Establishment of Cell Lines with High and Low Metastatic Potential from A549 Human Lung Adenocarcinoma

This article reports the establishment of variant cell lines with high and low metastatic potential by the dilution plating technique. Two clones with high metastatic potential, 2S Lu-4 and 11S Lu-1 and two clones with low metastatic potential, 8S and 16S were established from A549 human lung adenocarcinoma. The high-metastatic cell lines produced enhanced lung metastases, but the low- metastatic cell lines did not produce lung metastasis after injection into the tail vein of 5-week-old BALB/c nude mice. The primary tumors produced by the two high-metastatic cells grew fast and showed enhanced angiogenesis. The high-metastatic cells were small and flat-shaped, while the low- metastatic cells were large and flat-shaped. When the four variant cell lines and original A549 cells were embedded in collagen gels, the colonies of 2S Lu-4, 11S Lu-1 and A549 grew actively, whereas almost of all the colonies of 8S and 16S did not survive after 35 days in culture. These four cloned cell lines originated from heterogeneous populations of the parental A549 cells should be an excellent tool for studying the process of metastasis of lung cancer.     

Establishment of cell lines with high- and low-metastatic potential from PC-14 human lung adenocarcinoma.

This article reports the establishment of variant cell lines with high and low metastatic potential by repeated selection and the dilution plating technique. Five clones with high metastatic potential, Lu-2, Lu-7, Lu-4, Lu-1 and Lu-5, and four clones with low metastatic potential, 3S, 7S, 8S and 13S, were established from PC-14 human lung adenocarcinoma. The high-metastatic cell lines produced enhanced lung metastases, but the low-metastatic cell lines did not produce lung metastasis by injection into the tail vein of 5-week-old BALB / c nude mice. The high-metastatic cell lines produced enhanced tumors on both visceral and parietal pleurae, and enhanced metastases to the mediastinum and contralateral pleural cavity. The low-metastatic cell lines produced reduced tumors on both visceral and parietal pleurae and reduced metastases to the mediastinum and contralateral pleural cavity after injection into the left preceral cavity of the nude mice. When the nine variant cell lines and original PC-14 cells were embedded in collagen gels, the PC-14 cells and the low-metastatic cell lines gave rise to colonies with a dendritic morphology, and cells were tightly associated. The high-metastatic cell lines were more loosely associated and scattered into three-dimensional colonies. These nine cloned cell lines originated from heterogeneous populations of the parental PC-14 cells should be useful tools for studying the process of metastasis of lung cancer.     

Establishment of Cell Lines with High- and Low-metastatic Potential from PC-14 Human Lung Adenocarcinoma

This article reports the establishment of variant cell lines with high and low metastatic potential by repeated selection and the dilution plating technique. Five clones with high metastatic potential, Lu-2, Lu-7, Lu-4, Lu-1 and Lu-5, and four clones with low metastatic potential, 3S, 7S, 8S and 13S, were established from PC-14 human lung adenocarcinoma. The high-metastatic cell lines produced enhanced lung metastases, but the low-metastatic cell lines did not produce lung metastasis by injection into the tail vein of 5-week-old BALB/c nude mice. The high-metastatic cell lines produced enhanced tumors on both visceral and parietal pleurae, and enhanced metastases to the mediastinum and contralateral pleural cavity. The low-metastatic cell lines produced reduced tumors on both visceral and parietal pleurae and reduced metastases to the mediastinum and contralateral pleural cavity after injection into the left preceral cavity of the nude mice. When the nine variant cell lines and original PC-14 cells were embedded in collagen gels, the PC-14 cells and the low-metastatic cell lines gave rise to colonies with a dendritic morphology, and cells were tightly associated. The high-metastatic cell lines were more loosely associated and scattered into three-dimensional colonies. These nine cloned cell lines originated from heterogeneous populations of the parental PC-14 cells should be useful tools for studying the process of metastasis of lung cancer.     

