Sequential degradation of heparan sulfate in the subendothelial extracellular matrix by highly metastatic lymphoma cells

A highly metastatic variant (ESb) of a methylcholanthrene-induced T lymphoma elaborates a heparan sulfate (HS) degrading endoglycosidase (heparanase) to a much higher extent than its non-metastatic parental subline (Eb). Whereas a serum-free medium conditioned by either subline contained a trypsin-like serine protease, heparanase activity was detected only in the ESb-conditioned medium (CM). ESb CM was incubated with a naturally produced, sulfate-labelled subendothelial extracellular matrix (ECM) or with a soluble, high-MW labelled proteoglycan first released from the ECM by incubation with Eb CM or with the partially purified ESb protease. Sulfate labelled degradation products were analyzed by gel filtration on Sephrose 6B. The optimal pH for degradation of ECM-bound HS was 6.2 as compared to pH 5.2 for degradation of the soluble proteoglycan. Heparanase-mediated degradation of both ECM-bound and soluble HS was inhibited by heparin. Addition of either trypsin, plasmin or to a lower extent, the purified ESb protease, stimulated between 5- and 20-fold the ESb CM-mediated degradation of ECM-bound HS but had no effect on heparanase-mediated degradation of the soluble proteoglycan. This stimulation was inhibited in the presence of heparin or protease inhibitors. These results indicate that both a protease and heparanase are involved in the ESb-mediated degradation of ECM-bound HS and that one enzyme produces a more accessible substrate for the next enzyme. This sequential cleavage is characteristic of degradation of a multimolecular structure such as the subendothelial ECM and hence cannot be detected in studies with its isolated constituents.     

MHC class I genes controlling the metastatic phenotype of tumor cells.

Metastatic clones of murine tumors that manifest impaired expression of class I MHC antigens do not induce an antitumor CTL activity. Transfection of H-2Kb genes into D122 carcinoma and B16 melanoma clones converted these cells to low metastatic immunogenic clones that can be used to protect in vivo against metastases of parental clones. Amplification of the protective effect can be achieved by combination of syngeneic and allogeneic MHC class I genes. Studying the mechanisms involved in MHC class I suppression in tumor cells, we found that changes in H-2 promoter activity were the cause of low expression. Proteins that might be involved were demonstrated by migration retardation methods. The involvement of the fos-jun complex in regulation of MHC expression is discussed.     

不同转移能力肿瘤细胞间转移相关特征的比较研究

目的 进一步明确肿瘤细胞相关生物学特性与肿瘤细胞转移能力间的关系。方法 应用遗传背景相同的３个人结肠癌（ＨＣＣ）细胞系在裸大鼠体内建立转移模型,通过三维球体培养、末端脱氧核苷酸转移酶介导的ｄＵＴＰ缺口末端标记（ＴＵＮＥＬ）凋亡细胞和免疫组化等技术,比较分析不同肿瘤细胞间同种细胞凝集力、靶器官定植力、细胞增殖和细胞凋亡现象、癌基因及肿瘤相关抗原表达等生物学特征。结果 高转移细胞具有高靶器官定植力和低     

In vitro motility of BSp73 rat tumor cells with different metastatic ability

The highly metastatic and nonmetastatic variants of the rat tumor BSp73 have been tested for motility in vitro by a phagokinetic assay. Surprisingly, the nonmetastatic variant is locomotional, whereas the highly metastatic variant exhibited stationary motility only. Soluble factors of normal rat serum and medium conditioned by regenerating normal rat lung, or solid constituents of the extracellular matrix material which was exudated by normal rat lung fibroblasts or bovine cornea endothelial cells as well as collagen type III could not stimulate locomotory ability in these tumor cells. However, by contact to rat lung cells they acquired passive mobility. The significance of these cellular properties for their metastatic behavior is discussed.     

Metastatic phenotype of murine tumor cells expressing different cooperating oncogenes.

