Malignant tumours cause sickness and death largely because they invade and metastasize. Such spread is made possible by many cellular properties, including the ability of neoplastic cells to move and to release degradative enzymes. These properties enable tumour cells to break free of the primary tumour, penetrate blood or lymphatic vessels and, after being transported to distant sites, pass out of the vessels to establish new tumours. Not all cells in a tumour, however, are able to metastasize, so the process tends to select for greater malignancy in the secondary tumour. The heterogeneity of tumours probably accounts for the difficulty of providing effective treatment, in that the various subpopulations of cells arising from each tumour vary in their responses to chemotherapeutic agents. We do not yet understand the process sufficiently to treat cancer patients by interfering selectively with the metastatic mechanisms.
SPARC is known to be important in development and tissue remodelling. Here, we examined the effects of SPARC (secreted protein, acidic and rich in cysteine; osteonectin) derived from a rat osteosarcoma cell line on migration of renal cell carcinoma (RCC) by a Boyden chamber assay. YCR RCC cells migrated through type IV collagen-coated filters without stimuli (basal level). SPARC in the lower compartment stimulated chemotactic activity to 120% of the basal level, whereas premixing of YCR with purified SPARC before inoculation reduced their migration to 72% of the basal level. Furthermore, SPARC mixed with type IV collagen more efficiently stimulated their migration in a concentration-dependent manner (up to 170% of the basal level). This suggests that SPARC bound to type IV collagen plays a role in tumor invasion.
This study examines the Helix pomatia lectin (HPA) binding characteristics of metastases arising from primary breast cancer, and compares HPA binding patterns with binding of Dolichos biflorus lectin (DBA), Limax flavus lectin (LFA), and a monoclonal antibody against the Tn epitope. Of 81 blocks of metastases in a range of tissues, taken at autopsy from 46 individuals, 79% were HPA positive. No site specificity with regard to HPA binding was observed. Both HPA-positive and -negative tumour deposits were seen within a single individual. HPA-positive tumours were commonly negative for binding of sialic acid specific lectin LFA (86% were negative). Binding patterns of alpha-GalNAc specific HPA and DBA, and a monoclonal antibody against Tn epitope (GalNAc-O-Ser/Thr) were markedly different.
Gangliosides are thought to be involved in tumor cell proliferation, migration and invasiveness as so far demonstrated by the addition of exogenous gangliosides to the culture medium. To better understand the direct influence that alterations in ganglioside synthesis can exert on these functional aspects of cell biology, in the present study, we investigated the behaviour of C6 rat glioma cells after stable transfection with the human CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4GlcCer alpha 2,8-sialyltransferase (SAT-II, EC 2.4.99.8) gene. The enzyme synthesizes ganglioside GD(3) by adding a sialic acid residue to ganglioside GM(3). Stable transfection of the constructs into C6 cells and expression of the human SAT-II gene were evaluated using PCR and RT-PCR amplification, respectively. Qualitative and quantitative analysis of the ganglioside profile was performed by conventional HP-TLC and identity of de novo synthesized species was assessed by TLC immunostaining. Results show that whereas C6 parental cells and C6 cells transfected with the empty expression vector synthesize, almost exclusively, ganglioside GM3, de novo synthesis of GD(3) is clearly observed in clones expressing the alpha 2,8-sialyltransferase. Subcutaneous grafting in athymic nude mice of cells expressing high levels of GD3 induces tumors growing faster and more aggressively than controls. In in vitro assays, the same cells demonstrate increased proliferation rate, motility and invasiveness. Chemotaxis and chemoinvasion were assayed using the modified Boyden chamber. Data obtained suggest that endogenously neosynthesized GD(3) is able to modify proliferation rate, motility and invasion of C6 rat glioma cells, enhancing the features of malignancy of this tumor cell line.
