Application Note

FLEXcyte 96
Chronic cardiotoxic effects of tyrosine kinase inhibitors
and anthracyclines analyzed with the FLEXcyte 96 on
human iPSC-derived cardiomyocytes
Summary
In cancer research, the intense developmentof targeted therapeutics such as tyrosine
kinase inhibitors (TKIs) has brought
tremendous improvement to the survival rateof cancer patients over the last two decades.
The goal to reduce diverse toxic side effectsof cancer treatment with targeted therapy has
been widely achieved in comparison totraditionalanti-cancer
treatments
likeanthracyclines.
Nevertheless,boththerapeutics, TKIs and anthracyclines, stilllead to adverse cardiotoxic side effects suchas left ventricular dysfunction and heart
failure. [1] [2]
The severity of these side effects depends on
dosage and time span of treatment whichbrings chronic assessment of cardiotoxicity
into focus.
However, acute testing (min to hours) of cell
models with low predictive value remains theprimary application so far, due to the inabilityof common cell-based assays to analyze
cellular behavior reliably over prolonged
periods of time.

Figure 1. FLEXcyte 96 device with FLEXcyte 96 plate

To ensure a safer transition of new drugcandidates into clinical stages, a modern
preclinical approach is of vital importance toevaluate chronic cardiotoxic side effects.
To meet this need, human induced pluripotent
stem cell-derived cardiomyocytes (hiPSCCMs) are employed for acute as well as
chronic evaluation of preclinical cardiotoxic
risk using the FLEXcyte 96 system. This newtechnology analyzes cardiac contractility withspecial flexible membranes as substrate forthe cells on a 96 well plate instead ofcommonly used stiff glass or plastic surfaces.
This physiological in vitro environment has aproven pro-maturation effect on the cells
leading to adult-like cardiac responses toknown compounds. [3] [4] [5]Here, we present the applicability of hiPSC-CM
contractility measurements for chronic
toxicological assessment using the highthroughput FLEXcyte 96 system. 15 kinase
inhibitors and 3 anthracyclines with wellknown cardiotoxic profiles were selected toevaluate the reproducibility of clinical data.
The results showed the expected cardiotoxiceffects, including negative inotropy andinduction of proarrhythmic events and ceased
beating in a dosage and timespan dependend
manner and proving the advantage of theFLEXcyte system over commonly used cellbased assays for preclinical cardiac risk
assessment.

Application Note FLEXcyte 96

A. Erlotinib

B. Vandetanib

110%

110%
* *

90% * *

**
*
*

70%
50%
30%70%

*

*

50%

30%

100%

* ***

*

*

*

*

*

amplitude*

amplitude

90%

amplitude

C. Idarubicin

10%-10% 0246-10% 040%0%246

*
*

60%

20%

10%

*80%

* ** ** ** ** *024control

10nM

control

10nM

control

10nM

100nM

1uM

100nM

1uM

100nM

1uM

10uM

10uM

610uM

Figure 2. Chronic cardiotoxic effects of kinase inhibitors and anthracyclines. Amplitude of hiPSC-CM (iCell® CM2, FCDI) cultured on
FLEXcyte 96 well plates after treatment with kinase inhibitors erlotinib (A), vandetanib (B) and anthracycline idarubicin (C). Graphs
show dose and time-dependent effects on hiPSC-CMs over the five days incubation span. Graphs represent mean ± SEM. Asterisksrepresent statistical significance with p < 0.05 (*) or p < 0.01 (**) (Wilcoxon-Mann-Whitney test, n = 4).

Results
Figure 2 shows the time and dose-dependent
effects of kinase inhibitors erlotinib,
vandetanib as well as athracycline idarubicin
treatment on hiPSC-CMs cultured on FLEXcyte
96 well plates.
Cardiotoxic effects were evaluated in atimeframe of 5 days of incubation. For thispurpose, hiPSC-CMs were incubated withconcentrations ranging from 10 nM to 10 μM
and beat parameters were monitored daily.
Erlotinib, an EGFR (epidermal growth factor
receptor) and tyrosine kinase inhibitor
generally regarded as non-cardiotoxic, had aminor dose and time-dependent effect on
hiPSC-CMs only at concentrations in themicromolar range (Fig. 2A). At 1 μM, adecrease in beat amplitude was observed
from day 4 on, reaching 60 ± 9% of control on
day 5 with a tendency to further decrease. At
the highest concentration of 10 μM, astatistically significant decrease in amplitude
to 70 ± 6% of control was observed from day 2on. On day 5, the amplitude reached a value of
60 ± 6% of control, comparable to results at
1 μM (Fig. 2A).

