Biological properties and gene expression associated with metastatic potential of human osteosarcoma

Lung metastasis has a great influence on the prognosis of patients with osteosarcoma. We previously established two high-metastatic sublines, M112 and M132, from the HuO9 human osteosarcoma cell line by in vivo selection. In this study, we newly isolated a high-metastatic subline, H3, and three low-metastatic sublines, L6, L12 and L13, from HuO9 by the dilution plating method. Three high-metastatic sublines produced more than 200 metastatic nodules in the lung, while three low-metastatic sublines produced no or few nodules after injection of 2 × 10⁶ cells into the tail vein of nude mice. There were significant differences in the motility and invasiveness between high- and low-metastatic sublines, whereas the growth rates in vitro and the tumorigenicity in vivo showed no correlation with their metastatic abilities. Early adherence to culture plates was significantly lower in two of three low-metastatic sublines, which occupied smaller surface areas on the culture plates than other sublines did. Comparison of the expression of 637 cancer-related genes by cDNA microarray revealed that seven genes were differentially expressed between high- and low-metastatic sublines. Among them, five genes (AXL, TGFA, COLL7A1, WNT5A, and MKK6) were associated with adherence, motility, and/or invasiveness. These results suggest that the differences in motility/invasiveness and adhesive abilities are key determinants of lung metastasis in osteosarcoma. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phenotypic heterogeneity of end-stage prostate carcinoma metastatic to bone

To better understand the clinical and pathologic features of end-stage, androgen-independent carcinoma of the prostate (CaP), we performed rapid autopsies on 14 men who died of progressive CaP and recorded relevant clinical data. The timing of tumor progression varied widely. The median time to androgen independence was 2 years (range, 4 months to 13.6 years). The median survival after androgen independence was 1 year (range, 1 month to 3.6 years). Because osseous metastases are prevalent in progressive CaP, up to 20 bone sites were systematically sampled in each patient. Bone metastases were widespread; tumor filled the marrow in an average of 14 bone sites. Tumor histology and expression of prostate-specific antigen (PSA) and chromogranin A (CGA) were examined in all metastases and were compared with the primary tumor. Five histological patterns of metastatic tumor were observed: solid (10 patients), macroacinar (1 patient), microacinar (1 patient), clear cell (1 patient), and comedocarcinoma (1 patient). Gleason grade of the primary tumor did not predict the histological pattern of the metastases. Although >70% of tumor cells expressed PSA, the fraction of PSA-positive cells varied widely in separate metastases in some patients (standard deviation >25). Likewise, the fraction of neuroendocrine (NE) (CGA-positive) tumor cells in different metastases varied widely. For example, between 0 and 95% of tumor cells in different metastases in 1 patient had a NE phenotype. The present study highlights the heterogeneity--histologically and immunophenotypically--of metastatic CaP. Consequently, therapy directed to the phenotype of 1 metastasis may have no effect on other metastases in the same patient because of phenotypic heterogeneity.     

Metastatic potential of human colorectal carcinoma SW1222 cells transfected with cDNA encoding carcinoembryonic antigen

In order to examine a role of carcinoembryonic antigen (CEA) in metastasis, cDNA encoding CEA was introduced into a clone of human colorectal carcinoma SW1222 cells. Western blot analysis revealed that all transfectants express CEA of 180 kDa while the parent clone does not. In the transfectants, the level of CEA expression in clone 3 was higher than that of clone 1. Clone 3 formed aggregates rapidly after suspended by trypsinization while clone 1 did not. In experimental metastasis assay where tumor cells were injected intrasplenically, clone 3 exhibited a higher liver-metastatic activity than clone 1. Fab fragment of anti-CEA antibody significantly inhibited both the cell aggregation and the liver metastases caused by clone 3. These findings suggested that CEA expressed on the cell surface may play an important role in hepatic metastasis from colorectal carcinoma, possibly through its cell adhesion activity.     