Four murine cellular tumor models expressing various combinations of oncogenes (SV40 large T and v-Ha-ras, SV40 large T and v-src, SV40 large T and neu, adenovirus EIA and v-Ha-ras) induce sarcoma when they are inoculated s.c. into the DBA/2 syngenic mice. The metastatic patterns, distribution and fate of these tumor cells transplanted by two different routes into syngenic DBA/2 mice have been studied. All the tumor cell lines except EIA-ras, induce massive overt artificial metastases principally in the lung after i.v. injection. In s.c. tumor-bearing mice, a few resting cells colonize the lung as micrometastases. When removed from this tissue context and injected s.c. these cells regain their proliferative potential and grow as local tumors which again give rise to occult pulmonary micrometastases.     

A functional heparan sulfate mimetic implicates both heparanase and heparan sulfate in tumor angiogenesis and invasion in a mouse model of multistage cancer

Heparan sulfate proteoglycans are integral components of the extracellular matrix that surrounds all mammalian cells. In addition to providing structural integrity, they act as a storage depot for a variety of heparan sulfate (HS)-binding proteins, including growth factors and chemokines. Heparanase is a matrix-degrading enzyme that cleaves heparan sulfate side chains from the core proteoglycans, thus liberating such HS-binding proteins, as well as potentially contributing to extracellular matrix degradation. Here, we report that heparanase mRNA and protein expression are increased in the neoplastic stages progressively unfolding in a mouse model of multistage pancreatic islet carcinogenesis. Notably, heparanase is delivered to the neoplastic lesions in large part by infiltrating Gr1+/Mac1+ innate immune cells. A sulfated oligosaccharide mimetic of heparan sulfate, PI-88, was used to inhibit simultaneously both heparanase activity and HS effector functions. PI-88 had significant effects at distinct stages of tumorigenesis, producing a reduction in the number of early progenitor lesions and an impairment of tumor growth at later stages. These responses were associated with decreased cell proliferation, increased apoptosis, impaired angiogenesis, and a substantive reduction in the number of invasive carcinomas. In addition, we show that the reduction in tumor angiogenesis is correlated with a reduced association of VEGF-A with its receptor VEGF-R2 on the tumor endothelium, implicating heparanase in the mobilization of matrix-associated VEGF. These data encourage clinical applications of inhibitors such as PI-88 for the many human cancers where heparanase expression is elevated or mobilization of HS-binding regulatory factors is implicated.     

The anticancer activity of lytic peptides is inhibited by heparan sulfate on the surface of the tumor cells

Background  

Cationic antimicrobial peptides (CAPs) with antitumor activity constitute a promising group of novel anticancer agents. These peptides induce lysis of cancer cells through interactions with the plasma membrane. It is not known which cancer cell membrane components influence their susceptibility to CAPs. We have previously shown that CAPs interact with the two glycosaminoglycans (GAGs), heparan sulfate (HS) and chondroitin sulfate (CS), which are present on the surface of most cells. The purpose of this study was to investigate the role of the two GAGs in the cytotoxic activity of CAPs.     

Themotaxis of metastatic tumor cells

Summary A number of factors have been identified which are chemotactic for tumor cells. Recent studies have shown that, in addition to inducing directional motility in the Boyden chamber assay, these factors also induce a number of other responses. Included among these responses are cell swelling and foreign surface adhesiveness. The adherence response has been studied in detail using the Walker 256 carcinosarcoma cells and several other cell types. In the Walker cells, treatment with the C5a-derived tumor cell chemotactic peptide, the synthetic tripeptide, N-formyl-methionyl-leucyl-phenylalanine or with 12-O-tetradecanoyl phorbol ester induces a rapid, transient adherence response. The response is completely inhibited by several agents known to block the activity of phospholipase A₂ or the metabolism of arachidonic acid through the lipoxygenase pathway but is not inhibited by inhibition of the cychlooxygenase pathway. This suggests that lipoxygenase metabolites of arachidonic acid may actually mediate the adherence response.It has been shown that chemotactic factor treatment of animals that are bearing circulating tumor cells induces a localization of these cells at the site of chemotactic factor injection. On the basis of these observations it has been hypothesized that tumor cells respond to chemotactic factors in much the same way that leukocytes do and that tumor cell localization at metastatic sites in vivo may be influenced by chemotactic factors in much the same way that leukocyte localization at inflammatory sites is.     

