Establishment of human osteosarcoma cell lines with high metastatic potential to lungs and their utilities for therapeutic studies on metastatic osteosarcoma

Relevant animal models for metastasis of osteosarcoma is needed to understand the biology and to develop the treatment modality of metastasis of human osteosarcoma. Therefore, we screened six human osteosarcoma cell lines for metastatic ability in nude mice. The HuO9 cell line was identified as being metastatic to the lung after intravenous injection. We established two sublines, HuO9-M112 and HuO9-M132, with high metastatic potential to the lung from the parental HuO9 cells by in vivo selection. There were no differences between these two sublines and the parental cells in the growth rate in vitro and the tumorigenicity after subcutaneous injection in nude mice, however, mice injected with the metastatic sublines became moribund earlier than mice injected with the parental HuO9 cells did. Thus, adriamycin (ADR) and recombinant interleukin-12 (IL-12) were administered to mice injected with the HuO9-M112 subline to suppress experimental lung metastases. Production of lung colonies was significantly suppressed and the prognoses of mice were significantly improved by both ADR and IL-12 treatments. These results indicate that both ADR and IL-12 are effective agents against pulmonary metastatic osteosarcoma, and that these sublines are useful for studies on the biological behavior and treatment of pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytoplasmic and/or nuclear staining of beta-catenin is associated with lung metastasis

Beta-catenin is involved in cell motility in the extracellular matrix, and is expressed in normal and neoplastic mesenchymal cells. In order to clarify whether beta-catenin expression in the cytoplasm and/or nucleus is associated with a propensity for pulmonary metastasis in osteosarcoma, the LM8 murine osteosarcoma cell line with a high metastatic potential to the lung was compared with original Dunn cells in terms of the beta-catenin expression level. Both osteosarcoma cell lines lost membrane localization of beta-catenin. However, beta-catenin gene had no mutation in exon 3 by direct sequence analysis. A large number of LM8 cells showed diffuse cytoplasmic and/or nuclear staining of beta-catenin (30.8 per high power field (HPF)), while a much smaller number of Dunn cells showed expression of beta-catenin (7.7 per HPF). Cells with positive staining of beta-catenin were frequently seen at the invasive front and in intravenous tumor deposits within the metastatic lesions to the lung. Thus, LM8 cells express a larger amount of the beta-catenin protein than Dunn cells, as judged by immunoblot analysis. In five resected cases of pulmonary metastasis, translocation of beta-catenin to the cytoplasm and/or nucleus of osteosarcoma cells was detected, although seven primary osteosarcomas cells that did not metastasize for more than five years did not show beta-catenin expression. These data indicate that the cytoplasmic and/or nuclear staining of beta-catenin is a biological marker of metastatic potential of osteosarcoma to the lung. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of gene expression between metastatic derivatives and their poorly metastatic parental cells implicates crucial tumor-environment interaction in metastasis of head and neck squamous cell carcinoma

Metastasis of human head and neck cancer is a multistep and highly heterogeneous process requiring activation and deactivation of multiple and specific genes. To identify these genes, we established highly metastatic head and neck squamous cell carcinoma (HNSCC) cell lines from poorly metastatic HNSCC cells through in vivo selection using a lymph node metastatic mouse model. The very close genetic relationship between these highly metastatic cell lines and the parental cell line provided an excellent model for differential gene expression analysis using cDNA microarrays. Comparison of 6 cell lines established individually from the lymph node metastases with their poorly metastatic parental cell line revealed 33 differentially expressed genes. Some of these genes are involved in cellular signal transduction and matrix modeling. Differences in expression of members of the tumor necrosis factor, interleukin, caspase, and matrix metalloproteinase families were also examined. We found that two upregulated genes participated in the NF-κB regulatory pathway. Furthermore, differences in gene expression between six cell lines derived from primary tumors and six cell lines derived from lymph node metastases in the mouse model were analyzed statistically. Tissue growth factor-β and tumor necrosis factor-related genes showed significantly altered expression in cells derived from lymph node metastases as compared with cells derived from primary tumors, suggesting that the differential growth advantage of metastatic cells requires more aggressive responses to their environment, such as a lymph node tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