In order to quantitate the metastatic burden of secondary tumor deposits in locoregional lymph nodes, we produced a green fluorescent protein (GFP)-expressing murine cell line (B16F1) metastatic to lymph nodes in immunocompetent mice. When implanted into the hindleg of mice, all animals developed paraaortic lymph node metastases. Fluorescence microscopy, immunohistochemistry, and RT-PCR confirmed the presence of metastatic cells in lymph nodes. Tumoral deposits occurred preferentially in marginal sinuses and spread superficially to the subcortical area. Western blot analysis showed that the local tumor burden could be quantitated in individual lymph nodes. This model system should be useful for quantitating metastatic invasion particularly of micrometastases and aid in the development of lymphotropic drugs to detect and/or treat lymph node metastases in advanced cancers.
Neutrophils are a key cell type of nonadaptive immune system and are the first phagocytic cell type that reaches mucosal inflammatory sites. On the last stage of their journey from the blood stream to a mucosal surface, neutrophils cross a generally sealed epithelium by migrating along the paracellular pathway to the luminal side of the epithelial layer. This last step involves a specific receptor-mediated adhesion event of the neutrophil to the epithelium, followed by a rapid and highly coordinated reversible opening of the epithelial intercellular junctions that allows the transmigration of the neutrophils. Although we do not yet understand the molecular mechanisms that mediate this transmigration process, the last years witnessed the discovery of the first neutrophil and epithelial cell surface proteins critically involved in transepithelial migration of neutrophils.
Tumor progression is characterized by stepwise accumulation of genetic alterations. To identify alterations associated with breast cancer metastasis, an analysis of comparative loss of heterozygosity (LOH) was performed on 38 primary sporadic breast carcinomas and 16 distant metastases. Two loci at 5q21 and 18q21 were chosen because of their reported increased deletion frequency in metastatic tumors. LOH at 17q21, 13q12–13, 17p13.1 and 11q22–23 was analyzed to determine whether there is a specific involvement of these breast cancer-associated gene loci in the metastatic process. Our data show that distant metastases are characterized by markedly increased LOH frequency at all loci examined. In both gene locus groups, significantly more distant metastases are affected by combined LOH. Furthermore, a significantly reduced postmetastatic survival time has been observed in patients with brain metastases affected by synchronous allelic loss at the four breast cancer-associated gene loci. Our results suggest that cumulative LOH of breast cancer-related gene loci is associated with a more aggressive phenotype of metastatic breast tumors.
To understand the mechanisms of tumor invasion and metastasis, model systems are required that isolate the individual steps of these complicated, multifaceted processes. We propose a new procedure to identify genes involved in cell invasion and/or motility that features the combined advantages of transient gene transfection and Matrigel invasion assays. Cancer cells were transiently cotransfected with two vectors expressing the gene of interest and luciferase, as a marker of transfected cells, and then assayed for Matrigel invasion. Luciferase cotransfection appeared to be a sensitive semiquantitative assay for transfected cells and was maximal throughout the invasion assay. The proposed transfection procedure, using calcium phosphate precipitation, did not affect cell invasiveness and allowed cellular coexpression of both genes. When applying this method, we found that transient expression of the unliganded and liganded human estrogen receptor α prevented invasiveness of MDA-MB-231 breast cancer cells. In conclusion, we propose rapid and versatile in vitro procedure for studying the effects of individual cloned genes on cellular processes, such as invasion and motility.
Squamous cell carcinomas of the head and neck (HNSCC) evolve from diploid epithelial cells of the mucosa. At the time of diagnosis about two thirds of clinically diagnosed HNSCC are non-diploid according to flow-cytometric (FCM) analysis, indicating that during tumour progression there must be an acquisition and accumulation of chromosomal aberrations. At diagnosis one third to one half of HNSCC have clinically positive neck nodes. The objective of the present study was to see whether the progression to a metastatic phenotype is reflected in the distribution of FCM DNA ploidy in node-negative and node-positive HNSCC. The series comprised 200 patients with HNSCC. Tumour samples were obtained from diagnostic biopsies or primary surgery. A multistep preparation method and propidium iodide staining of nuclear DNA content was used for FCM. One hundred and forty one (71%) of the tumours were non-diploid. Only two tumours were hypodiploid (DNA index 0.73 and 0.93, respectively). Ten of the tumours exhibited two non-diploid stem cell lines. The frequency of non-diploidy in node-negative tumours was 65% and in node-positive ones about 80%. The frequency distribution of non-diploid DNA indices clustered in the hypotetraploid region (with a modal value of 1.71–1.74) and did not differ between node-negative and node-positive tumours. The hypothesis that the disposition to metastasis is reflected in the frequency distribution of non-diploid DNA indices could thus not be verified.
