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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 3 of 13

concentrate (0.8 mg/mL) was diluted in saline to obtain the concentration of 0.02 mg/mL. Saline was used as
[8]
a vehicle control. MDA-MB231(SA)GFP were authenticated using short tandem repeat analysis (GenePrint10
system, Promega, Madison, WI, USA) at Institute for Molecular Medicine Finland (FIMM, Helsinki,
Finland).

In vivo study
The animal experiment was approved by the Animal Experiment Board of Finland and performed according
to European Union directive 2010/63/EU. Five-week-old female athymic nude mice (Hsd: Athymic nude-
nu, Harlan Laboratories B.V./Envigo, Horst, The Netherlands) were used. The mice were housed in groups of
five in Scantainer™ (Scanbur, Karlslunde, Denmark) and fed an autofluorescence-reducing chlorophyll-free
purified diet (AIN-76A, Special Diets Services, Witham, UK). The animals were weighed three times per week
5
and daily during the last five days. On day 0, 10 MDA-MB231(SA)GFP breast cancer cells were inoculated
into the left cardiac ventricle of the animals, as described previously [16,17] . The mice were anesthetized with 4
mg/kg xylazine (Rompun Vet 20 mg/mL, Bayer Animal Health GmbH, Leverkusen, Germany) and 75 mg/kg
ketamine (Ketalar 10 mg/mL, Pfizer, NY, USA) intramuscularly (im) for inoculations and imaging procedures.
Buprenorphine (Temgesic, Invidior UK Ltd, Slough, UK) was used as analgesic at 0.1 mg/kg subcutaneously
(s.c.) and was administered once before the intracardiac inoculations and twice a day during the last five days
of the study. The mice were randomized into four treatment groups (n = 8) on day 14 based on tumor growth
and osteolytic lesion size as well as body weight. Tumor growth and osteolytic lesion size were measured
by fluorescence imaging and X-ray analysis, respectively, on study day 14 and at sacrifice. The mice were
sacrificed on day 25, except the 2-3 mice per group, which were sacrificed earlier due to weight loss over 20%
or paraplegia. All the mice were included in the endpoint analyses. The mean day of sacrifice was very similar
in all groups ranging from 24.13 in the DOX group to 24.63 in the ZOL group.

The control group received vehicle 5 mL/kg s.c. on day 15 and intraperitoneally (i.p.) on days 15 and 22. The
DOX group received half of the maximal tolerated dose, DOX 2.5 mg/kg i.p. on days 15 and 22. The ZOL
group received a single dose of ZOL 0.1 mg/kg s.c. on day 15. The DOX + ZOL group received DOX (2.5 mg/kg
i.p.) on days 15 and 22 and a single dose of ZOL (0.1 mg/kg s.c.) 24 h after the first dose of DOX, i.e, on day
16 (schedule of the combination group dosing is shown in Figure 1).

Blood samples were collected from saphenous vein to 100 µL Microvette serum tubes (Sarstedt AG&Co,
Nümbrecht, Germany), on days 1, 9, 17 and 24. Terminal blood samples were obtained by cardiac puncture.
Five intact animals of the same strain and age were sacrificed on day 25 in order to obtain the baseline level
2+
for blood Ca (ionized calcium). Femur and tibia were collected for histomorphometric analyses.

Fluorescence imaging
Metastatic growth of disseminated breast cancer cells was followed by fluorescence imaging using the LT
9 GFP imaging system LT-MACIMSYSPLUSC (Lightools Research, Encinitas, CA, USA) on days 10, 14
(randomization), and after sacrifice. Autofluorescence was reduced using the chlorophyll-free diet. The
animals were anesthetized and imaged in both the ventral and dorsal position. The imaging settings (exposure
2.34 s, gain 6.7 and offset 238) were the same for all animals. The area of fluorescence was determined from
the images using MetaMorph™ software (Molecular Devices, Sunnyvale, CA, USA).

Radiography
Development of bone lesions was monitored by radiography on days 10, 14 (randomization), and prior to
sacrifice. The animals were anesthetized and radiographed (31 kV, 10 s, 2× magnification) in a prone position
using the faxitron specimen radiographic system MX-20 DC-2 and Specimen Radiography Beta 2.0.0
software (both from Faxitron Bioptics, LLC, Tucson, AZ, USA). Lesion number and area were determined in
tibias and femurs and the resulting data were analyzed using MetaMorph™ software.