Comparison of IgG diffusion and extracellular matrix composition in rhabdomyosarcomas grown in mice versus in vitro as spheroids reveals the role of host stromal cells

The tumour extracellular matrix acts as a barrier to the delivery of therapeutic agents. To test the hypothesis that extracellular matrix composition governs the penetration rate of macromolecules in tumour tissue, we measured the diffusion coefficient of nonspecific IgG in three rhabdomyosarcoma subclones growing as multicellular spheroids in vitro or as subcutaneous tumours in dorsal windows in vivo. In subcutaneous tumours, the diffusion coefficient decreased with increasing content of collagen and sulphated glycosaminoglycans. When grown as multicellular spheroids, no differences in either extracellular matrix composition or diffusion coefficient were found. Comparison of in vitro vs in vivo results suggests an over-riding role of host stromal cells in extracellular matrix production subjected to modulation by tumour cells. Penetration of therapeutic macromolecules through tumour extracellular matrix might thus be largely determined by the host organ. Hence, caution must be exercised in extrapolating drug penetrability from spheroids and multilayer cellular sandwiches consisting of only tumour cells to tumours in vivo.     

细胞外基质与肿瘤干细胞相互作用的研究进展

细胞外基质（extracellular matrix,ECM）是由蛋白质与糖类等生物大分子在细胞表面或细胞间构成的复杂网络结构。细胞外基质的过量沉积或结构形态异常是肿瘤微环境（tumor microenvironment,TME）的重要特征之一。肿瘤干细胞（cancer stem cells,CSC）指肿瘤内部一类具有无限增殖、自我更新及多向分化潜能的细胞亚群,参与肿瘤的启动、扩散、转移和复发。研究表明,肿瘤的发生是一个由细胞外基质和肿瘤干细胞相互促进、共同演化的过程。本文就ECM的组成成分、特性及其对CSC的生物学行为影响展开讨论,以期为肿瘤的临床治疗寻求新方向。     

Inappropriate production of collagen and prolyl hydroxylase by human breast cancer cells in vivo.

Thirty-two scirrhous cancers of breast have been examined to determine the origin of the collagen stroma in these tumours. Employing two immunohistochemical techniques it has been shown that the malignant epithelial cells in 30 of these tumours contain not only collagen but also prolyl hydroxylase, a key enzyme in collagen biosynthesis. Neither this enzyme nor collagen was detectable in the spindle cells in the stroma of these tumours. Neither the epithelium in normal breast, that in fibrocystic disease and in fibroadenomata, nor the malignant epithelium in two medullary cancers of breast contained either collagen or prolyl hydroxylase. These results strongly suggest that the malignant epithelium of scirrhous breast cancers produces its own collagen stroma and that the scirrhous reaction in these tumours is not a host response to tumour invasion. The production of collagen and prolyl hydroxylase by breast cancer cells (of the scirrhous type) therefore represents another example of inappropriate protein production by a human tumour.     

Integrins and metalloproteinases: an efficient collaboration in the invasive process

During the invasive process, tumor cells must move through the extracellular matrix. They have to adhere to the extracellular matrix components, then proteolyse them and migrate on their fragments. This implicates integrins and proteinases, namely metalloproteinases. Numerous experiments which had been performed on various models, namely malignant melanomas proved that integrins have a major role in the transduction of signals from the outside to the inside of the cells, such signals enhancing the expression of the metalloproteinases or, in the contrary, inhibiting it. The modifications of this expression are dependent of extracellular matrix components and may be induced by the linking of specific antibodies to integrins. In some instances, the integrins localized on the tumor cell surface may act as receptors for extracellular matrix proteins and metalloproteinases at once, that may give to tumor cells an higher efficiency in the invasive process. Such mechanisms may result in interesting clinical perspectives for the control of metalloproteinases regulation in pathological processes.     

