The secretory leukocyte protease inhibitor (SLPI) suppresses cancer cell invasion but promotes blood-borne metastasis via an invasion-independent pathway

An invasion-independent pathway has been proposed as a novel mechanism in blood-borne metastasis, where tumour cells enveloped by sinusoidal tumour vessels enter the circulation without vascular invasion. We previously identified the secretory leukocyte protease inhibitor (SLPI) as a candidate gene responsible for this pathway. In this study, the functional role of SLPI in metastatic dissemination was investigated. We transfected the SLPI gene into a poorly metastatic clone of the MCH66 mouse mammary tumour cell line. Over-expression of SLPI promoted in vivo growth and spontaneous metastasis to the lung, whereas it suppressed invasive activity in vitro. The inoculated tumours of SLPI-transfectants exclusively induced a sinusoidal vasculature and subsequently produced endothelial-coated tumour emboli, which are morphological indices of the invasion-independent pathway. In addition, exogenous SLPI inhibited the migration activity through Matrigel of both tumour cells and human umbilical vein endothelial cells (HUVECs). In vivo angiogenesis assays also demonstrated that SLPI suppressed the migration of newly formed blood vessels. These results suggest that an anti-migratory effect of SLPI on tumour-associated endothelial cells may induce vascular remodelling to form a sinusoidal architecture, and consequently promote invasion-independent metastasis. This study provides a new model for metastasis, based on the mechanism regulated by anti-invasive factors, such as SLPI.     

Sinusoidal tumor angiogenesis is a key component in hepatocellular carcinoma metastasis

Hepatocellular carcinoma (HCC) has a tendency for intravascular dissemination leading to a poor prognosis. The importance of the sinusoidal structure of the tumor vasculature in HCC has been implicated in the metastasis formation. To clarify the role of tumor angiogenesis in HCC metastasis, we morphologically investigated the interaction of HCC cells with blood vessels during the sequential process of metastasis. Autopsy specimens of 80 patients with HCC were examined with immunohistochemistry using a specific antibody against CD31, a marker for endothelial cells. The most frequent sites of metastasis were the liver (82.5%) and lung (43.8%). In most cases, the metastatic process was initiated by vascular involvement where tumor nests surrounded by sinusoidal vessels extend into the portal and hepatic veins. Subsequently, these endothelial-coated tumor emboli enter the circulation, embolize at distant organs, proliferate within the blood vessel and ultimately form metastatic foci. These steps are indicative of an invasion-independent pathway. Our findings in animal models and now in human cases suggest that sinusoidal angiogenesis may represent a novel target for therapeutic strategies to limit HCC metastasis. In combination with primary tumor treatment, perturbation of tumor emboli may reduce dissemination of disease.     

Synergistic inhibition of lung cancer cell invasion,tumor growth and angiogenesis using aptamer-siRNA chimeras

Early metastasis is one of the major causes of mortality among patient with lung cancer. The process of tumor metastasis involves a cascade of events, including epithelial-mesenchymal transition, tumor cell migration and invasion, and angiogenesis. To specifically suppress tumor invasion and angiogenesis, two nucleolin aptamer-siRNA chimeras (aptNCL-SLUGsiR and aptNCL-NRP1siR) were used to block key signaling pathways involved in lung cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Through nucleolin-mediated endocytosis, the aptNCL-siRNA chimeras specifically and significantly knocked down the expressions of SLUG and NRP1 in nucleolin-expressing cancer cells. Furthermore, simultaneous suppression of SLUG and NRP1 expressions by the chimeras synergistically retarded cancer cell motility and invasive ability. The synergistic effect was also observed in a xenograft mouse model, wherein the combined treatment using two chimeras suppressed tumor growth, the invasiveness, circulating tumor cell amount, and angiogenesis in tumor tissue without affecting liver and kidney functions. This study demonstrates that combined treatment of aptNCL-SLUGsiR and aptNCL-NRP1siR can synergistically suppress lung cancer cell invasion, tumor growth and angiogenesis by cancer-specific targeting combined with gene-specific silencing.     