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。     

Differential motility stimulation but not growth stimulation or adhesion of metastatic human colorectal carcinoma cells by target organ-derived liver sinusoidal endothelial cells

Liver is the most common distant metastatic site for colorectal cancers and when blood-borne colorectal cancer cells reach the liver, they first encounter hepatic capillary and sinusoidal endothelial cells. Thus we studied differences between highly (HT-29LMM) and poorly (HT-29P) liver-metastatic sublines of human colorectal cancer cells by examining the interactions between tumor cells and liver microvessel endothelial cells. Using hepatic sinusoidal endothelial (HSE) and lung microvessel endothelial (MLE) cell-conditioned medium we measured the growth and motility stimulating activities released from these endothelial cells and adhesion of these cancer cells to the endothelial cells. Differences in the ability of HSE-conditioned medium (HSE-CM) or MLE-conditioned medium (MLE-CM) to stimulate HT-29 cell growth were not observed. There was a small but significant increase in the rate of adhesion of highly metastatic HT-29LMM cells to HSE cell monolayers than poorly metastatic HT-29P cells, but there was no difference in adhesion to MLE cell monolayers. HSE-CM stimulated the motility of highly metastatic colorectal cancer cells to a greater extent than the poorly metastatic cells. Motility-stimulating activity for the colorectal cancer cell lines was not detected in MLE-CM. The HSE-CM motility-stimulating activity for human HT-29 cells was not removed using antibodies against hepatocyte growth factor (HGF/SF), complement component C3 or laminin, indicating that it is not related to these known liver-derived motility factors. The results suggest that the ability of highly metastatic HT-29LMM colorectal cancer cells to colonize the liver is related to their ability to respond to liver sinusoidal endothelial cell-derived motility factors and to a lesser degree to adhere to liver sinusoidal endothelial cells.     

Increased expression of human macrophage metalloelastase (MMP-12) is associated with the invasion of endometrial adenocarcinoma

To evaluate the association between the expression of human macrophage metalloelastase (matrix metalloproteinase-12, MMP-12) with cancer invasion and differentiation of endometrial adenocarcinoma, specimens from endometrial adenocarcinoma (n=61) of diverse stages and histologic types were collected from patients having undergone hysterectomy, and specimens from normal endometrium (n=38) were obtained from patients with benign diseases. The expression of MMP-12 was analyzed immunohistochemically, by Western blot, and RT-PCR. The positive rate of MMP-12 was significantly increased in endometrial adenocarcinoma (81.97%) as compared with that in normal endometrium (13.16%). The results showed that expression of MMP-12 correlated with stage (p=0.022) and grade (p=0.018) of endometrial cancer. MMP-12 immunoreactive proteins were found mainly on the glandular epithelial cells of endometrial adenocarcinoma. The macrophage infiltration detected by CD68 immunohistochemical staining in endometrial adenocarcinoma was also higher than that in normal endometrium. In this study, we show that in addition to macrophages, endometrial adenocarcinoma cells are able to express MMP-12. Increased MMP-12 expression tended to be associated with the extent of adenocarcinoma invasion accompanied by marked macrophage infiltration. Our results suggest that MMP-12 is an important oncogene in high-stage and high-grade endometrial adenocarcinoma.     

Anti-metastatic prostacyclins inhibit the adhesion of colon carcinoma to endothelial cells by blocking E-selectin expression

