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. 2023 Jul 3;37(4):1532–1539. doi: 10.21873/invivo.13238

Effects of Soft Tissue Sarcoma and Doxorubicin on Bone Metabolism in Mice

FUMIHITO KASAMA 1, HIROYUKI TSUCHIE 1, HIROYUKI NAGASAWA 1, MICHIO HONGO 1, YUJI KASUKAWA 1, KOJI NOZAKA 1, DAISUKE KUDO 1, RYO SHOJI 1, SHUN IGARASHI 1, SHUNTARO HARATA 1, KENTO OKAMOTO 1, KEITA OYA 1, NAOHISA MIYAKOSHI 1
PMCID: PMC10347941  PMID: 37369484

Abstract

Background/Aim: This study aimed to evaluate the effects of doxorubicin (Dox) on bone microstructure and metabolism in a mouse model of soft tissue sarcoma.

Materials and Methods: CCRF S-180Ⅱ cells (2-4×105 cells/0.2 ml) were injected subcutaneously into the back of mice. The mice were divided into four groups according to tumor and treatment status and were reared and sacrificed after 2 or 4 weeks. Micro-computed tomography (CT) was performed to calculate the architecture of the femoral bone. The proximal tibia was double stained with tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP), and bone morphometry was performed.

Results: Trabecular bone mass was significantly reduced in the Sarcoma and Sarcoma+Dox groups. Cortical bone thickness was reduced in the DOX group, with a stronger effect observed in the Sarcoma+Dox group. In bone morphometry, osteoclast number at the bone surface (Oc.N/BS) was significantly lower in the Dox, Sarcoma, and Sarcoma+Dox groups than in the Control group at 2 weeks. The osteoblast surface at the bone surface (Ob.S/BS) was significantly lower in the Dox and Sarcoma groups than in the Control group at 2 weeks. At 4 weeks, the differences were smaller for both Oc.N/BS and Ob.S/BS.

Conclusion: The use of doxorubicin alone worsened the cortical bone structure; however, the presence of both soft-tissue sarcoma and doxorubicin use worsened both cortical and trabecular bone structures from an early stage.

Keywords: Soft-tissue sarcoma, doxorubicin, osteoporosis

Soft-tissue sarcoma is a rare cancer, with a reported incidence of approximately 1.5% of all malignant tumors (1). It is estimated that 35-47 new patients per million people visit a hospital with soft-tissue sarcoma annually (2,3), and 12,608 cases of soft-tissue sarcoma were registered in Japan during the 10 years between 2006 and 2015 (4). Although soft-tissue sarcomas of various histological types are generally treated surgically with extensive resection, chemotherapy with anti-cancer agents such as doxorubicin and radiotherapy are also used, depending on the histological type and progression of the lesion. The 5-year overall survival rate of patients with soft-tissue sarcoma is between 55 and 77% (4-6). In recent years, advances in surgical treatment and an increasing number of available chemotherapeutic options, such as pazopanib, trabectedin, and eribulin, have improved life expectancy, though doxorubicin remains the first choice for chemotherapy.

The predilection age for many histological types of soft-tissue sarcomas is middle age or older, which increases the frequency of various complications. Osteoporosis is a typical age-related metabolic disease, with a reported prevalence of 23.1% in women and 11.7% in men (7), affecting an estimated 12.8 million people in Japan (8). It is generally known that postmenopausal estrogen deficiency accelerates bone resorption. However, it has become clear recently that rheumatoid arthritis, inflammatory bowel disease, chronic obstructive pulmonary disease, cardiovascular disease, and cancer are the secondary causes (9). The American Society of Clinical Oncology guidelines recommend bone mineral density testing in patients with non-metastatic cancer with risk factors for osteoporosis (10). The presence of cancer causes low nutrition (11) and reduces physical activity levels (12), and is thought to predispose patients to osteoporosis, which is considered one of the causes of secondary osteoporosis (13). Furthermore, most cancer treatments, including hormone therapy, chemotherapy, and radiotherapy, are associated with cancer treatment-induced bone loss (CTIBL) (14). A 2- to 3-fold increase in fracture risk in patients with CTIBL have been observed and the importance of osteoporosis assessment and medication considerations have been discussed (15). To prevent CTIBL associated with breast and prostate cancer treatment, zoledronic acid and denosumab have been reported to increase bone mineral density and reduce the occurrence of fractures (16-19).

