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Page 10 of 13 Suominen et al. J Cancer Metastasis Treat 2019;5:14 I http://dx.doi.org/10.20517/2394-4722.2018.64

MB231BO2 or intracardiac MDA-MB231 models. In contrast, we observed clear induction of apoptosis with
DOX alone, but we did not observe any potentiation of apoptosis induction with the sequential combination.
Furthermore, the sequential DOX + ZOL treatment did not decrease tumor area in our study, despite of
increased apoptosis of cancer cells induced by DOX. The absence of any observed decrease in the GFP
positive area representing total tumor burden supports the histological data.

The metastatic behavior between cell sublines can be strikingly different. Sublines of the MDA-MB231 cells
derived through in vivo selection for bone metastases behaved differently, some formed metastases in adrenal
glands and others in bone, some were fast with a high frequency and others were slow with a low frequency
[26]
of metastases . However, the bone or adrenal metastatic sublines of the MDA-MB231 had similar growth
rate when inoculated subcutaneously demonstrating that the cells were not generally more aggressive.
[26]
Importantly, the bone metastatic subline cells exclusively induced osteoclast mobilization and recruitment .
The MDA-MB231(SA) model is more aggressive and progresses faster than the MDA-MB231BO2 model, as
[11]
[8]
the median survival time is 25 days in the former and 60 days in the latter , with the same number of
inoculated cells. However, the different inoculation route is likely to play a role in the disease progression.
It is possible that more cells are seeded to the bone marrow through the intracardiac route, than through
the intravenous route, where the cells have to pass through the lung capillaries first. The MDA-MB231(SA)
cells have been found to express, e.g., more serine and interleukin-11 compared to the parental MDA-MB231
cells [27,28] , whereas the MDA-MB231BO2 cells were found to have decreased expression of the miRNA30
[29]
family regulating several osteomimicry genes, compared to the parental cells . Although the history of the
two sublines is very different, the BO2 subline being derived through in vivo selection and the cells from San
Antonio having occurred spontaneously, a common finding for both MDA-MB231(SA) and -BO2 sublines
compared to the parental line is the strong downregulation of miRNAs 200a and 429 [28,29] . These miRNAs
[30]
are involved in the epithelial-to-mesenchymal transition . Positive effects on tumor growth and survival
have also been observed in a model of MDA-MB436 intratibial tumors using intensive, weekly sequential
[14]
administration of DOX + ZOL . The MDA-MB436 cells are triple negative like MDA-MB231 cells, and
both represent the claudin-low subtype.

As expected, ZOL efficiently prevented the progression of osteolytic lesions as both monotherapy and in
combination with DOX. However, trabecular bone area was not statistically significantly higher in the DOX
+ ZOL group compared to the control group. The lesions visible in X-ray images present mainly lesions in
cortical bone, whereas the reduction of trabecular bone is typically not so clearly observed in the X-ray
images. This could partly explain the difference in radiography and histological data. Furthermore, a lesion
has lost 50% of the bone volume by the time it is visible with planar X-ray imaging. MicroCT analysis would
provide volumetric information of both cortical and trabecular compartments. Unfortunately, this imaging
modality was not available for this study. Furthermore, there were no statistically significant differences
in the numbers of osteoclasts at the tumor-bone interface between the groups. The variation of osteoclast
number measurement was high, affected by the variable amount of measurable tumor-bone interface in
the samples. The reason for not observing significant reduction in numbers of osteoclasts at the tumor-
bone interface might be that the ZOL-induced reduction in the number of osteoclasts has been reported to
be transient, observed three days after ZOL dosing to mice, but non-observable already five days after ZOL
[31]
administration due to the formation of new osteoclasts . Consistently, serum TRACP 5b was decreased
three days post-dosing compared to the control group. Serum TRACP 5b remained low at later time
[32]
points in the groups treated with ZOL, suggesting that the osteoclasts may have been smaller , or that
[12]
the number of osteoclasts along normal bone surface at tumor-free areas was lower. Indeed, Brown et al.
have reported decreased number of osteoclasts in tumor-free bone but not in tumor-bearing bone 13 days
after administration of ZOL. Very importantly, however, the function of the osteoclasts at the tumor-bone
interface was inhibited, as shown by the decreased osteolytic lesion area in the ZOL and DOX + ZOL groups,
the increased trabecular bone area in the ZOL group, and the prevention of hypercalcemia in the ZOL and
DOX + ZOL groups.