In the present study, the effect of in vitro cyclosporin A (CsA) treatment on IL-2R expression and the metastatic behavior of B16F10 melanoma cells has been reported. CsA treatment was found to increase the percentage of B16F10 cells expressing the alpha-subunit of IL-2R on the cell surface and also at the mRNA level. Moreover, CsA treated B16F10 cells also express the beta-subunit of IL-2. In vivo experiments showed that CsA increases the affinity of B16F10 metastazing cells for the liver and decreases that for the lung. CsA modulated the expression of MHC class I and class II antigens, but no significant differences in the resistance of CsA-treated B16F10 cells to NK lysis were observed. Finally, proliferation of B16F10 cells in the presence of several doses of CsA did not vary and CsA increased the amount of IL-1beta mRNA expression. These results suggest that CsA, through the modulation of cytokines and MHC antigen expression on B16F10 cells, could have an effect upon the metastatic progression of the B16F10 melanoma.
Expression of matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 was studied in non-small-cell lung cancer (NSCLC). Activity of MMP-2 and MMP-9 by gelatin zymography and expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were examined in 11 lung cancer cell lines which included six small-cell lung cancer (SCLC) cell lines. Localization of MMP-2, MMP-9, TIMP-1, and TIMP-2 was examined by immunohistochemistry in 43 resected NSCLC (22 adenocarcinomas, 17 squamous cell carcinomas, 4 large cell carcinomas) using specific anti-human monoclonal antibodies. Expression of MMP-2 mRNA was detected in 5 (100%), MMP-9 in 1 (20%), TIMP-1 in 4 (80%), and TIMP-2 in 5 (100%) of 5 NSCLC cell lines examined. MMP-2 gelatinolytic activity also was detected in all five NSCLC cell lines, whereas MMP-9 activity was detected in only one cell line. In 43 patients, MMP-2, MMP-9, TIMP-1, and TIMP-2 immunoreactivity was demonstrated in 19 (44%), 9 (21%), 15 (35%), and 29 (67%) excised tumors, respectively. All stromal fibroblasts in tumor samples stained positive for MMP-2. There was a correlation between TIMP-2 immunoreactivity and disease stage (42% stage I versus 88% stages II, III, and IV) (p = 0.0024). Both cancer cell lines and NSCLC tumor samples frequently expressed MMP-2, MMP-9, TIMP-1, and TIMP-2; MMP-2 in particular was highly expressed in malignant cells and surrounding fibroblasts. These findings suggest that MMP-2 plays a more important role in invasion of NSCLC than MMP-9 and that TIMP-2 may have clinical relevance in NSCLC.
The S100A4(mts1) is a gene associated with generation of metastatic disease. In order to analyze the consequences of alteration of the pattern of expression of the S100A4(mts1) gene we obtained strains of transgenic mice bearing the S100A4(mts1) gene under the control of a ubiquitous and constitutive 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) gene promoter. In transgenic animals the expression of the transgene RNA was detected in all organs, but only some of the organs showed elevated levels of the protein. Expression of the S100A4(Mts1) protein was downregulated in the organs that normally do not express the gene in the wild-type animal. The transgene RNA is detected in the polysomes indicating that it could be translated into the S100A4(Mts1) protein. The specificity of the S100A4(Mts1) protein expression is determined by a complex mechanism including regulation of translation and/or posttranslational degradation.
