Elsevier

Cancer/Radiothérapie

Volume 25, Issue 2, April 2021, Pages 126-134
Cancer/Radiothérapie

Original article
In vitro effects of Trastuzumab Emtansine (T-DM1) and concurrent irradiation on HER2-positive breast cancer cells

https://doi.org/10.1016/j.canrad.2020.06.028Get rights and content

Abstract

Background

To determine the effects of concurrent irradiation and T-DM1 on HER2-positive breast cancer cell lines.

Methods

Five human breast cancer cell lines (in vitro study) presenting various levels of HER2 expression were used to determine the potential therapeutic effect of T-DM1 combined with radiation. The toxicity of T-DM1 was assessed using viability assay and cell cycle analysis was performed by flow cytometry after BrdU incorporation. HER2 cells were irradiated at different dose levels after exposure to T-DM1. Survival curves were determined by cell survival assays (after 5 population doubling times).

Results

The results revealed that T-DM1 induced significant lethality due to the intracellular action of DM1 on the cell cycle with significant G2/M phase blocking. Even after a short time incubation, the potency of T-DM1 was maintained and even enhanced over time, with a higher rate of cell death. After irradiation alone, the D10 (dose required to achieve 10% cell survival) was significantly higher for high HER2-expressing cell lines than for low HER2-expressing cells, with a linearly increasing relationship. In combination with irradiation, using conditions that allow cell survival, T-DM1 does not induce a radiosensitivity.

Conclusions

Although there is a linear correlation between intrinsic HER2 expression and radioresistance, the results indicated that T-DM1 is not a radiation-sensitizer under the experimental conditions of this study that allowed cell survival. However, further investigations are needed, in particular in vivo studies before reaching a final conclusion.

Résumé

Objectif de l’étude

Déterminer les effets du T-DM1 avec une irradiation concomitante sur des lignées cellulaires de cancer du sein HER2-positives.

Matériel et méthodes

Cinq lignées cellulaires humaines de cancer du sein (étude in vitro) présentant différents niveaux d’expression de HER2 ont été utilisées pour déterminer l’effet thérapeutique potentiel du T-DM1 combiné à l’irradiation. La toxicité du T-DM1 a été évaluée à l’aide d’un test de viabilité et une analyse du cycle cellulaire a été effectuée par cytométrie de flux après incorporation de BrdU. Les cellules HER2 ont été irradiées à différents niveaux de dose après exposition au T-DM1. Les fractions de survie ont été déterminées par des tests de croissance cellulaire (après 5 temps de doublement de la population).

Résultats

Les résultats ont révélé que le T-DM1 induisait une létalité significative due à l’action intracellulaire de la DM1 sur le cycle cellulaire avec un blocage significatif de la phase G2/M. Même après une courte période d’incubation, la toxicité du T-DM1 a été maintenue et même renforcée au fil du temps, avec un taux de mort cellulaire plus élevé. Après irradiation seule, la D10 (dose nécessaire pour obtenir une survie cellulaire de 10 %) était significativement plus élevée pour les lignées cellulaires à forte expression de HER2 que pour les cellules à faible expression de HER2, avec une relation linéairement croissante. En combinaison avec l’irradiation, dans des conditions qui permettent la survie cellulaire, le T-DM1 n’induit pas de radiosensibilité.

Conclusion

Bien qu’il existe une corrélation linéaire entre l’expression intrinsèque de HER2 et la radiorésistance, les résultats indiquent que le T-DM1 n’est pas un sensibilisateur au rayonnement dans les conditions expérimentales de cette étude qui permettent la survie cellulaire. Cependant, des recherches supplémentaires sont nécessaires, en particulier des études in vivo avant de parvenir à une conclusion finale.

Introduction

Anti-HER2 (Human Epidermal Growth factor Receptor 2) drugs have become standard treatment for HER2-positive breast cancer patients and are routinely used in adjuvant and neoadjuvant therapy, and for metastatic disease [1], [2]. Trastuzumab emtansine (T-DM1, Kadcyla®), is an ADC (Antibody-drug conjugate) which combines trastuzumab (an anti-HER2 monoclonal antibody) covalently linked via a non-reducible linker to the maytansinoid DM1, a powerful mitotic spindle inhibitor [3]. T-DM1 improves overall survival in patients with HER2-positive metastatic breast cancer [4], [5], [6]. T-DM1 binds the extracellular domain of HER2 via the trastuzumab component and enters into the cell. The HER2-T-DM1 complex is then internalized and cleaved by lysosomal degradation, allowing intracytoplasmic release of the cytotoxic agent DM1, a potent inhibitor of tubulin polymerization [7].