Metastatic heterogeneity of cells from Lewis lung carcinoma

To allow investigations of the role of tumor cell proteases in invasion and metastasis, an attempt was made to obtain well-defined homogeneous populations of Lewis Lung carcinoma cells differing widely in their metastatic potential. From a single Lewis lung carcinoma, a parental line of cells was established and subsequently cloned so as to provide 18 clonal tumor cell lines. These clones differed in their ability to produce spontaneous, macroscopically visible metastases in the lung after i.m. inoculation into syngeneic C57BL/6 mice. Several of them were less metastatic than the parental line. The parental line expressed a metastatic behavior close to that of the high-metastatic cell subpopulations that it contained. There was, within certain limits, a good correlation between the potential for spontaneous lung metastases arising from a primary tumor and that for "artificial" lung colonies obtained after i.v. injection of the Lewis lung carcinoma cells. Although positively correlated with the growth rate of the tumor cells, the metastatic ability of the clones could not be considered as a mere reflection of the proliferation rates of the cells constituting the primary tumors. Differences in metastatic behavior observed among clones persisted in several cases after the cells had been maintained in culture for prolonged periods. However, this stability of the clones in vitro was not absolute. Indeed, some subclones isolated from the low-metastatic clone H122 displayed metastatic abilities which were lower than that of the parent clone. Furthermore, a significant increase in metastatic potential was once observed after a prolonged culture period of that same clone, H122. Thus, new metastatic phenotypes can emerged under in vitro culture conditions. However, the relative rarity of this event suggests that some metastatic heterogeneity already preexisted in vivo among the tumor cells.     

Proteolytic enzymes in tumor metastasis. I. Plasminogen activator in clones of Lewis lung carcinoma and T10 sarcoma

Plasminogen activator (PA; urokinase) levels were studied in metastatic and nonmetastatic clones of the Lewis lung carcinoma (3LL) and of the T10 sarcoma. Tests of clones grown in vitro revealed that the cell content and secretion of PA correlated positively with the metastatic capacity of the clones of both tumors. When cell-associated activities were examined in cloned cell populations grown subcutaneously in vivo, the apparent activities in the solid tumors produced by low-metastatic clones were equal to or even higher than those in solid tumors produced by high-metastatic clones. This finding was attributed to the observation that solid tumors produced by low-metastatic clones, but not those produced by high-metastatic clones, were highly infiltrated with macrophages. Subsequent tests indicated that the ip inoculation of X-irradiated or mitomycin-treated tumor cells of low-metastatic clones elicited a significantly greater peritoneal infiltration of macrophages than did tumor cells of high-metastatic clones. Such "tumor-associated" macrophages manifested high levels of PA, whereas resident (nonactivated) peritoneal macrophages did not. These findings suggest that although PA may cause the initial detachment from the local tumor of both nonmetastatic (via the macrophage PA) and metastatic cells (via their own PA), the PA secreted by the metastatic cells may enable them to complete subsequent stages of the metastatic process that may be PA-dependent.     

The relationship between motility factor receptor internalization and the lung colonization capacity of murine melanoma cells

The in vitro motility of B16-F1 melanoma cells is enhanced by incubation with a monoclonal antibody against gp78, previously characterized as a motility factor receptor. This antibody was used to study the relationship between motility stimulation in vitro and metastatic ability in vivo in the B16-F1 and K-1735 murine melanoma systems. While both high- and low-metastatic variants exhibited enhanced in vitro motility in response to the anti-gp78 monoclonal antibody, only the high-metastatic cells exhibited an increased metastatic ability. Surface immunofluorescence of low-metastatic cells was distributed more diffusely compared to a highly localized patching of gp78 on high-metastatic cells, suggesting that the directed endocytosis of gp78 to form a single leading edge is related to the metastatic ability of a cell, while fluorescence-activated cell sorter analysis revealed decreased gp78 surface expression in high-metastatic clones. Priming of cells by preventing internalization of gp78-antibody complexes by pertussis toxin resulted in a marked enhancement of pulmonary metastases by the treated cells which was directly correlated with decreased surface expression of gp78 following washout of pertussis toxin. These results suggest that cell motility induced by motility factor receptor occupancy may play a role in the process of metastasis and that the ligand-receptor complex internalization from the cell surface is involved in control of cell kinesis during metastasis.     

