Case study

Immune cell - mediated killing of tumor
cells with the AtlaZ platform

Tools:AtlaZ

Dr. Michael Dudek, Ph.D.
featured by Nanion Technologies
Michael Dudek, Technical University of Munich,kill cancer cells, such as blood cancers. In general, identifying

knows: attack wins games, defense wins

T cells that kill cancer cells in vivo or monitoring CAR T cellchampionships. Equally, the immune system

activity in vitro is critical to the development of successful cellalways wins under normal conditions, andtherapies. In vitro assays are key for evaluating the potency

the goal of his scientific career is to betterof a treatment and also to understand the interactions

understand auto-immune related diseases

between immune (effector) cells and cancer (target) cells.

and to use the knowledge for discovering newtreatment options.

Classical potency assays detect the ability of effector cells
to kill target cells by flow cytometry or metabolic viability
measurements (e.g. MTT or Alamar Blue (resazurin) dye)or membrane integrity (e.g. the chromium-release assay

Michael Dudek sees the immune system as a fascinatingmasterpiece of every living being. The tight regulatedbalance between resolving an infection or cancer but notdamaging unaffected tissue, impressed him since he has
started to study the immune system in more detail. CD8 T
cells are equipped with a broad spectrum of weapons tofight against pathogens or cancer but they could also be
responsible for deleterious tissue damage during chronic
inflammation. In his recent studies he provided mechanistic
insights into the tight balance of the functionality of CD8 T
cells in organs like the liver and how this balance is out-ofcontrol during diseases like non-alcoholic steatohepatitis
which could reach pandemic-like dimensions in the future (1).
In-depth understanding of efficacies and responses against
healthy or malfunctioning target cells like tumor cells toimmune cells like CD8 T cells is highly relevant.
In the last decade immunotherapy, particularly T-cell therapy
with chimeric antigen receptor (CAR) revolutionized thefield of cancer treatments (2) because this “living drug” uses
genetically modified T cells from the same patient to find andNanion’s AtlaZ platform
AtlaZ allows for cellular research on cell adhesion
and proliferation, cytotoxicity, GPCR, morphology
and barrier function, label-free and in realtime. In up to six 96-well plates simultaneously orindependently.

developed in 1968). However, these assays are limited to asingle readout only, because the same sample cannot bemeasured at more than one time point.
Due to this limitation of endpoint assays, developing
immune cell-based therapies has been notoriously difficult.
With the AtlaZ platform, cell behavior and viability can bequantitatively measured rather than qualitatively. Readouts

“The AtlaZ has a clean user interface with a cool design. The
system allows for a robust and consistent data generation andanalysis and there is no fear to miss out on any relevant data.”
Michael Dudek, Ph.D., Technical University of Munich (TUM)

are in real-time, continuous and label free. Furthermore thedetect impedance signals at a huge spectrum of frequencies

system enables robust in vitro kinetic assays to accurately

ranging from 0.1 kHz – 100 kHz. This allows a multitude of effects

predict the in vivo behavior of therapies. Dr. Dudek recognizedin cells to be detected, for example Transepithelial electricalthe great potential of the AtlaZ platform because theresistance (TEER) signals representing barrier integrity can be

device does not only allow to precisely quantify effector

recorded in parallel to cell adherance or cytolysis.

responses against target cells, but also to detect novel killingmechanisms of immune cells that would not have been

Here the human A549 cell line was used (Figure 1A), which

possible with other technologies.

is a widely utilized epithelian lund adenocarcinoma cell line
derived from a primary lung tumor. The aim was to investigte

Reading the impedance of planar gold-film electrodes

immune cell - mediated cytolysis of A549 cells.

that are used as growth substrate for adherent cells revealschanges in electrode coverage or cell behavior. Electrical

To investigate CD8 T cell mediated cytotoxicity, A549 cells

impedance spectroscopy (3,4) as the methodology behind

were cultured for 4 days until they reached 80-90% confluency.

the AtlaZ system, in combination with the throughput of 6 x

With a seeding density of 40 k cells/well they were then directly

96-wells allows for a so far unmet quantity and richness ofplated on AtlaZ sensor plates without coating. The effector

information which can be gained from cells. AtlaZ allows tocells were obtained from the blood of healthy donors.

AB

100

Untreated
1:2

Cytolysis (%)

1:1
2:1

500C
untreated
1:2
1:1
2:1
3:1

Cell Signal

Kill Time 50% (h)520Time (h)30400N/A
1:2

1:1

Immune cell - mediated killing of A549 cells. A A549 cells at day 4 post plating. B Effector
cells added at t = 24 h after plating of A549 cells. Increasing E:T cell ratios induced a ratiodependent reduction of the viability of A549 cells, represented as a reducing Cell Signal, data
are shown +/- SEM. C Cytolysis in percent (%) as calculated from the data seen in A. D Kill time
50 shows that 50% of A549 cells were killed after approx. 11 h (E:T = 1:1) or 4 h (E:T = 2:1 and3:1). Data are shown +/- SEM.

