Whitepaper

Monitoring senescence-mediated growth arrest of
murine and human cancer cells in vitro
Barbara Schoerg1, Francisco José Reche Pérez1, Yvonne Heneka1, Lars Strohben1, Martin Ziller2, Ulrich Thomas2, Max
Engelstädter2, Elena Dragicevic2, Michael George2, Giovanna De Filippi2, Niels Fertig2, Sonja Stoelzle-Feix2
1

Eberhard Karls University of Tübingen, Tübingen, Germany.

2

Nanion Technologies GmbH, Munich, Germany.

Significance•

Cellular senescence is a key factor in ageing.

•

Because of its intricate role in cancer, the investigation, detection and quantification of cellular senescence has
gained major interest.

•

Electric Cell-Substrate Impedance Sensing (0.1 kHz – 100 kHz) allows for the analysis of the dynamics of cellproliferation, e.g. analysis of growth arrest during senescence induction.

Introduction
Ageing, cellular growth and cancer development appear to

immune system and promote tumor clearance, but canbe different processes, but they may have common origins.

also be immunosuppressive and contribute to the growth

The accumulation of cellular damage over time is widelyand increased stemness of neighboring cells15. Cancer cells

regarded as a general cause of ageing and also of cancer.

can override the senescence-associated cell-cycle arrest

In addition, uncontrolled cellular growth or hyperactivity

and escape from the non-proliferative compartment due tois not only involved in cancer, but also in aging-related

their genomic instability and potential to acquire additional

pathologies such as atherosclerosis and inflammation1,2.

mutations16,17. Because of its intricate role in cancer, theinvestigation, detection and quantification of cellular

Cellular senescence is a key factor in ageing and cancer. It

senescence has gained major interest.

is characterized by a permanent and generally irreversible
cell cycle arrest3 with a senescence-associated secretory

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

phenotype (SASP)4, macromolecular damage (DNA) andanalysis system allowing real-time and label-free monitoringaltered metabolism5,6.

of cell adhesion and proliferation, cytotoxicity, GPCR,
morphology changes and barrier function. Recordings can

In cancer, oncogene expression, cancerous mutations as

be performed in up to six 96-well plates simultaneously orwell as radiation, chemotherapy and other cytotoxic drugs7

independently. To test and implement the AtlaZ system forand even inflammation-related cytokines like interferon-γ

the investigation of cellular senescence, we analyzed theand TNF87,9,10 can drive tumor cells into senescence.

strength of growth arrest induced by established treatments

While therapy-induced cellular senescence can lead toin murine and human cancer cell lines. Cancer cells werepermanent cessation of tumor growth, recent findings

seeded to the 96-well plates and allowed to settle andhad contradicted the idea that senescence only has a

attach to the bottom (Figure 1) before the senescence-

positive effect on suppressing tumor growth11,12. Senescentinducing treatments were added to the wells. Here, themalignant and non-malignant cells can drive tumor growth

impedance was measured over a period of up to 5 days.

by secreting a plethora of pro-inflammatory cytokines,

In our assay, the murine as well as the human cancer cells

chemokines, growth factors and the SASP13,14. Furthermore,

showed an arrest in cell proliferation upon treatment. The

the SASP has a double-edged nature as it can activate thecells increased in size but stopped proliferating which wasdetected by a stable arrest of the Cell Signal. We observed

plates with integrated electrodes as used in the AtlaZ

a time- and treatment-dependent decrease of the Cellsystem enable long-term measurements over several weeks.

Signal, representing proliferation arrest of murine and humanThus, real-time data on cell adhesion, proliferation andcancer cells.

compound effects can be acquired.

The impedance of planar gold-film electrodes that are used

Changes in cell morphology can be qualitatively

as growth substrate for adherent cells reveals changes incharacterized by size, shape, and structural alterations.

electrode coverage or cell behavior, e.g. upon treatment

Such physical rearrangements in cells result in corresponding

with senescence-inducing drugs and/or cytokines. Real-

alterations in their electrical properties as the cell acts

time impedance data provide insights in the temporal

as both, a resistor and a capacitor. Electrochemical

dynamics of senescence induction, such as changes of cell

impedance spectroscopy (EIS) is an analytical method tomorphology and proliferation, even over prolonged periods

measure such electrical properties18,19 and this methodologyof time.

is employed in Nanion´s cell monitoring technology
implemented in the AtlaZ platform.

