Page 10 - Read Online

Cheng et al. J Cancer Metastasis Treat 2021;7:17 https://dx.doi.org/10.20517/2394-4722.2021.27 Page 5 of 18

Serum markers of bone turnover or estradiol
Serum markers of bone formation [rat/mouse P1NP EIA; Immunodiagnostic Systems (IDS), United
Kingdom] or bone resorption (mouse CTX-1; IDS) were measured in fasting serum collected 2 weeks after
the start of E supplementation (vs. age-matched controls) using commercially available ELISA kits [18,28] as
2
[34]
previously described . E levels in serum collected 2 weeks post pellet placement were assayed by the
2
University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core using a
commercially available 17β-estradiol ELISA developed for use in mice (Calbiotech, El Cajon, CA) . All sera
[56]
were stored at -80 °C prior to assay.
PTHrP assay
To analyze PTHrP secretion from ER+ tumor cells and its E dependency, ER+ MCF-7 cells or ER+ tumor
2
cells isolated from MCF-7 BMET were plated in 24-well plates at a density of 1.3 × 10 cells/well in E -
5
2
depleted media [phenol red-free DMEM (Invitrogen), 10% charcoal-stripped FBS (Valley Biomedical,
Winchester, VA), 1% penicillin/streptomycin (Thermo Fisher), and 200 mM L-Glutamine (Sigma Aldrich,
St. Louis, MO)] for 4 days, during which time cell number did not change for any cell line (data not shown),
prior to treatment with E (10 -10 M, as indicated; Sigma Aldrich), an ERα specific agonist propyl
-6
-11
2
pyrazole triol (PPT; 10 M; Tocris, Minneapolis, MN), an ERα specific antagonist methyl-piperidino-
-8
-6
pyrazole hydrate (MPP; 10 M, Tocris), or vehicle control for 48 or 52 h, as indicated. Conditioned media,
stored at -80 °C after addition of protease inhibitors (Sigma Aldrich), were assayed for secreted PTHrP
using a commercial immunoradiometric assay (Beckman Coulter, Brea, CA). A lack of treatment effect on
cell number during the 48 or 52-h incubation was verified using a commercial MTT assay (ATCC).
Statistical analyses
Unless otherwise noted, data are reported as mean ± SEM, with statistical significance of 2-sided P-values
defined as P ≤ 0.05. Statistical differences were determined using Prism 8.0 software (Graphpad, San Diego,
CA) for 1- or 2-way analyses of variance (ANOVA) with post-hoc testing as well as tests for log-rank,
mixed-effects, and t-test, as indicated. Analyses of skeletal parameters in tumor naive mice were not
corrected for multiple comparisons, using Fisher’s LSD test, to maximize the possibility of detecting dose-
dependent E effects (although none were found) .
[57]
2
RESULTS
E -dependent osteolytic ER+ MCF-7 BMET progression in young vs. skeletally mature E (0.72 mg)-
2 2
supplemented mice
Osteolytic BMETs were not detected in the absence of E supplementation when young (5-week-old) mice
2
inoculated with MCF-7 cells were followed for up to 8 months (data not shown). When supplemented with
an E dose (0.72 mg) supporting in vivo MCF-7 orthotopic tumor growth [17,58] , radiographic osteolytic breast
2
cancer lesions were evident as early as 2 weeks post-MCF-7 tumor cell inoculation of young (5-week old)
mice (Figure 1A and inset), reaching a maximal incidence of 69% within 4 weeks with continuous size
increases over the 6-week course of the experiment [Figure 1B], without evidence of metastases at non-bone
sites. BMET formation in E (0.72 mg)-supplemented MCF-7-inoculated mice contrasted with results in 5-
2
week-old mice inoculated with T47D or ZR-75-1 cells, where no osteolytic BMETs were noted (data not
shown). When skeletally mature (16-week-old) mice supplemented with the same 0.72 mg E dose were
2
inoculated with MCF-7 cells, the progression time course and incidence of osteolytic BMET lesion
formation were the same as those in 5-week mice [Figure 1A]; however, osteolytic lesion size was
significantly smaller [Figure 1B]. Radiographs documenting proximal tibial and distal femoral osteolytic
lesions, also common sites for ER- BMETs , were also notable for clear evidence of E -driven, albeit
[59]
2
possibly differential, increases in bone density in mice of both ages [Figure 1C]. This observation raised
questions about possible contributions of E effects on the bone microenvironment (vs. direct effects on ER+
2