Flow cytometric analysis of proteoglycan expression on murine tumor cells with different metastatic capacity.

Proteoglycan epitopes recognized by poly- or monoclonal antibodies were studied at the cell surface of 3LL murine tumor cell lines with different metastatic capacity. A decreased expression of heparan sulphate and chondroitin sulphate core protein epitopes was observed in the highly metastatic variant HM compared to the low metastatic counterpart LM. Interestingly, the biochemical analysis demonstrated an increased glycosaminoglycan-especially heparan sulphate-production in highly metastatic HM cells. A similar decrease in heparan sulphate proteoglycan core epitope expression was observed in the highly metastatic B16F10 cell line compared to its low metastatic variant F1. The under-representation of proteoglycan core protein epitopes at the cell surface of highly metastatic murine tumor cell lines could be interpreted by the increased glycosylation, or by the loss of those particular epitopes.     

Expression of the CD44v2-10 isoform confers a metastatic phenotype: importance of the heparan sulfate attachment site CD44v3

We expressed the full-length CD44v2-10 isoform in SKHep1 cells, a nonmetastatic human hepatocellular carcinoma cell line that does not express any endogenous CD44v isoforms. In SCID mice, expression of CD44v2-10 by SKHep1 cells had no effect on s.c. primary tumor development but caused pulmonary metastases in 41% (7 of 17) of animals compared with control SKHep1 cells (0 of 16; P < 0.01). CD44v2-10 expression by SKHep1 cells resulted in enhanced heparan sulfate (HS) attachment and an enhanced capacity to bind heparin-binding growth factors. Mutation of the v3 domain to prevent HS attachment and growth factor binding abolished the metastatic phenotype, demonstrating that HS modification of CD44v2-10 plays a critical role in the development of metastases in this model. However, in vitro proliferation, motility, and invasion were not altered by CD44v2-10 expression.     

Cell surface heparan sulfate proteoglycans control adhesion and invasion of breast carcinoma cells

Background

Cell surface proteoglycans interact with numerous regulators of cell behavior through their glycosaminoglycan chains. The syndecan family of transmembrane proteoglycans are virtually ubiquitous cell surface receptors that are implicated in the progression of some tumors, including breast carcinoma. This may derive from their regulation of cell adhesion, but roles for specific syndecans are unresolved.

Methods

The MDA-MB231 human breast carcinoma cell line was exposed to exogenous glycosaminoglycans and changes in cell behavior monitored by western blotting, immunocytochemistry, invasion and collagen degradation assays. Selected receptors including PAR-1 and syndecans were depleted by siRNA treatments to assess cell morphology and behavior. Immunohistochemistry for syndecan-2 and its interacting partner, caveolin-2 was performed on human breast tumor tissue arrays. Two-tailed paired t-test and one-way ANOVA with Tukey’s post-hoc test were used in the analysis of data.

Results

MDA-MB231 cells were shown to be highly sensitive to exogenous heparan sulfate or heparin, promoting increased spreading, focal adhesion and adherens junction formation with concomitantly reduced invasion and matrix degradation. The molecular basis for this effect was revealed to have two components. First, thrombin inhibition contributed to enhanced cell adhesion and reduced invasion. Second, a specific loss of cell surface syndecan-2 was noted. The ensuing junction formation was dependent on syndecan-4, whose role in promoting actin cytoskeletal organization is known. Syndecan-2 interacts with, and may regulate, caveolin-2. Depletion of either molecule had the same adhesion-promoting influence, along with reduced invasion, confirming a role for this complex in maintaining the invasive phenotype of mammary carcinoma cells. Finally, both syndecan-2 and caveolin-2 were upregulated in tissue arrays from breast cancer patients compared to normal mammary tissue. Moreover their expression levels were correlated in triple negative breast cancers.

