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Adhesive and invasive features in gliomas
PAntOlOGY RESEARCH AND PRACTICE o urban & Fischer Verlag
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Adhesive and Invasive Features in Gliomas Dominique S. Tews Division of Neuropathology, Medical Center, Johannes Gutenberg University, Mainz, Germany
Summary This study aims at the in situ identification of factors mediating glioma celJ invasion requiring adhesion. extracellular matrix degradation, and migration. Fortyfive gliomas (astrocytomas, glioblastomas, oligodendrogliomas, and mixed gliomas) were investigated for the immunohistochemical expression of the membrane protein CD44s, the basal lamina proteins laminin, collagen IV, and fibronectin, the lectin galectin-3 recognizing tenascin and N-CAM, as well as for the matrix-degrading enzymcs metalJoproteinases MMP-2, MMP-9, and cathepsin D. Besides vessels expressing basallamina proteins, tenascin, MMP-2, MMP-9, and galectin-3, tumor cells revealed strong immunoreactivity for CD44s, tenascin, galectin-3, and N-CAM, which was restricted to solid tumor masses. Single invading cells displayed distinct expression of MMP-2 and MMP-9, also found in solid tumor areas, as well as of cathepsin D. Restricted expression of CD44s. galectin-3, tenascin, and N-CAM in solid tumor masses seems to contribute to homotypical tumor celJ adhesion. However, switching to an invasive phenotype, single tumor cells lack this expression pattern and acquire degrading and phagocytic activities by expressing cathepsin D, MMP2, and MMP-9, which arc also expressed by solid tumor masses faci litating the loosening and invasion of single neoplastic cells. The blocking of these factors may be of potential benefit in anti-invasive therapy.
Key words: Cell adhesion - Degradation - Extracellular matrix - Gliomas - Invasion
ami nation reveals a diffuse single tumor cell invasion leading to the failure of local therapies, resulting in tumor recurrence, progression, and death. Invasion is a three-step process initially requiring the adhesion of the tumor cell to resident cells or matrix components, followed by the degradation of this matrix and invasion of the infiltrating cell into this new intercellular space. During invasion, homotypic adhesion to other tumor cells is reduced, with a concomitant increase in heterotypic adhesion to other ce lls or to extracellular matrix (ECM) molecules accompanied by the up and down regulation of specific cell adhesion molecules also involved in the rearrangemcnt of the cytoskeleton. In recent years, a growing number of receptors and ligands have been described in gliomas [4, 6, 10, 23, 26, 43, 45, 54, 55, 65]. CD44, an integralmembrane glycoprotein, has been implicated in the binding of hyaluronate, one of the most abundant molecules in the ECM of the brain, and in a wide range of other ECM components such as laminin, collagen, and fibronectin {4, 61]. Galectin-3, a p-galactoside-binding lectin, has been shown to bind to some neural recognition molecules such as N-CAM and tenascin {13, 54, 58]. Brain tumors are generally located in a nonpermeable environment. However, tumor cells are able to produce proteolytic enzymes mediating invasion by degrading the ECM. Among these are the matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) from a homologous family of zinc-dependent endopeptidases which havc broad specifities for ECM proteins such as collagen, fibronectin, and laminin {5, 12, 30, 36, 5 1]. Other
Address for correspondence: Dominique S. Tews, Division
Introduction
of Neuropathology, Medical Center, Johannes Gutenberg Uni-
Although the borders of gliomas sometimes appear to be well-delineated macroscopically, histological ex-
verSity, LangcnbcckstraBe I,D - 55 101 Mainz, Germany. Tel.: ++49 (0) 61 31117 73 08. Fax: ++49 (0) 61 311176606. E·mail: [email protected] z.de
Pathol. Res. Pract. 196: 701-711 (2000)
0344-033812000/196/10-70 1 $15.00/0
702 . D. S. Tews
putative factors in ECM degradation are cathepsins such as cathepsin D, an endosome-lysosomal aspartatic protease [64]. The activity of these proteases is regulated by a complex interplay of multiple factors and processes [71]. In vitro studies show the migration of glioma cells under a broad panel of substrates and conditions [10, 21,26,42,45,48,49,53]. However, the migration behavior of tumor cells in situ may differ from cell lines under culture conditions {21, 51, 56]. Although there is a large number of studies investigating the multi-step process of invasion, the distribution and expression of these factors at the invading edge between solid tumor masses and adjacent brain structures have not been well documented. The present study aims at identifying factors involved in adhesion, degradation, and migration of glial tumor cells in situ, concentrating on the infiltrative edge which contributes to growth and spread in different types of gliomas.
Materials and Methods Tissue Archival formalin-fixed and paraffin-embedded tumor tissue from 45 patients was examined. The material consisted of 5 pilocytic astrocytomas (age: 26.4 ± 9.7 years), 5 astrocytomas WHO II (age: 23 ± 5.6 years), 9 anaplastic astrocytomas WHO III (age: 47.8 ± 6.9 years), 12 glioblastomas (age: 56.6 ± 3.9 years), 4 oligoendrogliomas WHO II (age: 43.8 ± 9.0 years), 4 anaplastic oligodendrogliomas WHO III (age: 46.0 ± 7.4 years), 3 mixed gliomas (oligo-astrocytomas) WHO II (age: 47.0 ± 4.2 years), and 3 anaplastic mixed gliomas (oligo-astrocytomas) WHO III (age: 51.7 ± 3.2 years), from the files of the Neuropathology Division at Johannes Gutenberg University Hospital. Nannal brain tissue from 5 adult autopsy cases (age: 69.8 ± 3.9 years) with no neurological disorders was used as controls. Furthermore, the following entities were studied: three adult autopsy cases with brain infarction (stages 1- 3) (age: 76.7 ± 6.4 years), two with
Alzheimer's disease (age: 81.5 ± 2.5 years), and one with acute contusion injury (age: 49 years). These autopsy brains had also been fixed in formalin and tissue embedded in paraffin wax. Tissue sections were sectioned (5 11m) onto "SuperFrost Plus" slides (Menzel) and stored at room temperature.
Immunohistochemical preparations The sections were dewaxed, pretreated with 2% H20 2 in methanol for 30 min and rinsed in phosphate-buffered saline (PBS). The slides were then either placed in 10 mM citrate buffer, pH 6.0, and irradiated for 15 min in a microwave oven or incubated with proteinase K (14 ~g/ml 10 mM (TRIS/HCI, pH 7.4-8.0) for 20 min at room temperature. After washing in PBS, the sections were covered with I% horse or goat serum (Vector Laboratories) in PBS for 20 min. Then, the first antibody was applied (Table I) and incubated at room temperature. After intervening washes following each step, the secondary biotinylated anti-mouse (raised in horse) or anti-rabbit (raised in goat) (all Vector laboratories) antibody was applied to all sections, and in the third step, it was connected to the avidin-biotin-peroxidase-complex (Vectastain ABC-Kit, Vector laboratories), all diluted 1:100 and incubated for 30 min. Finally, the sections were incubated with diaminobenzidine substrate solution (DAB tablets, Sigma), washed, and COUIlterstained with hematoxylin. In control sections, the primary antibody was either omitted or substituted with nOll specific mouse or rabbit immunoglobulin.
