Combined analysis of tumor growth pattern and expression of endogenous lectins as a prognostic tool in primary testicular cancer and its lung metastases

The aim of this study was to glyco- and immunohistochemically analyze expression of distinct growth/adhesion-related markers of primary testicular carcinomas and their lung metastases in relation to the risk of developing lung metastases and survival of patients, and to correlate immunohistochemical staining profile and syntactic structure analysis in order to delineate new prognostic parameters for this tumor type. Clinical features of 50 patients with primary testicular carcinomas and their corresponding lung metastases were evaluated and compared to those of a control cohort of 25 cases. The set of eight probes including labeled galectins-1 and -3, specific non-cross-reactive antibodies against galectins-1, -3, and -8 as well as anti-Ki-67, anti-bcl-2, and anti-p53 was applied to formalin-fixed, paraffin-embedded tumor sections of both primary and metastatic lesions. Syntactic structure analysis computed staining intensities and structural features of the tumor cells. These parameters were set into relation separately and in combination to clinical data including tumor stages, smoking habits, applied cytostatic therapy, disease-free interval, and survival. The risk of testis cancer patients to develop lung metastases depends in descending order on the tumor cell type (non-seminoma versus seminoma), tumor cell heterogeneity (mixed versus monomorphous cell type), age of patients, and pT stage. The extent of differential expression of galectin-related features between primary and secondary lesions was pronounced. Prognostic correlations for distinct galectin-related features were delineated in combination with data from syntactic structure analysis, for example cluster radius of galectin-3-positive tumor cells and post-surgical and total survival. Lengths of disease-free interval and total survival of patients were also correlated to characteristics obtained by syntactic structure analysis and their combination with galectin data in the first place, then to smoking habits, percentage of proliferating cells in the primary and secondary tumors, and finally to expression of certain galectins and of p53. Patients with non-seminoma testicular cancer should be thoroughly controlled for lung metastases. Regarding marker selection, our study underscores that further investigation of the growth-regulatory network of galectins is clearly warranted.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site--WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily alpha-fucosylated complex type chains with alpha2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells.     

Expression Profiling of Primary and Metastatic Ovarian Tumors Reveals Differences Indicative of Aggressive Disease

The behavior and genetics of serous epithelial ovarian cancer (EOC) metastasis, the form of the disease lethal to patients, is poorly understood. The unique properties of metastases are critical to understand to improve treatments of the disease that remains in patients after debulking surgery. We sought to identify the genetic and phenotypic landscape of metastatic progression of EOC to understand how metastases compare to primary tumors. DNA copy number and mRNA expression differences between matched primary human tumors and omental metastases, collected at the same time during debulking surgery before chemotherapy, were measured using microarrays. qPCR and immunohistochemistry validated findings. Pathway analysis of mRNA expression revealed metastatic cancer cells are more proliferative and less apoptotic than primary tumors, perhaps explaining the aggressive nature of these lesions. Most cases had copy number aberrations (CNAs) that differed between primary and metastatic tumors, but we did not detect CNAs that are recurrent across cases. A six gene expression signature distinguishes primary from metastatic tumors and predicts overall survival in independent datasets. The genetic differences between primary and metastatic tumors, yet common expression changes, suggest that the major clone in metastases is not the same as in primary tumors, but the cancer cells adapt to the omentum similarly. Together, these data highlight how ovarian tumors develop into a distinct, more aggressive metastatic state that should be considered for therapy development.     

The structure of the oligosaccharides of N-cadherin from human melanoma cell lines

N-cadherin is calcium-dependent cell adhesion molecule that mediates cell-cell adhesion and also modulates cell migration and tumor invasion. N-cadherin is a heavily glycosylated protein. Many studies have demonstrated that malignant transformation of a number of cell types correlates with changes of cell surface N-linked oligosacharides. We have studied the carbohydrate profile of N-cadherin synthesized in human melanoma cell lines and the effect of this protein and complex N-glycans on in vitro migration of melanoma cells from the primary tumor site—WM35 and from different metastatic sites WM239 (skin), WM9 (lymph node), and A375 (solid tumor). N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterization of its carbohydrate moieties was carried out by SDS-PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and on-blot deglycosylation using PNGase F for glycan release. N-glycans were separated by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and their structures identified by the computer matching of the resulting masses with those derived from a sequence database. The assay of in vitro chemotaxic cell migration was performed using QCM? Cell Invasion Assay (Chemicon). N-cadherin from WM35 (primary tumor site) possessed high-mannose and biantennary complex type glycans with α2–6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines possessed mostly tri- or tetra-antennary complex type glycans. In addition, N-cadherin from WM9 (lymph node metastatic site) and A375 (solid tumor metastatic site) contained heavily α-fucosylated complex type chains with α2,3 linked sialic acid. Blocking of N-cadherin-mediated intercellular interaction by N-cadherin-specific antibodies significantly (of about 40%) inhibited migration of melanoma cells. Inhibition of synthesis of complex type N-glycans by swainsonine (mannosidase II inhibitor) led to 50% decrease of cell migration. The results indicated differences between N-cadherin glycans from primary and metastatic sites and confirmed influence of N-cadherin and complex -type N-glycans on in vitro migration of melanoma cells. Published in 2004.     

