Tumor progression and metastasis in murine D2 hyperplastic alveolar nodule mammary tumor cell lines

We have examined tumor progression and metastatic properties of three clonal murine mammary tumor cell lines of recent origin (D2A1, D2.OR and D2.1). These lines were derived from spontaneous mammary tumors which originated from a D2 hyperplastic alveolar nodule (HAN) line. D2A1 cells were more malignant than D2.OR or D2.1 cells, whether measured by experimental metastasis assays after intravenous injection in nude mice or chick embryos,in vivo growth rate of primary tumors following mammary fat pad injection in nude mice, or spontaneous metastasis assay from primary tumors growing in mammary fat pads. D2A1 cells also were more invasivein vitro in a Matrigel invasion assay than D2.1 cells, while the D2.OR cells were non-invasive in this assay. The increased invasiveness and malignancy of D2A1 cells were associated with increased levels of mRNA for the cysteine proteinase cathepsin L. Levels of osteopontin (OPN), nm23, int-1 and int-2 mRNAs were also examined. Nm23 levels were highest in the most malignant cell line. These cell lines provide a model for studying the tumorigenic and metastatic ability of mammary tumor cells and offer several advantages: they were cloned from mammary tumors that originate from a common source of preneoplastic cells (D2HAN); they are of relatively recent origin; and they have spontaneously arrived at different stages of tumor progression.     

Dynamic analysis of lung metastasis by mouse osteosarcoma LM8: VEGF is a candidate for anti-metastasis therapy

Osteosarcoma (OS) is the most common malignant bone tumor and the prognosis depends on pulmonary metastases, which arise from multi-step progression of malignant tumors. We herein aimed to clarify the critical step of pulmonary metastasis using the syngeneic mouse spontaneous highly metastatic OS LM8 and parental Dunn cell lines, to identify new candidate molecules to suppress pulmonary metastasis. We first investigated the chronological detection of circulating tumor cells (CTCs) from mice with either cell line. LM8 CTCs appeared faster, at a higher rate and with a greater number compared to Dunn CTCs. Cultured cells from CTCs of LM8 showed higher proliferative ability than cells from the primary site in suspension culture, which mimicked the environment of the bloodstream for CTCs. The proliferative ability of LM8 cells was also higher than that of Dunn cells in 3D collagen culture with low stiffness (?150 Pa; close to conditions in the lung). We next focused on the extravasation step. LM8 showed higher migration ability compared to Dunn with transendothelial migration assay. We also found a disruption in endothelial barrier function throughout co-culture with LM8 using time-lapse imaging. In addition, LM8 secreted high levels of vascular endothelial growth factor (VEGF), while VEGF signal inhibition with a small molecule tyrosine kinase inhibitor (pazopanib) decreased disruption of the vascular barrier and transendothelial migration of LM8. Finally, daily oral administration of pazopanib reduced the rate and size of pulmonary metastasis in vivo. Collectively, these results show anti-VEGF therapy as a candidate for pulmonary metastasis of OS.     

GFP-transfected tumor cells are useful in examining early metastasis <Emphasis Type="Italic">in vivo</Emphasis>, but immune reaction precludes long-term tumor development studies in immunocompetent mice

To develop effective therapeutic strategies aimed at treating tumor metastasis, critical steps in this process must be better understood. For this purpose we have established a new model to visualize and quantify early metastasis. Murine CT-26 colon adenocarcinoma cells were stably transfected with green fluorescent protein (GFP). Tumor cells were intraportally delivered to the liver of Balb/c mice and subsequently tracked by intravital fluorescence microscopy. Coinjection of fluorescent beads and in vivo propidium iodide staining allowed examination of initial tumor cell arrest, extravasation, viability and proliferation. Results showed that GFP-transfection compared to conventional labeling procedures (Calcein, cytoplasmic microspheres) did not alter early metastatic properties. However, the long-term development of liver metastases expressing GFP was markedly reduced compared to wild type CT-26 tumor cells. An increase in the size and the number of liver metastases in T- and B-cell-deficient SCID mice suggested an immune response to the GFP transfected cells responsible for the reduced metastatic growth in wild-type mice. Based on our findings, this model can be used to examine the early steps of metastasis in vivo. However, in immunocompetent mice, the use of GFP-labeled tumor cells should be limited to tracking cell arrest and extravasation, whereas evaluations of long-term metastatic growth should be performed in immunodeficient mice. This revised version was published online in July 2006 with corrections to the Cover Date.     