Semiquantitative analysis For quantitative analysis, a representative area comprising 10,000 tumor cells was defined. In 16 patients, the tissue specimen contained fewer than lO,OOO tumor cells~ in those cases, an area containing between 1,000 and 9,000 tumor cells was analyzed. In the representative areas, positive tumor cells were determined and scored in a semiquantitative fashion (no positive cells, + 1-25% positive cells, ++ 26--50% positive cells, +++ 51-75 % positive cells, ++++ 76--100% positive cells). For tenascin, extracellular expression was similarly quantified as a percentage of immunoreactive vital tumor tissue in the representative area in 1-25%, ii in 26--50%, iii in 51-75%. iiii in 76--100% of vital tumor tissue).
ct
Table I. Antigen
Antibody
Dilution, pretreatment, incubation
Source
Tenascin-C Fibronectin Laminin, A chain Collagen IV Galectin 3 CD44 CD56INCAM Cathepsin D MMP-2 MMP-9
monoclonal, mouse polyclonal, rabbit monoclonal, mouse monoclonal, mouse monoclonal, mouse monoclonal. mouse monoclonal, mouse monoclonal, mouse monoclonal, mouse monoclonal, mouse
I: 100, PK, overnight 1:600, PK, overnight 1:30, PK, overnight 1:75, MV, 60 min 1:200, MV, 60 min 1:500, MV, 60 min 1:100, MV, 60 min 1:100. MV, 60 min 1:25, MV, 90 min 1:100, MV, 90 min
Dako Dako Dako Dako Novocastra R&D Systems Novocastra Novocastra Calbiochem Calbiochem
PK ;;;; proteinase K, MV ;;;; microwave
Glioma Invasion . 703 munoreactivity 10 the perivascular stroma. [n contrast, CD56/N-CAM was found in the plasma membranes of neurons, revealing a fine tibrillar meshwork particularly in the gray matter. Cathepsin D displayed strong cytoplasmic granular expression in neurons. Neither CD56/NCAM and galectin-3 nor cathepsin D and MMP-9 were found in glial cells. There was no virtual expression of MMP-2 in any central nervous system structure.
Results Normal control brain tissue
In normal control brain tissue, vessel basal laminas clearly expressed tenascin-C, fibronectin, laminin, collagen IV, and galectin-3, all of which were expressed in every vessel wall except for tenasein, which was found in only 10 to 50% of the vessels. Endothelial cells also revealed a weak granular staining pattern for MMP-9. There was a diffuse expression of CD44s (Fig. I A) and tenascin-C (Fig. IB) within the white matter. While tenascin was confined to the ECM, CD44s was expressed by the plasma membrane of glial cells and their fibrillary end feet on capillaries, effecting strong CD44s-im-
Disease control brain tissue
There was strong expression of galectin-3 and occasionally of cathepsin D in macrophages in patients with brain infarction and acute contusion injury. In these patients, galectin-3 as well as CD44s were strongly ex-
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PA - pilocytic astrocytoma. A - astrocytoma, OB - glioblastoma. 0 - glioblastoma, 0 - oligodendroglioma, MG - mixed glioma; * T in 1-25%, tt in 26-50%, ttt in 51-75%, t 1'1'1' in 76-100% of vital tumorti ss ue/number of cases; ** - no positi ve celis, + 1-25% positive cells, ++ 26-50% positive cells. +++ 5 1-75% positive ce ll s. ++++ 76--100% positive cells/numher of cases; v - vessels
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Glioma Invasion . 70S pressed by activated glial cells surrounding ischemic and traumatic lesions. Furthermore, in patients with Alzheimer's disease, tangles and neuritic plaques displayed strong immunoreactivity for tibroncctin. The expression patterns of all other factors studied resembled that of normal control brain tissue. Pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas (Table 2)
Tumor vessels in gliomas also clearly expressed basal lamina proteins such as fibronectin , laminin, and collagen IV. There was strong immunoreactivity for tenasein-C, but not in all the vessels; only between 10 and 90% percent of the vessels expressed tcnascin-C. A glomerula-like endothelial proliferation, in particular, which was occasionally apparent in pilocytie astrocytomas, but mostly found in glioblastomas, revealed strong immunostaining for tenascin-C (Fig. lC), MMP9, and, occasionally, for galectin-3 and MMP-2. A distinct and constant upregulation in solid tumor tissue was revealed by the cell adhesion molecule C044s, which was expressed on the plasma membrane of tumor cells (Fig. lO), Although the fibrillary end feet of astrocytic tumor cells affect strong perivascular immunoreactivity for C044s, the endothelial cells themselves were negative. C044s was also present in lymphocytic infiltrates predominantly found in glioblastomas. Glial tumor cells also showed expression of galectin-3, revealing strong cytoplasmic staining (Fig. 1E). Although there was a slight increase of galectin-3-positive tumor cells in glioblastomas, a clear correlation with tumor malignancy regarding individual tumors was not seen. Expression of galcctin-3 was confined to solid tumor areas and often found in gcmistocytic tumor cells, particularly in anaplastic astrocytomas and glioblastomas (Fig. IE). In adjacent brain tissue, only activated astrocytes displayed strong galectin-3 immunoreactivity. Tenascin expression was also restricted to solid tumor tissue (Fig. IG). Tenascin-C displayed a focal, extracellular staining pattcrn of stroma and intercellular spaccs, found in all glioma grades but more pronounced in high grade tumors. Occasionally, tumor cells exhibited cytoplasmic immunoreactivity for tenascin-C as well. A distinct upregulation in solid tumor masses was also found for the cell adhesion molecules C056/N-CAM, which,
like C044s, were expressed on the plasma membrane of tumor cells. Also, small tumor cell accumulations at the invasive edge confluent with the solid tumor mass were often C0561N-CAM-positive (Fig. IH). Neither the expression pattern of C044s nor that of C056/N-CAM was related to the degree of malignancy. In contrast, cathepsin 0 was constantly expressed in anaplastic astrocytomas and glioblastomas, while only 40% of the pilocytic astrocytomas and 60% of astrocytomas WHO II showed neoplastic cathepsin 0 expression. However, with cathepsin 0 being present in a tumor, the number of positive tumor cells revealed no correlation with the degree of malignancy. Cathepsin 0 was cytoplasmatically expressed and predominantly found in gemistocytic tumor cells as well as in glioma cells diffusely infiltrating central nervous system structures adjacent to tumor tissue (Fig. 2A). The expression of MMP-2 and MMP-9 in glioma and endothelial cells increased with tumor malignancy. MMP-2 was not present in pilocytic astrocytomas and astrocytomas WHO II, but nearly 50% of anaplastic astrocytomas and glioblastomas displayed MMP-2-positive tumor cells. MMP-9 was present in all tumor specimens investigated, with positive tumor cells increasing markedly with the grade of malignancy. It was strongly expressed cytoplasmatically, particularly by gemistocytic tumor cells (Fig. 28). However, the distribution of MMPs within solid tumor tissue was heterogeneous. Tumor cells invading adjacent brain areas displayed distinct expression for both metalloproteinases (Figs, 2C, 0). There was also strong MMP-9 immunoreactivity in lymphocytic infiltrations. Rosenthal fibers were negative for all proteins studied. Oligodendrogliomas
Thc expression patterns of the antigens studied in oligodendrogliomas were similar to those observed in astrocytic tumors and glioblastomas (Fig. 2E). The most striking difference was the abscnce of galcctin-3 in oligodendroglial neoplastic cells (Fig, 2F). Mixed gliomas
Qualitative and quantitative expression profiles of the antigens investigated resembled those of astrocytomas and oligodendrogliomas.