BRI基因的表达与非小细胞肺癌发生及转移潜能的关系

目的 探讨BRI基因在人非小细胞肺癌中的差异表达情况及其与肺癌发生及转移能力形成的相关性。方法 应用逆转录-聚合酶链反应(RT-PCR)、Northern印迹杂交方法分析BRI基因在一对遗传背景相同、转移能力不同的人肺腺癌细胞系AGZY83-a和Anip-073中的差异表达,并进一步检测BRI基因在另外6个非小细胞肺癌细胞系(SPC-A-1,A549,95D,TKB-18,GLC-82,PAa)及30例临床肺癌标本中的表达情况。结果 BRI基因在2细胞系中存在明显的差异表达,在具有高转移潜能的Anip973中高表达。在其他6个细胞系中BRI的表达与肺癌转移潜能可能有关。同时发现与同一病例的正常肺组织相比,BRI基因在肺癌组织中高表达,其过表达在有淋巴结转移的肺癌为6／8,无转移者为45．5％(10／22),并且BRI基因在肺癌与正常肺组织之间存在转录本的差异。结论 BRI基因在肺癌中表达上调,其1．6kb转录本表达的增加可能与肺癌的发生和转移能力的形成有关。     

高转移性人成骨肉瘤细胞系在裸小鼠体内的筛选和维持

目的：筛选人成骨肉瘤高转移细胞并探讨维持其高转移性状的方法。方法：采用低转移性成骨肉瘤细胞系在裸小鼠腹腔内连续传代和体外培养肺转移灶筛选高转移亚系,通过体内、外交替传代维持其高转移性状,并用流式细胞仪、染色体分析等方法,研究该高转移亚系的细胞周期、形态、增殖等生物学特性。结果：筛选所得腹水型高转移亚系自发性肺转移率达100％。在细胞形态、增殖速度、染色体数目等方面与母系有较大差别,经体内、外交替传代高转移性状得以维持,且出现较高比例的脑、骨、肌肉转移。结论：腹腔连续传代培养转移灶和体内、外交替传代是筛选和维持高转移细胞的可靠方法。     

与骨肉瘤预后相关的基因研究进展

骨肉瘤肺转移是临床患者死亡的主要原因,近年来对骨肉瘤预后相关基因的发现、研究增进了人们对骨肉瘤转移机制的认识,对于骨肉瘤的个体化治疗和患者预后有一定的指导意义,本文就几个与骨肉瘤预后相关的热点基因进行综述。     

Integrin expression in human melanoma cells with differing invasive and metastatic properties

During the process of tumor cell invasion and metastasis, tumor cells are known to interact with extracellular matrix proteins, endothelial cells, platelets and other organ-specific structures. Integrins are cell surface molecules which mediate cell-matrix and cell-cell interactions and are likely to be important for tumor cell survival and dissemination. The purpose of this study was to characterize the integrin and proteolytic enzyme repertoire from low (A375P), medium (A375M) and high metastatic (A375SM) human melanoma cell lines. These cell lines are also invasive through human amniotic membranes in vitro and their invasiveness parallels the reported metastatic phenotype. The types and levels of expression of the various integrin receptors were analysed by quantitative immunoprecipitation using a panel of monoclonal antibodies directed to known integrin subunits. In addition, cDNA probes to the integrin subunits were used in quantitative northern blot analysis. These data show that the integrin _v3 increases 50- to 100-fold as these cells progress to a more metastatic phenotype. ₄₁ levels also appeared to increase several fold, while other ₁ integrins did not differ in their expression levels. The increased _v3 expression in the more metastatic cells resulted in an increased adhesion to vitronectin and fibrinogen substrates in cell attachment assays. However, _v- and ₃-specific antibodies did not inhibit A375 cell invasion through the amnion. Each cell line was found to release similar quantities of a 72-kDa gelatinase/type IV collagenase and tissue type plasminogen activator. These results suggest that during the progression of these tumor cells from a low to high metastatic phenotype, marked changes in integrin expression occurred which may facilitate interactions with platelets, endothelial cells and specific extracellular matrix proteins to promote metastasis.     