Proteoglycans and neoplasia

There is a growing realization that the whole tumor cell-matrix complex must be investigated in order to fully understand the process of cancer growth and metastasis. Proteoglycans are intrinsic constituents of the cell surface, extracellular matrix, and basement membrane, three logistically and functionally important structures involved in most cellular interactions. Proteoglycans influence the behavior of normal and malignant cells by virtue of their expanded configuration, polyanionic nature and, most of all, by their ability to interact with a variety of cellular products. Consequently, they have been implicated in a number of biological processes including proliferation, recognition, adhesion, and migration. They can serve as links between the extracellular and intracellular environment and thus transduce key biological signals. They can act as receptors for interstitial collagens and other matrix proteins and thus contribute to the organization of pericellular matrix. During neoplastic development there is a profound structural rearrangement of these macromolecules at both the plasma membrane and the pericellular level. Qualitative and quantitative abnormalities in proteoglycan metabolism may contribute to the establishment of some well-known neoplastic properties, including lack of cohesiveness, abnormal assembly of extracellular matrix, abnormal growth, and invasion. The present work will focus on recent advances in our understanding of these complex macromolecules and on some of the alterations associated with the neoplastic phenotype, and will then attempt to elucidate some of the mechanisms regulating these changes.     

Uptake of IgG in osteosarcoma correlates inversely with interstitial fluid pressure, but not with interstitial constituents.

The uptake of therapeutic macromolecules in solid tumours is assumed to be hindered by the heterogeneous vascular network, the high interstitial fluid pressure, and the extracellular matrix. To study the impact of these factors, we measured the uptake of fluorochrome-labelled IgG using confocal laser scanning microscopy, interstitial fluid pressure by the 'wick-in-needle' technique, vascular structure by stereological analysis, and the content of the extracellular matrix constituents collagen, sulfated glycosaminoglycans and hyaluronan by colourimetric assays. The impact of the microenvironment on these factors was studied using osteosarcomas implanted either subcutaneously or orthotopically around the femur in athymic mice. The uptake of IgG was found to correlate inversely with the interstitial fluid pressure and the tumour volume in orthotopic, but not subcutaneous tumours. No correlation was found between IgG uptake and the level of any of the extracellular matrix constituents. The content of both collagen and glycosaminoglycans depended on the site of tumour growth. The orthotopic tumours had a higher vascular density than the subcutaneous tumours, as the vascular surface and length were 2-3-fold higher. The data indicate that the interstitial fluid pressure is a dominant factor in controlling the uptake of macromolecules in solid tumours; and the site of tumour growth is important for the uptake of macromolecules in small tumours, extracellular matrix content and vascularization.     

Effects of glycosaminoglycans on cell proliferation of normal osteoblasts and human osteosarcoma cells depend on their type and fine chemical compositions

Osteoblastic cells produce a complex extracellular matrix (ECM) composed of a mixture of proteoglycans (PGs), collagens and non-collagenous proteins. The interaction of proteoglycans with matrix effector macromolecules via either their glycosaminoglycan (GAG) chains or their protein core is critical in regulating a variety of cellular events. Alterations in the structural composition of the GAG/PG component of the ECM may have important consequences on cell proliferation and/or differentiation. Human osteoblasts and two osteosarcoma cell lines, able to produce galactosaminoglycan (GalAGs) and heparan sulphate (HS)-containing proteoglycans, were treated with their main GAG chain types, and the effects on cell growth were examined. Chondroitin sulphate (CSA) and dermatan sulphate (DS) inhibited cell proliferation of all osteoblastic cell lines at high concentration (100 microg/ml). DS showed the stronger inhibitory effect, probably due to the presence of flexible IdoA residues that provide a greater variety in conformation to these macromolecules. Heparin strongly inhibited the proliferation rates of both normal osteoblasts and transformed osteoblastic cells at concentrations > or = 1 microg/ml. The presence of large amounts of IdoA-derived trisulphated disaccharides, responsible for the overall negative charge of heparin, should be considered as a critical factor for the inhibition of cell proliferation. The obtained results suggest that matrix GAGs are factors which affect cell growth of both malignant and normal cells of the osteoblastic lineage in a concentration-dependent manner. This effect is closely related to the fine chemical structure of GAGs, i.e. the presence of L-iduronic acid and the degree of sulphation.     