Fig. 1. A: Normal brain revealing CD44s expression in the white matter with strong perivascular immunoreactivity. (AntiCD44s, x60). B: Expression of tenascin-C in normal brain white matter and vessels. (Anti-tenascin-C, x60). C: Glioblastoma showing strong vasc ular positivity for tenascin-C. (Anti-tenascin-C, x IIO). D: Anaplastic astrocytoma with abundant CD44s immunoreactivity restricted to tumor cells and not found in proliferating vessels. (Anti-CD44s, xI40). E: Pilocytic astrocytoma showing strong cytoplasmic expression of galcctin-3 in the typical bipolar tumor cells. (Anti-galectin-3. x280). F: Galectin-3 expression in solid tumor masses of an anaplastic astrocytoma and in activated astrocytes in adjacent brain tissue. (Anti-ga]ectin3, x60). G: Anaplastic astrocytoma with tenascin-C up-regulation confined to solid tumor areas and lacking in the infiltrative
margin. (Anti-tenascin-C, xl 10). H: Invading edge of a glioblastoma with numerOUSCD56/N-CAM-positive tumor lesions connuating with each other and with the solid tumor mass. (Anti-CD56/N-CAM, xI40).
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Glioma Invasion . 707
Discussion Tumor growth and spread are related to two main factors: proliferation and local invasion. While tumor cell proliferation has been shown to correlate well with tumor malignancy, the degree of local infiltration is usually not correlated with the tumor grade, as low grade astrocytomas also reveal local invasion [33}. Tumor spread appears to he due to the following two possibilities: single cell invasion and continuous growth of solid tumor masses, both facilitated by secretion of ECM protein-degrading enzymes [27]. Both anaplastic gliomas and glioblastomas showed distinct up-regulation of ECM-degrading proteases such as MMP-2 and 9; this concurs with various other ill vitro [2, 49 J and in vivo studies [5, 20, 44, 50, 51, 62, 67]. Metalloproteinases were expressed in neoplastic glioma cells and in endothelial cells. While the expression of MMP-2 was restricted to anaplastic gliomas and glioblastoms, MMP-9 was found in tumors of all grades. It has been suggested that the increased expression in high-grade gliomas is mostly attributable to prominent vasculature {SOl and is not found in cells of low-grade glioma. However, in this study, MMP-9-positive tumor cells were found in all types and grades of gliomas contributing to the clinical picture of diffuse invasion also found in low-grade gliomas. Nevertheless, there was a clear increase in the rates of MMP-9-positive glial tumor cells, correlating with tumor malignancy. Immunohistochemical analysis, however, gives no information about latent or activated forms of MMPs. However, it has been shown that up-regulated immunohistochemical expression of MMPs is associated with enhanced gelatinolytic activity {49, 50, 77 J. MMP-2 degrades denatured collagens (gelatins), collagen type III, IV, V, fi bronectin, and laminin, while MMP-9 cleaves gelatins, collagen type I, III, IV, and V. It is suggested that the upregulation of MMP-9 in endothelial cells contributes in particular to angiogenesis and neovascularization, initiated by endothelial cells that degrade the basement membrane surrounding their vessel and that migrate through the ECM into the surrounding tissue toward the source of the angiogenic stimulus [15, 20, 36, 72]. Furthermore, MMPs also facilitate spread on myelin in the white matter [lJ and disruption of the glia limitans extema membrane to mediate leptomeningeal involvement and spread into the cerebrospinal t1uid [2].
It remains unclear why solid tumor areas and single cells expressed high levels of degrading enzymes. Pericellular proteolysis, however, seems to be a multifactorial event. Expression of ECM-degrading enzymes indicates random proteolysis at the periphery of a solid growing tumor mass mediating the loosening of adhesion of cellular and extracellular structures by invading the surrounding brain tissue, rendering this environment more favorable for single tumor cell dissemination. Single tumor cells then transform into an invasive phenotype and start to migrate into the surrounding tissue. Becoming more motile, the tumor cells also acquire the phagocytic capacity to remove ECM and cell debris by endophagocytosis for tinal degradation by intracellular enzymes such as cathepsin D, which was strongly expressed by infiltrating tumor cells, particularly in anaplastic astrocytomas and glioblastomas, as reported previously [64, 69]. Nevertheless, another study reported down-regulation of cathepsin D with increased malignancy which, however, correlated with decreased GFAP expression [74]. Associated with the expression of ECM-degrading enzyme are changes in the integrin expression pattern [II, 55}. The initial event in the process of invasion before migration into a proteolytically modified matrix is the recognition and attachcment of tumor cells to specific ECM molecules. Hyaluronate, one of the most abundant molecules in the brain , is recognized by CD44s {4 /. It showed distinct expression in gliomas of all grades and was found in white matter glial cells in normal brains [3, 2S, 34, 39, 43, 57]. As described previously, there was no correlation between CD44s expression and grade of malignancy [39, 53, 57, 61]. It was shown that CD44s is the most abundant form of CD44, while other specific splicc-variants arc found only in small amounts or arc lacking entirely. This may contribute to the usually non-metastatic behavior of malignant gliomas {IS , 63, 7SJ. This interaction of CD44s with hyaluronate is suggested to contribute substantially to the invasive character of primary brain tumors {40, 4S, 59, 60, 61]. Nevertheless, CD44s was found in both normal and activated astrocytes, indicating that CD44s up-regulation may be a common astrocytic response in a variety of disease states [29]. CD44s, however, abu binds to a broad panel of basal lamina proteins such as laminin, fibronectin, and collagen IV, classically found in vessels both in the normal brain and in gliomas [5 ,
Fig. 2. A: Glioblastoma with cathepsin-D-posiiivc tumor cells perivascular (arrow) and infiltrating adjacent brain tissue (arrow-
heads) (Anti-cathepsin-D. x2(0). B: Gemistocytic tumor cells in a glioblastoma displaying strong MMP-9 immunoreactivity. (Anti-MMP-9, x280). C: Infiltrative margin of a glioblastoma with numerous MMP-9-positive invading tumor cells. (AntiMMP-9, xI40). D: Infiltrating glioblastoma cells also displaying strong expression of MMP-2. (Anti-MMP-2, xl40). E: Distinct expression of CD44s in an oligodendroglioma, anti-CD44s (x140). F: Galectin-3 negaLivity in an oligodendroglioma, ex-
cept in the activated astrocytes diffusely distributed among the oligodendroglial tumor cells. (Anti-galectin-3, x280).