肺癌高转移和低转移细胞株中差异基因的分析

目的:以人肺癌低转移细胞株SPC-A-1为对照,分析人肺癌高转移细胞株SPC-A-1sci的分子转移机制及其相关的信号通路,寻找肺癌转移的关键基因.方法:应用芯片技术检测人肺癌低转移细胞株SPC-A-1和高转移细胞株SPC-A-1sci的差异基因.采用显著性通路分析和构建信号转导网络等生物信息学分析方法寻找肿瘤转移相关的潜在关键基因和信号通路.结果:与SPC-A-1细胞比较,SPC-A-1sci细胞共有上调表达的差异基因2 892个,下调表达的差异基因3 248个;上调差异基因参与的显著性信号转导通路共有48条,下调差异基因参与的显著性信号转导通路共65条.网络中的关键基因主要是丝裂原活化蛋白激酶1、表皮生长因子受体、AKT1、AKT3、PIK3CD( phosphoinositide-3-kinase,catalytic,delta polypeptide)、PIK 3R1 [phosphoinositide-3-kinase 3,regulatory subunit 1 (alpha)]、PIK 3R3、KRAS和胰岛素样生长因子1受体等.结论:人肺癌高转移细胞株SPC-A-1sci的基因芯片检测和生物信息学分析为肺癌转移的基础研究和临床防治提供了依据.     

Constitutive upregulation of hypoxia-inducible factor-1alpha mRNA occurring in highly metastatic lung carcinoma cells leads to vascular endothelial growth factor overexpression upon hypoxic exposure

Neoangiogenesis is crucial for tumor growth and metastasis and is regulated by various angiogenic factors including vascular endothelial growth factor (VEGF). However, little is known whether highly metastatic cells express higher level of VEGF in response to various stimuli, thereby increasing neoangiogenesis compared to low-metastatic cells. Here we report that hypoxia markedly induced the expression of VEGF mRNA in the cell lines with high-metastatic potential (A11 and D6 cells) compared to the cell lines with low-metastatic potential (P29 and P34 cells) established from Lewis lung carcinoma. A11 cells exhibited higher VEGF gene-promoter activity, produced a larger amount of VEGF and showed higher activity to induce neoangiogenesis than P29 cells. Although the degradation rate of VEGF mRNA under hypoxic conditions was similar in both cell lines, hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA, but not HIF-1beta mRNA, was found to be constitutively upregulated in A11 cells compared to P29 cells. Accordingly, the level of HIF-1alpha protein in response to hypoxia was higher in A11 cells than in P29 cells. Upregulation of HIF-1alpha mRNA was also observed in D6 cells but not in P34 cells. Thus, the high-metastatic cells produced a larger amount of VEGF under hypoxic conditions through constitutive HIF-1alpha mRNA upregulation compared to the low-metastatic cells, thereby leading to extensive neoangiogenesis.     

Insulin and Insulin-like Growth Factor 1 Stimulate Proliferation of Metastatic Variants of Colon Carcinoma 26

The proliferation rate of malignant cells in vivo is one of the important factors which affect the formation of tumor metastasis. A highly metastatic variant of mouse colon adenocarcinoma 26 (NL- 17) grew more rapidly than a low-metastatic variant (NL-44) both in vitro and in vivo. The effect of growth factors on the proliferation of NL-17 and NL-44 cells was examined in serum-free medium. Among growth factors examined, human insulin and insulin-like growth factor 1 (IGF-1), which were produced by gene engineering techniques, stimulated the growth of metastatic NL-17 and NL-44 cells as determined by thymidine incorporation and cell counts. DNA synthesis and cell proliferation of the high-metastatic NL-17 was stimulated to a greater extent by insulin and IGF-1 than those of the low-metastatic NL-44. These findings suggest that circulating growth factors could enhance the formation of tumor metastasis. Scatchard analysis of [¹²⁵I]IGF-1 binding to NL-17 and NL-44 showed that each cell line had an almost equal number of IGF-1 receptors (1.37 × 10⁵/cell and 1.26 × 10⁵/cell, respectively), which had similar dissociation constants (8.94×10⁻¹⁰ M and 9.54×10⁻¹⁰ M , respectively). Since the number and affinity of IGF-1 receptors are equivalent between low- and high-metastatic cells, the intracellular events which result in the cell growth after binding of IGF-1 may differ between NL-17 and NL-44 cells.     