Histopathological study of lymphatic invasion in squamous cell carcinoma (O-1N) with high potential of lymph node metastasis

The process of lymph node metastasis was studied in an animal model (termed O-1N) that was successfully established using a metastatic tumor to the submandibular lymph node from a chemically induced squamous cell carcinoma of the hamster tongue. The model has been maintained by serial transplantation of metastatic tumors into the buccal pouch. Lymphovascular invasion of transplanted O-1N in the tongue was examined in serial histologic sections. Lymphatic vessels were distinguished from blood vessels by Masson's trichrome stain for vascular smooth muscle, BSA-I lectin binding for vascular endothelium, and laminin and type IV collagen immunostaining for the vascular basement membrane. Transplanted tumors enlarged progressively with invasion of surrounding tissues of the tongue and resulted in lymph node metastasis in all animals with successful takes. Local growth of the tumors in the tongue was accompanied by stromal proliferation with abundant dilated lymphatic vessels which contained clusters of tumor cells. On serial sections, the carcinoma cell clusters in lymphatics in the close proximity of tumor nests were in continuity with adjacent tumor nests, whereas such continuity was not recognized in those occurring apart from tumor nests. The formation of isolated carcinoma cell clusters resulting from disintegration of elongated processes of tumor nests with invasion of lymphatics and subsequent transport in lymphatics and deposition in lymph nodes in clusters were well demonstrated in other serial sections. The key step of lymph node metastasis therefore appears to be direct invasion of lymphatic vessels by tumor cells, similar to their invasion of adjacent tissues but different from the way that blood cells escape through vessel walls. Proliferation of lymphatics around tumor nests and transport of tumor cells in clusters would also contribute to the production of metastatic deposits in lymph nodes.     

Endothelial cells expressing Bcl-2 promotes tumor metastasis by enhancing tumor angiogenesis, blood vessel leakiness and tumor invasion

Metastatic spread of tumor cells to vital organs is the major cause of mortality in cancer patients. Bcl-2, a key antiapoptotic protein, is expressed at high levels in a number of human tumors. We have recently shown that Bcl-2 is also overexpressed in tumor-associated blood vessels in head-and-neck cancer patients. Interestingly, enhanced Bcl-2 expression in tumor blood vessels is directly correlated with metastatic status of these cancer patients. In addition, endothelial cells (ECs) expressing Bcl-2 showed increased production of interleukin-8 (IL-8) resulting in significantly enhanced tumor cell proliferation and tumor cell invasion. Therefore, we hypothesized that Bcl-2 expression in tumor-associated ECs may promote tumor metastasis by enhancing tumor cell invasiveness and release in the circulation. To test our hypothesis, we coimplanted tumor cells along with ECs expressing Bcl-2 (EC-Bcl-2) in the flanks of SCID mice. Our results demonstrate that incorporation of EC-Bcl-2 in primary tumors significantly enhanced tumor cell metastasis to lungs and this EC-Bcl-2-mediated tumor metastasis was independent of primary tumor size. In addition, Bcl-2-mediated tumor metastasis directly correlated with increased tumor angiogenesis. Bcl-2 expression in ECs also promoted transendothelial cell permeability, blood vessel leakiness and tumor cell invasion. EC-Bcl-2-mediated tumor cell proliferation and tumor cell invasion were significantly mediated by IL-8. These results suggest that Bcl-2, when expressed at higher levels in tumor-associated ECs, may promote tumor metastasis by enhancing tumor angiogenesis, blood vessel leakiness and tumor cell invasiveness.     

人肺癌细胞LALU血管生成过程的形态学观察及意义

目的：观察人肺癌细胞血管生成过程的病理形态学、超微结构特点及意义。方法：采用人肺癌细胞LALU皮下移植瘤模型,以病理动态形态学及透射电镜不同时期肿瘤血管生成状态。结果：光镜显示,人肺癌移植瘤第2天至第10天之间可分为血管生成前期和血管形成期,在肿瘤血管生成的第20天出现肺转移灶。电镜显示,人肺癌移植瘤第2天出现成血管细胞,第4－10天,不成熟血管内皮细胞逐渐形成务管腔伴有较完整的新生基底板,内皮细胞趋向成熟发展,第20天肿瘤血管内皮细胞更成熟,部分区域新生毛细血管基底板发育不全或缺陷。在以上全过程中癌细胞突起直接与成血管细胞、血管内皮细胞及血管壁相连。结论：人肺癌细胞可诱导自身肿瘤血管生成,病理形态学及电镜有其特征性形态学改变,且与转移密切相关,为肺癌的血管导向治疗提供了重要依据。     