A nude mouse model of human osteosarcoma lung metastases for evaluating new therapeutic strategies

The purpose of these studies was to develop a metastatic osteosarcoma nude mouse model to evaluate the in vivo efficacy of new therapeutic compounds. Human SAOS-2 osteosarcoma cells (10⁶ cells) were injected i.v. into nude mice. Cells isolated from a rare pulmonary metastases 6 months later were established (SAOS-LM1) in culture and re-injected. This procedure was repeated 5 additional times to produce the SAOS-LM6 cell line. Visible pulmonary nodules were present 8 weeks following i.v. injection of 10⁶ SAOS-LM6 cells as compared to 17 weeks using SAOS-LM2 cells. Microscopic SAOS-LM6 pulmonary metastases were demonstrated at 6 weeks. Administration of adriamycin on week 9 resulted in regression of macroscopic SAOS-LM6 lung tumors. The ability of the model to be used to evaluate the effectiveness of a biologic agent against microscopic disease was also verified. It was concluded that this model can assess therapeutic efficacy and therefore, may have a role in investigating the potential of novel approaches aimed at eliminating pulmonary metastatic osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Increased metastatic potential in human prostate carcinoma cells by overexpression of arachidonate 12-lipoxygenase

Arachidonate 12-lipoxygenase (LOX) converts arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid (HETE), a bioactive lipid implicated in tumor angiogenesis, growth, and metastasis. Alteration in 12-LOX expression or activity has been reported in various carcinomas including prostate carcinoma. However, little is known about the impact of the altered expression or activity of 12-LOX on tumor metastasis. In the present study, we examined whether or not an increase in 12-LOX expression in human prostate carcinoma cells can modulate their metastatic potential. We report that increased expression of 12-LOX in PC-3 cells caused a significant change in cell adhesiveness, spreading, motility, and invasiveness. Specifically 12-LOX transfected PC-3 cells were more adhesive toward vitronectin, type I and IV collagen, but not to fibronectin or laminin, than cells transfected with control vector. Increased spreading on vitronectin, fibronectin, collagen type I and IV also was observed in 12-LOX transfected PC-3 cells when compared to control PC-3 cells. The increased spreading of 12-LOX transfected PC-3 cells was blocked by treatment with 12-LOX inhibitors, baicalein and CDC. 12-LOX transfected PC-3 cells were more invasive through Matrigel than cells transfected with control vector. In vivo, tumor cell invasion to surrounding muscle or fat tissues was more frequent in nude mice bearing s.c. tumors from 12-LOX transfected PC-3 cells than in those from control vector transfected cells. When injected via the tail vein into SCID mice with implanted human bone fragments, there was an increase in tumor metastasis to human bone by 12-LOX transfected PC-3 cells in comparison to control vector transfected cells. Taken together, our data suggest that an increase in 12-LOX expression enhances the metastatic potential of human prostate cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

miR-30a抑制人骨肉瘤细胞143B的迁移、侵袭和活力

目的探讨miR-30a对人骨肉瘤细胞143B侵袭、迁移和细胞活力的影响。方法过表达或抑制miR-30a分别处理人骨肉瘤细胞143B。划痕实验观察细胞划痕愈合能力;Transwell实验检测143B细胞迁移和侵袭能力;MTT实验检测细胞活力;定量PCR实验确认过表达miR-30a腺病毒有效性并且检测RUNX2 mRNA表达;Western blot检测细胞中RUNX2总蛋白表达。结果过表达miR-30a抑制了骨肉瘤细胞143B迁移和侵袭(P0.05),在72 h时,miR-30a明显抑制细胞活力(P0.01);抑制miR-30a的内源性表达后,143B细胞的迁移和侵袭能力增加(P0.05),细胞活力也表现出上升水平(P0.01);同时过表达miR-30a可以抑制RUNX2的蛋白表达,抑制内源性miR-30a后RUNX2蛋白水平表达增加(P0.05)。荧光素酶活性检测,miR-30a可以靶向于RUNX2(P0.01)。结论 miR-30a抑制骨肉瘤细胞143B的迁移、侵袭和活力,其作用可以能是通过抑制RUNX2的表达来实现。     

An orthotopic model of murine osteosarcoma with clonally related variants differing in pulmonary metastatic potential

