A gelatin sponge model for studying tumor growth: quantitation of tumor cells and leukocytes in the CHO tumor

A gelatin sponge model system for tumor cell inoculation and retrieval of tumor-associated leukocytes is described. Gelatin sponges pre-implanted in nude mice harboring tumorigenic Chinese hamster ovary cells (line CHO) were examined at 2 and 11 days after injection of tumor cells for tumor cell content and leukocyte accumulation after digesting the sponge matrix in collagenase solution. The data indicate a progressive influx of host cells consisting primarily of macrophages, neutrophils and lymphocytes. The total number of viable tumor cells as well as the fraction of surviving tumor cells with clonogenic potential also increased with tumor age. Blank sponges not harboring tumor cells elicited an inflammatory response in the animals which did not change appreciably with length of sponge residence. However, when the sponges were harboring tumor cells, the accumulation of host leukocytes far exceeded that which occurred in blank sponges. This observation suggests a host response directed toward the tumor which is absent in animals bearing blank sponges. Apart from providing anchorage for injected cells, the gelatin sponge, by virtue of its digestibility in collagenase, makes possible the easy retrieval and precise quantitation of tumor-associated host cells.     

Myosin regulation in the migration of tumor cells and leukocytes within a three-dimensional collagen matrix

The migration of cells is a complex regulatory process which results in the generation of motor forces through the reorganization of the cytoskeleton. Here we present a comparative study of the expression and involvement of myosin in the regulation of the physiological migration of leukocytes and the pathological migration of tumor cells. We show that the involvement of myosin in the migration is distinct in these two cell types. In leukocytes, the activity of non-muscle myosin II is essential for both the spontaneous (matrix-induced) migration and the migration induced by ligands to G protein-coupled receptors, i.e. chemokines and neurotransmitters. In contrast, spontaneous tumor cell migration is largely independent of non-muscle myosin II activity, whereas the norepinephrine-induced migration is completely inhibited by either direct inhibition of non-muscle myosin II or of the kinases phosphorylating the myosin light chain, namely ROCK or the calcium/calmodulin-dependent myosin light-chain kinase.Received 31 August 2004; accepted 26 October 2004     

Circular proteoglycans from sponges: first members of the spongican family

Species-specific cell adhesion in marine sponges is mediated by a new family of modular proteoglycans whose general supramolecular structure resembles that of hyalectans. However, neither their protein nor their glycan moieties have significant sequence homology to other proteoglycans, despite having protein subunits equivalent to link proteins and to proteoglycan monomer core proteins, and glycan subunits equivalent to hyaluronan and to the glycosaminoglycans of hyalectans. In some species, these molecular components are assembled into a structure with a circular core formed by the link protein- and hyaluronan-like subunits. Besides their involvement in cell adhesion, these sponge proteoglycans, for which we propose the term spongicans, participate in signal transduction processes and are suspected to play a role in sponge self-nonself recognition. Their in vivo roles and the mild methods used to purify large amounts of functionally active spongicans make them ideal models to study the functions and possible new applications of proteoglycans in biomedical research. Received 21 May 2002; received after revision 5 July 2002; accepted 10 July 2002 RID="*" ID="*"Corresponding author.     

Efficacy of tumor cell vaccine after incorporating monophosphoryl lipid A (MPL) in tumor cell membranes containing tumor-associated ganglioside

Murine B16 melanoma expresses the ganglioside. GM₃. GM₃ shed from tumor cells is immunosuppressive and promotes tumor growth¹. Reduction or elimination of the shed GM₃ could be therapeutic, and the anti-GM₃ antibodies may reduce and clear the shed ganglioside. To test this hypothesis, mice were challenged with tumor cells, with or without inducing anti-GM₃ antibody response. Since gangliosides are poor immunogens and T-cell independent antigens, an adjuvant (monophosphoryl lipid A (MPL), a non-toxic lipid A ofSalmonella), directed against B-cells, was employed. MPL was incorporated onto liposomes and into the surface membrane of B16 mouse melanoma cells; both are rich in GM₃. C57BL/6J mice immunized with MPL-liposomes or MPL-B16 cells responded with elevated levels of anti-GM₃ IgM. Non-immunized mice or mice immunized with B16 cells alone or ganglioside GM₃ alone (without MPL) elicited poor anti-GM₃ IgM response, confirming the GM₃'s immunologic crypticity and MPL's immunopotentiating effect. MPL's immunopotentiating effect was improved by coupling it to melanoma cell membranes C57BL/6J mice were immunized with irradiated B16 alone or MPL alone or MPL-conjugated irradiated B16. After three weekly immunizations, each mouse received a challenge dose of viable syngeneic B16. Neither MPL alone nor B16 alone had a significant effect on tumor growth or host survival; however, administration of MPL-conjugated B16 cells significantly prevented tumor growth and prolonged survival. Our results indicate that MPL-incorporated B16 cells augment the anti-GM₃ IgM response, which may reverse GM₃-induced immunosuppression by eliminating tumor-derived GM₃, and restore immunocompetence.     

