List of Abbreviations Used: VEGF vascular endothelial growth factor
Osteosarcoma is a highly malignant bone tumor that most commonly affects adolescents and young adults. Advances in multimodality treatments consisting of aggressive chemotherapy and wide resection of the tumor have markedly improved the prognosis for patients with osteosarcoma. 25 However, pulmonary metastasis occurs in approximately 50% of the patients and still is the major cause of mortality in patients with osteosarcoma. 27
Similar to other patients with solid organ cancer, 3,29 more than 50% of the patients with osteosarcoma have pulmonary metastasis at the time of diagnosis of the primary lesion, although such dormant micrometastases often are asymptomatic and are clinically undetectable. Some of these patients have a relapse with pulmonary metastasis within months after removal of the primary lesion. Therefore, to improve the prognosis of patients with osteosarcoma, the prevention of progression of pulmonary metastasis postoperatively is essential and one of the key points in therapeutic strategy for osteosarcoma.
Concomitant tumor resistance is the phenomenon by which primary large tumors are able to inhibit the growth of smaller metastatic tumors. 21,23 Several animal experiments showed that resection of the primary tumor leads to rapid progression of metastatic foci suggesting the presence of concomitant tumor resistance in several kinds of cancer. 19,30 However, there are no studies that show the presence of this concomitant tumor resistance in osteosarcoma.
Although the mechanism of progression of remote metastasis postoperatively has not been well understood, experimental data indicate that removal of the primary tumor leads to downregulation of circulating levels of antiangiogenic factors and that the following enhancement of systemic angiogenesis causes loss of suppression of micrometastatic disease with subsequent growth and promotion of gross nodular disease. 8 If the progression of pulmonary metastasis is associated with elevation of systemic angiogenesis postoperatively even in patients with osteosarcoma, antiangiogenic therapy may suppress progression of pulmonary metastasis and lead to improvement in the prognosis for patients with osteosarcoma.
In the current study, the authors examined the effects of primary osteosarcoma tumor removal for progression of pulmonary metastasis to clarify whether the presence of primary osteosarcoma tumor suppresses the progression of pulmonary metastasis. In addition, whether removal of the primary osteosarcoma tumor enhanced systemic angiogenic activity was assessed, and whether subcutaneous delivery of TNP-470, an angiogenesis inhibitor, suppressed the progression of pulmonary metastasis after removal of the primary osteosarcoma was investigated.
MATERIALS AND METHODS
Female BALB/c nu/nu mice, which originated from the Central Institute for Experimental Animals (Kawasaki, Japan) were obtained from CLEA Japan Inc (Tokyo, Japan). Six-week-old mice that weighed approximately 20 g were used. All animals were pair fed.
Cells and Cell Culture
A mouse osteosarcoma cell line, LM 8 (Riken Bank, RCB1450), was provided by the Department of Orthopedic Surgery, Osaka University Medical School, Osaka, Japan. 1 Cells were maintained at 37° C at 5% CO2 in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS), penicillin, and streptomycin.
TNP-470 was a gift from Takeda Chemical Industries Ltd (Osaka, Japan). In in vivo experiments, TNP-470 was suspended in a vehicle composed of 0.5% ethanol plus 5% gum arabic in saline.
Pulmonary Metastasis Assay
Female 6- to 7-week-old nude mice were inoculated subcutaneously with murine LM8 osteosarcoma (1 × 105 cells/0.1 mL phosphate buffered saline). Two weeks after tumor inoculation, the animals were divided into two groups. In one group, the mice were anesthetized and the primary tumor was removed surgically. In the other group, the mice were anesthetized and subjected to a sham operation leaving the primary tumor intact. The lungs were removed 2 weeks after surgery and the number of macroscopic pulmonary metastases was counted. For treatment with antiangiogenic drugs, mice whose tumors had been removed received injection of 30 mg/kg of TNP-470 or vehicle alone every other day starting on the day of tumor removal. Five animals were used in each group and each experiment was done in triplicate.
Matrigel Plug Assay for Evaluation of Angiogenesis
Mice whose tumors were removed and whose tumors were intact received subcutaneous injections of 0.5 mL of Matrigel (Becton Dickinson, Bedford, MA) containing 200 ng of bFGF (Upstate Biotechnology, Lake Placid, NY) at the time of the operation. Matrigel pellets were harvested 1 week after the inoculation and reliquefied by incubation at 4° C overnight in 300 μL phosphate buffered saline. Matrigel neovascularization was determined quantitatively by measuring the hemoglobin content of the liquefied pellets using the Drabkin’s method and Drabkin reagent kit 525 (Sigma Chemical Co, St Louis, MO). 20,24 The concentration of hemoglobin was calculated from a known amount of hemoglobin assayed in parallel. Protein content of the supernatant fluid was determined using the BioRad protein assay methods (Bio-Rad Laboratories, Hercules, CA). In brief, the absorbance of the mixture of protein assay dye reagent and Drabkins’s solution with reliquefied Matrigel plug was measured at a wave length of 595 nm. Hemoglobin concentration was obtained directly from the calibration curve of absorbance value versus blood hemoglobin. Five mice were used in each group and each experiment was done in triplicate.