Uterine cervical adenocarcinoma typically is an aggressive neoplasm with a propensity for early invasion and dissemination; however, the regulatory mechanism of invasive activity of cervical adenocarcinoma cells has not been fully understood. In this study, biological effects of epidermal growth factor (EGF) and transforming growth factor (TGF)-α on invasion and proteinase expression of human cervical adenocarcinoma OMC-4 cells were investigated. Tumor cell migration along a gradient of substratum-bound fibronectin and invasion into the reconstituted basement membrane were stimulated by 0.1–10 nM EGF and TGF-α in a concentration-dependent manner. Their effects on tumor cell migration were also confirmed by wound assay. The zymography of tumor-conditioned medium showed that the treatment of OMC-4 cells with EGF and TGF-α resulted in the increase of matrix metalloproteinase (MMP)-2 and urokinase-type plasminogen activator (uPA). Matrilysin (MMP-7), also secreted by OMC-4 cells, was not affected by these growth factors. These results suggest that EGF and TGF-α act as positive regulators on the invasion of cervical adenocarcinoma cells, which may be associated with their stimulatory effects on tumor cell motility and the induction of type IV collagenase and uPA secreted by tumor cells.
Cancer cell attachment to and invasion of the extracellular matrix has been associated with the metastatic potential of cell lines of the Dunning R-3327 rat prostatic adenocarcinoma model. We investigated the cell-matrix interactions of prostate tumor cells by comparing the invasive ability through reconstructed extracellular matrix and attachment upon EHS NATRIX (natural extracellular matrix), fibronectin, laminin, and collagen Type IV. We observed a correlation between metastatic potential and substrate dependence of attachment in prostate cancer cells. Nonmetastatic AT-1 cells possessed a higher adhesive potential to extracellular matrix components than the highly metastatic cells (ML, MLL and AT-3). It was also found that the invasive potential of the three highly metastatic cell lines was significantly higher than that of the nonmetastatic cell line. Here, it is reported that the ability to traverse a matrigel matrix correlates with their metastatic potential. These observations suggest that the extracellular matrix components are highly involved in influencing prostate cancer cell activities. In addition, we investigated the effects of two differentiation agents, retinoic acid (RA) and difluoromethylornithine (DFMO), on the adhesive and invasive profiles of the tumor cells. After treatment with both agents, adhesion was increased to levels not different from nonmetastatic cells. Furthermore, the ability of highly metastatic cells to traverse a matrigel barrier was significantly reduced after treatment with both differentiation agents. These results suggest that RA and DFMO are capable in reversing the metastatic potential of prostate cancer cells in vitro and may give a possible insight into their role as potential therapeutic agents in vivo.
Angiogenesis is required for tumor formation and growth; inhibition of angiogenesis is a promising new approach in cancer therapy. UCN-01, a protein kinase C (PKC) inhibitor, induces growth arrest and apoptosis in cancer cells and was recently introduced in a phase I clinical trial. We demonstrate that UCN-01, at concentrations lower than those necessary to inhibit cancer cell growth, inhibit proliferation of human endothelial cells in vitro. Moreover, UCN-01, at concentrations as low as 32 nM, prevent microvessel outgrowth from explant cultures of rat aortic rings. Since hypoxia activates hypoxia-inducible factor (HIF-1)-dependent transcription in cancer cells that, in a paracrine fashion, drive tumor angiogenesis, we investigated the effects of UCN-01 on HIF-1-responsive promoter constructs. We report that, in addition to direct inhibitory effects on endothelial cell growth, UCN-01 abrogates hypoxia-mediated transactivation of HIF-1-responsive promoters in a prostate cancer cell line. We conclude that UCN-01, at clinically relevant concentrations, exerts an anti-neovascularization effect by blocking two important steps in vessel formation: (1) the response of cancer cells to hypoxia, and (2) endothelial cell proliferation.
Tumour-bearing rats were randomized to a 70% partial hepatectomy or a sham operation. At days 1, 3 or 14, portal and systemic serum was obtained and colon carcinoma cells were cultured in the presence of 5, 10, 20 or 50% serum. Proliferation and epidermal growth factor receptor (EGFr) expression was measured in tumour cells. Proliferation was 25-40% higher in tumour cells cultured with portal serum after hepatectomy than after sham operation when using serum obtained at day 3, but not days 1 and 14 after operation. In cultures with serum obtained at day 14 after operation CC 531 cells showed a 30% higher proliferation rate with systemic hepatectomy serum than CC 531 cells with sham systemic serum. These effects were not mediated by a change in EGFr mRNA and protein levels as the used colon carcinoma cells did not reveal EGFr activity by any of the three detection methods used.