Conclusion

Cell surface proteoglycans, notably syndecan-2, may be important regulators of breast carcinoma progression through regulation of cytoskeleton, cell adhesion and invasion.     

Repression of IFN regulatory factor 8 by DNA methylation is a molecular determinant of apoptotic resistance and metastatic phenotype in metastatic tumor cells

Apoptotic resistance is often associated with metastatic phenotype in tumor cells and is considered a hallmark of tumor progression. In this study, IFN regulatory factor 8 (IRF8) expression was found to be inversely correlated with an apoptotic-resistant and metastatic phenotype in human colon carcinoma cell lines in vitro. This inverse correlation was further extended to spontaneously arising primary mammary carcinoma and lung metastases in a mouse tumor model in vivo. Exogenous expression of IRF8 in the metastatic tumor cell line restored, at least partially, the sensitivity of the tumor cells to Fas-mediated apoptosis, and disruption of IRF8 function conferred the poorly metastatic tumors with enhanced apoptotic resistance and metastatic capability. DNA demethylation restored IRF8 expression and sensitized the metastatic tumor cells to Fas-mediated apoptosis. Analysis of genomic DNA isolated from both primary and metastatic tumor cells with methylation-sensitive PCR revealed hypermethylation of the IRF8 promoter in metastatic tumor cells but not in primary tumor cells. Taken together, our data suggest that IRF8 is both an essential regulator in Fas-mediated apoptosis pathway and a metastasis suppressor in solid tumors and that metastatic tumor cells use DNA hypermethylation to repress IRF8 expression to evade apoptotic cell death and to acquire a metastatic phenotype.     

Overexpression of Merlin in B16F10 mouse melanoma cells reduces their metastatic activity: role of the cell surface heparan sulfate glycosaminoglycans

Merlin, the protein product of the neurofibromatosis type 2 gene (NF2) acts as a tumor suppressor in mice and humans. In this study, melanoma B16F10 cells were engineered to overexpress the NF2 gene by establishing stable transductants. A cell line overexpressing Merlin (B16F10-M) was generated. When compared to the parental cells, the B16F10-M cells demonstrated differences in their cell surface organization. The overexpressing strain changed its ability to grow in soft agar as well as its cell motility properties. B16F10-M cells were then examined in the in vivo mouse melanoma tumor growth and tumor metastasis models. While tumor growth was marginally affected, the presence of increased Merlin severely reduced the metastastatic ability of the cells. When isolated using specific enzymes with distinct substrate specificity, the cell surface heparan sulfate glycosaminoglycans (HSGAGs) from the overexpressing B16F10-M cells, inhibited the metastatic properties of the parental B16F10 cells. The results obtained provide a causal link between the reorganization/changes to the cell surface HSGAGs by the overexpression of Merlin and the inhibition of the metastatic activity of the mouse melanoma B16F10 cells in vivo.     

Tumor metastasis-associated heparanase (heparan sulfate endoglycosidase) activity in human melanoma cells

We discovered previously a tumor metastasis-associated heparan sulfate (HS)-degrading endoglycosidase in mouse melanoma cells that is a unique endo-beta-glucuronidase (heparanase) capable of specifically cleaving HS at intrachain sites (Nakajima et al., J. Biol. Chem., 259 (1984) 2283. Using unmodified- and chemically modified-HS and heparin substrates we demonstrate that human Hs939 and mouse B16 melanoma cells possess a HS-degrading endoglycosidase of similar specificity. Melanoma heparanase showed high activity against N-desulfated N-acetylated HS, and we therefore synthesized a solid-phase heparanase substrate crosslinking partially N-desulfated N-[14C] acetylated HS to agarose gel beads via one covalent linkage. Using this solid-phase substrate 15 human malignant melanoma cell lines were assayed for heparanase activity. All of the melanoma cells tested had heparanase activity, and almost all possessed activities comparable or greater than that of the murine B16-F1 melanoma line. Human A375 melanoma variants of high lung metastatic potential in athymic nude mice had significantly higher heparanase activities than did A375 parental cells of low metastatic potential.     

