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Page 6 of 18 Cheng et al. J Cancer Metastasis Treat 2021;7:17 https://dx.doi.org/10.20517/2394-4722.2021.27

Figure 1. Comparison of osteolytic ER+ BMET progression in young vs. skeletally mature mice supplemented with 0.72 mg E . (A)
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Osteolytic lesion incidence and (B) osteolytic lesion area in young (5-week-old) and skeletally mature (16-week-old) mice
supplemented with 0.72 mg E and inoculated with ER+ tumor cells (n = 8-13/group). Inset, representative immunohistochemical (IHC)
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images demonstrating cytokeratin+ (left panel; brown), ERα+ (right panel; brown) human breast cancer tumors in tibiae. ***P ≤ 0.001,
****P ≤ 0.0001 young vs. skeletally mature mice, by 2-way ANOVA with Sidak’s post-test. There was no significant difference (n.s.) in
osteolytic lesion incidence by Log-rank (Mantel-Cox) test. (C) Representative hind limb radiographs in young (top) vs. mature (bottom)
age-matched control (left panels), naive E (0.72 mg)-supplemented (middle panels), or tumor cell-inoculated and E (0.72 mg)-
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supplemented mice (right panels) 6 weeks post-inoculation and E supplementation. Osteolytic lesions are marked by arrows.
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tumors) in supporting ER+ MCF-7 BMET progression, a postulate further supported by findings in 5-week-
old mice inoculated instead with osteotropic ER- MDA-MB-231 cells, where treatment with the same 0.72
mg E dose led to an increase in osteolytic lesion size (3.5 ± 0.8-fold increased lesion size in E -supplemented
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(n = 12) vs. control mice (n = 10), P < 0.01), but unchanged incidence (91.6% vs. 80.0%, P > 0.05), consistent
with prior reports of pro-metastatic, anabolic E bone effects in ER- BMET models [35,36] .
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Differential effects of E (0.72 mg) on bone turnover in tumor-naive young vs. skeletally mature mice
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Because radiographs suggested that anabolic effects of E (0.72 mg) on bones in tumor-bearing mice could
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be age-dependent, direct bone effects of this E dose (0.72 mg) were quantified in tumor-naive mice of both
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ages to assess the postulate that E (0.72 mg)-driven ER+ BMET osteolytic lesion size was greater in mice
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whose bones yielded a greater anabolic E response (i.e., young mice). Total tibial aBMD increased
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significantly in response to E (0.72) over 6 weeks of supplementation in tumor-naive mice of both ages
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[Figure 2A], but with a larger increase in younger mice (68% vs. 23%), whose BMD was lower at baseline
and still increasing in untreated age-matched controls. Cross-sectional microCT images [Figure 2B]