Abstract
Background/Aim: Breast cancer frequently metastasizes to bone, and chemotherapy and hormone therapy can increase osteoporosis risk. Teriparatide (TPTD), an osteoporosis treatment that promotes bone formation, is contraindicated in patients with bone metastases due to concerns about osteosarcoma in animal studies. However, its effects on metastatic bone tumors remain unclear. This study aimed to evaluate TPTD’s effects on breast cancer bone metastases using a mouse model.
Materials and Methods: C57BL/6 mice were injected with E0771 breast cancer cells to establish bone metastasis and breast cancer models. Mice were assigned to the vehicle-treated group (control) or to the TPTD-treated group (80 μg/kg, subcutaneously three times weekly). Tumor weight, volume, bone destruction, pathological fractures, distant metastasis, tumor proliferation (Ki-67, BrdU), and bone microstructure were assessed at 4 and 6 weeks.
Results: In both models, no significant differences in tumor weight or volume were observed between the TPTD and control groups. In the bone metastasis model, bone destruction and pathological fractures were not significantly different. No distant metastasis was observed and there were no significant differences in the percentages of Ki-67-positive and BrdU-positive cells in both models. In the bone microstructure analysis at 6 weeks post-injection, bone volume/tissue volume and trabecular thickness increased in the bone metastasis model in the TPTD group (p=0.02 and p<0.01, respectively), and trabecular separation decreased in the TPTD group (p=0.01).
Conclusion: TPTD did not cause tumor growth, pathological fractures, or bone destruction in our in vivo models, indicating that it may be safe for use in breast cancer.
- Bone metastasis
- teriparatide
- breast cancer
- breast cancer mouse model
- breast cancer bone metastasis model
Introduction
Breast cancer is one of the most common malignancies worldwide, with millions of people affected each year. According to the GLOBOCAN 2018 report by the International Agency for Research on Cancer, there are 2.3 million new cases of breast cancer per year across 185 countries, and the incidence rate generally increases with age (1, 2). In addition, breast cancer has the tendency to metastasize to other organs, and the bones are one of the organs most prone to metastasis (3). For this reason, the proportion of elderly women with breast cancer that has metastasized to the bones is large, and the prevalence of osteoporosis is also generally high in elderly women (4, 5). Furthermore, in patients with breast cancer, bone mass loss is likely to be accelerated by the effects of the tumor itself, as well as by secondary estrogen deficiency caused by chemotherapy or treatments, such as aromatase inhibitors or glucocorticoids (6-8). Consequently, women with breast cancer are at a higher risk of bone fracture and are more likely to require treatment for osteoporosis.
Teriparatide (TPTD), a human parathyroid hormone (PTH) preparation, has been widely used in recent years to treat osteoporosis, which is associated with a high risk of fracture (9). TPTD is mainly used to treat severe osteoporosis because it increases bone mass and bone strength by promoting bone formation through intermittent administration (10). On the other hand, based on the results of animal experiments suggesting an increase in the incidence of osteosarcoma in rats after 2-year daily administration of TPTD, TPTD is not used in patients with primary malignant or metastatic bone tumors (11). However, the effects of TPTD on metastatic bone tumors were not evaluated. Therefore, there are still many unknowns about the effects of TPTD on metastatic bone tumors.
The main objective of this study was to evaluate whether TPTD promotes the growth of metastatic bone tumors by comparing the TPTD-treated group and the non-treated group in breast cancer bone metastasis mouse models. The secondary objective was to investigate whether TPTD promotes the growth of breast cancer itself in a breast cancer mouse model.
Materials and Methods
Cell cultures. E0771 (CH3 Biosystems LLC, NY, USA) cells were cultured in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Mediatech, Manassas, VA, USA) and 100 μg/ml kanamycin sulfate (Meiji Seika Pharma, Tokyo, Japan). These were maintained in a humidified atmosphere of 5% CO2 and a temperature of 37°C (12). Before mouse injections, the cells were confirmed to be mycoplasma free using a PCR-based method (ICLAS Monitoring Center, Kawasaki, Japan).