Metastatic profiling of HER2-positive breast cancer cell lines in xenograft models

Most studies on breast cancer metastasis have been performed using triple-negative breast cancer cells; thus, subtype-dependent metastatic ability of breast cancer is poorly understood. In this research, we performed intravenous injection (IVI) and intra-caudal arterial injections using nine human epidermal growth factor receptor-2 (HER2)-positive breast cancer cell lines for evaluating their metastatic abilities. Our results showed that MDA-MB-453, UACC-893, and HCC-202 had strong bone metastatic abilities, whereas HCC-2218 and HCC-1419 did not show bone metastasis. HER2-positive cell lines could hardly metastasize to the lung through IVI. From the genomic analysis, gene signatures were extracted according to the breast cancer subtypes and their metastatic preferences. The UACC-893 cell line was identified as a useful model for the metastasis study of HER2-positive breast cancer. Combined with our previous result on brain metastasis ability, we provide a characteristic metastasis profile of HER2-positive breast cancer cell lines in this study.

     

Establishment and characterization of three novel human gastric cancer cell lines with differentiated intestinal phenotype derived from liver metastasis

Gastric cancers with liver metastasis are fatal diseases with rapid progression and poor patient outcome. To date, however, the molecular basis of their growth and metastasis remains essentially unknown, largely because of the presence of few available gastric cancer cell lines established from liver metastasis. In the present study, we developed two novel cultured cell lines (designated GLM-1 and GLM-2) and one transplantable line in nude mice (designated GLM-3) derived from liver metastasis of gastric cancer patients. These GLM cell lines share unique biological features such as differentiation, growth and metastasis. They form moderately differentiated tumors with CD10 positive and MUC2 negative intestinal absorptive phenotype when injected into nude mice. Their growth is stimulated by EGF and TGF-α in vitro like other gastric cancer cell lines. However, GLM cells differ from conventional gastric cancer cell lines in their high apoptotic rate, even in the absence of apoptosis inducing stimuli as revealed by Caspase3/7 assay and the TUNEL method. This apoptosis is further enhanced by phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), but not by MEK1/2 inhibitor (U0126), indicating the strong dependency of their survival on PI3K/Akt pathway rather than MAPK pathway, the major downstream signaling pathways of EGFR. GLM-1 cells can metastasize to the liver after intrasplenic injection, and GLM-3 cells have spontaneous lung metastatic potential after subcutaneous transplantation, respectively. These results indicate that the GLM series are the first cell lines reflecting the intestinal-type differentiated adenocarcinoma, a major subtype of gastric cancer with liver metastasis. Therefore, they would be excellent models for understanding the mechanism of metastatic growth and the development of a new molecular targeting therapy for gastric cancer with liver metastasis.     

Lectin-resistant variants of mouse Lewis lung carcinoma cells. I. Selection and in vivo properties