Visualization of nascent tumor angiogenesis in lung and liver metastasis by differential dual-color fluorescence imaging in nestin-linked-GFP mice

Nestin regulatory-element-driven green fluorescent protein (ND-GFP) transgenic mice highly express GFP in proliferating endothelial cells and nascent blood vessels. In the present study, we visualized angiogenesis in experimental lung and liver metastases by GFP imaging in the ND-GFP transgenic mice. The murine melanoma cell line, B16F10 expressing red fluorescent protein (RFP), was injected i.v. in ND-GFP mice. ND-GFP was highly expressed in proliferating nascent blood vessels in the tumors that developed in the lung after tail vein injection, and in the tumors that developed in the liver after portal vein injection of RFP-expressing melanoma cells. Liver metastasis and angiogenesis were imaged intravitally. Doxorubicin significantly decreased metastatic angiogenesis in the liver. These results demonstrate a new imageable model of angiogenesis in metastasis in the liver and the lung. This new model should enable further understanding of the onset of angiogenesis in metastasis and its effect on metastatic growth. The model will serve as a unique screen for inhibitors of angiogenesis of metastatic tumors. The fact that liver-metastasis angiogenesis can be imaged in the live animal enables real-time studies of the effect of angiogenesis inhibitors.     

抗血管生成药物在非小细胞肺癌中的应用及研究进展

新生血管的形成是肿瘤生长、进展、转移的基础,此过程涉及各类受体介导的细胞信号通路,其中刺激血管生成作用最强的生长因子是血管内皮生长因子（VEGF）。随着VEGF作用的深入认识和血管靶向治疗的临床实践,以抗新生血管为核心的治疗策略取得显著成效。现将抗血管药物的作用机制及其在治疗非小细胞肺癌中的研究进展作简要综述。     

Therapeutic potential of renin angiotensin system inhibitors in cancer cells metastasis

Metastasis is a complex process which contributes to the dissemination of cancer cells to other organs and forms new tumor sites. The proliferation of tumor cells is a necessary step for the initiation and progression of cancers and is associated with the formation of new vessels.In the latter stages of metastasis, cancer cells may spread into the extracellular matrix and may form metastatic nodules. Despite efforts to prevent this, effective therapies are limited in the treatment of some malignancies. Among the different tumor properties which could be usefully employed as a cancer target, metastasis may be one suitable target.The renin- angiotensin system is a physiological pathway that contributes to the proliferation of tumor cells, angiogenesis and the inflammatory response in tumor tissue. Angiotensin II (ANGII), a key peptide of this pathway, induces cell proliferation through the activation of two cellular pathways (mitogen-activated protein kinase (MAPK)-STAT3 and phosphoinositide 3-kinase (PI3K) –AKT pathway). AT1-R increases angiogenesis via the elevation of angiogenic factors expression (vascular endothelial growth factor (VEGF) and matrix metallopeptidases (MMPs)). The local activation of the RAS pathway increases the expression of ICAM, VCAM and MMPs genes that are involved in the late steps of the metastasis process.There is some evidence that RAS components are expressed in metastatic tumors and RASIs (renin-angiotensin system inhibitors) could be used to reduce cancer metastasis by affecting the mechanisms involved in several different cancers. Therefore, we have summarized the effects of RASIs, observed in pre-clinical and clinical studies of cancer cell metastasis.     