The cells were diluted in phosphate-buffered saline (PBS) to obtain a final concentration of 1.0×104/μl. The survival rate of the tumor cells was evaluated using the trypan blue dye exclusion method with a hemocytometer (Kayagaki, Tokyo, Japan) under an optical microscope (Olympus BH-210, Tokyo, Japan ×400).
Mouse models of breast cancer and bone metastasis. Six-week-old female C57BL/6 mice (Charles River Laboratory Inc., Kanagawa, Japan) were housed in a specific pathogen-free environment and divided into groups to develop both models: the bone metastasis model (n=40) and breast cancer model (n=40). Before tumor cell administration, the mice were anesthetized using a combination of medetomidine (0.3 mg/kg), midazolam (4.0 mg/kg), and butorphanol (5.0 mg/kg) via a subcutaneous injection. The bone metastasis model was induced by creating a bone socket in the femur and injecting E0771 cells (1.0×105/10 μl) into the femoral cavity (13). To create the breast cancer model, E0771 cells (5.0×105/50 μl) were injected into the right fourth mammary fat pad, as previously described (14). Tumor progression and effects of TPTD were assessed at week 4 and 6 post-injection.
Treatment of mice with TPTD. In both models, the mice were divided into two groups of 20 mice each: the control (vehicle administration) and TPTD (TPTD administration) groups. Two weeks after the tumor cell injection, subcutaneous injections of PBS 0.2 ml or TPTD 80 μg/kg (h-PTH; Asahi Kasei Pharma Corp., Tokyo, Japan) were administered three times/week in the control and TPTD groups, respectively. The dose of TPTD was determined based on previous studies (15-17). The mice in the TPTD and control groups were euthanized at 4 and 6 weeks after tumor cell administration (n=10 each) (Figure 1).
The figure depicts the experimental procedure of the study. We developed bone metastasis and breast cancer models in C56/BL/6 mice (n=40 in each model). Two weeks after tumor cell administration, mice in each model were treated with vehicle (control group; n=20) or with TPTD (n=20) and were euthanized at week 4 and week 6. TPTD, Teriparatide.
Evaluation of tumor growth, bone destruction, and distant metastasis. Before euthanasia, the rate of bone destruction and presence of pathological fractures in the femur metastasis model were evaluated using a micro-focus X-ray computed tomography (CT) system, CosmoScan GX II (Rigaku Corporation, Tokyo, Japan). To calculate the rate of bone destruction, we first measured the length of the femur from the femoral head to the femoral condyle in the sagittal section. The axial section was used to identify the location where cortical bone destruction was partially observed, and the sagittal section was used to confirm the length of the portion of the femur with bone destruction. The following formula was used: length of part of femur with bone destruction/femur length×100 (14). Pathological fractures were defined as those with displacement accompanied by abnormal alignment on micro-CT. The weight and volume of the tumor in the femur metastasis model were defined as those of the right hind limb with the tumor minus those of the left hind limb without the tumor. The volume of the tumor was measured using a caliper and was calculated as the short diameter2×long diameter×0.5, in accordance with previous literature (18). The presence or absence of distant metastasis was confirmed by observation of the lung field using micro-CT, macroscopic observation of the thoracic and abdominal viscera during dissection, and fluorescent labeling with acridine orange (19).
Immunohistochemistry. Tissue samples were prepared by fixing the excised tumors in 10% neutral buffered formalin (Wako Chemical Industries, Osaka, Japan), embedding them in paraffin, and then slicing them into 5 μm-thick sections. In the femur metastasis model, the sections were sliced in the sagittal plane. For histological evaluation, immunostaining was performed for Ki-67, a tumor growth marker, and BrdU, an indicator of DNA synthesis, and the results were visualized using 3,3′-diaminobenzidine (DAB; ab64238, Abcam, Cambridge, MA, USA). The primary antibodies used for staining were Ki-67 (ab15580, Abcam) and BrdU (ab152095, Abcam). The blocking reagent, secondary antibody, and enzyme reagent were obtained from the Histofine SAB-PO Kit (424032, Nichirei Biosciences Inc, Tokyo, Japan). BrdU (ab142567, Abcam) was diluted in PBS to a concentration of 10 mg/ml, and a 100 mg/kg BrdU solution was injected intraperitoneally. The mice were euthanized and fixed within 30 min to 1 h after the intraperitoneal injection of BrdU. The all-in-one BZ-X800 fluorescence microscope (KEYENCE, Osaka, Japan) was used to observe the specimens. The number of cells was calculated by measuring three random fields within the regions where tumor cells were present and calculating the percentage of positive cells (20).