The availability of lectin-resistant cell lines with altered carbohydrate moieties in cell surface glycoproteins and glycolipids has greatly facilitated study of the involvement of cellular glycoconjugates in tumor growth and metastasis. We present here a new animal model for metastasis study based on mouse Lewis lung carcinoma LL₂ in vitro cell line [10]. From this line, five lectin-resistant variant sublines were selected with the following lectins: wheat germ agglutinin (WGA^R), Ricinus communis agglutinin II (RCA II^R) and Aleuria aurantia agglutinin (AAA^R). The correlation of the lectin resistance with their in vitro and in vivo growth properties, and especially lung colonizing ability, were investigated. Three WGA^R variants with well-preserved tumorigenicity revealed reduced metastatic ability, both spontaneous, after subcutaneous (s.c.) administration and experimental, after intravenous (i.v.) administration. The RCA II^R variant also possessed reduced spontaneous and experimental metastatic ability, but exhibited higher growth rate of local s.c. tumors. The AAA^R variant possessed reduced spontaneous metastatic ability but its ability to colonize the lungs after i.v. administration was five-fold higher than that of the parent LLZ line, whereas its tumorigenicity remained unchanged. The relative differences among WGA^R variants and parent LLZ line, concerning their experimental metastatic ability, remained similar in cyclophosphamide-modified mice to those in normal recipients.Abbreviations WGA wheat germ agglutinin - RCA II Ricinus communis agglutinin II - AAA Aleuria aurantia agglutinin - Lec^R lectin-resistant - EMEM Eagle's minimal essential medium - FCS fetal calf serum - CY cyclophosphamide - S.C. subcutaneously - i.v. intravenously - i.p. intraperitoneally - PBS phosphate buffered saline     

Variable expression of cathepsin B and D correlates with highly invasive and metastatic phenotype of oral cancer

The expression levels of cathepsins B, D, and L in oral cancer surgical specimens were determined using immunocytochemical analysis. Cathepsins B and D are frequently overexpressed in squamous cell carcinomas, whereas their overexpression was less frequent in verrucous carcinoma and basaloid squamous cell carcinomas. Elevated level of cathepsin B in oral carcinomas was significantly associated with advanced tumor stage (P < .05) and poor histologic malignancy grade (P < .001). Increased expression of cathepsin D correlated significantly with the presence of metastasis (P < .05), poor histologic malignancy grade (P < .001), and high proliferation rate (P < .05). Cathepsin L was less frequently overexpressed in oral cancers than cathepsin B and D. These findings indicate that there is a strong cause/effect relationship between the expression levels of cathepsin B and D in oral cancers and their local invasive and metastatic growth patterns. Thus, cathepsins B and D are useful prognostic markers as well as promising gene therapy targets for oral cancer.     

The role of extracellular matrix in human astrocytoma migration and proliferation studied in a microliter scale assay

Ligands in the extracellular matrix (ECM) are known to mediate migration of normal as well as tumor cells via adhesion molecules such as the integrin receptor family. We developed a microliter scale (15-20 fu total volume) monolayer migration assay to investigate the ability of astrocytoma cells to disperse on surfaces coated with purified human ECM protein ligands. In this system the rate of radial migration of the cell population was constant over time. For human astrocytoma cell lines U-251 and SF-767, laminin and collagen type IV supported a migratory phenotype; fibronectin and vitronectin only minimally supported migration. The different ECM proteins also influenced growth rate: cells on laminin and collagen had a protracted lag phase. Furthermore, migrating cells seeded on laminin or collagen showed a lower labeling index than did stationary cells in the central, crowded region on the same substrate. This micro-scale migration assay should enable detailed molecular and biochemical studies of the determinants of migration.     

Cell migration and actin organization in cultured human primary, recurrent cutaneous and metastatic melanoma. Time-lapse and image analysis.

Random cell migration and actin organization in seven human primary, recurrent cutaneous, and metastatic melanoma cell lines were studied by time-lapse video recording and image analysis. The migration of over 800 randomly selected cells from the cell lines were recorded using an inverted microscope with an attached incubator housing. The fraction of cells with random migration rates greater than 10 microns/hour was 8% in an established primary melanoma cell line, 2% and 34% in two recurrent cutaneous melanoma cell lines, and 5%, 30%, 31%, and 60% in four metastatic cell lines. The three metastatic cell lines with significantly higher mean migration rates (P less than 0.001) were derived from lymph node metastases, whereas the fourth metastatic cell line was derived from a visceral metastasis. The cellular morphology and presence of cell nests in the original tissue correlated with in vitro cell morphology and the formation of colonies. The ability of cells to organize actin into stress fibers directly correlated with significantly higher random migration rates and lack of colony formation. Characterization of random migration rates and actin organization of human melanoma cells that are isolated from different stages of tumor progression may lend insight into metastasis.     

