The results of the current study show for what to our knowledge is the first time the exact cellular and tissue localization of TN-C immunoreactivity in human control brain tissue and in a large number of different brain tumors. Immunoreactivity for TN-C was found predominantly in the following structures: In control brains, TN-C was found predominantly in the white matter of the frontal, temporal, parietal, and occipital lobes and in the hippocampus, with a significant difference in the expression of TN-C noted when comparing gray matter with white matter using either Western blot analysis or immunohistochemistry. In tumor tissue, the most constant TN-C immunopositivity was seen in the extracellular matrix of the fibrotic stroma of highly malignant tumors and along the tumor border, especially in high-grade astrocytomas. Tumor cells were usually TN-C negative.
The Normal Human Brain
With regard to the control brains, in general, the results of the current study confirm the previously published results. Authors described strong expression of TN-C restricted to the ECM and no TN-C expression in the blood vessels of the normal adult brain compared with the developing central nervous system.1, 4, 8, 13 Ventimiglia et al. 29 reported a detectable amount of TN-C (0.04 μg/mg brain tissue) in the temporal lobe and other cortical areas of adult brains. Perides et al. 30 described TN-C in the white matter of the central nervous system, comparing human and bovine brain tissues. We were able to confirm these observations by analyzing separately the gray matter and white matter of various regions from 21 control brains using Western blot analysis and immunohistochemistry with three different MoAbs against TN-C. The most consistent findings in control brains were a low-to-moderate TN-C positivity of the ECM in the white matter of nearly all specimens and the lack of TN-C immunoreactivity in intracerebral vessels. Furthermore, TN-C was expressed focally only in the first layer of the gray matter; this finding was confirmed by Western blot analyses, in which a weak signal for the gray matter homogenate could be seen at 230 kilodaltons. The expression of TN-C in the ECM of the normal brain tissue was interpreted as peritumoral in origin by some authors who used control brain specimens from surgical interventions for GBM.4, 13, 14 However, when using archival frozen tissues (n = 5 specimens) and formalin-fixed, paraffin-embedded blocks (n = 21 specimens) from control brains, we were able to detect TN-C expression constantly in the ECM of the white matter, a finding that is consistent with recently published data by Tews and Nissen.31 Based on these results, we conclude that, in the adult human brain, there still is a critical amount of total TN-C in the ECM of the white matter. This finding is highly important in the clinical setting, because it restricts systemic therapeutic approaches and forces us to develop highly specific MoAbs for radioimmunotherapy as applied in a locoregional therapeutic approach.
TN-C in Brain Tumors
Invasion and tumor cell-migration play crucial roles in disease progression and are biologic hallmarks of malignant tumors. These are the major causes of disease-associated morbidity and mortality. Cell biology of migrating fetal cells and invading neoplastic cells seems to possess similarities,32 because both cell types use the ECM and its proteins (e.g., TN-C) for attachment and motility.2, 7 It remains unknown whether the dissemination of glioma cells occurs along white fiber tracts because these tracts are the pathway of minor resistance or because of specific ligands located along these tracts.
It is well known that TN-C expression is up-regulated in tumor tissues.1, 4, 8, 13 In the current study, a comparison of the TN-C localization was made between tumors of astrocytic origin and tumors of other origin (meningiomas, metastases to the brain) by Western blot analysis. Furthermore, the TN-C distribution pattern in different regions of control brains and in a large variety of brain tumors using immunohistochemistry was used to establish whether there was an entity-specific localization of TN-C in tumor cells, blood vessels, or the ECM.
We found heterogeneous TN-C expression in the ECM or the perivascular area along fibrotic gliomesenchymal areas of nearly every examined nonastrocytic tumor entity (see Table 1). These expression patterns lead to the conclusion that TN-C is not important for malignant transformation of nonastrocytic tumors but may be involved in the ECM reaction around the tumor to prevent further tumor cell spread. It is well known that expression of TN-C is up-regulated actively by stimulated astrocytes in traumatic brain lesions.33, 34
Zagzag et al.35 observed significantly higher levels of TN-C in homogenates of GBM than in normal brain and observed enhanced TN-C expression in vascular hyperplasia in 59 astrocytomas. Those authors found a significant association between elevated TN-C expression and higher tumor grade. We also detected high levels of TN-C in GBM lesions and negative to low levels in tumors of nonastrocytic origin using Western blot analysis. Furthermore, we detected heterogeneous TN-C distribution in the ECM and in the perivascular compartment of different brain tumor entities, but the most consistent finding was observed in astrocytomas: Elevated TN-C expression was seen around blood vessels of the lesion of all tumor grades but was more pronounced around hyperplastic vessels in high-grade astrocytomas. Furthermore, there was a significant difference in the TN-C expression around vessels among the different astroglioma grades. Zagzag et al. observed elevated TN-C expression in the ECM at the infiltration zone of malignant glial tumors (astrocytoma, WHO Grade 2–4). We were able to confirm this observation in several specimens (Fig. 1D); in addition, we found TN-C immunopositivity in the cytoplasm of activated astrocytes in the peritumoral, edematous areas embedded in a TN-C negative ECM (Fig. 1C). Therefore, we conclude that TN-C generally may be up-regulated at the border of the tumor. Based on the adhesive activity of TN-C,36 the elevated TN-C expression in this zone leads to the hypothesis that the elevated expression of TN-C in areas of reactively transformed ECM may have an antiinfiltrative effect and may prevent further infiltrative tumor cell migration.
