector cells. This

Cytolysis (t, %) =D( ___k,t )DG noeffectorcells

D k,t( ___)
+DG targeteffector. 100cellswhere 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

allows to specifically investigate target cell cytotoxicity andaddition. This means that Cytolysis (t, %) is the effect of theis an advantage over other assays where the presence of aeffector cells on the target cells at a certain time t, displayed

second cell type like effector cells could induce backgroundin percent. Δk,t (k=killing; t= time) contains information onsignals.

the time-dependent cell death based on the impedancechange before and after treatment and ΔG considers the cell

Workflow of AtlaZ immune cell-mediated killing assay

proliferation and growth (g = growth) post-seeding.

Target cells are seeded on AtlaZ sensor plates and they

Next to Cytolysis (%) AtlaZ Control software also displays Kill

adhere on the surface with embedded gold electrodes

Time 50 values which are as well determined through the(Figure 4, 1). Each well of the 96-well plate contains 1 center

impedance measurements.

gold electrode with 0.6 mm in diameter and one referenceFigure 4: Workflow of immune cell-mediatedAdherenttarget cell

Goldelectrode

Cell Signal

1

killing assay. Target cells are seeded on AtlaZ
sensor plates, adhere and proliferate (1), thennon-adherent effector cells are added (2). The
cytolysis of the target cells is measured andProliferation

displayed as Cell Signal over time.

Time (h)Non-adherent
effector cell+ Effector cells

Cell Signal

2Time (h)Effector : Target ratio
neg. cntrl

Cell Signal

3

1:2
1:1

Time (h)3:1pos. cntrl

References
1. https://www.who.int/news-room/fact-sheets/detail/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 CD8 Tcells to kill A549 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