Although secondary osteoporosis and CTIBL have been reported in carcinomas such as breast and prostate cancer, there have been no studies on the effects of soft-tissue sarcomas on bone metabolism. Moreover, the effects of chemotherapy have only been reported in a limited number of carcinomas, such as breast cancer, whereas soft-tissue sarcomas have not been investigated.

This study aimed to evaluate the effects of doxorubicin, which is used as a first-line drug for many soft-tissue sarcomas, on bone microstructure and metabolism in a mouse model of soft-tissue sarcoma.

Materials and Methods

Cell culture. CCRF S-180Ⅱ (JCRB Cell Bank, Osaka, Japan) cells were cultured in Eagle’s Minimum Essential Medium (EMEM; 055-08975; Wako Chemical Industries, Osaka, Japan) supplemented with 5% fetal bovine serum (Mediatech, Manassas, VA, USA) and 0.5% penicillin-streptomycin (15140-122; Life Technologies, Carlsbad, CA). These were maintained in a humidified atmosphere of 5% CO2 in air at 37˚C. The cells were verified to be mycoplasma-free before mouse injection using a PCR-based method (ICLAS Monitoring Center, Kawasaki, Japan). The cells were diluted in phosphate-buffered saline (PBS) to a final concentration of 2.0-4.0×105 cells/0.2 ml. 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; Olympus, Tokyo, Japan; ×400).

Protocol. Ten-week-old female BALB/c mice (Jackson Laboratory Japan, Inc., Kanagawa, Japan) were housed in a specific pathogen-free environment. Air pouches were generated according to the method described by Franco-Molina et al. (20). Notably, 5 days before cell transplantation, an area of the back skin was shaved to provide a pouch site, and 5 ml of air was injected subcutaneously using a 26-gauge needle and a 5 ml syringe. Similarly, 3 ml of air was injected subcutaneously 3 days before cell transplantation. Mice were divided into 4 groups as follows: Control group with only solvent injected in the air pouch (Control), Dox group with only solvent injected in the air pouch followed by weekly intraperitoneal injection of doxorubicin (Dox), Sarcoma group with a suspension of CCRF S-180Ⅱ cells (2-4×105 cells/0.2 ml) injected in the air pouch (Sarcoma), Sarcoma+Dox group with the suspension injected in the air pouch followed by weekly intraperitoneal injection of Dox (Sarcoma+Dox). All mice in the Sarcoma and Sarcoma+Dox groups developed a subcutaneous mass of approximately 10 mm in diameter after 1 week. In the Dox and Sarcoma+Dox groups, Dox was injected intraperitoneally at 3 mg/kg weekly starting 1 week later (21,22). Mice in the four groups were sacrificed after 2 or 4 weeks of captivity (Figure 1).

Figure 1. Experimental groups and time schedule. The mice were divided into four groups. (1) Control group (n=24); (2) Dox group (n=24); (3) Sarcoma group (n=24); and (4) Sarcoma+Dox group (n=24). The Sarcoma and Sarcoma+Dox groups received local injections of tumor cells at 10 weeks of age. Each group was sacrificed after 2 or 4 weeks of captivity.

Figure 1

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Evaluation. Body weight (BW) was measured at the beginning of the experiment and at the time of sacrifice. Tumor volume and weight were measured in the Sarcoma and Sarcoma+Dox groups after the mice were sacrificed. Tumor volume was measured using calipers and expressed using the following equation: tumor volume (mm3)=(2×short diameter×long diameter)×0.5 (23). The weight at sacrifice, excluding tumor weight, was calculated, and compared among the four groups.

Before sacrifice, mice were secured in a sample holder. Micro-CT was performed in the abdominal region using a Cosmo Scan GX II (Rigaku Corporation, Tokyo, Japan) according to the manufacturer’s instructions, with an isotropic voxel size of 120 μm, energy of 90 kVp, and current of 88 μA. Captured images were analyzed using fat analysis software (Rigaku Corporation, Tokyo, Japan) to measure the visceral fat volume in the L4-L5 region (24). The right femur was fixed in 10% neutral buffered formalin (Wako Chemical Industries, Osaka, Japan) and secured in a sample holder for micro-CT, which was 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 based on the cortical area (Ct.Ar), total cross-sectional area (Tt.Ar), relative cortical bone area to tissue area (Ct.Ar/Tt.Ar), cortical thickness (Ct.Th) in the middle of the femur, bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and connectivity density (Conn.D) at the distal femur.