Vandetanib, a VEGFR (vascular epidermal
growth factor receptor), EGFR and RET(receptor tyrosine kinase) inhibitor had a dosedependent but not time-dependent effect on
hiPSC-CMs (Fig. 2B). At the highest
concentration of 10 μM, cells ceased beatingtransiently between initial compound additionand day 2. Subsequently, the beat amplitude
was constant at 20 ± 1% of control. At 1 μM,
the amplitude was relatively constant at 50 ±1% of control during the timeframe ofobservation. The two lowest doses of 100 nM
and 10 nM resulted in a decrease of amplitude
to 80 ± 5% and 90 ± 3% of control, respectively(Fig. 2B). The effect of idarubicin, ananthracycline chemotherapy agent, was bothtime and dose dependent (Fig. 2C). At thehighest concentration of 10 μM, the cells
ceased beating already after 2 h of incubation.
At 1 μM and 100 nM, the beat amplitudedecreased to arrest over a time period of 3 and4 days, respectively. At the lowestconcentration of 10 nM, the amplitudedecreased to 60 ± 8% of control over theperiod of 5 days (Fig. 2C).

Application Note FLEXcyte 96

Figure 3. Heatmap of chronic cardiotoxic effects of kinase inhibitors and anthracyclines.hiPSC-CM analyzed after 1 d, 3 d and 5 dof compound treatment on the FLEXcyte 96. Shown parameters are amplitude, beat rate and beat duration. The heat map colours
indicate increasing effects (green) of hiPSC-CMs, stable conditions (yellow) as well as decreasing reactions (red) up to ceasing
effects (deep red). Erlotinib, imatinib, everolimus, sirolimus and temsirolimus are known compounds with low cardiotoxic potential
and served as negative control. Anthracyclines are highlighted in dark grey, TKIs in grey and mTOR inhibitors in light grey.

In total, 15 kinase inhibitors and 3
anthracyclines
wereanalyzed
uponcardiotoxic side effects using hiPSC-CMs on
the FLEXcyte 96. Figure 3 shows a heatmap
summarizing the results regarding effects on
amplitude, beat rate and beat duration.
Chosen time points are 1 d, 3 d and 5 d afteradministration of compounds ranging
between 1 nM – 10 µM to reveal time anddose-depended effects. Known cardiotoxic
anthracyclines such as Doxorubicine andEpirubicine show expected toxic effects evenat lower concentrations after longer
incubation times (deep red). Negative controls
with known low cardiotoxic impact such as
erlotinib, imatinib, everolimus, sirolimus andtemsirolimus only show toxic side effects athigh non-physiological concentrations in atime-dependend manner.

Application Note FLEXcyte 96

The FLEXcyte 96 device employing hiPSCCMs results in a pre-clinical cardiotoxicity
approach that unites cost effective highthroughput for acute and particular chronic
analysis of cardiotoxic side effects.
Methods
Human iPSC-CMs were purchased from
Fujifilm Cellular Dynamics International(Madison, WA, USA).
The cells were cultured on FLEXcyte 96 wellplates according to manufacturers’ guidelines
in 200 µL maintenance medium per well. Cells
were seeded 6 days before compound
treatment at 100k (iCell® CM², FCDI) per wellto allow proper monolayer and network
formation. A final media change wasconducted 4-6 hours before drug application.
For the experiments, 50 µL of the cell culture
medium was removed and replaced with 50 µL
mediumcontaining
4x
concentrated
compound, resulting in the desired final
compound concentration. Measurements
were performed over a period of 5 days.

The CardioExcyte/ FLEXcyte Control software
enables direct analysis of contractility
parameters. An adaptive signal detection
algorithm extracts the positions and values ofbeating events. Beat intervals, amplitudes,
rising and falling time, pulse witdhs aredetected as well as integrals and arrhythmia.
Acknowledgments
iCell® CM² were sponsored by John Hopkins
University Center for Alternatives to Animal
Testing (CAAT).
References[1] Kerkelä et al., Nat Med 12(8), pp. 908-16,2006.
[2] Chu et al., Lancet 370(9604), pp. 20112019, 2007.
[3] Gossmann et al., Cell Physiol Biochem
38, pp. 1182-1198, 2016.
[4] Gossmann et al., J Pharmacol ToxicolMethods, 2020.
[5] Sala et al., Circ. Res. 122 (3), pp. e5-e16,
2018.

Notes

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