Ultrastructural and morphometric characteristics of neutrophilic granulocytes of blood

Ultrastructural and morphometric characteristics of neutrophilic granulocytes (NG) of blood were investigated. It was found that every cell size fraction distinguished by its own morphologic type of NG and its specified quantitative and qualitative characteristic of primary granules. There is a close interrelation between morphometric and ultrastructural characteristics, and such parameters as perimeter, area of NG, and area of primary granules can be considered as criteria of the NG functional state.     

Bone metastases: prognostic applications of cytometric DNA analysis

OBJECTIVE: To examine DNA parameters as prognostic factors for developing metastases. STUDY DESIGN: Image cytometry was used to determine DNA content of 21 tumors and 28 metastases. DNA ploidy status, 2c deviation index (2cDI) and DNA malignancy grade (DNA-MG) (based on the variation of nuclear DNA content of tumor cells around the normal DNA [2c] peak) were examined for their prognostic value. RESULTS: Twenty of 21 tumors showed aneuploid content, and 1 tumor showed diploid DNA content. Twenty-one bone metastases showed aneuploid cells. In 6 cases both euploid and aneuploid cells were detected. In 1 metastasis only euploid cells were present. DNA-MG was increased in bone metastases (mean, 2.4) as compared to the corresponding primary tumor (mean, 2.2) in most of the cases. The mean value of the 2cDI was 30.07 in primary tumors and 42.5 in metastases. Twelve bone metastases had a higher 5cEE than did the primary tumor. CONCLUSION: Diploid and aneuploid cells were able to leave a tumor and establish metastases. DNA-MG and 2cDI were increased in metastases in comparison with the primary tumor, but even tumors with lower DNA-MG had metastatic potential.     

NIH/3T3 cells transfected with human tumor DNA containing activated ras oncogenes express the metastatic phenotype in nude mice.

NIH/3T3 cells transfected with DNA from malignant human tumors produced experimental and spontaneous metastases in nude mice. In contrast, parent or spontaneously transformed NIH/3T3 cells failed to metastasize. The transfected clones contained either activated c-Harvey-ras or N-ras oncogenes. A representative clone (T71-17SA2) which was used to assess selected cellular and host factors relevant to the metastatic process produced lung metastases in 100% of the NIH nude mice recipients, secreted augmented levels of type IV collagenase, and invaded human amnion basement membrane in vitro. Expression of the metastatic phenotype was not related to decreased sensitivity to natural killer cells or macrophage-mediated cytotoxicity. Analysis of the cellular DNA from the T71-17SA2 transfectant and its corresponding metastases, both of which contained activated N-ras oncogenes, revealed a twofold increase in the N-ras-specific DNA sequences in the metastatic cells. Thus, transfection with human tumor DNA containing activated ras oncogenes can induce the complete metastatic phenotype in NIH/3T3 cells by a mechanism apparently unrelated to immune cell killing.     

Relation of the ultrastructural organization of mouse mammary gland cancer to its ability to metastasize]

A quantitative ultrastructural investigation of cancer in mice mammary glands and of its metastases to the lungs was carried out. It was established that in tumors showing no metastatic spreading in experiment the area of membrane surface of the endoplasmic reticulum and the number of bound ribosomes are greater than those in tumor spreading metastases. At the same time the number of ribosomes organized into polysomes is higher in the metastasized tumors. The comparison of the tumors with their metastases revealed differences in the structure of the mitochondrial apparatus: the surface area of cristae is larger in the metastatic foci. The evidence obtained shows that tumors spreading metastases have a lower level of cellular differentiation. The primary and metastatic foci do not exhibit any difference in the level of differentiation.     

Application of morphometric methods to the cytologic study of intradermal nevi.

Twenty-one intradermal nevi were studied by morphometric methods in an attempt to morphologically characterize the two types of nevus cell--epithelioids, type A, and fusiforms, type C--and to quantify the differences between them. Morphometric parameters of the intradermal nevi were compared with similar parameters of melanocytes and melanoma cells so that the maturation rates of the nevi cells could be established and to see if the parameters might indicate the degree of malignancy. Superficial nevus cells were differentiated from deep cells by their larger size and larger nuclear area. Nuclear area appeared to have potential for differentiating benign from malignant tumors. Decrease in cellular area appeared to indicate maturation rather than atrophy. Melanoma cells were differentiated by their larger size. Cell nuclear perimeter appeared to have confirmatory value, while cell perimeter was inconclusive.     

Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tumor growth fraction, expression of estrogen and progesterone receptors, p53, bcl-2 and cathepsin D activity in primary ductal invasive breast carcinoma and their axillary lymph node metastases

The aim of this paper is to determine similarities and differences between tumor cell subclones in cases of ductal invasive breast carcinoma, and which occupy primary tumor and local axillary lymph metastases. The tumor growth fraction evaluated by Ki-67 was analyzed along with the expression level of estrogen and progesterone receptors, protein p53, proto-oncogene protein bcl-2 and cathepsin D in 60 patients. Metastatic lymph node in axilla has a higher growth fraction of the tumor cells than the primary tumor (p = 0.045), as well as the higher level of bcl-2 overexpression (p = 0.014). No statistically significant difference was found in the presence of immunohistochemically identified estrogen receptors (p = 0.161) and progesterone receptors (p = 0.081) between the primary tumor and the metastatic lymph node in axilla. Likewise, no difference was found between the immunohistochemical evaluation of p53 (p = 0.356) and cathepsin D activity (p = 0.928). A higher growth fraction of the tumor cells and the higher level of bcl-2 overexpression in metastatic tumor cells indicate the more aggressive cell subclones. This study does not support the routine testing of both primary tumor and locoregional metastasis to evaluate the breast cancer hormone receptor status.     

Predicting hepatitis B virus-positive metastatic hepatocellular carcinomas using gene expression profiling and supervised machine learning

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Its high mortality rate is mainly a result of intra-hepatic metastases. We analyzed the expression profiles of HCC samples without or with intra-hepatic metastases. Using a supervised machine-learning algorithm, we generated for the first time a molecular signature that can classify metastatic HCC patients and identified genes that were relevant to metastasis and patient survival. We found that the gene expression signature of primary HCCs with accompanying metastasis was very similar to that of their corresponding metastases, implying that genes favoring metastasis progression were initiated in the primary tumors. Osteopontin, which was identified as a lead gene in the signature, was over-expressed in metastatic HCC; an osteopontin-specific antibody effectively blocked HCC cell invasion in vitro and inhibited pulmonary metastasis of HCC cells in nude mice. Thus, osteopontin acts as both a diagnostic marker and a potential therapeutic target for metastatic HCC.     

Distinguishing Tumor and Stromal Sources of MicroRNAs Linked to Metastasis in Cutaneous Melanoma

MicroRNA (miRNA) dysregulation in cancer causes changes in gene expression programs regulating tumor progression and metastasis. Candidate metastasis suppressor miRNA are often identified by differential expression in primary tumors compared to metastases. Here, we performed comprehensive analysis of miRNA expression in The Cancer Genome Atlas (TCGA) skin cutaneous melanoma (SKCM) tumors (97 primary, 350 metastatic), and identified candidate metastasis-suppressor miRNAs. Differential expression analysis revealed miRNA significantly downregulated in metastatic tumors, including miR-205, miR-203, miR-200a-c, and miR-141. Furthermore, sequential feature selection and classification analysis identified miR-205 and miR-203 as the miRNA best able to discriminate between primary and metastatic tumors. However, cell-type enrichment analysis revealed that gene expression signatures for epithelial cells, including keratinocytes and sebocytes, were present in primary tumors and significantly correlated with expression of the candidate metastasis-suppressor miRNA. Examination of miRNA expression in cell lines revealed that candidate metastasis-suppressor miRNA identified in the SKCM tumors, were largely absent in melanoma cells or melanocytes, and highly restricted to keratinocytes and other epithelial cell types. Indeed, the differences in stromal cell composition between primary and metastatic tumor tissues is the main basis for identification of differential miRNA that were previously classified as metastasis-suppressor miRNAs. We conclude that future studies must consider tumor-intrinsic and stromal sources of miRNA in their workflow to identify bone fide metastasis-suppressor miRNA in cutaneous melanoma and other cancers.     