To evaluate the effect of TPTD on normal trabecular bone, the left femur, which had not been manipulated, was fixed in 10% neutral buffered formalin and secured in a sample holder to be examined by micro-CT performed with an isotropic voxel size of 10 μm, energy of 90 kVp, and a current of 88 μA. Captured images were analyzed using TRI/3D BON software (Ratoc System Engineering Co., Ltd., Tokyo, Japan). The bone architecture of the mice was evaluated by measuring the following: bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp) at the left distal femur. Trabecular bone measurements in the left femur were taken 0.5 mm proximally from the distal epiphyseal growth plate, with a height of 1 mm (21).
The protocols for all the animal experiments were approved in advance by the Animal Experimentation Committee of our institute (permit No. a-1-0429), and all subsequent animal experiments adhered to the Guidelines for Animal Experimentation of our institution and were conducted in accordance with the ARRIVE guidelines (22).
Statistical analysis. Continuous variables were expressed as mean±standard deviation, and the Welch’s t-test was used for intergroup comparisons. Categorical data are expressed as count and percentages and were analyzed using Fisher’s exact test. Statistical significance was set at p<0.05. All statistical analyses were performed using R software (version 4.2.2; R Development Core Team, 2022).
Results
There were no significant differences in tumor weight and tumor volume between the TPTD and control groups of both models, at either 4 or 6 weeks (Table I, Figure 2). There was no difference in bone destruction rate and pathological fractures in the bone metastasis model between the two groups at 6 weeks. No distant metastases were observed in any of the specimens of both models. There was no significant difference in the percentages of Ki-67-positive cells and BrdU-positive cells between the TPTD and control groups at either 4 or 6 weeks (Table I, Figure 3 and Figure 4).
Summary of comparison between TPTD and control groups.
Micro-CT 3D image of the right femur in the bone metastasis model at 6 weeks. Representative images of the femur of mice in the TPTD (A) and control (B) groups show that similar bone destruction was observed in both groups. TPTD, Teriparatide; CT, computed tomography.
Ki-67 expression in tumor tissues at week 4 after treatment. Representative histological images show Ki-67 expression in tumor tissues from mice with bone metastasis treated with TPTD (A) or vehicle (B), as well as from mice with breast cancer in the TPTD (C) and control (D) groups (×400 magnification). TPTD, Teriparatide.
BrdU staining in tumor tissues at week 4 after treatment. Representative histological images show BrdU-positive cells in tumor tissues of TPTD (A) and control (B) mice of the bone metastasis model and of TPTD (C) and control (D) mice of the breast cancer model (×400 magnification). TPTD, Teriparatide.
In the bone metastasis model at 6 weeks, BV/TV and trabecular number (Tb.N) were increased (p=0.02 and p<0.01), and Tb.Sp was decreased (p=0.01) in the TPTD group compared to the control group. In the breast cancer model at 6 weeks, BV/TV, Tb.Th, and Tb.N were increased (p <0.01, p<0.01, p=0.03), and Tb.Sp was decreased (p=0.03) in the TPTD group compared to the control group (Table II).
Trabecular bone microstructure of the left distal femur.
Discussion
Bisphosphonates and denosumab, a monoclonal antibody that targets the osteoclast differentiation factor, RANKL, are generally used to suppress the progression of bone metastases (23, 24). These drugs are used to treat osteoporosis; however, high doses or long-term administration is associated with an increased risk of osteonecrosis of the jaw and atypical femoral fractures (25-27). Apart from bone resorption inhibitors, TPTD is another drug against osteoporosis, which acts by promoting bone formation. A limitation of its use in patients with cancer is its association with increased incidence of osteosarcoma. Herein, we investigated the effects of TPTD on the tumor growth in a breast cancer model and a breast cancer cell-derived bone metastasis mouse model.