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

The power of Electric Cell-Substrate Impedance Sensing

technique, is the continuity of cell monitoring. Endpoint(ECIS) resides in its ability to detect changes in cell layer

assays using dyes or labels are labor-intensive and lack

integrity in a label-free, real-time manner ranging fromkinetic information of the recorded effects. Unique culture

seconds to days.

secreted factors
due to SASP

Healthycell

Induction of
growth arrest

Senescent
cell

Cell Signal

cell confluency
compound-induced effectcell proliferation

cell shape changes

Electrode
Electric current

cell adhesion

Suspended cellTime (h)Adherend cell

Figure 1 Impedance measurements offer information on e.g. cell adherence, proliferation, membrane integrity or cell death. Cell-specific profilesof different growth and cell behavior patterns such as cell size changes in late senescent cells can be detected and are for example relevant incancer research.

Materials and methods for cell culture
We explored the feasibility of detecting the induction
and dynamics of senescent cells through impedance
measurements. To achieve this, we induced senescencein two different cell lines, a primary murine ß cell cancerline (RT2 ß-cc) and a human colon carcinoma cell line
(HCT116) using established senescence-inducing protocols.
Over the whole experiments, the cells were cultured on 96well plates with planar gold film electrodes (NSP-Z, Nanion
Technologies) and analyzed with the AtlaZ measurement
unit (Nanion Technologies) which was positioned in ahumified incubator (Binder) at 37°C with 5% CO2.
Murine cancer cells were derived from an endogenous
murine mouse model of ß-cell cancer (RIP1-Tag2, RT2) andcultured in growth medium (Dulbecco’s Modified Eagle’s
Medium; DMEM supplemented with with 10% fetal bovine
serum, FBS, Sigma-Aldrich; MEM Amino Acids, 0.5x, SigmaAldrich; 1 mM Sodium pyruvate, Sigma-Aldrich; 10 mM HEPES
buffer, Bio&Sell; 1 mM L-Glutamine, Sigma-Aldrich; 100 U
ml-1 and 0.1 mg/ml Penicillin-Streptomycin, Sigma-Aldrich;
50 µM 2-mercaptoethanol, Sigma-Aldrich). Senescenceinduction was achieved as described previously (Braumuller,
et al., 2013, Muller-Hermelink et al., 2008). Briefly, adherent
RT2 ß-cancer cells were washed in DPBS (Invitrogen of
Thermo Fisher, USA), trypsinized with 0.05% trypsin/EDTA
solution (gibco), and counted. Cells were seeded in growth
medium in a 96-well NSP-Z plate (5.000 cells per well) andplaced into the AtlaZ – measurement unit for monitoringof the impedance signal overnight. After 12 h, media
was replaced with fresh growth medium which was either
supplemented with a combination of 100 ng/ml murine
IFN-γ and 10 ng/ml murine TNF (IFN-γ+TNF, n = 11) (R&D
Systems), with 5 µM palbociclib isethionate (palbo, n=12)(Selleckchem), or with a combination of IFN-γ, TNF andpalbo (PIT, n = 12). Control cells remained untreated (nonsupplemented growth media, media control, n = 11). The
recording plate with the RT2 ß-cancer cells was placedback into the AtlaZ system and the impedance spectrum(1 kHz - 100 kHz, every 4 minutes) was recorded for a periodof 5 days. For final analysis, the Cell Signal at 70 kHz wasused, being the most sensitive recording frequency (seeFigure 2 for the concept).
The human colon carcinoma cell line (HCT116 cells)

was originally acquired from the American Type Culture
Collection (ATCC, Manassas, Virginia). Cells weremaintained in McCoy’s 5A medium (Sigma of Merck, USA)supplemented with penicillin-streptomycin (1% v/v) andinactivated FBS (10% v/v) (Sigma of Merck). Before seedingfor experiments, cells were freshly thawed and passaged
at least once maintaining the same passages in every
single experiment. Adherent HCT116 cells were washed in
DPBS, trypsinized with trypsin/EDTA solution, and counted.
In the experimental setup, cells were seeded in replicates(8x) to the 96-well NSP-Z plate (5.000 cells per well) andwere allowed to settle for 24 h before being incubated in
the presence of 250 nM doxorubicin (BB Pharma) for 24 h.
This protocol is correlated with a notable in vitro expressionof β-galactosidase, a commonly used marker for cellularsenescence. In line with the murine cells, HCT116 control
cells were treated with media only (Ctrl.). After 24 h, culture
media was replaced, and both control and doxorubicin
treated HTT116 cells were further cultured for an additional
four days in the absence of the drug. Throughout this entire
process, HCT116 cells were monitored by measuring theimpedance (here at 50 kHz, every 20 seconds) using the
AtlaZ system.