Possible origin of zeaxanthin in the marine sponge,Reniera japonica

As part of a study to clarify the origins of biologically active substances in marine sponges, the carotenoids produced by two species of marine bacteria,Flexibacter sp. strain number DK30213 and DK30223, associated with the marine sponge,Reniera japonica, were investigated. Both bacteria were found to produce zeaxanthin [(3R, 3R)-dihydroxy-,-carotene] which is widely distributed in marine organisms. This carotenoid was also detected in the host sponge, suggesting the transport of zeaxanthin from the microorganisms to the host. As zeaxanthin plays the role of a quencher and scavenger for active species of oxygen, it is presumed that the sponge accumulates the bacterial product as a defense substance against the active oxygen species produced under irradiation by strong sunlight. It is thought that the bacteria are symbionts of the host sponge and act by obtaining the solid substrate and medium needed for settlement and growth from the host, and by producing and transmitting the biologically active substance to the host. Zeaxanthin-producing bacteria are also considered to have potential for practical uses by the aquacultural, pharmaceutical and food industries.     

Scavenger receptor family proteins: roles for atherosclerosis, host defence and disorders of the central nervous system

In this review, we summarize the structure and function of the scavenger receptor family of proteins including class A (type I and II macrophage scavenger receptors, MARCO), class B (CD36, scavenger receptor class BI), mucinlike (CD68/macrosialin, dSR-CI) and endothelial (LOX-1) receptors. Two motifs have been identified as ligand-binding domains a charged collagen structure of type I and II receptors, and an immunodominant domain of CD36. These structures can recognize a wide range of negatively charged macromolecules, including oxidized low-density lipoproteins, damaged or apoptotic cells, and pathogenic microorganisms. After binding, these ligands can be either internalized by endocytosis or phagocytosis, or remain at the cell surface and mediate adhesion or lipid transfer through caveolae. Under physiological conditions, scavenger receptors serve to scavenge or clean up cellular debris and other related materials, and they play a role in host defence. In pathological states, they mediate the recruitment, activation and transformation of macrophages and other cells which may be related to the development of atherosclerosis and to disorders caused by the accumulation of denatured materials, such as Alzheimer's disease. Received 17 September 1997; received after revision 16 March 1998; accepted 17 March 1998     

Cytochemical study of leucocytes of DBA/2 Mice after leukemia L1210 tumor graft]

The growth of L 1210 leukemia in DBA/2 strain mouse, provokes in the grafted animals intraleucocytic enzymatic modifications. As increase of acid phosphatase and a decrease of beta-glucuronidase were observed in the lymphocytes, the level of polymorphonuclear non-specific esterase being decreased. The implications of these modifications in the host response toward the tumor is discussed.     

Metastasis: cell-autonomous mechanisms versus contributions by the tumor microenvironment

The fatality of cancer predominantly results from the dissemination of primary tumor cells to distant sites and the subsequent formation of metastases. During tumor progression, some of the primary tumor cells as well as the tumor microenvironment undergo characteristic molecular changes, which are essential for the metastatic dissemination of tumor cells. In this review, we will discuss recent insights into pro-metastatic events occurring in tumor cells themselves and in the tumor stroma. Tumor cell-intrinsic alterations include the loss of cell polarity and alterations in cell-cell and cell-matrix adhesion as well as deregulated receptor kinase signaling, which together support detachment, migration and invasion of tumor cells. On the other hand, the tumor stroma, including endothelial cells, fibroblasts and cells of the immune system, is engaged in an active molecular crosstalk within the tumor microenvironment. Subsequent activation of blood vessel and lymph vessel angiogenesis together with inflammatory and immune-suppressive responses further promotes cancer cell migration and invasion, as well as initiation of the metastatic process. Received 4 July 2005; received after revision 3 November 2005; accepted 14 November 2005     

Agglutinins from aquatic insects—tumor cell agglutination activity

Agglutinins were identified in whole body extracts of aquatic insects by means of murine tumor cell agglutination, using sarcoma 180 ascites, Ehrlich, and MM-46 cells. Screening revealed agglutinins in 5 of 10 of the larvae tested, and in 2 of 6 of the water-dwelling adult insects;Gerris paludum insularis andGyrinus japonicus. Only the agglutinin from adultG. paludum also agglutinated human erythrocytes. An ascites tumor was converted into a solid form in vivo after administration ofG. paludum agglutinin. The observation that these aquatic insect agglutinins preferentially agglutinate tumor cells has considerable implications in terms of anti-tumor effects such as inhibition of cell proliferation and metastasis.Deceased.     