The right tibia was decalcified with neutral 10% ethylenediaminetetraacetic acid for approximately 4 weeks and then embedded in paraffin. Subsequently, 3-μm-thick, mid-frontal slices were obtained and stained with a tartrate-resistant acid phosphatase (TRACP) and alkaline phosphatase (ALP) double-stain kit (Takara, Tokyo, Japan) for bone histomorphometry. Bone histomorphometric analysis of the proximal tibia was performed at 400×magnification using a semiautomatic graphic system (Histometry RT CAMERA; System Supply, Nagano, Japan). Measurements were obtained in an area bounded by 0.2-1.4 mm caudal to the lowest point of the growth plate and 0.1 mm medial to the cortical bone (25). The histomorphometric trabecular bone volume per tissue volume (BV/TV, %), eroded surface per bone surface (ES/BS, %), osteoid surface per bone surface (OS/BS, %), osteoclast number at the bone surface (Oc.N/BS, N/mm), and osteoblast surface at the bone surface (Ob.S/BS, %) were compared among the four groups.

Statistical analysis. All data are expressed as means±standard deviation (SD). The Kolmogorov–Smirnov test showed that all data were normally distributed. Comparisons of data among the four groups were performed using Student’s t-test, one-way analysis of variance (ANOVA), Kruskal-Wallis test, and Tukey’s post hoc test. All statistical analyses were performed using Easy R (26), a modified version of the R commander designed to add statistical functions frequently used in biostatistics. Statistical significance was set at p<0.05.

The protocol for animal experiments was approved in advance by the Animal Experiment Committee of the Akita University School of Medicine, and all subsequent animal experiments were conducted in accordance with the “Animal Experiment Guidelines” of Akita University.

Results

The starting weights did not differ between groups. The Sarcoma and Sarcoma+Dox groups were significantly heavier than the Dox group in the 2-week group (p<0.05), whereas in the 4-week group, the sarcoma group was significantly heavier than the Control, Dox, and Sarcoma+Dox groups (p<0.05), and the Sarcoma+Dox group was significantly heavier than the Control and Dox groups (p<0.05). Tumor weights were not significantly different between the Sarcoma and Sarcoma+Dox groups in either the 2- or 4-week groups. Tumor weight subtracted from body weight at sacrifice was significantly higher in the Control and Sarcoma groups than in the Sarcoma+Dox group at the 2-week group (p<0.05), and in the sarcoma group than in the Dox and Sarcoma+Dox groups at the 4-week group (p<0.05). Tumor volumes did not differ significantly between the Sarcoma and Sarcoma+Dox groups at 2 or 4 weeks (Table I).

Table I. Comparison of tumor weight and size.

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Values are means±standard deviation. Dox: Doxorubicin. ap<0.05 vs. the Control group. bp<0.05 vs. the Dox group. cp<0.05 vs. the Sarcoma+Dox group.

Visceral fat volume was smaller in the Sarcoma and Sarcoma+Dox groups than in the Control and Dox groups at 2 and 4 weeks (p<0.05). Regarding bone microstructure in the cortical bone of the femoral diaphysis, Ct.Th was smaller in the Sarcoma+Dox group than in the Dox group at 2 weeks (p<0.05) and in the Sarcoma+Dox group than in the Control and Dox and Sarcoma groups at 4 weeks (p<0.05). Ct Ar was smaller in the Sarcoma+Dox group than in the Dox and Sarcoma groups at 2 weeks (p<0.05) and in the Dox and Sarcoma+Dox groups at 4 weeks (p<0.05). Ct.Ar/Tt.Ar was smaller in the Sarcoma+Dox group than in the Dox group at 2 weeks (p<0.05) and in the Sarcoma+Dox group at 4 weeks than in the Control, Dox, and Sarcoma+Dox groups. The Sarcoma+Dox group was smaller than the Control, Dox, and Sarcoma+Dox groups (p<0.05). Regarding bone microstructure in the trabecular bone of the distal femur, BV/TV was smaller in the Sarcoma group than in the Dox group at 2 weeks (p<0.05), smaller in the Sarcoma+Dox group than in the Control and Dox groups (p<0.05), and at 4 weeks the Sarcoma+ Dox group was smaller than in the Control, Dox, and Sarcoma groups (p<0.05). Tb.Th was lower in the Sarcoma+Dox group than in the Control and Dox groups at 4 weeks (p<0.05). Tb.Sp was greater in the Sarcoma+Dox group than in the Control and Dox groups at 2 and 4 weeks (p<0.05). Conn.D was lower in the Sarcoma+Dox group than in the sarcoma group at 2 weeks (p<0.05) (Figure 2) (Table II).

