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Page 10 of 18 Cheng et al. J Cancer Metastasis Treat 2021;7:17 https://dx.doi.org/10.20517/2394-4722.2021.27

Assessing possible E dose-dependency of ER+ tumor cell dissemination to bone
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Because E pellets were placed 3 days prior to tumor cell inoculation to allow for stabilization, studies were
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undertaken to assess possible dose-dependent E effects on ER+ tumor cell dissemination to bone.
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Following inoculation of DiD-labelled MCF-7 cells, DiD+ tumor cells detected in the proximal tibia - while
trending slightly higher in E -treated vs. control mice 24 h post-inoculation [Supplementary Figure 2] - were
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not statistically different. Most importantly, there was no evidence of a dose-dependent E effect in mice
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treated with the lowest (0.05 mg) vs. highest (0.72 mg) E doses tested [Supplementary Figure 2]. There was
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also no evidence of an age effect, when comparing bone disseminated DiD+ MCF-7 cells in young vs.
skeletally mature mice treated with 0.72 mg E [Supplementary Figure 2].
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Assessing possible E dose-dependency of ER+ tumor burden and proliferation in bone
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Because proliferative effects of E on ER+ MCF-7 cells are well described in vitro and in vivo at orthotopic
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sites [17,58] , a possible E dose-dependency for histologic tumor burden (size) and tumor cell proliferation in
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bone were assessed 6 weeks post-inoculation, when osteolytic lesion size was still increasing. While the
mean area for cytokeratin+ ER+ breast cancer tumors in bone tended to be smaller for lower E doses, the
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range of tumor sizes was similar across doses without a statistical difference in mean values [Figure 4A]; nor
was there a significant linear trend for increasing doses. Tumor burden in 0.72 mg E -pelleted young vs.
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skeletally mature mice was also not statistically different [Figure 4A]. Tumor cell proliferation, assessed by
Ki67-positivity, was notable in E -supplemented mice (> 60%), but again was without E -dose or age-
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dependency [Figure 4B].
Assessing E dose-dependency of ER+ tumor-associated osteolysis
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Having eliminated differential tumor cell dissemination or proliferative effects of E as being responsible for
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the E dose-dependence of ER+ osteolytic BMET lesion progression, dose-dependent effects of E on tumor-
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associated osteolysis-specific mechanisms were next assessed. While E suppresses osteoclast numbers in
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estrogen-deficient bone , in ovary-intact tumor-naive mice, neither the highest (0.72 mg) nor the lowest
[60]
(0.05 mg) E dose altered osteoclast numbers per bone surface at 2 weeks [Table 1] or 6 weeks (data not
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shown). However, consistent with E dose-dependent increases in ER+ BMETs osteolytic lesion size and
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incidence [Figure 3B-C], the number of bone-resorbing osteoclasts at the tumor-bone interface of ER+
tumor cell-inoculated mice treated with the highest (0.72 mg) E dose was significantly greater than that in
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mice treated with the lowest (0.05 mg) E dose, where osteoclast numbers on bone surfaces interfacing with
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tumors [Figure 5A] were not different from those in age-matched, tumor-naive control mice [N.Oc/BS, 10.9
± 1.8 (n = 6), P > 0.05]. The osteolytic factor, parathyroid hormone-related protein (PTHrP), which is
expressed in most clinical breast cancer BMET [8,11,59,61-63] , was secreted constitutively from ER+ MCF-7 tumor
cells used for inoculation, while constitutive PTHrP secretion from ER+ tumor cells isolated from MCF-7
BMET lesions was 2- to 3-fold higher (P ≤ 0.05) [Figure 5B]. In both inoculated and BMET-derived cells,
tumoral PTHrP secretion was further increased (P ≤ 0.05) in response to E treatment, resulting in 2-fold
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higher levels of E -induced PTHrP secretion from BMET-derived (vs. inoculated) ER+ tumor cells. As with
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in vivo BMET-associated osteolysis, E -inducible PTHrP secretion in vitro was also dose-dependent
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[Figure 5C]. Moreover, E induction of PTHrP secretion was ERα-mediated; MPP, an ERα-specific
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[64]
antagonist that did not alter tumoral PTHrP secretion (data not shown), blocked E -induced PTHrP in
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BMET-derived tumor cells (Figure 5D; P ≤ 0.01). Furthermore, PPT, an ERα specific agonist with an affinity
[65]
for ERα similar to that of E (and 410-fold higher for ERα vs. ERβ) , significantly induced PTHrP to
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identical levels as compared to an equimolar concentration of E [Figure 5D].
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DISCUSSION
Anti-estrogen hormone therapies and bisphosphonates each have a proven benefit in reducing the
development and progression of osteolytic ER+ BMETs; however, BMETs still occur in ~80% of women