Vascular Endothelial Growth Factor and Endostatin Production
Serum levels of VEGF and endostatin were measured by using an enzyme-linked immunosorbant assay kit (VEGF, IBL, Fujioka, Japan; endostatin, Cytimmune Science Inc, College Park, MD) at the time of the evaluation of pulmonary metastasis. Five mice were used in each group and each experiment was done in triplicate.
Lung metastasis, hemoglobin concentration, and the serum levels of VEGF and endostatin were analyzed using the Student’s t test. Statistical significance was defined as a probability less than 0.05.
Effects of Removal of Primary Osteosarcoma on Progression of Pulmonary Metastasis
Whether the removal of the primary osteosarcoma enhanced the progression of macroscopic pulmonary metastasis in vivo was examined first. The number of macroscopic pulmonary metastases after the removal of the primary tumor were counted and compared with those of the sham operation groups. As shown in Figure 1, the number of macroscopic pulmonary metastases significantly increased in groups in which the primary tumor was removed compared with the control groups (p < 0.05). Severe weight loss was not observed in all animals at the end of the experiments (tumor intact group, 23.2 ± 0.3; tumor removed group, 22.0 ± 1.5; not significant in Student’s t test). These findings showed that the resection of the primary osteosarcoma resulted in the enhancement of the progression of macroscopic pulmonary metastasis.
Promoted Angiogenic Activity In Vivo After Removal of Primary Osteosarcoma
To clarify the mechanisms of how tumor resection enhanced the progression of pulmonary metastasis, whether tumor removal promoted angiogenic activity in vivo using a Matrigel plug neovascularization assay was examined. Matrigel plug neovascularization was quantitated by measuring the hemoglobin content of the pellets using Drabkin’s methods. 21,25 The hemoglobin concentration of Matrigel plugs inoculated into the nude mice whose tumors were removed was significantly higher than in mice whose primary tumor was intact (p < 0.05) (Fig 2). These results indicated that the primary tumor could suppress the systemic angiogenic activity and that the resection of the primary tumor resulted in elevation of the systemic angiogenic activity of nude mice.
Downregulation of Serum Levels of Vascular Endothelial Growth Factor and Endostatin After Removal of Tumor
The serum levels of a known angiogenesis stimulator (VEGF) and inhibitor (endostatin), which are involved in angiogenesis were measured. As shown in Table 1, VEGF and endostatin concentrations were lower in the serum from the animals in which the tumor was removed than in the serum from the animals in which the tumor was intact.
TNP-470, an Angiogenic Inhibitor, Suppresses Establishment of Pulmonary Metastasis After Removal of Primary Osteosarcoma Tumor
To investigate whether elevation of angiogenic activity after removal of the primary tumor induces progression of pulmonary metastasis, whether the administration of an exogenous angiogenesis inhibitor could suppress the progression of pulmonary metastasis after resection of the subcutaneous osteosarcoma tumor was examined. TNP-470 is an analog of fumagillin with well-described antiangiogenic activity. 9,12,13 Mice whose tumors had been removed were treated with TNP-470 or vehicle alone and the number of macroscopic pulmonary metastases was counted. As shown in Fig 3, the increase in the number of macroscopic pulmonary metastases after removal of the primary tumor was inhibited efficiently by administration of TNP-470 (p < 0.05). In addition, postoperative elevation of the systemic angiogenic activity was suppressed significantly by administration of TNP-470 (Fig 2). The administration of TNP-470 had no effect for the serum levels of VEGF and endostatin in the mice whose tumors had been removed (Table 1).
Patients with osteosarcoma often have a relapse with pulmonary metastasis soon after the primary tumor is removed. Although this phenomenon suggests that concomitant tumor resistance is present even in patients with osteosarcoma, direct evidence of concomitant tumor resistance in osteosarcoma has not been reported. In the current study, the authors showed that removal of the primary osteosarcoma enhanced the establishment of pulmonary metastasis. Furthermore, this malignant progression was caused by activation of systemic angiogenesis. These findings indicate that the presence of a primary osteosarcoma tumor can retain the pulmonary metastasis dormant state through angiogenic suppression and can be evidence for the presence of concomitant tumor resistance even in osteosarcoma.