Figure 2. μCT image of the distal and diaphysis femur at 4 weeks. The figure shows CT images of the distal and diaphysis femur in the Control (a, a’), Dox (b, b’), Sarcoma (c, c’), and Sarcoma+Dox (d, d’) groups. The Sarcoma+Dox group shows a smaller trabecular bone volume and cortical bone thickness than the Control, Dox, and Sarcoma groups.

Figure 2

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Table II. Comparison of visceral fat and bone architecture of right femur.

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Values are means±standard deviation. Dox: Doxorubicin; Ct.Th: cortical thickness; Ct.Ar: cortical area; Tt.Ar: total area; Ct.Ar/Tt.Ar: cortical area/total area; BV/TV: bone volume/tissue volume; Tb.Th: Trabecular thickness; Tb.Sp: trabecular separation; Conn.D: connectivity density. ap<0.05 vs. the Control group. bp<0.05 vs. the Dox group. cp<0.05 vs. the Sarcoma group.

For bone morphometry of the proximal tibia, BV/TV was significantly smaller in the Sarcoma and Sarcoma+Dox groups than in the Control group at 2 weeks (p<0.05), and at 4 weeks, the Sarcoma+Dox group was significantly smaller than the Control, Dox, and Sarcoma groups. Oc. N/BS was significantly smaller in the Dox, Sarcoma, and Sarcoma+Dox groups than in the Control group only at 2 weeks (p<0.05). Ob.S/BS was significantly smaller in the Dox and Sarcoma groups than in the Control group at 2 weeks. ES/BS and OS/BS were not significantly different among the four groups (Table III).

Table III. Comparison of bone metabolism of right tibia.

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Values are means±standard deviation. Dox: Doxorubicin; BV/TV: bone volume/tissue volume; ES/BS: eroded surface/bone surface; OS/BS: osteoid surface/bone surface; Oc.N/BS: osteoclast number at bone surface; Ob.S/BS: osteoblast surface at bone surface. ap<0.05 vs. the Control group. bp<0.05 vs. the Dox group. cp<0.05 vs. the Sarcoma group.

Discussion

In the present study, the presence of soft-tissue sarcoma and the use of doxorubicin resulted in a significant reduction in bone mass. In the group with soft-tissue sarcoma, a reduction in visceral fat mass (i.e., the presence of malnutrition) was observed, which may have affected bone metabolism. Moreover, the use of doxorubicin reduced the cortical bone thickness, the effect of which was strengthened by the presence of soft-tissue sarcoma. The use of doxorubicin and the presence of soft-tissue sarcoma alone had little effect on the trabecular bone; however, the combination of both resulted in significant bone loss. Several studies have investigated the relationship between bone metabolism and malignancy. In mouse models of colorectal cancer, it has been reported that bone mass and trabecular number in the distal femur are reduced, along with a decrease in body weight, skeletal muscle, and fat mass (27). It has also been reported that in a mouse model of pancreatic cancer, the expression of the osteoclast-specific markers NFAT2, TRAP, CTSK, and MMP-9 is increased, and bone mass and trabecular thickness in the distal femur are reduced (28). In a mouse model of breast cancer, the use of doxorubicin has been reported to decrease bone mass, and anti-TGFβ antibodies prevent bone loss (29). These are all reports on cancer cells, and no previous reports have investigated the effects of sarcomas on bone metabolism. Our study showed that doxorubicin has a strong effect on bone loss and that soft-tissue sarcoma, similar to other carcinomas, may influence bone loss, though the effect is small in the presence of soft-tissue sarcoma alone.

Cortical and trabecular bones differ in structure and metabolism, with cortical bone being a low-porosity, metabolically inactive tissue, whereas trabecular bone is a honeycomb-like trabecular network with a large remodeling area and high turnover rate (30). These features are closely related to the different roles of cortical bone, including mechanical strength and trabecular bone mineral homeostasis (31). Most previous reports on cancer types have evaluated the trabecular bone and all of them reported a decrease in bone mass (27,28). However, the only evaluation of cortical bone has been a report of reduced bone strength in the femoral diaphysis in a mouse model of colorectal cancer using a three-point bending test, with no reports evaluating cortical bone tissue (27). It has been reported that the use of doxorubicin in a mouse model of breast cancer results in a reduction in trabecular bone volume in the distal femur but no change in the thickness of the trabecular bone (29). In the present study, the presence of soft-tissue sarcoma and the use of doxorubicin reduced both bone volume and trabecular thickness. Doxorubicin alone also reduced cortical bone thickness and area, with a marked reduction in the presence of soft-tissue sarcoma. No other studies have examined both trabecular and cortical bones in animal models in which malignancy is present, as in the present study, and it should be noted that bone structure is compromised in both trabecular and cortical bones.