Disorganisation of cytoskeleton in cells resistant to photodynamic treatment with decreased metastatic phenotype

The appearance of cells resistant to photodynamic therapy (PDT) is crucial for the outcome of this antitumoral treatment. We had previously isolated two sublines resistant to PDT derived from the mammary adenocarcinoma LM3 [A. Casas, C. Perotti, B. Ortel, G. Di Venosa, M. Saccoliti, A. Batlle, T. Hasan, Induction of murine tumour cell lines resistant to ALA-mediated Photodynamic Therapy, Int. J. Oncol. 29 (2006) 397-405.]. These clones have severely impaired its metastatic potential in vivo together with decreased general anchorage-dependent adhesion and invasion. In the present work we analyzed the differential expression and distribution of cytoskeleton and adhesion proteins in these cell lines. In both resistant clones, loss of actin stress fibers and disorganization of the actin cortical rim was observed. E-cadherin and beta-catenin and vinculin distribution was also disorganized. However, Western blot assays did not show differential expression of actin, E-cadherin, vinculin or beta-catenin. It was demonstrated that PDT strongly affects cell-cell and cell-substrate adhesion through the reorganization of some cytoskeletal and adhesion proteins. Changes in the metastasis phenotypes previously found are likely to be ascribed to these differences.     

转移性循环肿瘤细胞的特征

循环肿瘤细胞（circulating tumor cells,CTCs）是肿瘤转移的潜在种植者。这些引起肿瘤转移的CTCs具有干性、转化、簇群、亲器官性和变形等特征,最终在靶器官形成转移灶。研究CTCs的特征可能揭开肿瘤转移的机制。     

Nonmetastatic tumor cells acquire metastatic properties following somatic hybridization with normal cells

Summary Somatic cell hybridization between nonmetastatic tumor cells and normal cells of the lymphoreticular system results in hybrid cells manifesting metastatic properties of defined target organ specificity. Thus, fusion of the nonmetastatic BALB/c originated NSI plasmacytoma with C57BL B lymphocytes resulted in hybridomas, each of which were metastatic. Of 10 hybridomas, 7 generated metastases in the spleen and liver, whereas 3 generated liver metastases. The generation of liver metastases by hybridomas which homed to both spleen and liver, but not by those which homed to the liver only, was controlled by the spleen. The acquisition of metastatic properties via somatic cell fusion seems to represent a general principle, in which the normal partner determines the target organ specificity for the metastatic growth. Thus, fusion of SP2/O myeloma cells with syngeneic B lymphocytes also resulted in a hybrid cell metastasizing to the spleen and liver, yet a somatic hybrid between NSI and a macrophage or dendritic-like cell metastasized to the lung. Cell surface molecules encoded by the genome of the normal partner was demonstrated to control the target organ specificity: antibodies against MHC-encoded antigens of the normal B cell partner prevented the generation of metastases by hybridomas metastasizing to the spleen and liver, but not by those metastasizing to the liver only. This is in accordance with the function of MHC molecules on lymphocytes in controlling their homing to lymphoid organs. Hybridomas of T cell lymphomas also manifested metastatic properties. Analysis of the cell surface Thy-1 antigens of a hybridoma (DCH10), produced via somatic fusion between BW5145 lymphoma and a putative macrophage cell indicated that cells of liver metastases (DCH10-Li) generated by the hybrid cells might have undergone further somatic cell fusion in vivo with host (T?) cells. These cells have acquired new metastatic properties, generating metastases in spleen, liver and kidneys. In fact, even the inoculation of the parental BW lymphoma cells resulted in a case of liver metastasis (BW-Li). Such BW-Li cells, upon reinoculation, also generated metastases in the spleen, liver and kidneys. Analysis of the Thyl phenotype indicated that BW-Li cells may also have undergone somatic cell fusion in vivo with host (T?) cells, resulting in the acquisition of metastatic properties. The pattern of cell-cell interactions (adhesion, infiltration) with liver cell monolayers of BW-Li cells and of DCH10-Li (T-cell lymphomas) was identical, and differed from cells of liver metastases of the myeloma-B cell hybridomas which might be based on responses to liver growth signals. Accordingly, the morphology of liver metastases generated by the two categories of hybridomas was different. It appears therefore, that (a) the acquisition of metastatic properties following somatic cell fusion with normal lymphoreticular cells is of a general significance; (b) somatic cell fusion provides an experimental system for the analysis of molecular properties determining the acquisition of metastatic capability; and (c) it may also represent a mechanism for tumor progression in vivo.     