At least three hypotheses have been proposed to explain concomitant tumor resistance: (1) the primary tumor induces an immunologic response against metastasis; (2) depletion of available nutrients by the primary tumor; and (3) production of antimitotic factors by primary tumor cells that directly inhibit proliferation of tumor cells in metastasis. 22,26 However, none of these hypotheses has provided the basis for a molecular mechanism by which small metastatic tumor growth is suppressed by the primary large tumor mass. Recently, another hypothesis has been proposed to explain this phenomenon. Several reports showed that this resistance depends on circulating antiangiogenic factors secreted from the primary tumor using several animal experimental models. 4,8–19 In the current study, removal of the primary tumor resulted in the downregulation of serum levels of not only VEGF but also of endostatin. When VEGF and endostatin are shed from the tumor bed into circulation, levels of VEGF decrease rapidly whereas levels of the more stable endostatin rise relatively, which creates a systemic antiangiogenic environment that prevents small metastatic tumors from inducing neovascularization. As a result, these incipient tumors in the remote site remain small and dormant. Although a previous report showed that doses as high as 20 mg/kg/day of endostatin were required for an antitumor effect, 17 its absolute serum levels to block neovascularization have not been determined. As the systemic angiogenic activity depends on the balance of angiogenic stimulator and angiogenic inhibitor, the ratio of VEGF to endostatin may be an important factor.
Once the primary tumor has been removed, endostatin levels decrease and systemic angiogenic suppression by primary tumor can be weakened. 3,8 Angiogenesis inducers such as VEGF, which have been secreted from metastatic tumors, can promote vascularization in the small metastatic tumor and the growth of dormant metastasis is rapid. In the current experiment, however, the status of other angiogenic factors such as angiostatin 19 or thrombospondin-1 30 were not evaluated. Therefore, it is not known which angiogenic inhibitor is dominant for establishment of concomitant tumor resistance. Furthermore, unknown angiogenic factors may be involved in this phenomenon.
Currently, treatment for osteosarcoma consists of a combination of chemotherapy and tumor resection. Although this therapeutic strategy improves the prognosis for many patients, some patients still die of postoperative pulmonary metastases. 16 Resection of the primary tumor is mandatory to treat osteosarcoma successfully. However, the current data indicate the possibility that resection of the primary osteosarcoma tumor enhances progression of pulmonary metastasis through activation of systemic angiogenesis and makes the prognosis for patients with osteosarcoma poor. Therefore, suppressing postoperative elevation of angiogenic activity is critical to improving the prognosis for these patients. Angiogenesis is essential for establishment of remote metastasis leading to the concept of angiosuppression as a new therapeutic strategy for treatment of patients with cancer. 7,10 Several clinical trials using antiangiogenic reagents have begun. 11,28 Regarding osteosarcoma, two reports showed that TNP-470 could suppress the establishment of pulmonary metastasis in animal osteosarcoma models. 14,15 The current authors also showed that postoperative administration of TNP-470 could suppress progression of pulmonary metastasis. In contrast, the clinical significance of antiangiogenic therapy has been diminished by the failure of antiangiogenic reagents to lead to tumor shrinkage in clinical trials. 11,28 However, antiangiogenic reagents still have the potential to prevent additional tumor growth, 28 implying their therapeutic role in retaining the micrometastases as a dormant state during the postoperative period. Postoperative antiangiogenic therapy may enable patients with osteosarcoma to coexist with dormant metastasis.
The current findings show the possible molecular mechanism for postoperative progression of pulmonary metastasis in patients with osteosarcoma. The findings also indicate the basis for therapeutic strategy targeting angiogenesis to osteosarcoma. The withdrawal of angiogenic suppression after resection of the primary tumor is associated highly with progression of pulmonary metastasis postoperatively. Therefore, antiangiogenic therapy should be done immediately after primary tumor removal. This therapeutic strategy may improve the prognosis for patients with osteosarcoma.
Preoperative chemotherapy is expected to prevent progression of the primary tumor and often leads to a limb salvage procedure. In parallel, preoperative chemotherapy also is considered to have a cytotoxic effect for the dormant micrometastases. In the current study, however, the effects of the combination of antiangiogenic agents and chemotherapeutic agents were not investigated. The clinical significance of the combination of antiangiogenic agents and chemotherapeutic agents should be investigated.
The authors thank Dr. H. Yoshikawa for supplying tumor cell lines, M. Ono and M. Naka for secretarial assistance, and M. K. Barrymore for comments on the manuscript.
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