肝素酶和碱性成纤维细胞生长因子与非小细胞肺癌转移和预后的关系

目的 探讨肝素酶(heparanase)和碱性成纤维细胞生长因子(bFGF)在人非小细胞肺癌(NSCLC)组织中表达的临床意义及其与肺癌转移和预后的关系。方法 采用免疫组织化学、原位杂交和Western blot方法,检测115例人NSCLC石蜡切片和45例新鲜肺癌及对应癌旁正常组织中肝素酶和bFGF的表达情况。采用χ^2检验、t检验、生存曲线和Cox比例风险回归等方法分析肝素酶和bFGF分别表达及共表达的意义。结果 免疫组织化学染色证实肝素酶(91／115)和bFGF(89／115)主要表达在癌细胞质和(或)细胞膜中,在正常肺泡上皮和支气管上皮中则呈阴性表达。Western blot也证实肝素酶在肺癌中的表达明显增高(P=0．041)。统计分析结果显示：肝素酶和bFGF的表达具有明显的一致性(P：0．0001),二者单独表达和共表达均与肺癌的分期、血管侵袭、淋巴结转移、微血管密度和预后有关,其中,二者共表达时与分期和微血管密度的相关性更显著;另外,bFGF还与肺癌的分化程度有关。多因素分析结果显示,肺癌的分化程度、血管浸润、淋巴结转移和bFGF的表达可以作为判断肺癌预后的危险因素,但肝素酶不是影响预后的独立因素。结论 肝素酶和bFGF均与肺癌的转移、血管生成和预后密切相关。     

Significance of lymphatic invasion on regional lymph node metastasis in early gastric cancer using LYVE-1 immunohistochemical analysis

It has been reported that lymphatic invasion is a predictor for lymph node metastasis in early gastric cancer (EGC); however, it has been impossible to differentiate between lymphatic invasion and blood vessel invasion using current staining techniques. We studied the significance of lymphatic invasion on regional lymph node metastasis in EGC by using human lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) antibody, specific to lymphatic vessels, and von Willebrand factor (vWF) antibody, specific to the blood vessels, to clearly distinguish these vascular tissues.EGC tissues were obtained from 66 node-positive and 66 node-negative subjects and were matched by age and sex. These tissues were immunostained with antibodies against LYVE-1 and vWF. Multivariate logistic regression analysis demonstrated that lymphatic invasion was a significant independent predictor for regional lymph node metastasis (odds ratio, 4.667; P = .0094), whereas blood vessel invasion was not. Thus, lymphatic invasion identified by LYVE-1 antibody could predict the existence of regional lymph node metastasis in EGC.     

Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment

Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.     

Angiogenesis dependent characteristics of tumor observed on rabbit VX2 hepatic carcinoma

Objective: To evaluate angiogenesis dependent characteristics of carcinoma proliferation, metastasis and to found if there is tumor vascularity architecture defect. Methods: 36 rabbits were random divided into 2 groups: Experimental group: 18 rabbits liver were implanted with VX₂ tumor by surgery operation; Control group: 18 experimental rabbits performed the same surgery operation without tumor implantation, the course of tumor growth and blood vessel invasion was observed by autopsy. One slide was used for hematoxylin-eosin (HE) staining, one slide was used for elastic fiber staining by Victoria blue and Ponceau’s histochemical staining, and one slide was used for vascular endothelial cell immunohistochemical staining with biotinylated-ulex europaeus agglutinin I (UEA I); all three slides were observed under an optical microscopic. One additional slide was systematically observed by electron microscopy. SPSS 19.0 software was used for the statistical analyses of the data. Results: The tumor grew acceleration after tumor angiogenesis, volume of original tumors was correlated with vascular caliber. The central tumor found necrosis without enough blood supply while tumor grew rapidly after tumor angiogenesis. The tumor infiltrated into liver blood sinus, blood vessels in hepatic interstitial tissue, the liver capsular vein and important organs metastasis such as lungs, kidneys, abdominal cavity caused rabbits died. The average vascular density count of 18 experimental rabbits under 400 times light microscope were 43.17 ± 8.68/vessels/High Power Field; Tumor vascular diameter all within 200 μm. Vascular elastic fiber staining presented tumor blood vessels internal, external elastic plate intact, vascular endothelial cells organelles of tumor were integrity without endothelial cells architecture defect found by pathologic observation. Conclusion: Proliferation and metastasis of rabbit VX₂ hepatic carcinoma was correlated with tumor angiogenesis and no tumor vascular architecture defect was found by pathologic observation, it need further exploration by other methods.     