Prostacyclins have long been shown to have anti-metastatic activity. One hypothesis is their modulation of cell adhesion molecule (CAM) expression by target organ endothelial cells. We have postulated that prostacyclin, its analogs, and mechanistic mimics decrease colon carcinoma adhesion to cytokine-stimulated endothelial cells by blocking endothelial expression of the adhesion molecule E-selectin, but not the vascular cell adhesion molecule-1 (VCAM-1). Cultured human microvascular endothelial cells (HDMEC) were pre-incubated with prostacyclin (PGI₂), dibutyrl-CAMP (dbcAMP), forskolin (FOR), and/or iso-methylbutylxanthine (IBMX) for 15 min, then co-incubated with the cytokine tumor necrosis factor (TNF) for 4 h. HDMEC surface expression of E-selectin and VCAM-1 was evaluated by flow cytometry and ELISA. Adherence of ⁵¹Cr-labeled colon carcinoma cells to HDMEC monolayers was then determined. In parallel assays, HDMECs were incubated with anti-E-selectin and anti-VCAM-1 monoclonal antibody (1:100) prior to the addition of tumor cells. Prostacyclins, its analogs, and mimics significantly reduced E-selectin expression by HDMEC, while the reduction of VCAM-1 expression was much less pronounced. Prostacyclins also significantly decreased colon carcinoma adherence to stimulated HDMECs. The inhibition of E-selectin expression, but not VCAM-1 expression, corresponded to the reduction of tumor cell adherence. Prostacyclin's effects on tumor adhesion were nullified by pre-incubation with E-selectin antibody. The inhibition of colon carcinoma adherence to cytokine-stimulated endothelial cells treated with prostacyclin, its analogs, and mimics appears to result from blocking endothelial E-selectin, but not VCAM-1, expression. These data support the hypothesis that prostacyclins may exert their anti-metastatic effect, in part, by inhibiting CAM-mediated adherence of colon carcinoma to endothelial cells in metastatic target organs.     

Neural cell adhesion molecule is upregulated in nerves with prostate cancer invasion

Currently postulated mechanisms of perineural invasion (PNI) include interaction between tumor cells and nerves. Neural cell adhesion molecule (N-CAM), one of the well-known members of the immunoglobulin super-family of adhesion molecules, was implicated in PNI and metastasis in various types of cancer. Tissue microarray technology was used to build 2 sets of tissue array (with versus without PNI) from 50 prostate cancers (PCa). The slides were stained immunohistochemically, and the results were evaluated semiquantitatively using a 0 to 3+ scoring system. N-CAM staining was observed in all nerves with variable intensity. N-CAM expression was upregulated in 73% (31 of 42) of the nerves with PNI compared with nerves without PNI (P >.001). The results suggested that N-CAM is probably involved in PNI in PCa. It is conceivable that cancer cells, through a yet-to-be-established paracrine loop, signal the nerve to increase N-CAM production and increase adhesion. N-CAM upregulation in nerves may also facilitate cancer cells to migrate toward nerves and promote the process of perineural spread through increased survival using the nuclear factor kappa B pathway.     

Inhibition of cancer cell motility and invasion by interleukin-12

Tumour cell motility and attachment are crucial requirements in the formation of metastatic lesions. These properties are affected by a number of cytokines including hepatocyte growth factor/scatter factor (HGF/SF) and several immunoregulatory proteins, including interleukin-12 (IL-12). Although IL-12 has been reported to exhibit potent anti-tumour effects in vivo, a direct effect of IL-12 on cancer cells has not been reported. We show here that IL-12 directly inhibited the attachment of the human colon cancer cell lines HRT18, HT29 and HT115 to Matrigel, HGF/SF-stimulated cell motility and HGF/SF-induced cell invasion through a reconstituted basement membrane. IL-12 did not affect the growth of these cell lines. Flow cytometry, Western analysis and immunohistochemistry revealed an up-regulation of E-cadherin cell-surface adhesion molecules. These direct effects of IL-12 on colon cancer cells suggest a potentially important role for IL-12 in metastasis.     

Anin vitro invasion model for human renal cell carcinoma cell lines mimicking their metastatic abilities

We developed a modifiedin vitro invasion assay system using monolayers of vascular endothelial cells. A type I collagen gel was formed in plastic dishes, and overlaid with type IV collagen. Calf pulmonary arterial endothelial (CPAE) cells were seeded onto these plates, and incubated until they reached confluence. Five human renal cell carcinoma cell lines with various metastatic potentialsin vivo were then seeded on the monolayer CPAE cells, and their colony formation and invasion activities were examined for 9 days. At day 4, the highly metastatic cell lines increased the number of colony foci on monolayer CPAE cells several fold higher than their poorly metastatic counterpart. The horizontal spreading patterns were also different between poorly and highly metastatic cell lines. On day 9, the number of carcinoma foci that penetrated the monolayer of CPAE cells and type IV collagen sheets into type I collagen gels in highly metastatic cell lines greatly increased as compared with that of poorly metastatic cell lines. Ourin vitro invasion assay using monolayer CPAE cells would be useful to evaluate protease activities and colony formation during invasion.     