A model of self-maintenance of in vivo malignant growth not dependent on tumour type or origin: syndrome of everlasting wound healing

The common mechanism of permanent growth maintenance in all types of malignant tumours is proposed. According to the model, the main event, stimulating tumour growth is the stochastic but permanent death of a proportion of tumour cells as a result of inherent genetic instability of tumour cell genome (chromosome fragility). Dead cells trigger the complex and multicomponent process of wound healing, manifesting in stimulation of cell proliferation, angiogenesis, cell migration and other events. Stimulation of proliferation of tumour cells leads to further production of dead (necrotic) cells and as a result to further stimulation of wound healing system etc. The nature of genetic instability of malignant cells, as proposed, is connected with arising of heritable uninemic (uniduplex) structures in pieces of some chromosomes or in whole chromosomes or sometimes even in whole genomes. Uninemic areas possess extremely high incidence of spontaneous chromosome aberrations due to failure of the mechanism of repair of DNA double-strand breaks in the absence of second DNA copy. The theory is based on the binemic structure of normal eukaryote chromosomes. Possible approaches to the mechanisms of cancer therapy are discussed.     

Basement membranes and tumor pathology

The distribution of four basement membrane components, type IV collagen (C IV), laminin (LM), heparan sulfate proteoglycan (HSP) and fibronection (FN) has been studied by indirect immunofluorescence using specific antibodies, in benign and malignant proliferations of the mammary gland and in soft tissue tumors. In breast carcinomas, specially intraductal cancers, there is a progressive and concomitant loss of these macromolecules around tumoral cells, preceding an overt tumoral invasion. In sarcomas, FN is frequently seen between malignant cells but the regular pericellular labeling observed around normal muscular cells, Schwann cells and adipocytes is absent. Nevertheless, the persistance of some pericellular staining with anti-C IV, anti-LM, anti-HSP and anti-FN antisera, in most differentiated territories of liposarcomas, leiomyosarcomas and neurifibrosarcomas can help to the diagnosis of such lesions.     

The fundamental role of the p53 pathway in tumor metabolism and its implication in tumor therapy

It is well established that the altered metabolism exhibited by cancer cells, including high rates of glycolysis, lactate production, and biosynthesis of lipids, nucleotides, and other macromolecules, and which may occur either as a consequence or as a cause of tumorigenesis, plays an essential role in cancer progression. Recently, the tumor suppressor p53 was found to play a central role in this process. Here, we review the role of p53 in modulating tumor metabolism. Specifically, we focus on the functions of p53 in regulating aerobic glycolysis, oxidative phosphorylation, the pentose phosphate pathway, fatty acid synthesis and oxidation, and glutamine metabolism, and we discuss the therapeutic strategy whereby p53 helps to prevent malignant progression.     

Tumor invasion and host extracellular matrix

In this review some of the major mechanistic pathways by which tumor cells are thought to invade host tissues are discussed. Tumor invasion has been conceived to be the result of pathological, close-range interactions between malignant cells and host stroma. The sequence of events that characterize invasion can be summarized as follows: (a) Tumor cell clusters break from the confinement of the primary tumor. Loss of intercellular junctions (desmosomes), alterations in the chemical composition and physical properties of the cell surface coat (loss of fibronectin and heparan sulfate; excessive amounts of hyaluronate), and loosening of cell-substrate interactions (loss of hemidesmosomes, fibronectin, and heparan sulfate), are among the most frequently listed causes of tumor cell shedding. (b) Increased proteolytic activities at the invasion front cause focal alterations in the surrounding extracellular matrix, thereby changing its physical properties. Collagenases and cathepsins, as well as elastase and other neutral proteinases are the enzymes most frequently associated with matrix destruction and invasion. In some tissues this process is effectively regulated by inhibitors of matrix-degrading, proteolytic enzymes. (c) Tumor cells migrate into the altered matrix, possibly moving as aggregates along guidance tracks provided by host structures (blood vessels, lymphatics, nerves) or matrix macromolecules (collagen and fibronectin tracks). Migration seems to be preceded by increased swelling of glycosaminoglycan (i.e., hyaluronate) in the matrix, ahead of the migrating cell population. Various host cell types (mast cells, fibroblasts, endothelial cells, macrophages, etc.) may participate in these events.     