Endogenous synthesis of peptidoglycan in eukaryotic cells; a novel concept involving its essential role in cell division,tumor formation and the biological clock

Degradation products of peptidoglycan, the universal bacterial cell wall constituent, were previously found in animal tissues and urine. Reassessment and quantitative analysis of available data lead to an original concept, i.e. that eukaryotic cells synthesize peptidoglycan. We present a model in which this endogenously synthesized peptidoglycan is essential for the processes of eukaryotic cell division and sleep induction in animals. Genes for peptidoglycan metabolism, like those for lysine biosynthesis in plants, are probably inherited from endosymbiotic bacteria, the ancestors of mitochondria and chloroplasts. Corollaries of this concept, i.e. roles for peptidoglycan metabolism in tumor formation and in the biological clock, are supported by abundant evidence. We propose that many interactions between bacteria and eukaryotes are conditioned by their common genetic heritage.     

Macrophage–tumor crosstalk: role of TAMR tyrosine kinase receptors and of their ligands

Ample clinical and preclinical evidence indicates that macrophages interact with tumor cells as well as with virtually all populations of host cells present in the tumor microenvironment. This crosstalk can strongly promote malignancy, but also has in principle the potential to inhibit tumor growth. Thus, it is of the utmost importance to improve our understanding of the mechanisms driving the pro- and antimalignant behavior of tumor-associated macrophages (TAMs) in order to develop better anticancer therapies. In this review, we discuss the biological consequences of reciprocal interactions between TAMs, cancer cells, endothelial cells, fibroblasts and other leukocyte subfractions within tumors. It was recently elucidated that tumors specifically educate macrophages to secrete growth arrest-specific gene 6 (Gas6), the common ligand of the Tyro3, Axl, Mer receptor (TAMR) family. In turn, Gas6 fosters tumor growth by promoting cancer cell proliferation. Therefore, the Gas6–TAMR axis might represent a novel target for disrupting tumor–macrophage crosstalk. We summarize here what is known about TAMR and their ligands in (human) cancer biology. In order to shed more light on the role of macrophages in human cancer, we additionally provide an overview of what is currently known about the prognostic impact of TAMs in human cancer.     

Cytotoxic activity of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide is underlain by DNA interchain cross-linking

Currently, chemical bifunctional cross-linkers are regarded as promising therapeutic agents capable of affecting cell metabolism. Depending on the nature of the active groups and on the length of their mediating spacer, these cross-linkers have been shown to influence mitochondrial functions, the cell cycle and cell death. The current study was aimed to assay cellular effects of a cross-linker with ‘zero’-length spacer, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). When added to cultures of transformed cells, EDC induced a G2/M blockade followed by cell death. Analysis of the molecular targets revealed that alteration of the cell cycle was caused by EDC-induced interchain cross-linking within double-stranded DNA. Administration of EDC to animals with experimental tumors increased their life span. The analysis of tumor cells from EDC-treated mice showed up-regulation of p21/WAF1, disturbance of tumor cell cytokinesis and, hence, cell death. Thus, both in vitro and in vivo, EDC exhibits cytotoxic activity, which may be of potential therapeutic use. Received 15 August 2005; received after revision 23 September 2005; accepted 15 November 2005     

Analysis of OCT4 expression in an extended panel of human tumor cell lines from multiple entities and in human mesenchymal stem cells

OCT4 is considered a main regulator of embryonic stem cell pluripotency and self renewal capacity. It was shown that relevant OCT4 expression only occurs in cells of embryonic pluripotent nature. However, several recent publications claimed to have demonstrated OCT4 expression in human somatic tumor cells, human adult stem or progenitor cells and differentiated cells.We analysed 42 human tumor cell lines from 13 entities and human bone marrowderived mesenchymal stem cells (MSC). To validate OCT4 expression we used germ cell tumor (GCT) cell lines, derived xenografts and GCT samples. Analysis by RT-PCR, western blotting, immunocytochemistry and immunohistochemistry was performed. With exception of typical embryonal carcinoma cells, we did not observe reliable OCT4 expression in somatic tumor cell lines and MSC. We suggest that a high level of expression of the OCT4 protein together with its nuclear localization still remains a reliable and definitive feature of cells with embryonic pluripotent nature. Received 30 September 2008; received after revision 05 November 2008; accepted 10 November 2008     