To provide an investigative tool for the study of osteosarcoma (OSA) biology we have developed a syngeneic (balb/c) murine model of OSA, using cell lines derived from a spontaneously occurring murine OSA (Schmidt et al. Differentiation 1988; 39: 151-60). This model is characterized by orthotopic primary tumor growth, a period of minimal residual disease, spontaneous pulmonary metastasis, and clonally related variants (K7M2 and K12) that differ in pulmonary metastatic potential. Primary tumor and pulmonary metastasis histology was consistent with OSA in human patients. Expression of bone sialoprotein, biglyan, decorrin, and osteopontin was suggestive of bone lineage cells. The development and use of a more aggressive OSA cell line (K7M2) resulted in spontaneous metastasis to the lungs in over 90% of mice, whereas metastases were seen in only 33% of mice when a less aggressive OSA cell line (K12; Schmidt et al. Differentiation 1988; 39: 151-60) was used. Death from metastasis occurred at a median of 76 days using K7M2 whereas no median was achieved after 140 days using K12. Angiogenic potential, characterized by CD31 and factor VIII staining of primary tumors and pulmonary metastases, was greater in the K7M2 model compared to the K12 model. No significant differences in the in vitro or in vivo expression of angiogenesis associated genes (flt1, flt4, TIE1, TIE2, and VEGF) was found between K7M2 and K12. This well characterized and relevant model of OSA will be a valuable resource to improve our understanding of the biology and treatment of metastasis in OSA. This revised version was published online in July 2006 with corrections to the Cover Date.     

人脑胶质瘤基因表达谱芯片的构建和初步验证

目的：构建人脑胶质瘤特异的基因表达谱芯片,促进大规模胶质瘤表达谱的研究。方法：采用389条基因探针,优化基因芯片制作的各个条件：片基、点样液、点样大小、紫外交联、采集信号强度等。使用自制芯片检测20例胶质瘤标本的基因表达谱,并与以前报道的实验结果对比,验证芯片的特异性和灵敏度。结果：构建了针对人脑胶质瘤的基因表达谱芯片,并用于脑胶质瘤的基因表达谱分析,证实本产品可以有效地高通量检测相关基因的表达水平。结论：构建的胶质瘤芯片有望用于胶质瘤基因表达谱分析。     

应用抑制性消减杂交技术鉴定肿瘤转移抑制相关基因

目的 克隆与肿瘤转移抑制相关的基因，探讨成骨肉瘤转移的分子机理。方法 采用抑制性消减杂交技术构建了人成骨肉瘤低转移细胞株SOSP－9607的消减文库，对部分克隆进行了测序和同源性分析，用Northern杂交及后转录PCR技术证实了克隆的表达情况。结果 在低转移细胞株SOSP－9607中高表达的克隆中有2个克隆与人端粒结合因子2（telomeric repeat binding factor 2,TERF2)同源。Northern杂交及反转录PCR证实这个基因只在低转移细胞中表达，而在高转移人成骨肉瘤细胞株SOSP－M和裸鼠肺转移结节中均未表达。结论 TERF2可能在维持肿瘤细胞自身相对稳定、抑制骨肉瘤演进中起重要作用。     

Tiam1基因过表达对结直肠癌细胞增殖和转移能力的影响

目的探讨Tiaml（T lymphoma invasion and metastasis1）基因转染对结直肠癌细胞增殖、转移能力的影响。方法采用裸鼠皮下接种结直肠癌细胞的方法观察Tiaml基因对结直肠癌细胞体内增殖能力的影响,利用外科原位移植技术观察Tiaml对体内增殖、转移能力的影响。结果接种HT29／Tiaml细胞（转染Tiaml基因）的裸鼠皮下肿瘤生长明显加快,肿瘤体积自皮下注射后的第7天起与接种HT29／mock细胞（转染对照）的裸鼠皮下肿瘤比差异有统计学意义,接种HT29／Tiaml细胞的裸鼠皮下肿瘤第20天的平均体积是接种HT29／mock细胞组的2．5倍。进行结直肠癌细胞外科原位移植6周后,接种HT29／Tiaml细胞的裸鼠结肠原位肿瘤重量明显大于接种HT29／mock细胞的裸鼠（t=-14．916,P〈0．01）。采用外科原位移植的方法比较HT29／mock和HT29／Tiaml细胞的体内转移能力。在HT29／Tiaml细胞组,7只裸鼠全都发生了腹腔转移,4只发生了肝转移,其中有1只裸鼠发生了广泛转移,包括脾、肺及淋巴结等转移。在HT29／mock细胞组,7只中只有2只裸鼠发生了腹腔转移,没有一只裸鼠发生远处转移。结论Tiaml基因是结直肠癌增殖、转移的促进基因,Tiaml表达可作为结直肠癌增殖、转移过程中一个有价值的指标。     