Loss of NOS1 expression in high-grade renal cell carcinoma associated with a shift of NO signalling

In normal human kidney, NOS1 and soluble guanylate cyclase (sGC) are expressed in tubular epithelial cells, suggesting a physiological autocrine NO signalling pathway. Therefore, we investigated both NOS1 and sGC expressions in benign and malignant renal tumours. In addition, we examined the pattern of protein tyrosine nitration in normal and tumour tissue. NOS1 expression and activity were found to be downregulated, correlating with the tumour grade, as shown by immunohistochemistry, quantitative RT-PCR analysis, and histochemical detection of the NADPH-diaphorase activity of nitric oxide synthases (NOS). These results show that the autocrine NO signalling pathway is maintained in benign tumours and lost in malignant tumours. In contrast, sGC expression was maintained in renal tumours whatever the tumour type, a finding showing that tumour cells remain sensitive to the bioregulatory role of exogeneous NO(*). Finally, the staining pattern of protein tyrosine nitration, assessed by immunohistochemistry, parallelled that of NOS1 expression in normal renal parenchyma and benign tumours, supporting the concept that protein nitration was accounted for by NOS1 activity. In contrast, in malignant tumours, protein tyrosine nitration was accounted for by the production of reactive nitrogen oxide species by the inflammatory infiltrate. Altogether, these findings argue for a pattern of NO signalling similar in normal kidney and benign renal tumours, whereas it is completely different in malignant renal tumours.     

Plasminogen activator system and its clinical significance in patients with a malignant disease

Urokinase (uPA) plays an essential role in the activation of plasminogen to plasmin, a serine protease participating in the activation of matrixmetaloproteinases, latent elastases, growth factors and cytokines involved in the degradation of extracellular matrix elements. Together with its receptor (uPAR), tissue activator (tPA) and urokinase inhibitors (PAI-1, PAI-2, PAI-3 and protease nexin), it forms the plasminogen activator system (PAS), a component of metastatic cascade importantly contributing to the invasive growth and angiogenesis of malignant tumours. Plasminogen activator inhibitor 1 inhibits uPA-dependent invasiveness of some cancer cell lines. The vitronectin-PAI-1 complex inhibits migration of smooth muscle cells by binding alpha(v)beta3 integrin to vitronectin. PAI-1 or its deficiency interferes with signalling pathways such as PI3K/Akt and JAK/STAT and it is included in the processes of maintaining the integrity of the endothelial cells and thereby regulation of cell death. PAI-1 affects apoptosis by reducing cell adhesion and functioning of intracellular signalling pathways. The individual components of PAS undoubtedly play an important role in angiogenesis and metastasising of malignant tumours. In the near future, results of published studies with various types of cancer could be reflected in diagnostic and therapeutic algorithms and, at the same time, could serve as the goal for targeted therapies.     

Effects of altering surface glycoprotein composition on metastatic colonisation potential of murine mammary tumour cells