In the present study, TPTD improved the trabecular microstructure of the distal femur but did not promote the growth of breast cancer cell-derived bone metastases or breast cancer. Our results support that TPTD can be used in patients with breast cancer if there are no obvious bone metastases. Swami et al. reported that, in vitro, intermittent TPTD administration did not affect cell proliferation when administered to breast cancer cells, and that, in vivo, it reduced the incidence of breast cancer bone metastases. A suggested mechanism is that TPTD reduces the expression of VCAM-1, which is involved in breast cancer cell migration, and CXCL12, which is involved in breast cancer cell engraftment in the bone marrow, thereby reducing the incidence of breast cancer bone metastasis (28).
The effects of TPTD on tumor cells have been investigated in several hematological malignancies. In multiple myeloma cells, TPTD suppresses tumor cell growth by increasing the levels of osteoblast-derived anti-tumor factors (including decorin, lumican, and CYR61) (29). In addition, pretreatment with TPTD suppresses tumor cell engraftment in mice with chronic myeloid leukemia. On the other hand, in acute myeloid leukemia, TPTD pretreatment promotes tumor cell engraftment in mice, and in prostate cancer, TPTD increases the number of metastatic prostate cancer cells in the bone marrow, accompanied by an increase in the number of osteoblasts (30, 31). TPTD has been shown to induce osteoblast differentiation and alter the bone marrow microenvironment. These changes can modulate tumor behavior indirectly by affecting the secretion of osteoblast-derived factors and the structural characteristics of the bone niche (28). Its effects appear to differ depending on the type of tumor cell and the underlying mechanisms are not fully elucidated. Therefore, future studies are necessary to investigate the role of TPTD in various carcinomas.
In the present study, TPTD 80 μg/kg was administered to mice three times a week, which is assumed to be equivalent to the frequency of use in humans, which is approximately once a week (15-17). In previous reports involving myeloma, acute myeloid leukemia, chronic myeloid leukemia, and prostate cancer, mice were administered TPTD at a dose of 50-80 μg/kg daily, which is a higher dose than that used in the present study. Further investigation is necessary to determine whether higher doses or more frequent administration of TPTD affect breast cancer cell growth.
This is the first study to perform a detailed investigation of the effects of TPTD on bone metastases of breast cancer. However, some limitations must be acknowledged. First, the observation period was short. In this study, the animals were euthanized within a maximum of 6 weeks after tumor cell administration, and the maximum duration of TPTD administration was set at 4 weeks. This is because in the breast cancer femur metastasis model used in this study, the frequency of tumor death increases after 6 weeks of tumor cell administration. Second, bone metastasis is normally caused by multiple cellular mechanisms, including division, invasion, immune evasion, and control of the tissue microenvironment (32). However, the femur metastasis model used in this study was generated by directly implanting tumor cells into the femur; therefore, it does not reflect the physiological mechanism of metastasis. To resolve these issues, it is necessary to create an animal model with a longer prognosis by inducing bone metastases through physiological mechanisms.
Conclusion
In this study, TPTD administration in a breast cancer bone metastasis model did not exacerbate bone metastasis. Our results support that TPTD may be safe as a treatment for osteoporosis in patients with breast cancer; however, further research is needed to confirm this.
Footnotes
Authors’ Contributions
Conceptualization, Hiroyuki Tsuchie, Yuji Kasukawa, and Naohisa Miyakoshi; Data curation, Takashi Kawaragi; Investigation, Takashi Kawaragi, Fumihito Kasama, Keita Oya, Manabu Watanabe, and Kenta Tominaga; Methodology, Hiroyuki Tsuchie and Hiroyuki Nagasawa; Project administration, Hiroyuki Tsuchie; Resources, Manabu Watanabe; Supervision, Naohisa Miyakoshi; Validation, Michio Hongo, Yuji Kasukawa and Koji Nozaka; Writing – original draft, Takashi Kawaragi; Writing – review and editing, Hiroyuki Tsuchie.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received March 24, 2025.
- Revision received April 14, 2025.
- Accepted April 24, 2025.
- Copyright © 2025 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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