Carbohydrate moieties of N-cadherin from human melanoma cell lines

Expression of N-cadherin an adhesion molecule of the cadherin family, in tumor cells is associated with their increased invasive potential. Many studies suggested the role of N-linked oligosaccharides as important factors that contribute to metastasis by influencing tumor cell invasion and adhesion. N-cadherin is a heavily glycosylated protein. We have analysed the carbohydrate profile of this protein synthesized in human melanoma cell lines: WM35 from the primary tumor site and WM239, WM9, and A375 from different metastatic sites. N-cadherin was immunoprecipitated with anti-human N-cadherin polyclonal antibodies. Characterisation of its carbohydrate moieties was carried out by SDS/PAGE electrophoresis and blotting, followed by immunochemical identification of the N-cadherin polypeptides and analysis of their glycans using highly specific digoxigenin or biotin labelled lectins. The positive reaction of N-cadherin from the WM35 cell line with Galanthus nivalis agglutinin (GNA), Datura stramonium agglutinin (DSA) and Sambucus nigra agglutinin (SNA) indicated the presence of high-mannose type glycans and biantennary complex type oligosaccharides with alpha2-6 linked sialic acid. N-cadherin from WM239, WM9, and A375 cell lines gave a positive reaction with Phaseolus vulgaris leukoagglutinin (L-PHA) and lotus Tetragonolobus purpureas agglutinin (LTA). This indicated the presence of tri- or tetra-antennary complex type glycans with alpha-fucose. In addition, N-cadherin from WM9 (lymphomodus metastatic site) and A375 (solid tumor metastatic site) contained complex type chains with alpha2-3 sialic acid (positive reaction with Maackia amurensis agglutinin--MAA). The results demonstrated that N-glycans of N-cadherin are altered in metastatic melanomas in a way characteristic for invasive tumor cells.     

Eradication of metastatic renal cell carcinoma after adenovirus-encoded TNF-related apoptosis-inducing ligand (TRAIL)/CpG immunotherapy

Despite evidence that antitumor immunity can be protective against renal cell carcinoma (RCC), few patients respond objectively to immunotherapy and the disease is fatal once metastases develop. We asked to what extent combinatorial immunotherapy with Adenovirus-encoded murine TNF-related apoptosis-inducing ligand (Ad5mTRAIL) plus CpG oligonucleotide, given at the primary tumor site, would prove efficacious against metastatic murine RCC. To quantitate primary renal and metastatic tumor growth in mice, we developed a luciferase-expressing Renca cell line, and monitored tumor burdens via bioluminescent imaging. Orthotopic tumor challenge gave rise to aggressive primary tumors and lung metastases that were detectable by day 7. Intra-renal administration of Ad5mTRAIL+CpG on day 7 led to an influx of effector phenotype CD4 and CD8 T cells into the kidney by day 12 and regression of established primary renal tumors. Intra-renal immunotherapy also led to systemic immune responses characterized by splenomegaly, elevated serum IgG levels, increased CD4 and CD8 T cell infiltration into the lungs, and elimination of metastatic lung tumors. Tumor regression was primarily dependent upon CD8 T cells and resulted in prolonged survival of treated mice. Thus, local administration of Ad5mTRAIL+CpG at the primary tumor site can initiate CD8-dependent systemic immunity that is sufficient to cause regression of metastatic lung tumors. A similar approach may prove beneficial for patients with metastatic RCC.     

Tumor-host interactions contribute to the elevated expression level of alpha1-antichymotrypsin in metastatic breast tumor xenografts

We investigated alpha1-antichymotrypsin (ACT) gene expression in xenograft tumors generated by two isogenic human breast cancer cell lines derived from the same parent, MDA-MB-435, which display opposite metastatic behaviors. Microarray and real-time PCR experiments showed an overexpression of this serine protease inhibitor in the metastatic tumors (M-4A4T) and its derived metastases (M4-Mets) compared with the weakly metastatic tumors (NM-2C5T), and its release into the blood was confirmed by western-blotting. However, functional assays in vivo using genetically engineered tumor cells demonstrated that ACT up-regulation was not, by itself, responsible for the metastatic phenotype. We also made observations that ACT gene regulation was sensitive to tumor-host interactions: inoculation of these lines into the mouse mammary gland greatly increased ACT production and accentuated the intrinsic difference observed when they are cultured in vitro. Sensitivity of tumor cells to their environment was further analyzed by in vitro experiments, which demonstrated that a purified ECM environment and soluble components from normal host mammary cells were both able to significantly promote ACT expression. In addition, we took advantage of the xenogeneic nature of the model to measure ACT expression by the host cells (mouse) and the tumor cells (human) within the neoplasm using species-specific primers in real-time PCR experiments. It was found that the presence of tumor cells, irrespective of their metastatic capabilities, induced local ACT production by host cells at the primary and secondary tumor sites. Thus, this work indicates that there is a specific association of ACT overexpression with the metastatic phenotype in our breast cancer metastasis model. Moreover, because of the xenogeneic nature of our system, we were able to provide evidence of tumor-host reciprocal regulation of ACT production.