CXCR2-Blocking Has Context-Sensitive Effects on Rat Glioblastoma Cell Line Outgrowth (S635) in an Organotypic Rat Brain Slice Culture Depending on Microglia-Depletion (PLX5622) and Dexamethasone Treatment

In glioblastoma (GBM), the interplay of different immune cell subtypes, cytokines, and/or drugs shows high context-dependencies. Interrelations between the routinely applied dexamethasone (Dex) and microglia remain elusive. Here, we exploited rat organotypic brain slice co-cultures (OBSC) to examine the effects on a rat GBM cell line (S635) outgrowth resulting from the presence of Dex and pretreatment with the colony-stimulating factor receptor 1 (CSF1-R) inhibitor PLX5622: in native OBSC (without PLX5622-pretreatment), a diminished S635 spheroid outgrowth was observable, whereas Dex-treatment enhanced outgrowth in this condition compared to PLX5622-pretreated OBSC. Screening the supernatants of our model with a proteome profiler, we found that CXCL2 was differentially secreted in a Dex- and PLX5622-dependent fashion. To analyze causal interrelations, we interrupted the CXCL2/CXCR2-axis: in the native OBSC condition, CXCR2-blocking resulted in increased outgrowth, in combination with Dex, we found potentiated outgrowth. No effect was found in the PLX5622-pretreated. Our method allowed us to study the influence of three different factors—dexamethasone, PLX5622, and CXCL2—in a well-controlled, simplified, and straight-forward mechanistic manner, and at the same time in a more realistic ex vivo scenario compared to in vitro studies. In our model, we showed a GBM outgrowth enhancing synergism between CXCR2-blocking and Dex-treatment in the native condition, which was levelled by PLX5622-pretreatment

Function of carbonic anhydrase IX in glioblastoma multiforme

Carbonic anhydrase (CA) IX is over-expressed in glioblastoma; however, its functions in this context are unknown. Metabolically, glioblastomas are highly glycolytic, leading to a significant lactic acid load. Paradoxically, the intracellular pH is alkaline. We hypothesized that CAIX contributes to the extrusion of hydrogen ions into the extracellular space, thereby moderating intra- and extracellular pH and creating an environment conductive to enhanced invasion. We investigated the role of CAIX as a prognostic marker in patients with glioblastoma and its biological function in vitro. CAIX expression was analyzed in 59 patients with glioblastoma by immunohistochemistry. The expression levels were correlated to overall survival. In vitro, U251 and Ln 18 glioblastoma cells were incubated under hypoxia to induce CAIX expression, and RNA interference (RNAi) was used to examine the function of CAIX on cell attachment, invasion, intracellular energy transfer, and susceptibility to adjuvant treatment. High CAIX expression was identified as an independent factor for poor survival in patients with glioblastoma. In vitro, cell attachment and invasion were strongly reduced after knockdown of CAIX. Finally, the effects of radiation and chemotherapy were strongly augmented after CAIX interference and were accompanied by a higher rate of apoptotic cell death. CAIX is an independent prognostic factor for poor outcome in patients with glioblastoma. Cell attachment, invasion, and survival during adjuvant treatment are significantly influenced by high CAIX expression. These results indicate that inhibition of CAIX is a potential metabolic target for the treatment of patients with glioblastoma

Expression levels of melanoma inhibitory activity correlate with time to progression in patients with high-grade glioma

Melanoma inhibitory activity (MIA) is related to disease progression in patients with malignant melanoma and to invasion and metastasis of melanoma in vivo and in vitro. An alternative splice product termed MIA(splice) was described recently. In addition to melanoma, both proteins are expressed in a substantial subset of high-grade gliomas. We hypothesize that expression levels of both proteins correlate with early tumor progression and parameters of disseminated disease in patients with high-grade glioma. We examined the correlation of expression levels of MIA and MIA(splice) with time to progression and morphological and clinical markers of disseminated disease (defined as multifocal occurrence, gliomatosis, invasion or metastasis) in a series of 24 newly-diagnosed human high-grade gliomas. Homogenates of surgical specimens, cell cultures and blood samples were analyzed. Significant levels of MIA and MIA(splice) protein were detected in 71% of homogenates of high-grade glioma, but not in the related blood samples. Patients with early tumor progression had lower expression levels of MIA than patients with late progression, and the expression level of MIA was inversely related to time to progression. In addition, MIA expression correlated with a high fiber content of the extracellular matrix, suggesting a role in dissemination as known from malignant melanoma. Expression levels of MIA in homogenates of surgical specimen directly relate to a more benign clinical prognosis in patients with high-grade glioma. While a mechanistic relation has not yet been verified, factors such as a high fiber content of the extracellular matrix may explain this observation