Technological background
Impedance (|Z|) describes the electrical resistancein response to an AC signal. It takes into account thatcapacitors become more conductive with increasing AC
frequency. Thus, impedance spectroscopy that records
the impedance at a set of discrete AC frequencies alonga certain spectral window (here between 0.1 kHz and 100
kHz) enables a more diversified study of the passive electric
properties of the cell layer.
The optimal frequency is cell-type specific and has to be
determined empirically, but for most electrode types it
ranges between 1 kHz and 50 kHz20. As a general guideline,
the sensitive frequency for detection of changes in cell
layer properties can be determined by dividing theimpedance magnitude |Z| of a cell-covered electrode by
the impedance of a cell-free electrode (Figure 2). Whenthese ratios are plotted for each individual frequency one
obtains a curve from which the maximum ratio reflects thefrequency with the broadest range of relative change inimpedance (Figure 2C, the most sensitive frequency).

“The AtlaZ platform facilitates rapid and straightforward early
screenings in search of senescence-inducing drugs, particularly for
anticancer therapies.”
Barbara Schoerg, Postdoctoral Researcher, Eberhard Karls University Tübingen

B
Electric current

Adherent cell

C
Cell-covered electrodeI Z I (Ohm)

CulturemediumHigh AC frequency
Low AC frequency

Goldsubstrate

most sensitivefrequencyI Z Icell covered /I Z Icell-freeA

Culturesubstrate
Frequency (kHz)Frequency (kHz)

Figure 2 A The frequency dependent behavior of a cell-free and cell-covered electrode is presented in a Bode diagram, reflecting the impedance
increase due to the cell layer between ~ 1 – 100 kHz. For the specific experimental setup here, the signal is dominated by the paracellular cell layer
resistance between about 1 – 10 kHz (range indicated by light green frame) and is increasingly influenced by capacitive currents across the cell
membranes between 10 – 100 kHz (range indicated by darker green frame). B Schematic side view of a cell-covered electrode with current flow
at low and high AC frequencies. C The ratio of the normalized impedance - calculated by dividing |Z| for a cell-covered electrode by that of thesame cell-free electrode (as in Figure 2A) - is used to estimate the monitoring frequency with maximum sensitivity. Different experimental setups(e.g. various cell types within particular research areas) own specific optimal frequencies and the available frequency spectrum (0.1 – 100 kHz)
allows to exploit a multitude of experimental conditions.

At lower AC frequencies (100 Hz – 5 000 Hz, dependent on

the impedance-based assay to dynamically measure theelectrode surface area), the current is forced to flow around

growth arrest in murine and human cancer cells, monitoring

the cell bodies, between and beneath neighboring cells

in real time the effects induced after treating the cells with

(Figure 2B green arrows). At high frequencies (> 10 000 Hz),the established protocols mentioned above. As control,

the current couples transcellularly (Figure 2B, black arrows)

we used equal amounts of tumor cells which are allowed

and the impedance is dominated by the capacitance ofto grow constantly due to normal media conditions. Afterthe membrane. At intermediate frequencies the current

experimental senescence was induced by the respective

takes a mixed combination of these paracellular andtreatments, we detected a stabilization of the Cell Signaltranscellular routes.

in the treated wells of both cell types (human and murine
cancer cells), while the Cell Signal of the control cells was

Depending on the selected recording mode, the displayed

increasing constantly until a plateau was reached.

“Cell Signal” represents the impedance (Ohm) as acquired
at a recording frequency of 10 kHz (Cell Monitoring mode)

Figure 3 shows the proliferation curves of the murine RT2

or 50 kHz (Cytolysis mode), or the Resistance (Transepithelialß cancer cells measured at 70 kHz over a time period

Electrical Resistance, TEER) as detected at 1 kHz in the TEER

of 5 days post-plating. The impedance signal of the cellmode. This means that the “Cell Signal” is not a calculatedmonolayer was recorded every 4 minutes and is plottedvalue but represents raw data.

over time (Figure 3A). Cells adhered overnight within lessthan 24 h. Next, the media was replaced and cells were

NSP-Z transparent sensor plates (Figure 3) allow fortreated with the senescence-inducing agents (IFN-γ+TNF,

observation of the cells under a microscope or for making

Palbo or PIT). The senescence inducing agents resulted

use of the plates in a complementary imaging system.

in a treatment-dependent growth arrest which remained
stable over the recorded period of 5 days. This induction
and maintenance of senescence is represented by a nearly