An Orthotopic Model of Human Osteosarcoma Growth and Spontaneous Pulmonary Metastasis

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. In order to investigate the pathogenesis of human osteosarcoma, there is a great need to develop a clinically relevant animal model. Here we report the development of an osteosarcoma animal model using three related human osteosarcoma lines, the parental TE-85 and two derivative lines MNNG/HOS and 143B. In vitro characterization demonstrated that the 143B line had the greatest cell migration and the least cell adhesion activities among the three lines. The 143B line also exhibited the greatest ability for anchorage independent growth. When GFP-tagged osteosarcoma cells were injected into the proximal tibia of athymic mice, we found that 143B cells were highly tumorigenic and metastatic, and MNNG/HOS cells were tumorigenic but significantly less metastatic. TE85 cells were neither tumorigenic nor metastatic. The number of pulmonary metastases was found 50-fold higher in 143B injected animals than that in MNNG/HOS injected mice. No pulmonary metastases were detected in TE85 injected animals for up to 8 weeks. Primary tumors formed by MNNG/HOS and 143B cells could be visualized by whole body fluorescence imaging, while the pulmonary metastases were visualized on the necropsied samples. The GFP tagged 143B cells (and to a lesser extent, MNNG/HOS cells) were readily recovered from lung metastases. This clinically relevant model of human osteosarcoma provides varying degrees of tumor growth at the primary site and metastatic potential. Thus, this orthotopic model should be a valuable tool to investigate factors that promote or inhibit osteosarcoma growth and/or metastasis.     

LncRNA linc00460 sponges miR-1224-5p to promote esophageal cancer metastatic potential and epithelial-mesenchymal transition

BackgroundIncreasing studies highlight the crucial role of long non-coding RNAs (lncRNAs) in carcinogenesis of various human cancer types, including esophageal cancer (ESCA). Long intergenic non-coding RNA 00460 (Linc00460), a novel oncogenic lncRNA, has been reported to accelerate ESCA cell growth. This study aimed to investigate the role and possible regulatory mechanism of linc00460 in ESCA metastasis.MethodsBioinformatics analysis and quantitative real time polymerase chain reaction (qRT-PCR) were used to detect linc00460 expression in ESCA. Wound healing assay, Transwell assay and Western blot were utilized to examine migration, invasion and epithelial-mesenchymal transition (EMT) of ESCA cells. The direct binding effect between linc00460 and microRNA-1224-5p (miR-1224-5p) was evaluated by the dual luciferase reporter assay.ResultsIn this study, we discovered that lncRNA linc00460 was obviously over-expressed in ESCA, both in tissues and cell lines. Down-regulation of linc00460 significantly suppressed the metastatic potential (including cell migration and invasion) and EMT of ESCA cells. In addition, miR-1224-5p, a potential tumor suppressor, was negatively correlated with linc00460 in ESCA. Linc00460 and miR-1224-5p could bind directly in ESCA cells. Inhibition of miR-1224-5p partially abrogated the effects of linc00460 decrease on metastatic potential and EMT of ESCA cells.ConclusionsTaken together, linc00460 may function as a molecular sponge to adsorb miR-1224-5p, thereby promoting ESCA metastasis and EMT. Our findings suggest that linc00460/miR-1224-5p is a possible clinical target for ESCA.     