C-Met tyrosine kinase receptor expression and function in human and canine osteosarcoma cells

To further characterize the role of hepatocyte growth factor-scatter factor (HGF-SF) and its receptor (c-Met) in osteosarcoma (OS), human OS cell lines with low (SAOS-2) and high (SAOS-LM2) metastatic potential, and cell lines derived from spontaneous canine OS were studied. All cell lines were evaluated for c-Met and HGF-SF expression and receptor activation using Northern, RT-PCR, and Western blot analyses, respectively. Functional activity of receptor-ligand interaction was measured using c-Met phosphorylation status, proliferation assays (anchorage-dependent and -independent), Matrigel invasion, modulation of urokinase plasminogen activator (uPA) expression, and cell dispersion (scattering). All cell lines exhibited steady-state mRNA expression of c-Met. The canine OS cell lines also expressed HGF-SF mRNA as determined by RT-PCR analysis. Western analysis showed c-Met protein expression and HGF-stimulated (human) or constitutive (canine) receptor autophosphorylation. Treatment with recombinant human HGF resulted in enhanced proliferation in 3 of 5 OS cell lines and enhanced colony formation in 2 of 5 OS cell lines. Matrigel invasion was significantly enhanced in 3 of the cell lines and uPA levels were significantly increased in the SAOS-2 cells following HGF treatment. Scattering was enhanced in both the SAOS-2 and SAOS-LM2 cells. These data support the involvement of c-Met and HGF-SF in the growth and progression of human and canine OS, and may offer new targets for the development of therapeutic strategies for OS. This revised version was published online in July 2006 with corrections to the Cover Date.     

不同转移特性瘤细胞系的筛选及其生物学特性

目的：探讨与肿瘤转移相关的某些生物学特性。方法：将小鼠乳腺癌Ｃａ７６１－ＦＰ８／Ｌ和Ｃａ７６１－ＦＬ１０／Ｌ经体内筛选得到细胞系Ｃａ７６１－Ｐ５Ｂ和Ｃａ７６１＝Ｌ６Ｂ，并观察其瘤细胞与凝集反应、靶器官组织条件培养基对瘤细胞真挚化作用等，结果：Ｃａ７６１－Ｐ５Ｂ具有高肺转移、低淋巴结转移特性，Ｃａ７６１－Ｌ５Ｂ具有低肺、低淋巴结转移特性。两个瘤细胞系的细胞表面的糖基表达，对条件培养的趋化反应不同。结     

Correlation between spontaneous metastatic potential and type I collagenolytic activity in a human pancreatic cancer cell line (SUIT-2) and sublines

A human pancreatic cancer cell line (SUIT-2) and four sublines cloned in vitro (S2-007, S2-013, S2-020 and S2-028) were inoculated into nude mice for assessment of metastatic potentials. After 16 weeks of subcutaneous injection, the parent SUIT-2 line metastasized to the lungs and lymph nodes in three of six mice. S2-007 cells presented the highest metastatic potential in pulmonary (5/6) and lymph node (2/6) metastases among the four sublines. No metastasis was found in S2-028. The incidence of spontaneous pulmonary metastasis was correlated with that of pulmonary colonization after intravenous (i.v.) injection of cell clusters (r = 0.87, P = 0.056). Pulmonary colonization potential using single cells, however, did not always reflect a spontaneous metastatic ability. Type I collagenolytic activity in serum-free conditioned media of these cells was correlated effectively with the incidence of spontaneous pulmonary metastasis (r = 0.92, P = 0.026) and pulmonary colonization after i.v. injection of cell clusters (r = 0.95, P = 0.013). Thus, type I collagenolytic activity may possibly be essential to spontaneous cancer metastasis.