Results

stable Cell Signal which was for every treatment and after 3
days of senescence induction (t = 96 h) drastically reduced
compared to the signal of the proliferating cells (media

The induction of senescence in murine and human cancer

control, p<0.0001) (Figure 3B). The strongest effect wascells by IFN-γ+TNF and Palbociclib (ß cancer cells) as wellobserved as a consequence of the cytokine treatment (IFN-

as doxorubincin (human HTC116 cancer cells) was shown

γ+TNF +/- Palbo). Here, the dynamics of the proliferation waspreviously in vitro as well as in vivo8,10. Our aim was to use

calculated as the slope of a regression curve (Palbo: 0.45x;

IFN-γ+TNF: -0.45x; PIT:-0.46x; media control: 2.79x) (Figure

Finally, we assessed the strength of growth arrest, which is3C). In sharp contrast to the Cell Signal of the treated

represented by the slope of the regression curve (doxo: 0.02;

cells, we detected an increasing signal over a period of 3

media control: 1.23) (Figure 4C).

days deriving from the physiologically normal cells (media
control) which represents stable cell proliferation (Figure

Overall, the results for both the murine RT2 ß cancer cells as

3A, B). The induction of senescence was verified using

well as the HCT116 cells demonstrate senescence-mediatedenzymatic ß-gal staining (Figure 3D).

growth arrest which was validated by the well establishedprotocols as reported in the literature.

HCT116 cells were seeded on NSP-Z plates (5000 cells/well)and adhesion and proliferation was monitored with AtlaZ

In conclusion, these data show the applicability of theover the next 24 hours. At t = 24 h, the cells were treated

impedance technique as implemented by AtlaZ system towith with doxorubicin, a chemotherapeutic drug whichmonitor the dynamics of cellular senescence. This enables

is known to induce senescene at certain concentrations.

an easy comparison of the power of newly discovered or(Figure 4). Control wells were kept under normal conditions.

even clinically approved senescence inducing drugs.

After another 24 h, the media of the treated wells was
replaced with culture media (without doxorubicin). Cells

Summary

were cultured for another period of 3 days. The Cell Signalof the cell monolayer was recorded (measurement interval:

The interconnection between ageing, cancer development,

every 2 minutes) over the whole experiment (Figure 4A). The

and cellular growth suggests common origins despite

data shows a nearly steady signal in consequence of the

distinct outward processes. The accumulation of cellular

doxorubicin treatment. The Cell Signal was not increasing

damage is widely acknowledged as a primary cause

over time which most likely represents cellular growth arrestof ageing, potentially leading to abnormal cellularover the recorded period of 3 days. This growth arrest andadvantages (aberrant properties of cells, enabling them toinduction of senescence could be verified using enzymatic

bypass normal growth controls) and cancer. Uncontrolledß-gal staining (Figure 4D). In contrast to the doxorubicin

cellular overgrowth is implicated in age-related pathologies

treated cells, normal cell proliferation represented by an

like atherosclerosis and inflammation.

increasing Cell Signal was observed in wells which weretreated with media control (values, p<0.0001) (Figure 4A, B).

Media
IFN-γ+TNF
Palbociclib
PIT

Cell Signal

800
600
400

800

0

0

48

72

96

120

Time after seeding (h)Media Control

144

IFN-γ+TNF

96 h

Media
Palbociclib
IFN-γ+TNF
PIT

900
750

Y = 2.791*X + 441.7

600
Y = 0.4485*X + 476.5
Y = -0.4498*X + 513.7

450
300

24 h

Palbociclib

Induction

Palbociclib

400

200

24

Media

PIT

600

200

0

****

1000

Cell Signal

Induction
1000

D

C

B

Cell Signal

A

Impedance analysis of mammalian cells grown on planar

Y = -0.4607*X + 496.7

0

24

48

72

96

Time after seeding (h)IFN-γ + TNF

Palbociclib + IFN-γ + TNF

100 µmFigure 3 A Proliferation curves of the murine RT2 ß cancer cells measured at 70 kHz over a time period of 5 days post-plating. At t=24 h cells weretreated with the senescence-inducing agents IFN-γ+TNF, Palbo or PIT. B Bar graph plots of Cell Signal amplitudes at t= 24 and t=96 h. C Strength of
the senescence induction calculated as the slope of a regression curve (Palbo: 0.45x; IFN-γ+TNF: -0.45x; PIT:-0.46x; media control: 2.79x). D Inductionof senescence as verified using enzymatic ß-gal staining.