708 . D.S. Tews 47, 48, 61, 65 J, giving CD44 a central role in cell invasion by glioma cells, particularly along vessels which are a main route for neoplastic cell invasion, although glial tumor cells do not penetrate the vascular wall. However, this study failed to confirm the expression of the basal lamina proteins by glial tumor cells, a result that has been controversially discussed [5, II , 22, 68, 70]. All astrocytic tumors showed distinct expression of the ~-galactos ide-binding protein galectin-3 [31). However, although some anaplastic astrocytomas and glioblastomas showed exceptionally high p ercentages of galectin-3-positive tumor cells, this study failed to confirm a positive correlation of galectin-3 expression and malignancy, as described previ ously 18}. Nevertheless, oligodendroglial tumor cells wcre completely negative for galectin-3 [8J. Galectin-3 is involved in ho motypic cell-to-cell adhesive interactions by mediati ng cell-to-cell adhesion via bridging molecules on adjacent cells of the same type [38, 58J; thi s may explain the exclusive presence in solid tumor areas. Furthermore, galectin-3 binds to N-CAM and tenascin 158/.Interestingly, all these proteins are confined to solid tumor areas. As galectin-3 has been shown to be a substrate for MMP-2 and -9 152}, their activity may not only contribute to the migration of tumor cells in contiguous brain ti ssuc, but also to their detachment from solid tumor cell adhesion. The galectin-3 binding partner tenasc in , also called glioma-mesenchymal extracellular protein , later shown to be identical to tenascin-C [71, was focally expressed in all tumors and grades to a variable extent, as described previously [9, 35, 80). The present stud y a lso showed that the expression of tenascin-C was more pronounced in high grade tumors [9, 35, 79, 80]. As shown by others [35 , 47 J, tumor cells al so revealed occasionally cytoplasmic tenascin immunoreacti vity, indicating that there are two sources of tcnascin in human gliomas producing and secreting tenascin: one is thc tumor cells f7, 35, 79J, and the other the endothelial cclls of proliferating tumor vessels [79]. In gliomas, tenascin-C expression was confined to solid tumor areas. As it has been shown that tenascin inhibits migration of glial cells, its down-regulation may promote migration of tumor cells at the invasive edge [1 3, 24. 75]. Concerning proliferation, contradictory results have been achieved for tenascin, demonstrating that it both promotes [19/ and inhibits [141 proliferation. However, it is likely that proliferation is influenced by many different factors not allowing a simple correlation between single elements. Nevertheless, the tenascin expression in solid tumor masses may contribute to enhanced proliferation rates, particularly in high grade gliomas. The loss oftenascin expression in the infiltration and migratory zones may, on the other hand, contributc to decreased proliferation activity there, as migration and
proliferation have been described as exhibiting dichotomous behaviors [25 , 42J. Furthermore, enhanced endothelial tenascin expression may contribute to the proliferation of endothelial cells. Not all tumor vessels showed expression of tenascin, a finding that may be due to different steps in vascular neogenesis and maturation {73/; up-regulated vascularization, however, contributed to the up-regulated overall expression of tenasc in correlating with tumor malignancy [35, 37, 80/. In both our normal control specimens and normal brains, tenascin was constitutively expressed in the white matter. This contrasts with others who described tcnascin in the normal brain as being restricted to vessels or completely absent 135, 80/. However, our immunohistochemical finding may explain the unexpectedl y high tenascin levels found in the normal brain by immunoblotting in the stud y of Zagzag and coworkers [80/. Expression of N-CAM in the normal brain was confined to neuronal elements in the gray matter [28J, while it was also constantly found in glial tumor cells in all types of gliomas [41, 54, 66, 76J. N-CAM, serving as its own ligand, was predominantly expressed in solid tumor areas contributing to cell-to-cell contacts among neoplastic cells. Nevertheless, in some tumor specimens, N-CAM was clearly positi ve in single tumor cells near the invasive edge. It has been shown in culture that N-CAM is down-regulated in the migratory phase, promoting cell disaggregation. Furthermore, it shows both an inverse correlati on with ganglioside and metalloproteinase expression fac ilitating migration [16, 17,32,46]. Tumor cells might stop migrating, proliferate, and form their own lesion, which would finally merge with other lesions and the growing tumor mass. Therefore, it is likely that these N-CAM positive tumor cells stop migrating, but represent stationary cells formin g their own neoplastic island. Concerning different types of gliomas. the present study revealed that the expression profiles of oligodendrogliomas did not differ from those of their astrocytic counterparts, except for differences in the expression of galectin-3. To summarize, these findin gs indicate that thc expression of the adhesion molecules CD44s, tenascin, galectin-3. and N-CAM, which was confined to solid tumor areas, contributes to homotypic tumor cell aggregation and that it is down-regulated when single tumor cells switch to an invasive phenotype. The loosening and invasion of single tumor cells are mediated and facilitated by thc expression of ECM-degrading metalloproteinases on the part of solid tumor masses, and by the invading cells themselves, which also acquire phagocytic activity by intracellular degradation in endophagosomes through cathepsin D. Each of these three steps of invasion may he of potemial benefit in anti-invasive therapy. [t is important that most adhesion molecules may be bound and recognized by several
Glioma Invasion . 709 other receptors and ligands with different affinities. Therefore, the blocidng of onc factor may have no net cffect and does not allow conclusions regarding the permeability of a complex matrix 121, 27}. Thi s assumption is corroborated by the lack of a simple correlation between receptor and ligand expression and invasiveness . Anti-invasive therapies concerning glial tumor cell invasion may also be effective in the inhibition of tumor angiogenesis. Nevertheless, further studies dealing with the interdependence of these factors and their relevance are necessary to gain a better understanding of the mechanisms of glioma cell invasion ill vivo.
Acknowledgements: The author is grateful to Drs. J. Bohl and H. H. Goebel for their helpful suggestions and comments. and to R. Hanne for technical assistance and W. Meffen for photographic work.
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77.