Inflammatory cytokines and hormonal changes caused by cancer result in malnutrition due to anorexia, which can lead to osteoporosis (32). In young mice, calorie restriction reduced femoral diaphyseal cortical thickness and distal femoral trabecular bone volume. This finding is associated with reduced levels of serum leptin, an essential hormone, and IGF-1, a growth factor, resulting in reduced bone formation and increased bone resorption (33). However, the effects of calorie restriction on bone metabolism vary according to age, duration, and disease (33-36). It also remains unclear whether non-metastatic tumor growth is associated with bone loss (37). In limited reports, TGF-β is involved in reduced bone mineral density in mouse models of pancreatic cancer (38), and in mouse models of lung cancer, JAK/STAT activation and glucocorticoids cause bone loss by impairing the balance between osteogenic and adipogenic differentiation, along with a decrease in bone marrow mesenchymal stem cells (39). Doxorubicin suppresses osteoblast differentiation via the bmp-2/Smad signaling pathway by downregulating bmp-2, smad1/5/9 and OSX in vitro (40). In the present study, the presence of soft-tissue sarcomas may have resulted in reduction in bone resorption capacity and reduced bone formation capacity. Doxorubicin may also reduce bone formation capacity. Soft- tissue sarcomas and doxorubicin may cause low-turnover osteoporosis, a condition of reduced bone resorption and formation, leading to deterioration of bone microarchitecture. In contrast, there was no difference over time in bone resorption in soft-tissue sarcomas and bone resorption and bone formation in doxorubicin-treated cells. Additional investigation of the markers of osteoclast and osteoblast differentiation for transient changes in bone metabolism is warranted.

In the present study, the presence of soft-tissue sarcoma and the use of doxorubicin caused the greatest bone deterioration in both cortical and trabecular bones. However, the effect of doxorubicin on tumor shrinkage was not observed in this study, and if the effect of doxorubicin on tumor shrinkage had been achieved, the effect of the tumor itself and the low nutritional status could have been improved and bone metabolism reduced. Future studies should be conducted using soft-tissue sarcoma cells in which the antitumor effect of doxorubicin is strongly observed.

Osteoporosis drugs are broadly classified as inhibitors of bone resorption and stimulants of bone formation. The osteogenesis-promoting agent teriparatide should not be used to treat CTIBL associated with cancer therapy because of its potential to produce primary or metastatic malignant bone lesions (15). Therefore, bone resorption inhibitors such as bisphosphonates and denosumab are the mainstays of CTIBL treatment (16-19). However, the results of the present study showed a state of low bone metabolic turnover, mainly due to reduced osteogenic potential; therefore, it is difficult to expect the strong efficacy of bone resorption inhibitors. Further studies are needed to determine the effect of bone resorption inhibitors on CTIBL in soft-tissue sarcomas.

This study has some limitations. First, this study did not fully assess the effects on the bone over a long period because of the difficulty in keeping the animals for longer than 4 weeks owing to cancer progression. It is necessary to reduce the number of transplanted cells or create an animal model using different soft-tissue sarcoma cells to generate an animal model of soft-tissue sarcoma with a longer prognosis than that of the present study model. Moreover, this study could not distinguish between the effect of soft-tissue sarcoma itself and that of malnutrition on bone metabolism. The presence of soft-tissue sarcoma and the use of doxorubicin were found to significantly worsen bone metabolism; however, the mechanism of the interaction remains unclear, and a more detailed bone metabolism assessment is required in the future.

In conclusion, the use of doxorubicin alone worsened the cortical bone structure; however, the presence of both soft-tissue sarcoma and doxorubicin worsened both cortical and trabecular bone structures from an early stage. This study suggests that physicians should be aware of the development of osteoporosis early in patients with soft-tissue sarcomas, especially in those using anti-cancer drugs, and to consider initiating osteoporosis treatment if necessary. Because various osteoporosis drugs exist, further studies are needed to identify appropriate ones.

Conflicts of Interest

The Authors have no conflicts of interest directly relevant to the content of this article.

Authors’ Contributions

All Authors were involved in the planning and revising for this research. KF, TH and NH raised experimental animals and administered drugs. KF analyzed the raw data, and wrote this manuscript. HM, KY, NK, KD, SR, IS, HS, OK, OK and MN reviewed this manuscript.

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