A

Media
Doxorubicin

550

450

0

24

48

B

96

✱✱✱✱

800

Cell Signal

72

120

Time after seeding (h)

144

Media
Doxorubicin

600
400
200
0

21 h

130 h

750

Cell Signal

Media Control

650

700

Y = 1.227*X + 512.8

650
600
550

Y = 0.02295*X + 526.9

500
450

Doxorubicin

Cell Signal

750

C

D

0

24

48

72

Time (h)96

100 µm120

Figure 4 A Proliferation curves of the HCT116 cells measured at 50 kHz over a time period of 5 days post-plating. At t=24 h cells were treated withthe senescence-inducing doxorubicin. At t=48 h, the media was replaced with culture media without doxorubicin. Cells were cultured for anotherperiod of 3 days. B Bar graph plots of Cell Signal amplitudes at t= 21 h and t=130 h. C Strength of the senescence induction calculated as the slopeof a regression curve (Doxo: 0.02; Media Control: 1.23). D Induction of senescence as verified using enzymatic ß-gal staining.

film electrodes provides a label-free, non-invasive andrecorded ranging from 100 Hz – 100 kHz.

unbiased observation of cellular properties addressing thebiological response to putative senescence inducers, drugs,

In general, cell adhesion and proliferation assays suchtoxins or stressors in general21. Being label-free, automation

as immune-cell -mediated killing of cancer cells can be

and continuous monitoring of barrier function are among

successfully performed.

the most significant advantages offered by electrical
techniques in comparison to macromolecular solute

We exemplarily showed that:permeability studies20.
• senescence-mediated growth arrest can be detected
The label-free AtlaZ impedance recording system enables

using impedance measurements of cancer cells;

acute and chronic assessment of cellular toxicity as well as
senescence in a continuous fashion from living cells under• dynamics of senescence-mediated growth arrest, induced

physiological temperatures and without the confoundingby compound treatments, can be investigated using

effects of dyes that may affect cell function. The system

AtlaZ. A persistency of the effect during chronic exposure

uses 96-well plates with 96 parallel sensors offering a time

proves that not only acute but also chronic effects can be

resolution of down to 1 s for impedance measurements,

analyzed;thus allowing investigation of fast effects like GPCR related
morphology changes. A frequency spectrum can be• frequency spectrum function offers wide application ofimpedance measurements for cell structural changes, such5.

as cell size changes.
Multi-frequency impedance data allow for a non-invasive

production is necessary for cell senescence. Mol Syst Biol 6(1) 6(1):347.
6.

Rodier, F., et al., 2011. DNA-SCARS: distinct nuclear structures that sustain
damage-induced senescence growth arrest and inflammatory cytokine

continuous monitoring of cells with the option to zoom in on

secretion. Journal of Cell Science 124(1):68-81.

certain cell aspects or to tailor the readout to individual cell
types for improved data interpretation.

Passos, João F, et al., 2010. Feedback between p21 and reactive oxygen

7.

Kirkland, J. L., 2023. Tumor dormancy and disease recurrence. Cancer andMetastasis Reviews 42(1):9-12.

We conclude that the AtlaZ system provides a versatile
tool for in vitro cell monitoring addressing the demands for8.

Braumuller, H., et al., 2013. T-helper-1-cell cytokines drive cancer into
senescence. Nature 494(7437):361-5.

versatility, physiological relevance and throughput.
9.

Muller-Hermelink, N., et al., 2008. TNFR1 signaling and IFN-gamma signaling
determine whether T cells induce tumor dormancy or promote multistage
carcinogenesis. Cancer Cell 13(6):507-18.

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Contact Information
Dr. Barbara Schoerg
Postdoctoral Researcher

15. Carpenter, V.J., et al., 2021. Senolytics for Cancer Therapy: Is All That Glitters
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Eberhard Karls University of Tübingen, Germany

cancer stemness. Nature. 4;553(7686):96-100. doi: 10.1038/nature25167. Epubbarbara.schoerg@med.uni-tuebingen.de2017 Dec 20. PMID: 29258294.

Dr. Sonja Stoelzle-Feix

17. Saleh, T., et al. 2019. Tumor cell escape from therapy-induced senescence.

Director, Scientific Affairs

Biochem Pharmacol. 162:202-212. doi: 10.1016/j.bcp.2018.12.013. Epub 2018

Nanion Technologies GmbHDec 19. PMID: 30576620.

Sonja.Stoelzle-Feix@nanion.de
www.nanion.de

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Acknowledgments
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