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Received: January 12, 2000 Accepted: June 8. 2000
http://wv.Iw.urbanfischer.deljournalslprp
Adhesive and Invasive Features in Gliomas Dominique S. Tews Division of Neuropathology, Medical Center, Johannes Gutenberg University, Mainz, Germany
Summary This study aims at the in situ identification of factors mediating glioma celJ invasion requiring adhesion. extracellular matrix degradation, and migration. Fortyfive gliomas (astrocytomas, glioblastomas, oligodendrogliomas, and mixed gliomas) were investigated for the immunohistochemical expression of the membrane protein CD44s, the basal lamina proteins laminin, collagen IV, and fibronectin, the lectin galectin-3 recognizing tenascin and N-CAM, as well as for the matrix-degrading enzymcs metalJoproteinases MMP-2, MMP-9, and cathepsin D. Besides vessels expressing basallamina proteins, tenascin, MMP-2, MMP-9, and galectin-3, tumor cells revealed strong immunoreactivity for CD44s, tenascin, galectin-3, and N-CAM, which was restricted to solid tumor masses. Single invading cells displayed distinct expression of MMP-2 and MMP-9, also found in solid tumor areas, as well as of cathepsin D. Restricted expression of CD44s. galectin-3, tenascin, and N-CAM in solid tumor masses seems to contribute to homotypical tumor celJ adhesion. However, switching to an invasive phenotype, single tumor cells lack this expression pattern and acquire degrading and phagocytic activities by expressing cathepsin D, MMP2, and MMP-9, which arc also expressed by solid tumor masses faci litating the loosening and invasion of single neoplastic cells. The blocking of these factors may be of potential benefit in anti-invasive therapy.
Key words: Cell adhesion - Degradation - Extracellular matrix - Gliomas - Invasion
ami nation reveals a diffuse single tumor cell invasion leading to the failure of local therapies, resulting in tumor recurrence, progression, and death. Invasion is a three-step process initially requiring the adhesion of the tumor cell to resident cells or matrix components, followed by the degradation of this matrix and invasion of the infiltrating cell into this new intercellular space. During invasion, homotypic adhesion to other tumor cells is reduced, with a concomitant increase in heterotypic adhesion to other ce lls or to extracellular matrix (ECM) molecules accompanied by the up and down regulation of specific cell adhesion molecules also involved in the rearrangemcnt of the cytoskeleton. In recent years, a growing number of receptors and ligands have been described in gliomas [4, 6, 10, 23, 26, 43, 45, 54, 55, 65]. CD44, an integralmembrane glycoprotein, has been implicated in the binding of hyaluronate, one of the most abundant molecules in the ECM of the brain, and in a wide range of other ECM components such as laminin, collagen, and fibronectin {4, 61]. Galectin-3, a p-galactoside-binding lectin, has been shown to bind to some neural recognition molecules such as N-CAM and tenascin {13, 54, 58]. Brain tumors are generally located in a nonpermeable environment. However, tumor cells are able to produce proteolytic enzymes mediating invasion by degrading the ECM. Among these are the matrix metalloproteinases-2 (MMP-2) and -9 (MMP-9) from a homologous family of zinc-dependent endopeptidases which havc broad specifities for ECM proteins such as collagen, fibronectin, and laminin {5, 12, 30, 36, 5 1]. Other
Address for correspondence: Dominique S. Tews, Division
Introduction
of Neuropathology, Medical Center, Johannes Gutenberg Uni-
Although the borders of gliomas sometimes appear to be well-delineated macroscopically, histological ex-
verSity, LangcnbcckstraBe I,D - 55 101 Mainz, Germany. Tel.: ++49 (0) 61 31117 73 08. Fax: ++49 (0) 61 311176606. E·mail: [email protected] z.de
Pathol. Res. Pract. 196: 701-711 (2000)
0344-033812000/196/10-70 1 $15.00/0
702 . D. S. Tews
putative factors in ECM degradation are cathepsins such as cathepsin D, an endosome-lysosomal aspartatic protease [64]. The activity of these proteases is regulated by a complex interplay of multiple factors and processes [71]. In vitro studies show the migration of glioma cells under a broad panel of substrates and conditions [10, 21,26,42,45,48,49,53]. However, the migration behavior of tumor cells in situ may differ from cell lines under culture conditions {21, 51, 56]. Although there is a large number of studies investigating the multi-step process of invasion, the distribution and expression of these factors at the invading edge between solid tumor masses and adjacent brain structures have not been well documented. The present study aims at identifying factors involved in adhesion, degradation, and migration of glial tumor cells in situ, concentrating on the infiltrative edge which contributes to growth and spread in different types of gliomas.
Materials and Methods Tissue Archival formalin-fixed and paraffin-embedded tumor tissue from 45 patients was examined. The material consisted of 5 pilocytic astrocytomas (age: 26.4 ± 9.7 years), 5 astrocytomas WHO II (age: 23 ± 5.6 years), 9 anaplastic astrocytomas WHO III (age: 47.8 ± 6.9 years), 12 glioblastomas (age: 56.6 ± 3.9 years), 4 oligoendrogliomas WHO II (age: 43.8 ± 9.0 years), 4 anaplastic oligodendrogliomas WHO III (age: 46.0 ± 7.4 years), 3 mixed gliomas (oligo-astrocytomas) WHO II (age: 47.0 ± 4.2 years), and 3 anaplastic mixed gliomas (oligo-astrocytomas) WHO III (age: 51.7 ± 3.2 years), from the files of the Neuropathology Division at Johannes Gutenberg University Hospital. Nannal brain tissue from 5 adult autopsy cases (age: 69.8 ± 3.9 years) with no neurological disorders was used as controls. Furthermore, the following entities were studied: three adult autopsy cases with brain infarction (stages 1- 3) (age: 76.7 ± 6.4 years), two with
Alzheimer's disease (age: 81.5 ± 2.5 years), and one with acute contusion injury (age: 49 years). These autopsy brains had also been fixed in formalin and tissue embedded in paraffin wax. Tissue sections were sectioned (5 11m) onto "SuperFrost Plus" slides (Menzel) and stored at room temperature.
Immunohistochemical preparations The sections were dewaxed, pretreated with 2% H20 2 in methanol for 30 min and rinsed in phosphate-buffered saline (PBS). The slides were then either placed in 10 mM citrate buffer, pH 6.0, and irradiated for 15 min in a microwave oven or incubated with proteinase K (14 ~g/ml 10 mM (TRIS/HCI, pH 7.4-8.0) for 20 min at room temperature. After washing in PBS, the sections were covered with I% horse or goat serum (Vector Laboratories) in PBS for 20 min. Then, the first antibody was applied (Table I) and incubated at room temperature. After intervening washes following each step, the secondary biotinylated anti-mouse (raised in horse) or anti-rabbit (raised in goat) (all Vector laboratories) antibody was applied to all sections, and in the third step, it was connected to the avidin-biotin-peroxidase-complex (Vectastain ABC-Kit, Vector laboratories), all diluted 1:100 and incubated for 30 min. Finally, the sections were incubated with diaminobenzidine substrate solution (DAB tablets, Sigma), washed, and COUIlterstained with hematoxylin. In control sections, the primary antibody was either omitted or substituted with nOll specific mouse or rabbit immunoglobulin.
