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Page 4 of 18 Cheng et al. J Cancer Metastasis Treat 2021;7:17 https://dx.doi.org/10.20517/2394-4722.2021.27

tumor cell inoculation at gross necropsy for non-bone metastases, as determined by researchers and UA
veterinary staff: rare unanticipated deaths were attributable to anesthesia, weights were unchanged in E -
2
treated and/or tumor-bearing mice vs. controls, and urinary retention, a well-characterized side-effect of E -
2
supplementation in mice [45,46] that was not severe enough to warrant early termination, was observed in 20%-
30% of mice treated with higher E doses, as previously reported .
[34]
2
Radiographic determination of osteolytic BMET lesions and E effects on bone
2
To assess the size and incidence of radiographically-evident osteolytic BMET lesions, radiographs of mouse
hind limbs (Faxitron UltraFocus 1000, Faxitron Bioptics, Tucson, AZ) in E -supplemented tumor cell-
2
inoculated mice were obtained weekly over the 6-week course of experiments. Radiographic osteolytic
lesion incidence and area per mouse were determined in a blinded fashion with radiographic images
assessed by three independent investigators using ImageJ software (NIH) . Because E can induce
[34]
2
[34]
osteolytic osteosarcomas in nude mice , the identity of osteolytic BMETs in E -supplemented mice was
2
verified by correlating radiographic lesions in each hind limb bone with histologic evidence of cytokeratin-
positive breast cancer tumors [20,34] prior to calculating radiographic osteolytic BMET incidence or lesion area
per mouse. When calculating average osteolytic lesion size, mice lacking osteolytic BMET were excluded so
that E effects on lesion size could be assessed independent of effects on incidence. In parallel experiments
2
to determine E effects on bone in the absence of osteolytic BMET, dual-energy X-ray absorptiometry
2
(DXA) was performed weekly in E -supplemented mice not inoculated with tumor cells (tumor naive) to
2
[34]
assess changes in tibial areal bone mineral density (aBMD) (Faxitron UltraFocus 1000) . At termination of
these 6-week experiments, to examine E effects specific to trabecular bone, microcomputed tomography
2
(microCT) imaging was performed ex vivo in a subset of mice to assess proximal tibial metaphyseal
trabecular bone volume/total volume (BV/TV) by the Endocrine Research Unit at the San Francisco VA
Medical Center (Scanco microCT 50, Scanco Medical, Basserdorf, Switzerland) as previously described [34,47] .

Bone metastatic breast cancer tumor histology and bone histomorphometry
Hind limbs were removed either 2 weeks (bone histomorphometry) or 6 weeks (bone histomorphometry or
immunohistochemical analysis of Ki67, ER, or cytokeratin) post-tumor cell inoculation, fixed, decalcified,
and paraffin-embedded for histologic analyses of midsagittal (approximate depth of 400-500 µM) 5-6 µM
[48]
sections as previously described . For measuring histologic breast cancer tumor size (tumor burden),
epithelial MCF-7 breast cancer tumors were identified using a pan-cytokeratin primary antibody (#Z0622,
Agilent Dako, Santa Clara, CA) and continued expression of ERα was verified using a human ERα primary
antibody (#ab108398, Abcam, Cambridge, United Kingdom) using previously described
[48]
immunohistochemical (IHC) methods . Cytokeratin-positive breast cancer tumor area in hind limbs
bones was determined in a blinded fashion (expressed per leg as % of tumor area/bone area). Proliferating
breast cancer cells in bone were identified using a primary antibody directed against a human Ki67
proliferation marker (#D2H10, Cell Signaling, Danvers, MA). Breast cancer tumor cell proliferation in each
hind limb was assessed in a blinded fashion by calculating the average number of Ki67-positive tumor cells
in four high power fields per bone (expressed per hind limb as % of total tumor cells), with the mean for
each treatment group determined by averaging values for each limb. In addition, osteoblasts-identified as
hematoxylin-stained mononuclear cuboidal cells lining the bone surface-or multinucleated tartrate-resistant
acid phosphatase (TRAP)-positive osteoclasts were quantified in the tibial metaphyses of tumor-naive mice
or at the bone tumor interface of tumor-bearing mice, as per ASBMR nomenclature Committee Guidelines
and using standard methods, as previously described [34,41,49-53] . Osteoclasts or osteoblasts in tumor-naive mice
are reported as cell number per mm of bone surface (BS) or per tissue area (mm ), and osteoclasts in tumor-
2
bearing hind limbs of mice are reported as cell number per mm of bone at the tumor-bone
interface [34,41,49,54,55] . All images for immunohistology and bone histomorphometry were analyzed using
ImageJ software (NIH).