Hypoxia selects for high-metastatic Lewis lung carcinoma cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors

Low oxygen tension (hypoxia) is a common feature of solid tumors and stimulates the expressions of a variety of genes including those related to angiogenesis, apoptosis and endoplasmic reticulum (ER) stress response. Here we show a close correlation between metastatic potential and the resistance to hypoxia- and ER stress-induced apoptosis among the cell lines with differing metastatic potential derived from Lewis lung carcinoma. An apoptosis-specific expression profiling and immunoblot analyses revealed that the expression of antiapoptotic Mcl-1 increased as the resistance to apoptosis increased. Downregulation of the Mcl-1 expression in the high-metastatic cells by Mcl-1 small interfering RNA increased the sensitivity to hypoxia-induced apoptosis and decreased the metastatic ability. The hypoxia-induced apoptosis was not associated with p53 accumulation, although at present it is not possible to conclude that apoptosis-induced apoptosis is p53-independent. There was no correlation between the expression levels of ER stress-response proteins GADD153, GRP78 and ORP150 and the resistance to hypoxia or ER stresses. In vitro, small numbers of the high-metastatic cells overtook the low-metastatic cells after exposure to several rounds of hypoxia and reoxygenation. In solid tumors initially established from equal mixtures, the proportion of the high-metastatic cells to low-metastatic cells was significantly higher in hypoxic areas. Moreover, the high-metastatic cells were overtaking the low-metastatic cells in some of the tumors. Thus, tumor hypoxia and ER stress may provide a physiological selective pressure for the expansion of the high-metastatic cells overexpressing Mcl-1 and exhibiting reduced apoptotic potential in solid tumors.     

Insulin and insulin-like growth factor 1 stimulate proliferation of metastatic variants of colon carcinoma 26

The proliferation rate of malignant cells in vivo is one of the important factors which affect the formation of tumor metastasis. A highly metastatic variant of mouse colon adenocarcinoma 26 (NL-17) grew more rapidly than a low-metastatic variant (NL-44) both in vitro and in vivo. The effect of growth factors on the proliferation of NL-17 and NL-44 cells was examined in serum-free medium. Among growth factors examined, human insulin and insulin-like growth factor 1 (IGF-1), which were produced by gene engineering techniques, stimulated the growth of metastatic NL-17 and NL-44 cells as determined by thymidine incorporation and cell counts. DNA synthesis and cell proliferation of the high-metastatic NL-17 was stimulated to a greater extent by insulin and IGF-1 than those of the low-metastatic NL-44. These findings suggest that circulating growth factors could enhance the formation of tumor metastasis. Scatchard analysis of [125I]IGF-1 binding to NL-17 and NL-44 showed that each cell line had an almost equal number of IGF-1 receptors (1.37 x 10(5)/cell and 1.26 x 10(5)/cell, respectively), which had similar dissociation constants (8.94 x 10(-10) M and 9.54 x 10(-10) M, respectively). Since the number and affinity of IGF-1 receptors are equivalent between low- and high-metastatic cells, the intracellular events which result in the cell growth after binding of IGF-1 may differ between NL-17 and NL-44 cells.     

CCL21 promotes the migration and adhesion of highly lymph node metastatic human non-small cell lung cancer Lu-99 in vitro

To develop new therapy strategies for lung cancer, we established an animal model, which reflects the clinical features of mediastinal lymph node metastasis of lung cancer. This study was designed to determine whether CCL21 induced biological functions associated with the metastasis of highly lymph node metastatic human non-small cell lung cancer (NSCLC) selected by our model. Orthotopic intrapulmonary implantation of human NSCLC (Lu-99 and A549) was performed to analyze the metastatic characteristics of these cells. The expression of CCR7, which is a receptor of CCL21, was detected using CCL19 [also called EBI1-ligand chemokine (ELC)]-Fc chimera by flow cytometric analysis. The effects of CCL21 on the migration, adhesion and growth of human NSCLC were investigated. After orthotopic implantation of human NSCLC cell lines, Lu-99, but not A549, metastasized to mediastinal lymph nodes, forming large size nodules, and expressed CCR7 on the surface. Accordingly, its ligand CCL21 induced chemotactic migration and alpha4beta1-mediated adhesion to VCAM-1 of Lu-99. The expression of CCR7 and vigorous responses to its ligand CCL21 potentially account for lymph node metastasis of a human NSCLC line Lu-99.     