Suppressor mechanisms in tumor immunity

There are many parallels between T cell-mediated suppression of tumor immunity and suppression of immune responses to haptens and polypeptides. We propose a cell interaction model which takes this into account and outlines a regulatory pathway for suppression of immunity to tumor antigens. Free antigen or antigen/antibody complexes trigger an inducer T cell subset, Tsi, which is tumor-specific. This cell activates a non-immune T cell population, pre Tse, to generate effector suppressor cells, Tse. The Tse are specific for either the idiotype of Tsi or for antigen complexed with a soluble factor made by the Tsi, but the suppression they mediate is antigenically nonspecific. Tumor antigen-specific suppressor factors, TsF, play a major role in the communication between different suppressor cells. Characterization of polyclonal and monoclonal factors produced by Tsi, called TsFi, indicates that they both bind to tumor antigen and contain tumor-specific (idiotypic?) determinants.     

Leukozytenemigrationsfördernde Wirkung durch Leukozytenschädigung

Summary Leukocytic cells after slight damage produce stimulation of migration of other normal leucocytes; normal leukocytes inhibit to some extent migration of other normal leukocytes, killed leukocytes have no effect. The significance of this result is, that secondary accumulation of leukocytes with not primary chemotactic substances may be explained by the interaction of damaged leukocytes.     

The acidic microenvironment as a possible niche of dormant tumor cells

Although surgical excision, chemo-, and radio-therapy are clearly advanced, tumors may relapse due to cells of the so-called “minimal residual disease”. Indeed, small clusters of tumor cells persist in host tissues after treatment of the primary tumor elaborating strategies to survive and escape from immunological attacks before their relapse: this variable period of remission is known as “cancer dormancy”. Therefore, it is crucial to understand and consider the major concepts addressing dormancy, to identify new targets and disclose potential clinical strategies. Here, we have particularly focused the relationships between tumor microenvironment and cancer dormancy, looking at a re-appreciated aspect of this compartment that is the low extracellular pH. Accumulating evidences indicate that acidity of tumor microenvironment is associated with a poor prognosis of tumor-bearing patients, stimulates a chemo- and radio-therapy resistant phenotype, and suppresses the tumoricidal activity of cytotoxic lymphocytes and natural killer cells, and all these aspects are useful for dormancy. Therefore, this review discusses the possibility that acidity of tumor microenvironment may provide a new, not previously suggested, adequate milieu for “dormancy” of tumor cells.     

运用磁珠分选法建立免疫细胞共培养体系

目的以相互作用的几种免疫细胞上共同表达的抗原物质为标志,运用磁珠分选技术,从慢性乙型肝炎患者外周血单个核细胞中同时获得并建立体外多细胞共培养体系,体外观察细胞之间的相互作用.方法采用梯度离心法分离30例慢性乙肝病毒感染者外周血单个核细胞(PBMC),采用免疫磁珠分选法分离获得 CXCR5+细胞,通过流式细胞仪检测 CXCR5+细胞纯度和组成,并将 CXCR5+细胞进行体外培养.在分离得到的 CXCR5+细胞的基础上,将 CXCR5+细胞分为3组,分别为对照组:不加任何刺激;实验组一:加入 IL 21刺激培养,实验组二:与人肝癌细胞系(HepG2)2215细胞混合培养.体外培养一周后,流式细胞仪检测实验组与对照组细胞 B细胞数量变化,ELISA测定培养体系中上清液 HBe抗体的含量.结果1)用流式细胞仪鉴定 CXCR5+细胞的纯度和构成:CXCR5+细胞纯度为85.5±5.8%,其中 Tfh细胞占23.8±7.4%,B细胞占35.6±7.6%;2)培养7天后,IL 21刺激组 B细胞百分率为47.2±1.8%,与(HepG2)2215混合培养组 B细胞百分率为40.2±3.5%,空白对照组 B细胞百分率为36.6±7.5%,IL 21刺激组与对照组,(HepG2)2215混合培养组与对照组 B细胞百分率均有显著差异(p<0.05);3)ELISA检测培养液上清 HBeAb定量,IL 21刺激组为0.668±0.094pg/ml,空白对照组为0.378±0.088pg/ml,两组有显著差异(p<0.05).结论使用间接免疫磁珠分离法可成功建立 CXCR5+B淋巴细胞和 Tfh细胞的共培养体系,为进一步体外研究细胞之间相互关系奠定基础