This study has examined cells from naturally-occurring murine mammary tumours to ascertain whether cell surface glycoproteins play a significant role in colonisation of the lungs after intravenous inoculation. It was found that gel electrophoretic analysis of membrane extracts and lectin adsorption studies did not reveal any consistent differences in glycoprotein composition of cells from tumours which can heavily colonise the lungs relative to ones from tumours which cannot do so or to cells from pulmonary metastases. Also, alteration of structural and functional properties of surface glycoproteins by treatment with succinylated lectins or with drugs such as tunicamycin and swainsonine, which inhibit glycosylation of membrane proteins, had no specific effects on metastatic colonisation of the lungs. Tunicamycin apparently decreased capability to form experimental metastases but also diminished tumourigenicity on subcutaneous inoculation, although it did not affect tumour cell viability in vitro. This information supports earlier studies from this laboratory involving enzymic digestion of the surface of living tumour cells before inoculation and demonstrates that the pulmonary colonisation capability of these mammary tumour cells can withstand global disorganisation of membrane glycoprotein structure and composition. This implies that either the surface glycoproteins are not important in the colonisation process, or that these tumour cells have great capability for rapid repair of their surfaces. It is concluded that a clear answer to whether surface glycoprotein composition has a decisive role in pulmonary colonisation by these mammary tumour cells requires introduction of stable heritable traits into tumour cell populations by genetic manipulation.     

Transferrin receptor is a marker of malignant phenotype in human pancreatic cancer and in neuroendocrine carcinoma of the pancreas

Transferrin receptor (TFRC) is a membrane-bound protein expressed in larger amounts in proliferating, e.g., malignant, cells than in quiescent cells. The specific expression of TFRC can represent a diagnostic tool or a therapeutic target in solid tumours expressing this antigen. Whether TFRC is expressed in human pancreatic tumours is unknown. The aim of this study was the investigation of the expression of TFRC and transferrin in human pancreatic cancer and in neuroendocrine tumours of the pancreas. Fifty one specimens of human pancreatic cancer and 14 samples of pancreatic neuroendocrine tumours were obtained after surgery. The expression of TFRC, transferrin and cytokeratin was studied by standard immunohistochemistry. Flow cytometry was used for the investigation of TFRC expression in nine cell lines of ductal pancreatic cancer in vitro. In contrast to normal tissue, 93% of pancreatic tumour cells showed positive (82%) or heterogeneous (11%) expression of TFRC. It was strongly expressed by malignant epithelial cells; normal stromal and endothelial cells were not stained by anti-TFRC antibodies. Primary tumours and metastases showed a similar frequency of TFRC expression. Three neuroendocrine carcinomas showed positive expression of TFRC by malignant tumour cells. The expression of TFRC was negative in benign neuroendocrine tumours of the pancreas. The cell lines of pancreatic cancer were characterised by a low expression of TFRC in vitro. In contrast to normal pancreatic tissue and benign neuroendocrine tumours of the pancreas, pancreatic cancer and neuroendocrine carcinoma are therefore characterised frequently by high expression of TFRC. Hence, TFRC represents a marker of malignant transformation in the pancreas that could be applied as potential diagnostic and therapeutic target.     

Differences in the biosynthesis and localization of the fibronectin receptor in normal and transformed cultured human cells

We examined the biosynthesis and localization of the fibronectin receptor integrin from normal and transformed cultured human cells. Normal cells required a minimum of 20 h for the biosynthesis of completely mature fibronectin-receptor beta-subunit, while transformed cells required only 6-8 h. There was a correspondingly major decrease in the amount of the intracellular beta-chain precursor in the transformants. Immunostaining of normal fibroblastic cells with monoclonal antibodies indicated that both alpha- and beta-polypeptides of the fibronectin receptor are localized in cell surface streaks and focal contact areas. In contrast, both subunits lacked this clustering and had a more diffuse distribution on the surfaces of transformed cells, even though quantitative immunofluorescence experiments indicated that similar or larger amounts of each subunit were present on a per cell basis. Both immunostaining and biochemical analyses also indicated the presence of a relatively large intracellular pool of beta-polypeptides in normal fibroblasts that is not present in transformed cells. There was no major transformation-dependent change in total quantities of mature fibronectin receptor subunit expressed and inserted into the plasma membrane, when normalized to total protein synthesis. Our results indicate that malignant transformation of cultured human cells results in altered localization and processing of the fibronectin receptor. Such changes involving pathways of crucial cell surface molecules may contribute to alterations in their interactions with extracellular macromolecules, including during the process of cellular invasion.     

