Julia Cheng's Abstracts

Lay Abstract

The most common subtype of breast cancer is one where tumor cells removed from the
breast express the estrogen receptor (ER) on their surface (denoted as “ER+”). Advancedstaged
ER+ breast cancer tumors have a very high rate of spreading to the skeleton, a process
known as bone metastasis. When ER+ cells spread to bone, they form osteolytic lesions—the
bone is lysed or “chewed-up” at these sites, causing pain or fracture. While ER+ breast cancer
cells are more likely to spread to bone, scientists currently do not know why. To study this
mystery, a mouse model of human ER+ breast cancer bone metastasis is needed; however,
none are available. Our goal is to develop such a model using a human ER+ breast cancer
tumor cell line commonly used for research, called the MCF-7 cell line.
First, we assessed the ability of MCF-7 cells to form osteolytic bone metastasis when
injected into mice. Because we experimented with human cell lines, we used athymic, or “nude”,
mice with compromised immune systems to prevent any immune system rejection of the tumor.
Before we injected a mouse with tumor cells, it was implanted with an extended-release
estrogen (17β-estradiol) pellet to mimic optimal human growth conditions for the ER+ tumor
cells. As a comparison model, we also injected non-estrogen supplemented mice with a wellcharacterized
ER- tumor cell line. Once a week, we took x-rays of the mice’s hind legs to
observe the formation of osteolytic lesions and to measure bone mineral density (BMD).
Mice supplemented with 0.72 mg of 17β-estradiol and injected with MCF-7 cells formed
large osteolytic lesions by the end of the study, although non-estrogen supplemented mice with
ER- cells formed similar-sized lesions in a shorter duration. Closer assessment of the hind limbs
of MCF-7 tumor-bearing mice suggested that the given estrogen dose of 0.72 mg stimulated a
significant increase in bone mass, which resulted in loss of bone marrow space. This could
make it more difficult for tumor cells to propagate and/or form osteolytic lesions—akin to
termites attempting to chew through wood that continuously increases in density. Therefore, we
sought out to explore the estrogen-dependency of ER+ bone metastasis and BMD changes. We
injected MCF-7 cells into mice with different lower doses of estrogen pellets—from 0.72 to 0.05
mg—and assessed their osteolytic lesion formation and change in BMD. The results showed
that the incidence of osteolytic lesion formation was estrogen-dependent—the lower the
estrogen dose, the lower the number of mice in that group with visible osteolytic lesions.
However, there was no change in BMD between the different doses of estrogen
supplementation—even the lowest dose of estrogen increased BMD the same amount as the
highest dose.
In conclusion, human ER+ MCF-7 cells are able to form osteolytic bone metastases
when injected into estrogen-supplemented mice, despite the marked increase in bone density
caused by the 17β-estradiol pellets. This increase in BMD could be the reason why the ER+
model lags behind the ER- model in terms of osteolytic lesion formation. However, lower doses
of 17β-estradiol pellets did not mitigate the effects of estrogen-increased BMD, but they did
decrease the incidence rate of osteolytic lesion formation. Although there is room for
improvement, this is a novel mouse model to study human ER+ breast cancer bone metastasis,
which could lead to a better understanding of how the disease progresses and thus future
advancements in targeted therapies.

Full Abstract

Title: Characterization of an estrogen-dependent murine model of human estrogen receptorpositive
breast cancer bone metastasis
Authors: Cheng JN1, Frye JB2, Whitman SA2, Kunihiro AG3, and Funk JL2
Affiliations: 1Cancer Biology Graduate Interdisciplinary Program; 2Department of Medicine;
3Department of Nutritional Sciences, The University of Arizona, Tucson, AZ
Introduction: Breast cancer patients with estrogen receptor-positive (ER+) tumors have the
highest rate of developing clinically evident osteolytic bone metastasis. However, much of our
knowledge of breast cancer bone metastasis has been derived from ER- murine xenograft
models, as robust pre-clinical models of ER+ bone metastasis are currently lacking. Thus we
sought to develop and characterize a murine xenograft model of breast cancer bone metastases
using an estrogen-dependent ER+ MCF-7 cell line.
Methods: 28-day-old female nude mice were implanted with the following 60-day extendedrelease
17β-estradiol (E2) pellets: 0.72, 0.36, 0.18, 0.10, or 0.05 mg; with or without 1x105 MCF-
7 cells (ATCC) inoculated via the left cardiac ventricle. Non-estradiol-supplemented female
nude mice were also inoculated with either MCF-7 cells or a well-characterized, bone-tropic
human ER- MDA-MB-231 cell line (referred to as MDA-SA), used as a comparison model.
Radiographs and dual-energy X-ray absorptiometry’s (DXAs) of hind femurs and tibias were
obtained weekly to assess osteolytic lesion formation and BMD, respectively. Statistical
significance was analyzed using one-way ANOVA with Tukey’s post-hoc testing.
Results: Using E2 doses previously reported in the literature to promote MCF-7 orthotopic
tumors and bone metastases (0.72 mg 60-day 17β- E2 pellets), bone metastases developed
with 100% incidence (n = 13) by day 28 in the MCF-7 model, with osteolytic lesion area
increasing to 10.3±0.8 mm2 by day 42 (as compared with 100% incidence on day 21 in nonestrogen-
supplemented mice inoculated with MDA-SA (n=8) with osteolytic lesion area of
10.7±2.0 mm2). However, histologic assessment of hind limbs of MCF-7 tumor bearing mice
suggested that this estrogen dose stimulated a significant increase in bone mass. Therefore, the
estrogen-dependency of bone metastasis formation and changes in BMD were explored. Bone
metastasis incidence was estrogen-dependent; falling to less than 100% in mice with E2 doses
less than 0.72 mg (50-75%, n = 4-12). BMD of distal femurs of mice supplemented with
estradiol was significantly greater than naïve controls on day 28 (average increase of 65.3%,
p<0.05), and was not dose dependent over the range of doses tested (0.05-0.72 mg pellets).
Conclusion: Despite a marked increase in bone density, ER+ MCF-7 cells formed
radiographically detectable osteolytic lesions with 100% incidence by day 28 in 0.72 mg 60-day
17β- E2 supplemented nude mice. It is possible that estrogen-stimulated increases in BMD
contributed to the longer time required to achieve ER+ osteolytic lesion sizes comparable to
those in standard ER- models. However, lower doses did not mitigate effects of estrogen on
BMD in these 1-month-old mice, while lowering the incidence of bone metastasis formation.
While further optimization is planned, intracardiac inoculation of MCF-7 cells in female nude
mice provides a robust model of estrogen-dependent ER+ breast cancer bone metastases.
Source of Research Support:
R01 CA174926-01
R03 CA181893-01