Selection of more aggressive variants of the GI101A human breast cancer cell line: A model for analyzing the metastatic phenotype of breast cancer

In vivo models utilizing orthotopic injection of tumor cells into nude mice have proven valuable for the study of metastasis. However, breast cancers are among the more difficult of human tumors to grow in immunodeficient mice, with a relatively low tumor take. Fewer still develop spontaneous metastases. The injection of GI101A breast cancer cells into the mammary fatpad (mfp) produced lung metastases in 25% of tumor-bearing mice. Selecting cells from the lung metastases and recycling in vivo resulted in the isolation of a series of variant cell lines. These cell lines were tested for tumorigenicity and metastasis in nude mice following mfp injection compared with the original cell line, and in vitro expression of factors associated with the metastatic phenotype measured. The in vivo selected cell lines were more aggressive, with higher tumor take, faster local growth rate and increased incidence (≥85%) and extent of lung metastasis. However, the metastasis-selected variants showed no increases in expression of the growth factor receptors EGFR or HER-2, and the pro-angiogenic factors VEGF-A and IL-8. Immunohistochemistry of mfp tumors revealed no differences in microvessel density (counting CD-31 positive structures) and cell proliferation (PCNA-positive cells) comparing the GI101A line with selected variants. No TUNEL-positive cells were detected in the tumors of the metastasis-derived variant, with a small number of cells undergoing apoptosis detected in sections of GI101A tumors. In vitro, the metastasis-derived variants were found to have a more robust expression of phosphorylated PKB/Akt, with or without EGF or serum stimulation, suggesting an association between Akt activation and metastatic ability. This new series of isogenic cell lines may be valuable for identifying molecular mechanisms involved in the metastatic progression of breast cancer. This revised version was published online in July 2006 with corrections to the Cover Date.     

Experimental and clinicopathologic studies on the function of the HGF receptor in human colon cancer metastasis

The organ-specific metastasis characterizes several human cancers, including colon carcinoma, a disease that frequently involves metastases in the liver. The data on the molecular mechanisms of liver metastasis would therefore be highly useful for prognostic purposes. Although the upregulation/amplification of the hepatocyte growth factor (HGF) receptor, c-met, has been frequently observed in colon cancer metastasis, the actual functional significance of the feature in the liver metastatization is not yet known. We have used three human colon carcinoma cell lines (HT29, HT25 and WiDr), characterized by different liver metastatic potentials in SCID mice, to analyze the expression of c-met and the biological effects of HGF. We found that HGF induces scattering in in vitro liver-metastatic cell lines (HT25 and WiDr) only at doses which are non-mitogenic (1–20 ng/ml). Analysis of the c-met expression revealed that the metastatic cell lines express authentic c-met gene and protein material, unlike the non-metastatic HT29 cell line, which expresses only the c-terminal cytoplasmic domain of the c-met β-chain. Interestingly, c-met was found to be localized in the substrate-attached peripheral membrane and partially colocalized with phosphotyrosine-proteins in the metastatic cells only when kept on fibronectin. On the other hand, we have analyzed 86 primary human colon cancers in Dukes' B (invasive but non-metastatic) and C (invasive and lymph node metastatic) stages. Western blotting of the proteins isolated from the tumor tissues and immunohistochemical control study on the paraffin samples of a third of these cases (25/86) all indicated a significant upregulation of the c-met protein in the Dukes' C tumor glands compared to the Dukes' B stages (P<0.001 and P<0.05, respectively). Since the two stages differ in the involvement of the regional lymph nodes but not in the invasion depth, the clinicopathological data and our experimental findings further support the notion that the c-met expression in human colon cancer can be considered as a marker of the metastatic potential due to its involvement in the generation of the motility signal. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enhanced expression of cathepsin L in metastatic bone tumors.

Cathepsin L is a kind of cystein proteases which are known to facilitate the invasion and metastasis of tumor cells by degrading the components of basement membrane and extracellular matrix. This study was undertaken to investigate the expression of cathepsin L by Northern blot analysis with radiolabeled cDNA specific for cathepsin L in six normal tissues, two osteosarcoma cell lines, MG-63 and Saos-2, six primary bone tumors and six metastatic bone tumors. In six normal tissues, the highest level of cathepsin L was expressed in liver with the descending order of liver > lung > thymus > ovary > kidney > esophagus. One of the two osteosarcoma cell lines established from the primary sites expressed a high level of cathepsin L mRNA. Out of six primary bone tumors, three (50%) expressed cathepsin L mRNA, while all (100%) of six metastatic bone tumors expressed the mRNA. These results demonstrating the higher frequency of expression of cathepsin L in metastatic bone tumors suggest that cathepsin L may participate in tumor invasion and metastasis.     