Host factors important in immune surveillance against tumours

The nonspecific and specific arms of the immune response are described. The nonspecific components consist of three cell types - macrophages, natural killer and killer cells - and the specific components are antibody produced by B lymphocytes and regulatory and effector T lymphocytes. The evidence suggests that a surveillance system does operate, since at least some tumours are contained. Malignant lesions are more common in unselected autopsies and surgical biopsies of noncancer patients compared with the observed rate of clinical cases; and spontaneous cures of cancer do occur. There is general agreement that surveillance of all types of tumours by the specific components of the immune response does not occur; but there is direct evidence from some model systems and correlations from the incidence and class of tumours which arise in germ-free athymic mice, in immunosuppressed patients with transplants and in immunodeficient patients that the specific immune response, particularly by effector T cells, may control the expression of some tumours of lymphoreticular cells. Such surveillance may be superimposed on a more general system which is mediated by the nonspecific elements. One possible way of increasing the efficiency of the system would be to provide some of the soluble mediators of activated effector cells such as macrophages and T cells. Thus, administration of interferon has had some success, and there is a case for further purification of these and other lymphokines and monokines for administration to patients.     

Spectrum of matrix metalloproteinase expression in primary and metastatic colon cancer: relationship to the tissue inhibitors of metalloproteinases and membrane type-1-matrix metalloproteinase

The matrix metalloproteinases, MMP-2 and MMP-9, are capable of degrading components of the basement membrane, a vital barrier breached during the progression of colorectal cancer. The regulation of MMP-2 activation and subsequent targets is vital to understanding the metastatic process. MMP-2 was not expressed by colorectal cancer cells (C170 and C170HM(2)) in vitro but by stromal fibroblasts (46BR.1GI). There was induction of this MMP upon transwell co-cultivation of the colon cancer cells with the fibroblasts but in vivo growth did not lead to a similar increase in the metastatic tumour cells (C170HM(2)), MMP-2 again being attributed to the stromal cells. MMP-2 mRNA was overexpressed in human colorectal tumours compared to normal colorectal tissue, which correlated with Dukes' stage and immunolocalized to the stromal compartment of the tumour tissue. The active form of the MMP-2 enzyme was also present in the colorectal tumour tissue (7/8) but essentially absent in all normal colon samples examined (1/8). MMP-2 activation was not related to an increase in MT-1-MMP mRNA or a decrease in the specific inhibitor TIMP-2 in human tissue. There was however an increase in MMP-2/TIMP-2 ratio in tumour compared to normal. MMP-9, a target of active MMP-2, was present in the metastatic cell line but expression was down-regulated in the tumour cells in vivo, gelatin analysis revealed that MMP-9 was almost entirely attributable to the murine host, confirmed by PCR. There was no increase in mRNA for MMP-9 or its specific inhibitor TIMP-1 in colorectal tumour tissue compared to normal, MMP-9 protein localized to the inflammatory infiltrate. Fibroblast cells may provide malignant epithelial cells with a ready source of enzyme which is crucial to the metastatic process.     

Unraveling the microenvironmental influences on the normal mammary gland and breast cancer

The normal mammary gland and invasive breast tumors are both complex 'organs' composed of multiple cell types as well as extracellular matrix in three-dimensional (3D) space. Conventionally, both normal and malignant breast cells are studied in vitro as two-dimensional monolayers of epithelial cells, which results in the loss of structure and tissue function. Many laboratories are now investigating regulation of signaling function in the normal mammary gland using 3D cultures. However, it is also important to assay malignant breast cells ex vivo in a physiologically relevant environment to more closely mimic tumor architecture, signal transduction regulation and tumor behavior in vivo. Here we present the potential of these 3D models for drug testing, target validation and guidance of patient selection for clinical trials. We also argue that in order to get full insight into the biology of the normal and malignant breast, and to create in vivo-like models for therapeutic approaches in humans, we need to continue to create more complex heterotypic models to approach the full context the cells encounter in the human body.