Effects of ionizing radiation on the adhesive interaction of human tumor and endothelial cells in vitro

A centrifugation assay was used to determine the effects of ionizing radiation on the adhesive interaction of A549 human lung adenocarcinoma tumor cells and human umbilical vein endothelial cells (HUVEC). The tumor cells were fluorescently labeled and divided into control (sham-irradiated) and irradiated groups. The irradiated groups were exposed to irradiation levels ranging from 5 to 20 Gy using a Cs source. A specified number of these A549 tumor cells were then delivered into each well of 96-well cell culture plates containing confluent monolayers of human umbilical cord vein endothelial cells (HUVEC), and were given time to adhere to the endothelial cells. The wells were then sealed and were exposed to an acceleration field varying from 1 to 42 g (0-500 rpm) for 10 min. Finally, the wells were drained, and the number of tumor cells adhering to the endothelial monolayer were counted using a fluorescent microscope system. Our results indicate that the irradiation of A549 tumor cells significantly increased their adhesive interaction with endothelial cells (number of adhering irradiated cells/number of adhering control cells = 1.0, 1.3, 1.9, 2.2 for 0, 5, 10, 20 Gy respectively). In contrast, when endothelial cells were irradiated, rather than tumor cells, adhesive interaction decreased with an increase in the radiation dose (irradiated/control = 1.0, 0.9, 0. 8, 0.5 for 0, 5, 10, 20 Gy respectively). Simultaneous irradiation of both the tumor cells and the endothelial cells did not alter their adhesive interaction significantly. These findings may have important implications for the metastatic ability of irradiated tumor cells.     

Functional cloning of ARM-1, an adhesion-regulating molecule upregulated in metastatic tumor cells

Interactions of tumor cells with the endothelium and tissue stroma are considered to be critical steps in metastasis formation and progression of cancer. To identify cellular receptors that mediate the binding of tumor cells to endothelium, a murine T cell lymphoma-derived expression library was screened for adhesion-inducing cDNA clones. We identified a novel cell adhesion-promoting molecule, termed ARM-1 (adhesion regulating molecule-1), which is homologous to a human M _r 110.000 tumor-associated antigen. The ARM-1 cDNA codes for a type I transmembrane protein of 407 amino acids with potential O- and N-glycosylation sites that does not belong to any of the known families of cell adhesion molecules. Overexpression of ARM-1 in 293T human embryonic kidney cells significantly increased adhesion to different endothelial cells. ARM-1 expression in 293T cells did not alter integrin expression or β1-integrin-mediated cell adhesion. Northern blot analysis of human breast cancer cell lines revealed 3- to 5-fold elevated ARM-1 mRNA levels in metastatic as compared to non-metastatic cells. In conclusion, we have identified ARM-1 as a novel cell adhesion-promoting receptor that is upregulated in metastatic cancer cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Cytology of metastatic cervical squamous cell carcinoma in pleural fluid: Report of a case confirmed by human papillomavirus typing