Angiopoietin-2 is implicated in the regulation of tumor angiogenesis

We addressed the effect of angiopoietin expression on tumor growth and metastasis. Overexpression of angiopoietin-2 (Ang-2) in Lewis lung carcinoma and TA3 mammary carcinoma cells inhibited their ability to form metastatic tumors and prolonged the survival of mice injected with the corresponding transfectants. In contrast, angiopoietin-1 (Ang-1) overexpression had no detectable effect on the ability of either tumor type to disseminate. Tumors derived from Ang-2-overexpressing cells displayed aberrant angiogenic vessels that took the form of vascular cords or aggregated vascular endothelial cells with few associated smooth muscle cells. These vascular cords or aggregates were accompanied by endothelial and tumor cell apoptosis, suggesting that an imbalance in Ang-2 expression with respect to Ang-1 and vascular endothelial growth factor may disrupt angiogenesis and tumor survival in vivo. Our observations suggest that Ang-2 may play an important role in regulating tumor angiogenesis.     

Studies on anti-metastatic and anti-invasive effects of harmine using highly metastatic murine B16F-10 melanoma cells

Harmine is a beta-carboline alkaloid from the plant Peganum harmala. We evaluated the anti-metastatic activity of harmine using in vivo mouse lung metastasis and in vitro models. Lung metastasis was induced using B16F-10 melanoma cells in C57BL/6 mice by three different modalities of administration: simultaneous, prophylactic, and after tumor development. Harmine significantly inhibited tumor nodule formation in the lung tissue and decreased various biochemical parameters associated with lung metastasis. Higher expression levels of pro-metastatic genes such as matrix metalloproteinase-9 (MMP-9), extracellular signal[en]regulated kinase (ERK), and vascular endothelial factors (VEGFs), all of which play important roles in cancer cell migration and invasion, were observed in the metastatic group compared with normal, but were all down-regulated by treatment with harmine. Harmine was also able to inhibit tumor cell proliferation, invasion, and migration in vitro. In conclusion, harmine exerts anti-metastatic activity and this effect could be linked to the metastasis-related signaling pathway that includes ERK, VEGF, and MMPs.     

Chemokines, chemokine receptors, and cancer metastasis

It is clear from large clinical studies that selected chemokine receptors are often up-regulated in a large number of common human cancers, including those of the breast, lung, prostate, colon, and melanoma. Chemokine receptors and their corresponding chemokine ligands have been demonstrated to play a number of nonredundant roles in cancer metastasis to vital organs as well as regional lymph nodes, the most frequent site of cancer metastasis. Chemokine receptors may potentially facilitate tumor dissemination at several key steps of metastasis, including adherence of tumor cells to endothelium, extravasation from blood vessels, metastatic colonization, angiogenesis, proliferation, and protection from the host response via activation of key survival pathways such as phosphatidylinositol-3 kinase and Akt. It is interesting that many of these roles are reminiscent of their functions in leukocyte and stem cell trafficking. Lastly, we discuss therapeutic applications for chemokine receptor antagonists in cancer therapy.     

Alteration of angiogenic patterns on B16BL6 melanoma development promoted in Matrigel

Because the progression and metastasis of solid tumors depend on their local microcirculation, we sought to characterize tumor angiogenesis three dimensionally in a highly metastatic mouse melanoma model, B16BL6 (B16), injected with Matrigel into the subcutis in the skin on the back of syngeneic C57BL/6 mice. We found that B16 with Matrigel grew significantly faster than B16 alone and had altered tumor angiogenesis. Tumor vessels apparently grew vigorously in the opposite direction of the tumor without invading the tumor mass until at least day 10 of injection. In addition, vascular branching resulted not only from sprouting as was seen in B16 without Matrigel but also from vascular splitting, either because of compression from outside the vessels or from septum formation by endothelial cells. This phenomenon was characteristic of B16 cells, but not of other tumor cells, including Lewis lung carcinoma and ASH-1 hybridoma cell lines, both of which were tested under the same conditions. The reduction in various angiogenic factors in Matrigel did not affect the angiogenic patterns and tumor growth. We hypothesize that tumor vessels may vigorously alter their angiogenic patterns in response to the local microenvironment.     