Semiquantitative analysis For quantitative analysis, a representative area comprising 10,000 tumor cells was defined. In 16 patients, the tissue specimen contained fewer than lO,OOO tumor cells~ in those cases, an area containing between 1,000 and 9,000 tumor cells was analyzed. In the representative areas, positive tumor cells were determined and scored in a semiquantitative fashion (no positive cells, + 1-25% positive cells, ++ 26--50% positive cells, +++ 51-75 % positive cells, ++++ 76--100% positive cells). For tenascin, extracellular expression was similarly quantified as a percentage of immunoreactive vital tumor tissue in the representative area in 1-25%, ii in 26--50%, iii in 51-75%. iiii in 76--100% of vital tumor tissue).
ct
Table I. Antigen
Antibody
Dilution, pretreatment, incubation
Source
Tenascin-C Fibronectin Laminin, A chain Collagen IV Galectin 3 CD44 CD56INCAM Cathepsin D MMP-2 MMP-9
monoclonal, mouse polyclonal, rabbit monoclonal, mouse monoclonal, mouse monoclonal, mouse monoclonal. mouse monoclonal, mouse monoclonal, mouse monoclonal, mouse monoclonal, mouse
I: 100, PK, overnight 1:600, PK, overnight 1:30, PK, overnight 1:75, MV, 60 min 1:200, MV, 60 min 1:500, MV, 60 min 1:100, MV, 60 min 1:100. MV, 60 min 1:25, MV, 90 min 1:100, MV, 90 min
Dako Dako Dako Dako Novocastra R&D Systems Novocastra Novocastra Calbiochem Calbiochem
PK ;;;; proteinase K, MV ;;;; microwave
Glioma Invasion . 703 munoreactivity 10 the perivascular stroma. [n contrast, CD56/N-CAM was found in the plasma membranes of neurons, revealing a fine tibrillar meshwork particularly in the gray matter. Cathepsin D displayed strong cytoplasmic granular expression in neurons. Neither CD56/NCAM and galectin-3 nor cathepsin D and MMP-9 were found in glial cells. There was no virtual expression of MMP-2 in any central nervous system structure.
Results Normal control brain tissue
In normal control brain tissue, vessel basal laminas clearly expressed tenascin-C, fibronectin, laminin, collagen IV, and galectin-3, all of which were expressed in every vessel wall except for tenasein, which was found in only 10 to 50% of the vessels. Endothelial cells also revealed a weak granular staining pattern for MMP-9. There was a diffuse expression of CD44s (Fig. I A) and tenascin-C (Fig. IB) within the white matter. While tenascin was confined to the ECM, CD44s was expressed by the plasma membrane of glial cells and their fibrillary end feet on capillaries, effecting strong CD44s-im-
Disease control brain tissue
There was strong expression of galectin-3 and occasionally of cathepsin D in macrophages in patients with brain infarction and acute contusion injury. In these patients, galectin-3 as well as CD44s were strongly ex-
Table 2.
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++++ 15
+
14; II;
v
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v v v
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v v
+ II +++ I[ ++++ 13 + 12 ++ 13 +++ II ++++ 13 +++ 12 ++++ 110
+++
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t 1'l 13;
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viS
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+
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+
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+
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+++ 12 0: + /I +++ 12
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PA - pilocytic astrocytoma. A - astrocytoma, OB - glioblastoma. 0 - glioblastoma, 0 - oligodendroglioma, MG - mixed glioma; * T in 1-25%, tt in 26-50%, ttt in 51-75%, t 1'1'1' in 76-100% of vital tumorti ss ue/number of cases; ** - no positi ve celis, + 1-25% positive cells, ++ 26-50% positive cells. +++ 5 1-75% positive ce ll s. ++++ 76--100% positive cells/numher of cases; v - vessels
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Glioma Invasion . 70S pressed by activated glial cells surrounding ischemic and traumatic lesions. Furthermore, in patients with Alzheimer's disease, tangles and neuritic plaques displayed strong immunoreactivity for tibroncctin. The expression patterns of all other factors studied resembled that of normal control brain tissue. Pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas (Table 2)
Tumor vessels in gliomas also clearly expressed basal lamina proteins such as fibronectin , laminin, and collagen IV. There was strong immunoreactivity for tenasein-C, but not in all the vessels; only between 10 and 90% percent of the vessels expressed tcnascin-C. A glomerula-like endothelial proliferation, in particular, which was occasionally apparent in pilocytie astrocytomas, but mostly found in glioblastomas, revealed strong immunostaining for tenascin-C (Fig. lC), MMP9, and, occasionally, for galectin-3 and MMP-2. A distinct and constant upregulation in solid tumor tissue was revealed by the cell adhesion molecule C044s, which was expressed on the plasma membrane of tumor cells (Fig. lO), Although the fibrillary end feet of astrocytic tumor cells affect strong perivascular immunoreactivity for C044s, the endothelial cells themselves were negative. C044s was also present in lymphocytic infiltrates predominantly found in glioblastomas. Glial tumor cells also showed expression of galectin-3, revealing strong cytoplasmic staining (Fig. 1E). Although there was a slight increase of galectin-3-positive tumor cells in glioblastomas, a clear correlation with tumor malignancy regarding individual tumors was not seen. Expression of galcctin-3 was confined to solid tumor areas and often found in gcmistocytic tumor cells, particularly in anaplastic astrocytomas and glioblastomas (Fig. IE). In adjacent brain tissue, only activated astrocytes displayed strong galectin-3 immunoreactivity. Tenascin expression was also restricted to solid tumor tissue (Fig. IG). Tenascin-C displayed a focal, extracellular staining pattcrn of stroma and intercellular spaccs, found in all glioma grades but more pronounced in high grade tumors. Occasionally, tumor cells exhibited cytoplasmic immunoreactivity for tenascin-C as well. A distinct upregulation in solid tumor masses was also found for the cell adhesion molecules C056/N-CAM, which,
like C044s, were expressed on the plasma membrane of tumor cells. Also, small tumor cell accumulations at the invasive edge confluent with the solid tumor mass were often C0561N-CAM-positive (Fig. IH). Neither the expression pattern of C044s nor that of C056/N-CAM was related to the degree of malignancy. In contrast, cathepsin 0 was constantly expressed in anaplastic astrocytomas and glioblastomas, while only 40% of the pilocytic astrocytomas and 60% of astrocytomas WHO II showed neoplastic cathepsin 0 expression. However, with cathepsin 0 being present in a tumor, the number of positive tumor cells revealed no correlation with the degree of malignancy. Cathepsin 0 was cytoplasmatically expressed and predominantly found in gemistocytic tumor cells as well as in glioma cells diffusely infiltrating central nervous system structures adjacent to tumor tissue (Fig. 2A). The expression of MMP-2 and MMP-9 in glioma and endothelial cells increased with tumor malignancy. MMP-2 was not present in pilocytic astrocytomas and astrocytomas WHO II, but nearly 50% of anaplastic astrocytomas and glioblastomas displayed MMP-2-positive tumor cells. MMP-9 was present in all tumor specimens investigated, with positive tumor cells increasing markedly with the grade of malignancy. It was strongly expressed cytoplasmatically, particularly by gemistocytic tumor cells (Fig. 28). However, the distribution of MMPs within solid tumor tissue was heterogeneous. Tumor cells invading adjacent brain areas displayed distinct expression for both metalloproteinases (Figs, 2C, 0). There was also strong MMP-9 immunoreactivity in lymphocytic infiltrations. Rosenthal fibers were negative for all proteins studied. Oligodendrogliomas
Thc expression patterns of the antigens studied in oligodendrogliomas were similar to those observed in astrocytic tumors and glioblastomas (Fig. 2E). The most striking difference was the abscnce of galcctin-3 in oligodendroglial neoplastic cells (Fig, 2F). Mixed gliomas
Qualitative and quantitative expression profiles of the antigens investigated resembled those of astrocytomas and oligodendrogliomas.
