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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 7 of 13

A

B

C

D E F

Figure 3. Intraosseous tumor area, apoptosis and proliferation. Tumor burden in bone was analyzed in histological sections of the left hind
leg. Apoptotic tumor cells were stained using the TUNEL method and stained cells with apoptotic morphology were counted. Proliferation
index was analyzed from Ki-67 stained sections. A: Representative images of HE + Orange G stained sections showing tumors in bone
at sacrifice (day 25); B: representative images of the TUNEL stained sections; C: representative images of the Ki-67 stained sections; D:
intraosseus tumor area did not differ between the groups; E: apoptotic tumor cells relative to tumor area were increased in both groups
receiving DOX, but not in the ZOL group; F: proliferation index did not differ between the groups. Data are expressed as mean ± SD, n =
8 animals. Scale bars 1 mm (A); 50 μm (B and C). ***Significantly different from control group (P < 0.001); NS: non-significant; T: tumor;
M: marrow; Tr.B.: trabecular bone; HE: hematoxylin-eosin; TUNEL: terminal deoxynucleoitidyl transferase dUTP nick end labeling; DOX:
doxorubicin; ZOL: zoledronic acid

DISCUSSION
Direct anti-tumor effects of bisphosphonates on established tumors in bone have been reported in many
preclinical studies [5-7] . Decrease in tumor growth has been observed also in a model with defective
[19]
osteoclasts . However, findings have not been positive in all models, and clear clinical proof has been
lacking. Additive or synergistic effects when combined with chemotherapeutic agents observed in some
preclinical models of bone metastases as well as primary tumors have further fueled the interest in
the anti-tumor actions of bisphosphonates. Several clinical studies with combination of neoadjuvant