CD133(+) and CD133(-) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles

Although glioblastomas show the same histologic phenotype, biological hallmarks such as growth and differentiation properties vary considerably between individual cases. To investigate whether different subtypes of glioblastomas might originate from different cells of origin, we cultured tumor cells from 22 glioblastomas under medium conditions favoring the growth of neural and cancer stem cells (CSC). Secondary glioblastoma (n = 7)-derived cells did not show any growth in the medium used, suggesting the absence of neural stem cell-like tumor cells. In contrast, 11/15 primary glioblastomas contained a significant CD133(+) subpopulation that displayed neurosphere-like, nonadherent growth and asymmetrical cell divisions yielding cells expressing markers characteristic for all three neural lineages. Four of 15 cell lines derived from primary glioblastomas grew adherently in vitro and were driven by CD133(-) tumor cells that fulfilled stem cell criteria. Both subtypes were similarly tumorigenic in nude mice in vivo. Clinically, CD133(-) glioblastomas were characterized by a lower proliferation index, whereas glial fibrillary acidic protein staining was similar. GeneArray analysis revealed 117 genes to be differentially expressed by these two subtypes. Together, our data provide first evidence that CD133(+) CSC maintain only a subset of primary glioblastomas. The remainder stems from previously unknown CD133(-) tumor cells with apparent stem cell-like properties but distinct molecular profiles and growth characteristics in vitro and in vivo

Tenascin-C protein is induced by transforming growth factor-beta1 but does not correlate with time to tumor progression in high-grade gliomas

BACKGROUND: Tenascin-C is an extracellular matrix protein known to correlate with prognosis in patients with glioblastoma, probably by stimulation of invasion and neoangiogenesis. Transforming Growth Factor-beta1 (TGF-beta1) plays an important role in the biology of high-grade gliomas, partly by regulating invasion of these tumors into parenchyma. This study was designed to evaluate if TGF-beta1 induces the expression and deposition of Tenascin-C in the extracellular matrix of high-grade gliomas which may be pivotal for the invasion of these tumors into healthy parenchyma. METHODS: A series of 20 high-grade gliomas was stained immunohistochemically with Tenascin-C- and TGF-beta1- specific antibodies. Expression levels of both proteins were evaluated and correlated with each other, time to progression and molecular and morphological markers of invasion. A quantitative PCR assay was performed evaluating the induction of Tenascin-C mRNA by treatment with TGF-beta1 in vitro. RESULTS: Tenascin-C was expressed in 18 of 19 (95%) evaluable tumors, whereas 14 of 20 tumors (70%) expressed TGF-beta1 in a significant percentage of cells. Treatment with TGF-beta1 did induce the expression of Tenascin-C at the mRNA and protein level in vitro. The expression of Tenascin-C and TGF-beta1 did neighter statistically correlate with each other nor with time to progression. CONCLUSION: In our series, Tenascin-C and TGF-beta1 were expressed in the vast majority of high-grade gliomas. We could not detect a correlation of one of the proteins with time to progression. Nevertheless, we describe induction of Tenascin-C by TGF-beta1, possibly providing a mechanism for the invasion of high-grade gliomas into healthy parenchyma

Toll-like receptor triggered dendritic cell maturation and IL-12 secretion are necessary to overcome T-cell inhibition by glioma-associated TGF-beta2.

Contains fulltext : 52663.pdf (publisher's version ) (Closed access)Malignant gliomas are able to secrete large amounts of immunosuppressive cytokines like transforming growth factor beta 2 (TGF-beta2) and regularly escape from immune surveillance. Many strategies have been developed to induce potent anti-glioma responses, among those the use of dendritic cells (DC) as therapeutic vaccines. Here, we report that both mature DC and IL-12 secretion are necessary to overcome T-cell inhibition by TGF-beta2. Flow cytometric analyses showed that TGF-beta2 does not suppress the upregulation of MHC (major histocompatibility complex) class II molecules and the T cell stimulatory capacity of human DC that were stimulated with a strong cytokine cocktail containing tumor necrosis factor alpha (TNF-alpha), IL-1beta, IL-6 and prostaglandin E2 (PGE2). Moreover, TGF-beta2 signaling studies revealed that these cytokine-matured DC become unresponsive to TGF-beta2. Although both mature and immature DC expressed comparable amounts of the TGF-beta receptor type II, Smad2 phosphorylation and subsequent upregulation of Smad7 was inhibited in mature DC, but not immature DC. However, further analysis revealed that mature DC alone are not sufficient to mediate full T cell activation in the presence of TGF-beta2, unless IL-12 is added to the DC/T-cell coculture. Finally, we demonstrate that MHC class II expression and IL-12 secretion by DC are not disturbed by TGF-beta2 after DC stimulation with a modified maturation cocktail containing the Toll-like receptor (TLR)-ligands Poly I:C or R848, TNF-alpha, IL-1beta and INF-gamma. These data imply that mature DC retaining their capacity to produce IL-12 are of favorable use in glioma immunotherapy and suggest that TLR triggering of DC plays an important role to elicit a strong immune response in the immunosuppressive environment of malignant gliomas