High content in vitro cell monitoring effects of adjuvant chemotherapy inbreast cancer and cancer treatment-related cardiomyopathy
Ronald Knox1, Krisztina Juhasz2,3*, Oliver Reinhardt4*, George Okeyo1, Elena Dragicevic2, Leo Doer2, Matthias Beckler2, Sonja Stölzle-Feix2, Frauke
Alves4,5, Rodolfo Haedo1, Niels Fertig2
1Nanion Technologies Inc., 1 Naylon Place, Livingston, NJ, 07039, USA; 2Nanion Technologies, Munich, Germany;3Institute for Nanoelectronics, Technische Universität München, Germany; 4Translational Molecular Imaging Group, MPI of Experimental Medicine,Göttingen, Germany; 4Clinic of Hematology and Medical Oncology, University Medical Center Göttingen, 37075 Göttingen, Germany; * These authors contributed equally to this work.

Impedance changes of cell-covered electrodes give
profound insight into cell proliferation and contractility, evenover prolonged time periods, providing significant advantage
over standard mostly endpoint cytotoxicity assays. Here, thistechnology was used for monitoring murine breast cancer cell
growth after chemotherapy treatment in vitro. As theemerging field of cardio-oncology aims to find a balance
between oncologic efficacy and reducing adverse
cardiovascular effects, we tested the same treatment on
induced pluripotent stem cell-derived cardiomyocytes (iPSCCMs). One of the standard clinical regimens for breast cancer
is a combination of cyclophosphamide, Adriamycin(doxorubicin) and 5-fluorouracil (CAF) administered for 4months. Even though initially successful, tumor recurrence after
this therapy remains a major cause of mortality in breast
cancer patients, leaving the need for better treatment.

Long- term cell monitoring withCardioExcyte 96

Investigating therapy resistance of cancer cells in vitro“short term” measurement

100 mg/kg Cyclophosphamide
5 mg/kg Doxorubicin (Adriamycin)
100 mg/kg 5-Fluouracil

Tumor dissociation and cell isolation revealed eleven murine H8N8 mammary carcinoma cellline variants from 3 different groups. The murine H8N8 cells represent an immortal mammarycarcinoma cell line with tumor stem cell properties. H8N8 T1 cells represent untreated H8N8tumors, H8N8 T2 cells represent remitted tumors up to 9 days post CAF and the H8N8 T3 cells
represent a progressive tumor variant after regrowth initiating. Cell proliferation kinetics frommurine H8N8 and H8N8 T3.2 cells were investigated via an impedance-based cell monitoring
system (CardioExcyte 96, left) and an image-based live-cell analysis system (IncuCyte®, right).
The impedance signal recorded on the CardioExcyte 96 changes as a result of alterations inconfluency, cell contact (morphological shape) and conductivity of adherent cells andthereby provides a measure of cell proliferation and toxicity.

In this study, responses from murine H8N8 and isolated H8N8 cell variants were investigated by using the CardioExcyte 96 system. H8N8 T1 cells were established from a solid breast
tumor that received no CAF. H8N8 T2 and T3 cells were established from a solid breast tumor that received the CAF clinical regimen in vivo. For further in vitro tumor recurrence
investigation, the H8N8 and H8N8 T1 cells were treated with CAF for the first and the H8N8 T2 and T3 cells were treated for a second time (concentrations of C: 0.026 μg/ml up to 20μg/ml, D: 0.001 μg/ml up to 1μg/ml and F: 0,026 μg/ml up to 20 μg/ml). The impedance-based measurement allowed monitoring of cell viability over a time period of 168 h with a1 ms time resolution, under in vitro physiological conditions. Here, changes in impedance, and therefore confluency, were used as a measure of cytotoxicity. Intrinsic (dosedependent) effects of the standard clinical treatment regimen CAF could be identified. Furthermore, a dose- and treatment cycle-dependent proliferation of tumor cells could beobserved in some cell lines.

