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Suominen et al. J Cancer Metastasis Treat 2019;5:14 I http://dx.doi.org/10.20517/2394-4722.2018.64 Page 11 of 13

High variation was a problem in this study and may have masked treatment effects on osteoclast number.
Larger groups and more sensitive methods, such as µCT and luciferase labelled cells could have helped in
reducing the variation. Intraosseus tumor area can only be analyzed by histology, which was restricted to
analyzing the midsagittal sections in order to standardize the analysis. However, analyzing several levels
could have reduced the variation. In order to further clarify the reasons underlying the insensitivity of
the MDA-MB231(SA) tumors in bone to the combination treatment, as well as the unresponsive portion
of patient population, the ZOL uptake of the MDA-MB231(SA) cells after DOX administration requires
investigation. Longer treatment schedules with repeated cycles of sequential combinations would better
present the clinical situation, but such studies are unfortunately not possible in this model due to the time
frame of disease progression. Furthermore, the strong bone forming ability of the young, fast growing mice
used in this model presents a major difference to the elderly breast cancer patients. New bone formed after
the ZOL administration is not protected by ZOL, and is thus vulnerable to cancer induced degradation.
This could have mitigated the effects of ZOL in this study, but because the treatment period is so short, the
amount of new bone formed in that time period is not likely to influence the results.

In summary, we examined the anti-tumor effects of a single dose of ZOL alone and in sequential
combination with DOX on established bone metastases in the intracardiac MDA-MB231(SA) model. DOX
induced apoptosis in the tumor cells and ZOL prevented tumor-induced bone destruction. However, anti-
tumor effects of ZOL were not observed, nor additive or synergistic effects of the sequential combination of
DOX and ZOL.

DECLARATIONS
Acknowledgments
We gratefully acknowledge Nanna Merikoski, Johanna Örling and Suvi Suutari for their skillful technical
assistance and Aurexel Life Sciences Ltd. for the editorial support.

Authors’ contributions
Conception and design of the study, data analysis and interpretation: Suominen MI
Data acquisition, provided administrative, technical, and material support: Rissanen JP, Käkönen R, Halleen
JM
Revised the manuscript: Härkönen P, Käkönen SM

Availability of data and materials
The data used to support the findings of this study are available from the corresponding author upon
request.

Financial support and sponsorship
None.

Conflicts of interest
Suominen MI, Rissanen JP, Halleen JM and Härkönen P are voting stock holders of Pharmatest Services Ltd.
Käkönen SM and Käkönen R are voting stock holders of Aurexel Life Sciences. Halleen JM is a consultant of
and receipt of royalties from IDS Plc.

Ethical approval and consent to participate
All applicable international, national, and/or institutional guidelines for the care and use of animals were
followed.