In vitro, down-regulation or over-expression of HER2 in breast cancer cells induces changes in the radio-response. In HER2 transfected cells, the mechanisms of radioresistance are complex and have not been fully elucidated, but the PI3-K/Akt (Phosphatidylinositol-3-Kinase/Protein kinase B) pathway appears to play a major role in radioresistance by deregulating the cell cycle, accelerating DNA repair mechanisms, leading to resistance to apoptosis [8], [9], [10], [11], [12].

It has been previously reported that anti-HER2 therapies, and more specifically Trastuzumab and Lapatinib (inhibitor of tyrosine kinase activity in the intracellular domain of HER2), are specific radiosensitizing agents for HER2-positive cells [8], [9], [13]. These findings have led clinicians to deliver radiation concurrently with these anti-HER2 drugs [14].

We therefore conducted an in vitro preclinical study in HER2-positive breast cell lines to evaluate the potential radiosensitizing effect of T-DM1.

Section snippets

Cell lines

Five HER2-positive cell lines expressing different levels of HER2 amplification and one triple-negative (MDA-MB-231) human breast cancer cell line (TNBC) were used in this study (see characterics of these cell lines in supplementary table TS1). All cell lines, originally derived from the ATCC®, were kindly provided by Drs L. De Koning and T. Dubois (Institut Curie). Cells were routinely subcultured every 5 days and grown (37 °C, 5% CO2) as monolayers in RPMI (HCC1954 and ZR-75-1), DMEM/F12

T-DM1: a long and major cytotoxic effect depending on the level of HER2 expression (except for BT474)

The HER2 protein expression of cells lines was first demonstrated by western blots, confirming different levels of HER2 expression for the various cell lines (Fig. 1A and B). Cell lines were classified as follows: high (HER2 3+: HCC1954, SKBr3, BT474), moderate (HER2 2+: MDA-MB-453) and low (HER2 1+: ZR-75-1) HER2 expression. The triple-negative cell line MDA-MB-231 was used as negative control. The three HER2 3+ cell lines showed approximately the same levels of HER2. HER2 expression by

Discussion

This in vitro study provides three take home messages: The first is that T-DM1 presents a delayed toxic effect on HER-2 positive cell lines. The second is the linear correlation between intrinsic HER2-positive status and radioresistance (D10). The third is that concurrent combination of T-DM1 allowing cell survival with radiation does not radiosensitize HER2-positive breast cancer cells.

Despite improvement of the outcome associated with the use of anti-HER2 drugs, between 10 and 15% of patients

Conclusion

Our work indicated that:

  • within the first 24 hour-incubation time, the main determining factor is the presence of HER-2 receptors: the higher, the better (except for BT-474). For the moderate and low HER-2 expressing cell lines, T-DM1 uptake increased more slowly over time;

  • the G2/M blockade observed on the triple-negative (HER-2 negative) cell line leads us to think about a HER-2 independent trapping for T-DM1, even if we have no clear evidence of this mechanism;

  • the effects of T-DM1 on BT-474

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Disclosure of interest

The authors declare that they have no competing interest.

Funding

Academic study. This work was supported by financial aid from the Institut Curie, INSERM and CNRS.

Authors’ contributions

FM and FMC conducted the experiments. FM and FMC analyzed the data. PV, MPTF and YK read and approved the final manuscript.

Acknowledgements

The authors thank Genetech, USA Inc. (San Francisco, CA) for providing T-DM1 under Material Transfer Agreement (# OR-215963) and Drs Nora Ady-Vago and Fanny Bouquet (Roche Institute, France) for their help and continuous support. Members of the Institut Curie RadeXp platform is acknowledged. We want to thank Emilie Brun (Institut Curie, Genomics Platform) for DNA sequencing, Leanne de Koning (Institut Curie, RPPA Platform) and Thierry Dubois (Institut Curie, Breast Cancer Biology Group,

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