351001030Time (h)15500025D+ Effector cells

1000

3:1

2:1

3:1

CD8 T cells were positively selected and purified using

In summary, AtlaZ allows for label-free and real-time cellularmagnetic beads. After purification, the cells were stimulated

research on cell adhesion, proliferation and cytolysis.

with the cytokine IL-15 (10 ng/ml) for 2 days before being used

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

for the co-culture experiments on AtlaZ. Effector cells weresimultaneously or independently. Hence, multiple users canadded to the AtlaZ sensor plate at effector to target (E:T) ratios

run their dedicated measurements independently, or largerof 1:1, 2:1, and 3:1 24 hours after target cell seeding at thescreens in the same measruement mode can be executed.

desired final cell concentration.

Michael Dudek along with Percy Knolle have diverse ongoingprojects and collaborations with other departments, therefore

The impedance signal of the cell monolayer was recorded

the multi-user capability along with the outstanding capability

every 15 minutes, and is plotted using the unitless parameterto identify multiple diverse effects in cells over time in a 6 x 96

Cell Signal (Figure 1A). The data revealed that increasing

throughput format was key to decide on a longterm installation

E:T (Effector:Target) cell ratios induced a ratio-dependent

in the Institute of Molecular Immunology and Experimental

reduction of the viability of A549 cells seen as a decrease

Oncology of the Technical University of Munich (TUM). Theof the Cell Signal. Uninterrupted attachment and growth ofAtlaZ system provides a versatile tool for in vitro cell monitoring

A549 cells was observed in wells with only A549 cells (Figureaddressing the demands for versatility, physiological relevance

1A and Figure 1B light grey line). The Kill Time 50 values wereand throughput. Tackling the most significant challenge faced

calculated to investigate at what timepoint and which ratios

by not only TUM researchers when developing immune cell-of effector cells killed the cancer cells to 50% (Figure 1C). These

based therapies, namely predicting treatment efficacy andresults demonstrate the capability of CD8 T cells to kill A549

quantify responses, the AtlaZ allows for reliable kinetic assays

cancer cells.

overcoming these hurdles.

Sebastian Lacher, graduate student at the Institute of Molecular Immunology (IMI) TUM is setting up an AtlaZ
experiment. Apart from Dr. Dudek, many researchers and students use AtlaZ technology in their experiments.
Research at the Institute is diverse and agile - therefore in need of an easy, versatile and powerful technology indaily routines. AtlaZ is meeting that need. (Photo provided by: Michael Dudek).

“My experience with Nanion´s technology was great, achieving
highly reproducible data with the AtlaZ platform from day 1 wasconvincing for me.”
Percy Knolle, Prof. Dr., Technical University of Munich (TUM)

Contact Information

References

Michael Dudek, Dr.,1.

Dudek M, et al. Auto-aggressive CXCR6+ CD8 T cells cause liver immune

Postdoc, Research Group Knolle

pathology in NASH. Nature. 2021 Apr;592(7854):444-449. doi: 10.1038/s41586-021-

Institute of Molecular Immunology and Experimental

03233-8. Epub 2021 Mar 24. Erratum in: Nature. 2021 May;593(7860):E14. PMID:

Oncology, Technical University of Munich (TUM)

33762736.

Ismaningerstr. 2281675 München, Germany

2.

and Tomorrow. Cancer J. 2021 Mar-Apr 01;27(2):92-97. doi: 10.1097/
PPO.0000000000000514. PMID: 33750067.

Percy Knolle, Prof. Dr.,
Institute of Molecular Immunology and Experimental

Boucher JC, Davila ML. Chimeric Antigen Receptor Design Today

3.

Reiß, B. and Wegener, J. (2015) Impedance Analysis of different cell

Oncology, Technical University of Munich (TUM)

monolayers grown on gold-film electrodes. IEEE Eng. Med. Biol. 7079-7082.

Ismaningerstr. 22

org/10.1002/9780470015902.a0025710

81675 München, Germany
http://www.imi-muenchen.de/research-groups/knolle/research-group-knolle.html

4.

Lukic, S.; Wegener, J. (2015) Impedimetric monitoring of cell- based assays.
Encyclopedia of Life Sciences, Wiley & Co. https://doi.

Acknowledgments
We thank Percy and Michael for sharing their views on
immune cell mediated killing assays. We are gratefulfor your valuable insights and wonderful collaboration.

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