血管内皮生长因子C及其受体3在非小细胞肺癌组织中的表达及意义

目的　探讨血管内皮生长因子C(VEGF C)和受体 (VEGFR) 3在人非小细胞肺癌(NSCLC)组织中的表达及其与微血管、微淋巴管形成、淋巴转移、预后之间的关系。方法　对 76例人NSCLC及相应癌旁组织行VEGF C、VEGFR 3及CD34免疫组织化学染色链霉素抗生物素蛋白 过氧化物酶 (SP)法检测 ,进行淋巴管密度计数、微血管密度 (MVD)计数 ,并结合临床和病理资料进行分析。结果　NSCLC中 ,VEGF C的表达与肺癌分化程度负相关 (P =0 0 0 9)。VEGF C和VEGFR 3的表达水平与淋巴结转移呈正相关 (分别P =0 0 0 8,P =0 0 13) ,与淋巴浸润呈正相关 (分别P =0 0 2 7,P =0 0 2 0 )。VEGF C的表达与VEGFR 3在肺癌细胞中的表达呈正相关 (P =0 0 0 9)。VEGF C与淋巴管密度 (P =0 0 0 6 )、MVD(P =0 0 4 6 )呈正相关。淋巴管密度与淋巴结转移 (P =0 0 10 )、淋巴浸润 (P =0 0 19)、TNM分期 (P =0 0 15 )呈正相关 ,MVD与血行转移 (P <0 0 0 1)、TNM分期 (P <0 0 0 1)呈正相关。VEGF C阳性表达与生存时间、5年生存率呈负相关 (P <0 0 0 1)。结论　NSCLC中 ,VEGF C通过自分泌方式作用于受体VEGFR 3,促进肺癌组织生长 ,抑制分化。VEGF C促使肺癌内淋巴管形成 ,促进肺癌淋巴结转移。VEGF C和VEGFR 3表达增高、淋巴管密度增加     

EGFL7在乳腺癌中的研究进展

表皮生长因子样结构域7是表皮生长因子样蛋白家族的成员,是一种极为关键的血管生成因子和血管发育调节因子,在不同类型细胞中均有表达。EGFL7在早期胚胎的血管中表达较高,主要调节发育过程中的血管生成,促进内皮细胞增殖,形成有功能的血管网,促进组织增长和再生。与其在胚胎期较强的表达相比,在成人体内的表达量微乎其微,异常高表达的EGFL7常在多种恶性肿瘤中出现。目前越来越多的证据表明,EGFL7可调节肿瘤血管生成、促进肿瘤转移和侵袭。肿瘤组织中EGFL7高表达常常提示肿瘤生长和转移较为活跃及不良的预后。近年来,一系列研究表明,EGFL7的表达与乳腺癌的发生发展、转移过程密不可分,且在提示预后方面发挥着关键作用。本文就EGFL7在乳腺癌中的研究进展作一综述,为临床诊断及治疗提供新思路。     

Low-dose Methotrexate Inhibits Lung Metastasis and Lengthens Survival in Rat Osteosarcoma

Lung metastasis is the most crucial event affecting the treatment of osteosarcoma and is dependent on tumor angiogenesis. To improve the prognosis for patients with osteosarcoma, prevention of lung metastasis is essential. Low-dose methotrexate is a useful drug for treating a variety of diseases. Low-dose methotrexate reportedly plays a role in antiangiogenesis for the synovial blood vessels in rheumatoid arthritis. However, whether low-dose methotrexate is correlated with tumor angiogenesis and metastasis is unclear. We investigated the inhibitory effect of methotrexate on lung metastasis in a rat osteosarcoma cell line with high metastatic potential, S-SLM. Two weeks after inoculation of S-SLM cells into male Fischer 344 rats, low-dose methotrexate (1.2 mg/kg once or twice a week) or saline was intraperitonealy injected for 4 weeks and the antimetastatic effect was evaluated. Low-dose methotrexate significantly reduced the number of lung metastatic nodules and the wet weight of the lungs. Immunohistochemical staining showed a decrease in microvessel density in the metastatic nodules. We also evaluated the effect of methotrexate on the proliferation of endothelial cells and S-SLM osteosarcoma cells in vitro. Methotrexate significantly inhibited the proliferation of endothelial cells at a lower concentration than that of S-SLM osteosarcoma cells. These data suggest that low-dose methotrexate inhibited lung metastasis of osteosarcoma through its antiangiogenic activity. Our results indicate that low-dose methotrexate is a promising drug for tumor dormancy therapy in patients with osteosarcoma and lung metastasis.