The differential expression of H-2K versus H-2D antigens,distinguishing high- metastatic from low- metastatic clones,is correlated with the immunogenic properties of the tumor cells

Two clones of the 3LL Lewis lung carcinoma, a low-metastatic clone A9 and a high-metastatic clone D122, were studied for MHC expression and immunogenic properties. Using monoclonal antibodies, we demonstrated that the A9 clone expresses both the H-2K^b and the H-2D^b, whereas the D122 expresses only the H-2D^b, and lacks the expression of the H-2K^b encoded molecules. Cells of the low-metastatic clone A9 grew progressively in syngeneic (C57BL/6J) or in F₁ mice, but were rejected in allogeneic recipients. The high-metastatic D122 grew progressively in all mouse strains tested, yet metastases were formed only in syngeneic recipients. When H-2 recombinant mice were used, the A9 again manifested a significantly greater immunogenic potency than the metastatic D122, which grew in all 4 recombinants tested. Metastases, however, were formed in B10HTG and to a lesser extent in B10A(4R), thus indicating that metastasis formation is restricted by both C57BL background and H-2D^b sub region. We subsequently tested whether the higher immunogenicity of the H-2K^b-positive A9 cells is expressed also in syngeneic mice, to examine whether this could account for its low metastatic phenotype. We found that immunization by A9 cells significantly inhibited the growth of a subsequent A9 graft and even of D122, yet D122 did not retard the growth of secondary D122 or A9 cells. The increased immunogenic effect was expressed also in the generation of syngeneic cytotoxic lymphocytes by A9 but not by D122 cells. We suggest that expression of H-2K molecules on the 3LL clones, immunogenicity and the metastatic phenotype are causally related in this system.     

Comparison of 'spontaneous' and 'experimental' metastasis using rat 13762 mammary adenocarcinoma metastatic cell clones

Rat 13762NF mammary adenocarcinoma cloned cell lines were assayed at different in vitro passage numbers and compared for their abilities to form 'spontaneous' metastases by subcutaneous injection of cells and 'experimental' metastases by intravenous injection of cells. Tumor cell clones were established from locally growing tumor and spontaneous lung metastases, and these clones were found to possess heterogeneous metastatic potentials in both metastasis assays. The rank order of clonal metastatic potentials based on either the average number of lung tumor colonies or the average total lung tumor volume was generally equivalent for 'spontaneous' and 'experimental' metastases, but some differences were noted. Ranking of 'spontaneous' metastasis by average total lung tumor volumes more closely resembled the rank order of 'experimental' metastasis than by the average number of spontaneous metastases. The results demonstrated that in the 13762NF mammary adenocarcinoma system (i) there is heterogeneity in tumor cell clonal metastatic potential using either 'spontaneous' or 'experimental' assays; (ii) these two assay methods yield generally the same rank order of metastatic potential; (iii) the metastatic potential of each of the tumor cell clones drifts with time (passage number) in cell culture, and (iv) ranking by average tumor burden calculated from total lung tumor volumes may yield a better estimate of metastatic potential than ranking by the average number of lung tumor colonies.     

Altered expression of a third actin accompanying malignant progression in mouse B16 melanoma cells

The expression of actin was examined and compared in several mouse B16 melanoma cell lines with different metastatic ability, by the use of two-dimensional gel electrophoresis or horizontal isoelectric focusing. In the mouse B16 melanoma cell lines, the expression of newly found AX actin (Mr = 43,000, pI = 5.2) decreased with the increase in in vitro and in vivo selection cycles (F number) for high-metastatic cells. On the contrary, the metastatic ability of each mouse cell line, assessed by lung colony-forming ability following iv administration, increased with increase in the F number. The half life of AX actin was much the same as that of beta- and gamma-actin and the different expressions of AX actin between the low- (F = 1) and high-metastatic (F = 10) cell lines were attributed to differences in the rate of synthesis but not in the decay rate of AX actin. The AX actin was incorporated into the cytoskeletal fraction with the same efficiency as beta- and gamma-actin. The invasiveness of the cells, assessed in vitro using matrigel, was increased with the decrease in AX expression. The actin stress fibers, observed staining with rhodamine-conjugated phalloidin, were organized better in a low-metastatic cell line (F = 1) than in a high-metastatic one (F = 10). These results suggest to us that depression of AX actin is involved in disorganization of the cytoskeletal system, the cellular flexibility and motility are enhanced and there is a consequent increase in the invasiveness and metastatic potential.     