In patients with astrocytoma (WHO Grade 2; n = 19 patients) without TN-C expression around the vessels of the lesion, compared with patients who had TN-C positive astrocytoma, had a significant median survival benefit of 16 months (P = 0.04) according to Kaplan–Meier survival curves and log-rank test statistics. Among patients with GBM (WHO Grade 4; n = 99 patients), those with TN-C negative lesions (n = 12 patients) survived significantly longer (median: 16 months; P = 0.008) compared with patients who had elevated TN-C expression in the ECM of the lesion. Recently, Herold-Mende et al. 37 showed a significant correlation between a shorter disease free interval and perivascular staining for TN-C in patients with WHO Grade 2 and 3 gliomas
Because we observed a constant immunohistochemical finding of TN-C in the ECM of fibrotic changes in 33 different tumor entities, and especially elevated TN-C expression in the infiltrative tumor borders of astrocytic tumors, this antiinfiltrative TN-C up-regulation appears to be fatal in patients with astrocytic tumors, because these tumor cells spread easily—as in the developing brain—along TN-C boundaries into the nonlesioned brain. The postulated antiinfiltrative effect of elevated TN-C expression in nonastrocytic tumors protects the host brain from further nonastrocytic tumor cell invasion, whereas this antiinfiltrative effect seems to facilitate invasion of glioma cells at the tumor border in astrocytomas. This hypothesis is underlined by 1) the constant immunohistochemical finding of TN-C in the ECM of astrocytic tumor borders, 2) the intracytoplasmic TN-C expression of astrocytes in the peritumoral edematous areas of an untreated GBM (Fig. 1D), and 3) the obvious survival benefit of patients without TN-C expression in GBM.
Tumor cell invasion is defined as an active translocation of tumor cells through cell layers and ECM barriers of the host.38 In contrast to most malignant neoplasms, astrogliomas aggressively infiltrate the surrounding brain tissue as single cells but metastasize only very rarely.7 In astrogliomas, tumor cells most likely infiltrate and migrate to the neighboring brain parenchyma along the basement membrane of new endothelial proliferations as one possible pathway.7 Thus, the initiation of neovascularization is one critical step toward malignant progression.
Giese et al. 39 observed a five times higher migration rate of glioma cells on TN-C-monolayers than on collagen, fibronectin, or vitronectin layers using seven human glioma cell lines in a microliter scale assay. In vitro, endothelial cells attach to TN-C substrates and are able to elongate and extend as necessary for endothelial migration; in contrast, there was no spread on substrates, including fibronectin, collagen, vitronectin, or laminin.40 This suggests that, in gliomas, TN-C plays a critical role in modulating cell adhesion and motility in endothelial proliferation and tumor cell migration.
The observed histologic appearance of the TN-C staining in astrocytic tumors, with its strong immunopositivity along neovascularization and in the surrounding areas of highest cellular pleomorphism, the reduced TN-C expression in histologically lesser malignant areas, and the lack of TN-C immunostaining in vessels of control brains, lead to the conclusion that the adhesive/antiadhesive function of TN-C promotes endothelial cell motility and, thus, again plays a crucial role in glioma-induced neoangiogenesis. Current data show that endothelial cells of hyperplastic vessels can synthesize perivascular TN-C.35, 41 In vitro and in vivo experiments have shown the up-regulation of TN-C in the presence of basic fibroblast growth factor, transforming growth factor β, and platelet-derived growth factor.42, 43 Recently, Zagzag et al. 44 suggested that the expression of TN-C by migrating endothelial cells and the promotion of endothelial cell adhesion and migration by TN-C makes TN-C play a potential role in pathologic angiogenesis. Even if these factors stimulate angiogenesis and are associated with neovascularization in brain tumors, to date, it has not been proven that angiogenetic factors can stimulate TN-C up-regulation in brain tumors. Moreover, the elevation of TN-C expression seems to be a general reaction of the altered ECM of the brain induced by microglia and/or astrocytes, like what was seen with the elevated TN-C expression along fibrotic changes in the surrounding, nonaltered brain areas in four patients with bacterial meningoencephalitis.
The frequently observed infiltrative glioma growth in the cortex and especially the rapid glioma spread along white matter tracts, like the corpus callosum, anterior commissure, or optic radiation, could be mediated by TN-C, which was detected in the ECM of the white matter of nearly every examined region of the cortex in 21 control brains. Goldbrunner et al.7 postulated that gliomas obviously can produce their own ECM whenever necessary but also opportunistically use the host ECM whenever it is present and useful.
Varying degrees of TN-C expression are found in normal adult control brains and in astrocytomas with various degrees of malignancy. It appears the presence or absence of TN-C expression in the stroma of astrocytic tumors may play a not yet clearly understood role in patient survival. It is noteworthy that, in patients who had astrocytoma (WHO Grade 2; n = 19 patients) without TN-C expression around the vessels of the lesion, compared with patients who had TN-C positive astrocytoma, had a significant median survival benefit of 16 months (P = 0.04) according to Kaplan–Meier survival curves and log-rank test statistics. Among patients with GBM (WHO Grade 4; n = 99 patients), those with TN-C negative GMB (n = 12 patients) survived significantly longer (16 months; P = 0.008) compared with patients who had elevated TN-C expression in the ECM of the lesion.