Aberrations of growth factor control in metastatic follicular thyroid cancerin vitro

The aggressiveness of follicular thyroid cancer (FTC) varies widely, and metastasis is the primary cause of death. Uncontrolled proliferation of cancer cells may be associated with loss of growth factor control. We investigated the effects of stimulating (epidermal growth factor [EGF]; thyreotropin [TSH] in low concentrations) and inhibiting growth factors (transforming growth factor beta 1 [TGF beta 1]; TSH in high concentrations) on invasion and growth of FTC cell lines from the thyroid tumor (FTC133) and from the lymph node (FTC236) and lung (FTC238) metastases of the same patient. Invasion—penetration through an 8m pore membrane, covered by Matrigel (basement membrane)—and growth were measured using the MTT-method. EGF (10 ng/ml) and TSH in low concentrations (1 mU/ml) stimulated invasion and growth of all FTC cell lines, but the amplitude of stimulation differed significantly. The parental cell line FTC133 was considerably more responsive to growth factor stimulation than the metastatic clones. Invasion of FTC133 was enhanced by 42% (EGF;p<0.02) and 21% (TSH;p<0.01), invasion of FTC236 by 8% (EGF;p<0.02) and 8% (TSH;p<0.01), and invasion of FTC238 by 9% (EGF;p<0.02) and 8% (TSH;p<0.01). Conversely, invasion and growth of FTC133 were significantly more inhibited by TGF beta 1 (10 ng/ml) and supraphysiologic concentrations of TSH (100 mU/ml) than the cell lines from the lymph node and lung metastases. At day 7, invasion of FTC133 was inhibited by 32% (TGF beta 1;p<0.02) and 21% (TSH;p<0.01), invasion of FTC236 by 18% (TGF beta 1;p<0.02) and 11% (TSH;p<0.01), and invasion of FTC238 by 16% (TGF beta 1;p<0.02) and 12% (TSH;p<0.01). Moreover, we analyzed growth factor independence in minimally supplemented or unsupplemented medium. Growth, but no invasion was evident when cells were cultured completely unsupplemented over 7 days. These results suggest that metastatic FTCs may have developed by escaping from the normal control of TSH and other growth factors.     

Significance of the small subtelomeric area of chromosome 1 (1p36.3) in the progression of malignant melanoma: FISH deletion screening with YAC DNA probes

The short arm of chromosome 1 (1p), especially the subtelomeric region of 1p36, is a common site for abnormalities in malignant melanoma of the skin. In a recent study nodular melanomas displayed deletions of 1p36 in an augmented percentage of cases. To evaluate the dimension of these deletions and to study their significance for the progression of malignant melanoma we analyzed seven melanoma cell lines, 32 primary tumors, and 32 metastatic tumors by fluorescence in situ hybridization with the DNA probe D1Z2 in 1p36.3 and eight YAC DNA probes hybridizing to 1p36, 1p32, 1p31, and 1p21. All cell lines, 91% of the metastatic tumors and 63% of nodular melanomas showed a deletion of 1p36.3. In the YAC hybridization experiments, the most frequent deletions were found in 1p36 in all cell lines, in 13% of nodular melanoma, and in 44% of metastatic tumors. Deletions in 1p36 were mostly confined to a rather small area near the locus D1Z2. The frequent occurrence of this deletion in melanomas with a high metastatic potential and the abundant accumulation of this deletion in metastasis point to genes located on 1p36, which might be of significance for the metastatic capability of malignant melanoma. Received: 8 February 1999 / Accepted: 15 April 1999