Fig. 1. A: Normal brain revealing CD44s expression in the white matter with strong perivascular immunoreactivity. (AntiCD44s, x60). B: Expression of tenascin-C in normal brain white matter and vessels. (Anti-tenascin-C, x60). C: Glioblastoma showing strong vasc ular positivity for tenascin-C. (Anti-tenascin-C, x IIO). D: Anaplastic astrocytoma with abundant CD44s immunoreactivity restricted to tumor cells and not found in proliferating vessels. (Anti-CD44s, xI40). E: Pilocytic astrocytoma showing strong cytoplasmic expression of galcctin-3 in the typical bipolar tumor cells. (Anti-galectin-3. x280). F: Galectin-3 expression in solid tumor masses of an anaplastic astrocytoma and in activated astrocytes in adjacent brain tissue. (Anti-ga]ectin3, x60). G: Anaplastic astrocytoma with tenascin-C up-regulation confined to solid tumor areas and lacking in the infiltrative
margin. (Anti-tenascin-C, xl 10). H: Invading edge of a glioblastoma with numerOUSCD56/N-CAM-positive tumor lesions connuating with each other and with the solid tumor mass. (Anti-CD56/N-CAM, xI40).
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Discussion Tumor growth and spread are related to two main factors: proliferation and local invasion. While tumor cell proliferation has been shown to correlate well with tumor malignancy, the degree of local infiltration is usually not correlated with the tumor grade, as low grade astrocytomas also reveal local invasion [33}. Tumor spread appears to he due to the following two possibilities: single cell invasion and continuous growth of solid tumor masses, both facilitated by secretion of ECM protein-degrading enzymes [27]. Both anaplastic gliomas and glioblastomas showed distinct up-regulation of ECM-degrading proteases such as MMP-2 and 9; this concurs with various other ill vitro [2, 49 J and in vivo studies [5, 20, 44, 50, 51, 62, 67]. Metalloproteinases were expressed in neoplastic glioma cells and in endothelial cells. While the expression of MMP-2 was restricted to anaplastic gliomas and glioblastoms, MMP-9 was found in tumors of all grades. It has been suggested that the increased expression in high-grade gliomas is mostly attributable to prominent vasculature {SOl and is not found in cells of low-grade glioma. However, in this study, MMP-9-positive tumor cells were found in all types and grades of gliomas contributing to the clinical picture of diffuse invasion also found in low-grade gliomas. Nevertheless, there was a clear increase in the rates of MMP-9-positive glial tumor cells, correlating with tumor malignancy. Immunohistochemical analysis, however, gives no information about latent or activated forms of MMPs. However, it has been shown that up-regulated immunohistochemical expression of MMPs is associated with enhanced gelatinolytic activity {49, 50, 77 J. MMP-2 degrades denatured collagens (gelatins), collagen type III, IV, V, fi bronectin, and laminin, while MMP-9 cleaves gelatins, collagen type I, III, IV, and V. It is suggested that the upregulation of MMP-9 in endothelial cells contributes in particular to angiogenesis and neovascularization, initiated by endothelial cells that degrade the basement membrane surrounding their vessel and that migrate through the ECM into the surrounding tissue toward the source of the angiogenic stimulus [15, 20, 36, 72]. Furthermore, MMPs also facilitate spread on myelin in the white matter [lJ and disruption of the glia limitans extema membrane to mediate leptomeningeal involvement and spread into the cerebrospinal t1uid [2].
It remains unclear why solid tumor areas and single cells expressed high levels of degrading enzymes. Pericellular proteolysis, however, seems to be a multifactorial event. Expression of ECM-degrading enzymes indicates random proteolysis at the periphery of a solid growing tumor mass mediating the loosening of adhesion of cellular and extracellular structures by invading the surrounding brain tissue, rendering this environment more favorable for single tumor cell dissemination. Single tumor cells then transform into an invasive phenotype and start to migrate into the surrounding tissue. Becoming more motile, the tumor cells also acquire the phagocytic capacity to remove ECM and cell debris by endophagocytosis for tinal degradation by intracellular enzymes such as cathepsin D, which was strongly expressed by infiltrating tumor cells, particularly in anaplastic astrocytomas and glioblastomas, as reported previously [64, 69]. Nevertheless, another study reported down-regulation of cathepsin D with increased malignancy which, however, correlated with decreased GFAP expression [74]. Associated with the expression of ECM-degrading enzyme are changes in the integrin expression pattern [II, 55}. The initial event in the process of invasion before migration into a proteolytically modified matrix is the recognition and attachcment of tumor cells to specific ECM molecules. Hyaluronate, one of the most abundant molecules in the brain , is recognized by CD44s {4 /. It showed distinct expression in gliomas of all grades and was found in white matter glial cells in normal brains [3, 2S, 34, 39, 43, 57]. As described previously, there was no correlation between CD44s expression and grade of malignancy [39, 53, 57, 61]. It was shown that CD44s is the most abundant form of CD44, while other specific splicc-variants arc found only in small amounts or arc lacking entirely. This may contribute to the usually non-metastatic behavior of malignant gliomas {IS , 63, 7SJ. This interaction of CD44s with hyaluronate is suggested to contribute substantially to the invasive character of primary brain tumors {40, 4S, 59, 60, 61]. Nevertheless, CD44s was found in both normal and activated astrocytes, indicating that CD44s up-regulation may be a common astrocytic response in a variety of disease states [29]. CD44s, however, abu binds to a broad panel of basal lamina proteins such as laminin, fibronectin, and collagen IV, classically found in vessels both in the normal brain and in gliomas [5 ,
Fig. 2. A: Glioblastoma with cathepsin-D-posiiivc tumor cells perivascular (arrow) and infiltrating adjacent brain tissue (arrow-
heads) (Anti-cathepsin-D. x2(0). B: Gemistocytic tumor cells in a glioblastoma displaying strong MMP-9 immunoreactivity. (Anti-MMP-9, x280). C: Infiltrative margin of a glioblastoma with numerous MMP-9-positive invading tumor cells. (AntiMMP-9, xI40). D: Infiltrating glioblastoma cells also displaying strong expression of MMP-2. (Anti-MMP-2, xl40). E: Distinct expression of CD44s in an oligodendroglioma, anti-CD44s (x140). F: Galectin-3 negaLivity in an oligodendroglioma, ex-
cept in the activated astrocytes diffusely distributed among the oligodendroglial tumor cells. (Anti-galectin-3, x280).