Long-term cardiotoxicity monitoring using impedance

Orthotopic Tumor ModelContraction

Base impedance

H8N8 in vitro culture

functional inactivation of pRB and p53
due to binding by the large T-antigen

Formation of a H8N8 tumor

Orthotopic transplantation of murine H8N8 cells into the syngeneic WAP-T
mammary carcinoma mouse model generates triple negative breast cancer(TNBC) -like tumors similar to TNBC in the clinic. This mouse model serves assource of the murine H8N8 breast cancer cells. The murine H8N8 cells
represent an immortal mammary carcinoma cell line with tumor stem cellproperties.

Conclusions• The label-free CardioExcyte 96 impedance platform enables acute andchronic assessment of toxicity in a continuous fashion from living cells
without the confounding effects of dyes that may affect cell function.
• System uses 96 well plates with 96 parallel sensors offering high resolution
recordings (1 ms imp, 0.1 ms EFP). Recordings are made in a completely
controlled physiological environment.
• Cell adhesion and proliferation assays such as cancer immunotox assays
could be successfully performed.

orthotopic H8N8 tumor

Hormone independent expression of theSV40 T-Antigen in the murine H8N8 cells“long term” measurement

CAF Chemotherapy

Base impedance

CardioExcyte 96

The CardioExcyte 96 is a hybrid system
combining impedance and EFP (ExtracellularField Potential) recordings from the same cell.
System operates using 96 well plates with highresolution sensors (1 ms imp, 0.1 ms EFP). Highqualityconsumables
andcontrolledtemperature and environment, make thissystem ideal for short and long-term cell
monitoringandapplications
suchasproliferation and toxicity assays.
The CardioExcyte 96 comes with anautomated liquid handling system for cellseeding,compoundapplications
andmedium exchange.

In vivo tumor growth monitoring followed
by in vitro impedance recordings

Contraction

Introduction

In this in-vitro study, paclitaxel and the combination of cyclophosphamide, doxorubicin and 5-fluouracil (CAF) were investigated using human iPSC derived cardiomyocytes
via impedance recordings on CardioExcyte 96. Paclitaxel is a microtubule-stabilizing drug that is approved by the FDA for the treatment of ovarian, breast, and lung cancer.1
The CAF chemotherapy regimen if often used in the adjuvant setting for the treatment of nonmetastatic breast cancer or alone for the treatment of metastatic breast
cancer. However, apart from their benefits in fighting cancer, both treatments induce possible heart damage in the clinic, making cardiac toxicity an important side effectof anticancer therapies.2 Here, we monitored the acute and chronic effects of both paclitaxel and CAF on iPSC derived cardiomyocytes (left panels). Black arrows indicate
when the chemotheraphy solution was added. A washout with fresh medium was made after 48h (dashed lines). Cardiomyocyte´s viability and beating patterns were
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no or small effect, while doxorubicin shown significant toxic effects in all combinations (middle panels). We have also observed changes in the impedandce amplitude, here
depicted as the mean beat shape change after 48 hours of treatment (right panels), indicating a possible effect of the treatement on cardiomyocyte contraction. 1 Weaver
BA. How Taxol/paclitaxel kills cancer cells. Mol Biol Cell. 2014;25(18):2677-81.2 Florescu M, Cinteza M, Vinereanu D. Chemotherapy-induced Cardiotoxicity. Maedica (Buchar). 2013;8(1):59-67.

• In combination with murine mammary carcinoma cells, CardioExcyte 96
provides a novel tool for investigating therapy resistance of different H8N8cancer cell variants in vitro.
• Cardiotoxic effects can be reliably tested using CardioExcyte 96
impedance technology. Here we showed the effects of paclitaxel and
CAF on the Cor.4U iPSC derived cardiomyocyte (Ncardia).
• Additionally, the beating pattern of cardiomyocytes could be monitored.
Concentration and time depent decrease was observed afteradministration of paclitaxel and time dependent decrease in case of
doxorubucin combinations.
We thank Ncardia for kindly providing cardiomyocytes(Cor.4U®) on CardioExcyte 96 preplated plates.
We thank Miltenyi Biotec for isolating H8N8 cancer cellvariants from the dissociated H8N8 tumors.