Cervical squamous cell carcinomas are rarely the cause of malignant effusions. Their identification can be relatively easy when keratinizing atypical squamous cells are present, but may be very difficult when only nonkeratinizing malignant cells are present. We present the case of a 47‐year‐old woman who presented with a large left pleural effusion after having recently completed chemoradiation therapy for stage IIB cervical squamous cell carcinoma. Cytologic examination of the fluid showed a uniform population of single atypical cells with finely vacuolated cytoplasm, ectoendoplasmic demarcation, cell‐in‐cell arrangements, and short rows of cells with intervening “windows,” all features reminiscent of mesothelial cells. No keratinization or three‐dimensional cell clusters were identified. A panel of immunohistochemical stains was performed on the cell block material, and the atypical cells were positive for cytokeratin 5/6, p63, and p16 but not for cytokeratin 7, calretinin, WT1, or Ber‐EP4 or TTF1. These findings were consistent with metastatic squamous cell carcinoma. HPV DNA determination and typing by PCR confirmed the presence of HPV16 in an aliquot of pleural fluid. This is to our knowledge the first reported case of pleural fluid involved by metastatic squamous cell carcinoma where HPV DNA testing was used to confirm the origin of the metastasis. Despite its rarity, metastatic nonkeratinizing squamous cell carcinoma should be considered when a single cell population of large atypical cells is found in effusions. Immunoperoxidase stains and HPV testing can be performed to establish the diagnosis and confirm the origin from a cervical primary. Diagn. Cytopathol. 2009. © 2009 Wiley‐Liss, Inc.     

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.     

The metalloproteinase inhibitor TIMP-2 is down-regulated by androgens in LNCaP prostate carcinoma cells

Tissue inhibitors of metalloproteinases (TIMPs) have been shown to perform several biological functions in tumor promotion, principally by their action of inhibiting matrix metalloproteinases (MMPs) at different steps of the metastatic process. In particular, TIMP-2 is involved in a functional complex with the membrane-type 1 (MT1) MMP to convert the secreted MMP-2 progelatinase into the fully active proteolytic enzyme. We used the human, androgen-sensitive prostate carcinoma cell line LNCaP in coculture with the human osteosarcoma cell line OHS to experimentally address the possibility of androgen-dependent regulatory effects on the functional MT1-MMP/TIMP-2/MMP-2 complex upon interaction between prostate carcinoma and osteoblastic cells in metastasis of prostate cancer to bone. In the LNCaP cells a gradual, time-dependent decline in TIMP-2 mRNA expression was observed in the presence of the synthetic androgen analogue R1881 (100 nM), reaching ∼25% of the control level after 48 h of incubation. Consistent with this, the accumulation of secreted TIMP-2 in media from R1881-treated cells was significantly inhibited already after 3 h. Neither MMP-2 gelatinolytic activity nor expression of MT1-MMP was detected in LNCaP cells. In contrast, the OHS cells showed membrane-associated MT1-MMP expression as well as MMP-2 secretion. However, R1881 treatment of the LNCaP/OHS coculture model did not seem to change the overall proteolytic activity of the MT1-MMP/TIMP-2/MMP-2 complex. Hormonal control of TIMP-2 expression in prostate carcinoma cells has not been previously reported, but whether such regulation has any functional role in the development of osteoblastic metastases in prostate cancer is still unclear. This revised version was published online in July 2006 with corrections to the Cover Date.     

Increased resistance towards oxidative stress accompanies enhancement of metastatic potential obtained by repeated in vivo passage of colon carcinoma cells in syngeneic rats

The colon carcinoma cell line CC531 is metastatic to liver after splenic injection in syngeneic rats. After repeated in vivo passages, a subline was selected that produced liver metastases at a considerably higher rate than the original cell line. These cells were characterized by increased intracellular glutathione, proliferation and ability to restore glutathione after exposure to oxidative stress, thus indicating an elevated resistance to oxidative stress. Furthermore, the increased metastatic ability was also accompanied by increased proliferation rate, adhesion to extracellular matrix proteins and endothelial cells, and secretion of a 60 kD matrix metalloproteinase. When cultured in vitro for a prolonged time (more than 30 trypsinizations), the cells showed a reduced in vivo metastatic ability, reduced secretion of three metalloproteinases including the 60 kD proteinase, and reduced intracellular glutathione. These results indicate that metastatic ability can be influenced through several adaptive mechanisms, and that the cell's ability to resist oxidative stress and maintain intracellular glutathione are of central importance. This revised version was published online in July 2006 with corrections to the Cover Date.