708 . D.S. Tews 47, 48, 61, 65 J, giving CD44 a central role in cell invasion by glioma cells, particularly along vessels which are a main route for neoplastic cell invasion, although glial tumor cells do not penetrate the vascular wall. However, this study failed to confirm the expression of the basal lamina proteins by glial tumor cells, a result that has been controversially discussed [5, II , 22, 68, 70]. All astrocytic tumors showed distinct expression of the ~-galactos ide-binding protein galectin-3 [31). However, although some anaplastic astrocytomas and glioblastomas showed exceptionally high p ercentages of galectin-3-positive tumor cells, this study failed to confirm a positive correlation of galectin-3 expression and malignancy, as described previ ously 18}. Nevertheless, oligodendroglial tumor cells wcre completely negative for galectin-3 [8J. Galectin-3 is involved in ho motypic cell-to-cell adhesive interactions by mediati ng cell-to-cell adhesion via bridging molecules on adjacent cells of the same type [38, 58J; thi s may explain the exclusive presence in solid tumor areas. Furthermore, galectin-3 binds to N-CAM and tenascin 158/.Interestingly, all these proteins are confined to solid tumor areas. As galectin-3 has been shown to be a substrate for MMP-2 and -9 152}, their activity may not only contribute to the migration of tumor cells in contiguous brain ti ssuc, but also to their detachment from solid tumor cell adhesion. The galectin-3 binding partner tenasc in , also called glioma-mesenchymal extracellular protein , later shown to be identical to tenascin-C [71, was focally expressed in all tumors and grades to a variable extent, as described previously [9, 35, 80). The present stud y a lso showed that the expression of tenascin-C was more pronounced in high grade tumors [9, 35, 79, 80]. As shown by others [35 , 47 J, tumor cells al so revealed occasionally cytoplasmic tenascin immunoreacti vity, indicating that there are two sources of tcnascin in human gliomas producing and secreting tenascin: one is thc tumor cells f7, 35, 79J, and the other the endothelial cclls of proliferating tumor vessels [79]. In gliomas, tenascin-C expression was confined to solid tumor areas. As it has been shown that tenascin inhibits migration of glial cells, its down-regulation may promote migration of tumor cells at the invasive edge [1 3, 24. 75]. Concerning proliferation, contradictory results have been achieved for tenascin, demonstrating that it both promotes [19/ and inhibits [141 proliferation. However, it is likely that proliferation is influenced by many different factors not allowing a simple correlation between single elements. Nevertheless, the tenascin expression in solid tumor masses may contribute to enhanced proliferation rates, particularly in high grade gliomas. The loss oftenascin expression in the infiltration and migratory zones may, on the other hand, contributc to decreased proliferation activity there, as migration and
proliferation have been described as exhibiting dichotomous behaviors [25 , 42J. Furthermore, enhanced endothelial tenascin expression may contribute to the proliferation of endothelial cells. Not all tumor vessels showed expression of tenascin, a finding that may be due to different steps in vascular neogenesis and maturation {73/; up-regulated vascularization, however, contributed to the up-regulated overall expression of tenasc in correlating with tumor malignancy [35, 37, 80/. In both our normal control specimens and normal brains, tenascin was constitutively expressed in the white matter. This contrasts with others who described tcnascin in the normal brain as being restricted to vessels or completely absent 135, 80/. However, our immunohistochemical finding may explain the unexpectedl y high tenascin levels found in the normal brain by immunoblotting in the stud y of Zagzag and coworkers [80/. Expression of N-CAM in the normal brain was confined to neuronal elements in the gray matter [28J, while it was also constantly found in glial tumor cells in all types of gliomas [41, 54, 66, 76J. N-CAM, serving as its own ligand, was predominantly expressed in solid tumor areas contributing to cell-to-cell contacts among neoplastic cells. Nevertheless, in some tumor specimens, N-CAM was clearly positi ve in single tumor cells near the invasive edge. It has been shown in culture that N-CAM is down-regulated in the migratory phase, promoting cell disaggregation. Furthermore, it shows both an inverse correlati on with ganglioside and metalloproteinase expression fac ilitating migration [16, 17,32,46]. Tumor cells might stop migrating, proliferate, and form their own lesion, which would finally merge with other lesions and the growing tumor mass. Therefore, it is likely that these N-CAM positive tumor cells stop migrating, but represent stationary cells formin g their own neoplastic island. Concerning different types of gliomas. the present study revealed that the expression profiles of oligodendrogliomas did not differ from those of their astrocytic counterparts, except for differences in the expression of galectin-3. To summarize, these findin gs indicate that thc expression of the adhesion molecules CD44s, tenascin, galectin-3. and N-CAM, which was confined to solid tumor areas, contributes to homotypic tumor cell aggregation and that it is down-regulated when single tumor cells switch to an invasive phenotype. The loosening and invasion of single tumor cells are mediated and facilitated by thc expression of ECM-degrading metalloproteinases on the part of solid tumor masses, and by the invading cells themselves, which also acquire phagocytic activity by intracellular degradation in endophagosomes through cathepsin D. Each of these three steps of invasion may he of potemial benefit in anti-invasive therapy. [t is important that most adhesion molecules may be bound and recognized by several
Glioma Invasion . 709 other receptors and ligands with different affinities. Therefore, the blocidng of onc factor may have no net cffect and does not allow conclusions regarding the permeability of a complex matrix 121, 27}. Thi s assumption is corroborated by the lack of a simple correlation between receptor and ligand expression and invasiveness . Anti-invasive therapies concerning glial tumor cell invasion may also be effective in the inhibition of tumor angiogenesis. Nevertheless, further studies dealing with the interdependence of these factors and their relevance are necessary to gain a better understanding of the mechanisms of glioma cell invasion ill vivo.
Acknowledgements: The author is grateful to Drs. J. Bohl and H. H. Goebel for their helpful suggestions and comments. and to R. Hanne for technical assistance and W. Meffen for photographic work.
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Received: January 12, 2000 Accepted: June 8. 2000