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Cheng et al. J Cancer Metastasis Treat 2021;7:17 https://dx.doi.org/10.20517/2394-4722.2021.27 Page 3 of 18

effects were assessed in a murine model of osteolytic ER+ BMET using intracardiac (IC) injection of ER+
human breast cancer cells. Studies included an exploration of dose- and age-dependent effects of E with the
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goal of identifying conditions under which E effects on bone turnover could be accounted for separately
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from direct effects of E on ER+ tumor-mediated osteolysis. The primary objective of these studies was to
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determine whether tumoral ERα signaling, in addition to well-known proliferative effects that are not site-
specific, could be driving osteolysis within the bone microenvironment, thus potentially explaining the
greater proclivity of ER+ (vs. ER-) breast cancer cells to form clinically evident osteolytic BMET.

METHODS
Cell lines and culture
Human ER+ breast cancer tumor cell lines, MCF-7, T47D, and ZR-75-1 [American Type Culture Collection
(ATCC), Manassas, VA], or bone-tropic ER- human MDA-MB-231 (MDA-SA) cells [41,42] , generously
provided by Dr. Theresa Guise, were cultured in E -replete Dulbecco’s modified Eagle’s medium (DMEM;
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Invitrogen, Carlsbad, CA) or RPMI-1640 (Invitrogen), as per ATCC’s recommendation, containing 10% of
heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA), 1% of
penicillin/streptomycin (Thermo Fisher, Waltham, MA) in 37 °C, and 5% of CO in a humidified
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atmosphere. All human cell lines were authenticated, as previously described [41,43] , including MCF-7 BMET-
derived tumor cells used in parathyroid hormone-related protein (PTHrP) secretion experiments, which
were isolated from osteolytic BMET-bearing limbs 42-56 days post-inoculation and passaged twice to
remove non-immortalized and non-adherent murine cells prior to authentication.

Animal studies
All animal protocols were approved by the Institutional Animal Care and Use Committee at The University
of Arizona (UA) in accordance with the National Institutes of Health Guide for the Care and Use of
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Laboratory Animals. Four or 15-week-old female Foxn1 athymic nude outbred mice (Envigo, Indianapolis,
IN) were received and housed in plastic cages (maximum 5/cage) in laminar flow isolated hoods with ad
libitum access to water and autoclaved mouse chow (NIH-31 Modified diet, Envigo). The number of
animals required was determined a priori with the statistical goal of detecting a significant difference of
osteolytic lesion area between groups assuming a moderate effect size with α = 0.05 and β = 0.80 (G*Power
[44]
Software v3.1) . Mice (n = 8-13/group) were inoculated at approximately 5- or 16-week of age with 1 × 10
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human breast cancer cells (MCF-7, MDA-SA, T47D, or ZR-75-1) via the left cardiac ventricle (IC), as
previously described , either in estrogen-naive mice, or in estrogen-supplemented mice 3 days post-
[41]
placement of 60-day extended-release 17β-estradiol (E ) pellets (0.05, 0.10, 0.18, 0.36, or 0.72 mg, Innovative
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[34]
Research of America, Sarasota, FL) . In separate experiments, as indicated, mice not inoculated with tumor
cells (tumor-naive) were similarly treated with E pellets to determine effects on bone turnover independent
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of tumor-associated osteolysis. Additionally, to examine the possible influence of E supplementation on
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tumor cell dissemination to bone, mice 3-days post-E pelleting (vs. controls, n = 3-5/group) were
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inoculated with 8 × 10 MCF-7 cells freshly labeled with Vybrant DiD, as per the manufacturer’s
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instructions (Thermo Fisher), with fluorescent membrane staining remaining detectable for up to 7 days in
culture. Twenty-four hours post-inoculation, cells were isolated from each proximal (25%) tibia, the most
common and earliest site of BMET (data not shown), by flushing with media and repeated washing and
crushing of bone. Cells thus isolated were seeded into 48-well cell-culture plates (3 wells/tibia) and allowed
to adhere overnight prior to imaging of DiD-positive cells using the Cy5 filter of a Keyence BZ-X700
fluorescent microscope (Keyence Corporation of America, Itasca, IL). DiD-positive (DiD+) tumor cells,
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quantified using ImageJ software (National Institutes of Health, NIH), are reported as DiD+ cells/10 bone
marrow cells for each tibia. Similarly isolated cells from tumor-naive mice were included as negative
controls, and cultured MCF-7 cells 24 h-post DiD labeling were used as positive microscopy controls. No
changes in health status necessitating euthanasia occurred in mice, which were also examined 6-week post-