Metastatic potential of murine fibrosarcoma cells is influenced by cell surface laminin

In order to examine the role of cell surface laminin in tumor metastasis we have utilized four well-characterized murine fibrosarcoma cell lines. Two of these lines were highly metastatic when injected into syngeneic mice while the remaining two lines were significantly less metastatic. Using indirect immunofluorescence techniques, we detected cell surface laminin on the cell surface of both highly metastatic cell lines but not on the low-metastatic cell lines. Although the low-metastatic cell lines did not possess endogeneous cell surface laminin, they had the ability to specifically bind exogenous laminin to their surface in a time- and concentration-dependent manner, indicating the presence of laminin receptors on these cells. Incubation of the low-metastatic cells with exogenous laminin prior to injection into syngeneic animals significantly increased their metastatic potential. No such increase was observed when the highly metastatic lines were preincubated with exogenous laminin. On the basis of these results, we conclude that in this fibrosarcoma model, metastatic potential is influenced by cell surface laminin and that the presence of unbound laminin receptors on the cell surface is not alone sufficient to promote metastasis of these cells.     

Tumour-cell-induced platelet aggregation: studies with cloned metastatic and non-metastatic variants

Blood platelets have been suggested to play an important role in modulating the development of experimental metastases. Tumour cells can induce platelet aggregation in vitro and a number of mechanisms have been proposed to explain the in vivo and in vitro observations. In the present study, we used tumour cells cloned from B16 melanoma and mouse mammary tumour virus (MMTV) carcinoma polyclonal populations to check whether tumour cells with low- and high-metastatic behaviour in vivo had different quantitative and qualitative platelet-aggregating activity in vitro. We found no significant quantitative difference between platelet aggregation induced by the low- and the high-metastatic clones. Indeed both the high and the low metastatic B16 melanoma clones poorly aggregated platelets, while both the high and low metastatic MMTV carcinoma clones efficiently aggregated platelets. Both the B16 melanoma and the MMTV carcinoma parental cell lines, which can be classed as intermediate metastatic, aggregated platelets well. However, based on the results with heparin and creatine phosphate/creatine phosphokinase, it appeared that qualitative differences might exist in the mechanism of platelet aggregation by tumour cells. For the parental lines and highly metastatic clone C1 a thrombin-linked component was more important than an ADP-like component, which was nevertheless present, to promote platelet aggregation. For the low-metastatic clone C2, the ADP-like component appeared to be the most important.     

Follistatin suppresses the production of experimental multiple-organ metastasis by small cell lung cancer cells in natural killer cell-depleted SCID mice.

PURPOSE: Follistatin (FST), an inhibitor of activin, regulates a variety of biological functions, including cell proliferation, differentiation, and apoptosis. However, the role of FST in cancer metastasis is still unknown. Previous research established a multiple-organ metastasis model of human small cell lung cancer in natural killer cell-depleted SCID mice. In this model, i.v. inoculated tumor cells produced metastatic colonies in multiple organs including the lung, liver, and bone. The purpose of this study is to determine the role of FST in multiple-organ metastasis using this model. EXPERIMENTAL DESIGN: A human FST gene was transfected into the small cell lung cancer cell lines SBC-3 and SBC-5 and established transfectants secreting biologically active FST. The metastatic potential of the transfectants was evaluated using the metastasis model. RESULTS: FST-gene transfection did not affect the cell proliferation, motility, invasion, or adhesion to endothelial cells in vitro. I.v. inoculated SBC-3 or SBC-5 cells produced metastatic colonies into multiple organs, including the lung, liver, and bone in the natural killer cell-depleted SCID mice. FST transfectants produced significantly fewer metastatic colonies in these organs when compared with their parental cells or vector control clones. Immunohistochemical analyses of the liver metastases revealed that the number of proliferating tumor cells and the tumor-associated microvessel density were significantly less in the lesions produced by FST transfectants. CONCLUSIONS: These results suggest that FST plays a critical role in the production of multiple-organ metastasis, predominantly by inhibiting the angiogenesis. This is the first report to show the role of FST in metastases.