Microorganisms have evolved a variety of mechanisms designed to evade detection and/or destruction by the host. Many pathogens evade host defenses by invading cells, thus providing the bacterium with an environment free of competing microorganisms. Adherence and invasion are active processes in which microorganisms often use host proteins and enzymes to gain entry into the cell, thus stimulating their own uptake. The investigation of invasion by the periopathogen Porphyromonas gingivalis is in its infancy in comparison with that of the enteric pathogens. However, recent studies with P. gingivalis have revealed that these organisms have developed invasion strategies and mechanisms similar to those of the enteric pathogens for both epithelial and endothelial cells. The study and elucidation of the mechanisms by which microorganisms such as P. gingivalis persist in chronic infection will provide valuable insight into the pathogenesis of P. gingivalis-mediated periodontal disease. The ability to multiply in and to activate endothelial cells may be one of the pathogenic mechanisms exerted by P. gingivalis that may explain the recently described association between this organism and cardiovascular disease.
Cultured cells grown as spheroids provide an in vitro model that is closer to an in vivo tumour than conventional monolayer techniques. Previous work from our laboratory has demonstrated that spheroids formed from multidrug-resistant MCF-7 cells exhibit invasive characteristics which were not present in their sensitive counterparts. The treatment of these spheroids by all-trans-retinoic acid (ATRA), a potent inducer of in vitro and in vivo differentiation, decreases their proteolytic activity and ability to invade Matrigel-coated filters. The efficiency of ATRA is enhanced by its incorporation into low-density lipoprotein (LDL) (LDL-ATRA). Indeed, invasion through a reconstituted basement membrane was reduced by 73% with 10–6 M ATRA and 3 × 10–8 M LDL-ATRA. Furthermore, inhibition of invasion was correlated with a decrease in several factors: (1) secreted matrix metalloproteinase-9 and enzymes degrading type IV collagen and Matrigel films, and (2) tissue plasminogen activator. The results observed were found with a concentration of LDL-ATRA 30 times lower than that of ATRA. This could be due to the protective effect of LDL and to a better targeting of cancer cells through their LDL receptors. LDL-ATRA may therefore represent a new and potent inhibitor of invasion that could be developed for clinical trials.
A novel adhesion receptor/ligand pair was shown recently to mediate lung vascular arrest and metastasis of rat breast cancer cells. The interacting adhesion molecules are endothelial dipeptidyl peptidase IV (DPP IV) and tumor cell surface-associated, polymeric fibronectin (FN). A truncated DPP IV (DPP IV(31–767): amino acids 31–767) in which the FN-binding site is preserved is shown here to mask the breast cancer cell surface-associated FN complexes, causing a dose-dependent inhibition of adhesion to endothelial DPP IV and impeding lung colony formation by approximately 80%. Since surface accumulation of FN is chiefly occurring during dissemination in the blood and since many cancer cell types have surface receptors by which they may initiate FN accumulation on their surfaces, the present anti-metastatic treatment modality may extend its efficacy farther than appreciated by this study.
Low density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP) is a surface membrane endocytic receptor, one of whose many functions is the regulation of plasminogen activator-mediated cell migration. LRP is known to have a role in migration and invasion, but its direct involvement has been demonstrated only in non-tumour cells. We investigated six breast cancer cell lines and a normal mammary epithelial cell clone for surface and total cellular LRP expression, and confirmed that its presence corresponds to the ability to invade and migrate in vitro. We showed that LRP in the tumour cell lines is expressed at a wide range of levels: from ∼300 to ∼6,300 sites per cell. Four of the breast cancer cell lines expressed LRP at over 1,000 sites/cell and were markedly invasive in our assay, the remainder of the cell lines and the normal clone having far fewer LRP sites and lacking invasive ability. We further showed that the migratory and invasive abilities of a highly invasive breast cancer cell line are both inhibited by receptor-associated protein, a unique LRP ligand which normally has a solely intracellular distribution but which, when added to culture medium, can inhibit all other ligand interactions with this receptor.