不同表型肿瘤相关巨噬细胞在肿瘤进展中的作用

随着对肿瘤发生、侵袭和转移过程研究的不断深入,临床发现肿瘤相关巨噬细胞（tumor associated macrophage,TAM）在肿瘤微环境中扮演重要角色。由于经典活化的巨噬细胞（M1）/替代性活化的巨噬细胞（M2）理论过度简化了TAM在肿瘤微环境中的多种作用,目前临床大多根据表面蛋白表达的不同将TAM重新分为CD68+TAM、CD163+TAM、CD204+TAM、CD169+TAM、CCL18+TAM等。各型TAM表达的表面蛋白具有不同类型的配体并调控着不同的信号通路和细胞因子。因此,这些亚型的TAM具有促进或抑制肿瘤的类似作用,但其所牵涉的机制以及带来的临床表现均不相同。本文将就TAM各类表型对各类肿瘤的生长、转移、预后的影响及其临床关联进行综述。      

Interaction of metastatic tumor cells with bovine lens capsule basement membrane

The use of bovine lens capsule basement membrane as a model substratum for studies of invasion and extravasation by metastatic tumor cells is described. The abilities of three independently isolated pairs of metastatic variant cell lines to digest the purified substrates, laminin, type IV collagen, and type I collagen, were compared with their abilities to solubilize isotope from 125I-labeled lens capsule basement membrane matrix. The cell lines used were +SA and -SA mouse mammary adenocarcinoma cells, RT7-4bs and RT7-4b-Ls rat hepatocarcinoma cells, and B16-F1 and B16-F10 mouse melanoma cells. In general, imperfect correlations of lytic activity with metastatic ability were found for the purified substrate digestions, but, for each pair of variants, the more metastatic tumor cell line was always able to solubilize more surface-bound isotope from the lens capsule. Visual evidence of tumor cell-associated digestion of lens capsule basement membrane was obtained using transmission electron microscopy. Mouse mammary carcinoma cells attached more rapidly to lens capsule than to endothelial cell monolayers or tissue culture plastic. We next added endothelial cells to the model substrate. Aortic endothelial cells grew well on lens capsules without apparent synthesis of additional basement membrane matrix. In additional studies, the lens capsule was used in a chamber apparatus to demonstrate that cellular invasion of the full thickness of this basement membrane structure could be demonstrated and readily quantitated. Our results indicate that bovine lens capsule is a particularly versatile basement membrane structure useful for studies of tumor cell invasion and extravasation. In addition, the comparison of purified substrate digestions with lens capsule matrix digestion indicates the desirability of also using a matrix digest when correlating lytic abilities of tumor cells with their metastatic abilities.