Endothelial Indoleamine-2,3-Dioxygenase-1 is not Critically Involved in Regulating Antitumor Immunity in the Central Nervous System.

The vascular niche of malignant gliomas is a key compartment that shapes the immunosuppressive brain tumor microenvironment (TME). The blood-brain-barrier (BBB) consisting of specialized endothelial cells (ECs) and perivascular cells forms a tight anatomical and functional barrier critically controlling transmigration and effector function of immune cells. During neuroinflammation and tumor progression, the metabolism of the essential amino acid tryptophan (Trp) to metabolites such as kynurenine has long been identified as an important metabolic pathway suppressing immune responses. Previous studies have demonstrated that indoleamine-2,3-dioxygenase-1 (IDO1), a key rate-limiting enzyme in tryptophan catabolism, is expressed within the TME of high-grade gliomas. Here, we investigate the role of endothelial IDO1 (eIDO1) expression for brain tumor immunity. Single-cell RNA sequencing data revealed that in human glioma tissue, IDO1 is predominantly expressed by activated ECs showing a JAK/STAT signaling pathway-related CXCL11+ gene expression signature. In a syngeneic experimental glioma model, eIDO1 is induced by low-dose tumor irradiation. However, cell type-specific ablation of eIDO1 in experimental gliomas did not alter frequency and phenotype of tumor-infiltrating T cells nor tumor growth. Taken together these data argue against a dominant role of eIDO1 for brain tumor immunity.

Isocitrate-dehydrogenase-mutant lower grade glioma in elderly patients: treatment and outcome in a molecularly characterized contemporary cohort.

PURPOSE: Lower-grade glioma (LGG) is rare among patients above the age of 60 ("elderly"). Previous studies reported poor outcome, likely due to the inclusion of isocitrate dehydrogenase (IDH) wildtype astrocytomas and advocated defensive surgical and adjuvant treatment. This study set out to question this paradigm analyzing a contemporary cohort of patients with IDH mutant astrocytoma and oligodendroglioma WHO grade 2 and 3. METHODS: Elderly patients treated in our department for a supratentorial, hemispheric LGG between 2009 and 2019 were retrospectively analyzed for patient-, tumor- and treatment-related factors and progression-free survival (PFS) and compared to patients aged under 60. Inclusion required the availability of subtype-defining molecular data and pre- and post-operative tumor volumes. RESULTS: 207 patients were included, among those 21 elderlies (10%). PFS was comparable between elderly and younger patients (46 vs. 54 months; p = 0.634). Oligodendroglioma was more common in the elderly (76% vs. 46%; p = 0.011). Most patients underwent tumor resection (elderly: 81% vs. younger: 91%; p = 0.246) yielding comparable residual tumor volumes (elderly: 7.8 cm3; younger: 4.1 cm3; p = 0.137). Adjuvant treatment was administered in 76% of elderly and 61% of younger patients (p = 0.163). Uni- and multi-variate survival analyses identified a tumor crossing the midline, surgical strategy, and pre- and post-operative tumor volumes as prognostic factors. CONCLUSION: Elderly patients constitute a small fraction of molecularly characterized LGGs. In contrast to previous reports, favorable surgical and survival outcomes were achieved in our series comparable to those of younger patients. Thus, intensified treatment including maximal safe resection should be advocated in elderly patients whenever feasible.

Diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters (DGONC) - a molecularly defined glioneuronal CNS tumour class displaying recurrent monosomy 14.

AIMS: DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. PATIENTS AND METHODS: DNA methylation profiling was performed using the Infinium MethylationEPIC or 450 k BeadChip arrays (Illumina) and analysed using the 'conumee' package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. RESULTS: Genome-wide DNA methylation data from >25 000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. CONCLUSIONS: DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters.

Integrated molecular characterization of IDH-mutant glioblastomas.

AIMS: Mutations of isocitrate dehydrogenase (IDH)1/2 affect almost all astrocytomas of WHO grade II and III. A subset of IDH-mutant astrocytic tumours progresses to IDH-mutant glioblastoma or presents with the histology of a glioblastoma at first presentation. We set out here to assess the molecular spectrum of IDH-mutant glioblastomas. METHODS: We performed an integrated molecular analysis of a mono-centric cohort (n = 97); assessed through genome-wide DNA methylation analysis, copy-number profiling and targeted next generation sequencing using a neurooncology-tailored gene panel. RESULTS: Of these 97 IDH-mutant glioblastomas, 68 had a glioblastoma at first presentation ('de novo' IDH-mutant glioblastoma) and 29 emerged from a prior low-grade lesion ('evolved' IDH-mutant glioblastoma). Unsupervised hierarchical clustering of DNA methylation data disclosed that IDH-mutant glioblastoma ('de novo' and 'evolved') formed a distinct group separate from other diffuse glioma subtypes. Homozygous deletions of CDKN2A/B were found to be associated with shorter survival. CONCLUSIONS: This study demonstrates DNA methylation patterns in IDH-mutant glioblastoma to be distinct from lower-grade astrocytic counterparts but homogeneous within de novo and evolved IDH-mutant glioblastomas, and identifies CDKN2A as a marker for possible genetic sub-stratification.

Stem cell characteristics in glioblastoma are maintained by the ecto-nucleotidase E-NPP1.

Glioblastomas are highly aggressive brain tumours and are characterised by substantial cellular heterogeneity within a single tumour. A sub-population of glioblastoma stem-like cells (GSCs) that shares properties with neural precursor cells has been described, exhibiting resistance to therapy and therefore being considered responsible for the high recurrence rate in glioblastoma. To elucidate the underlying cellular processes we investigated the role of phosphatases in the GSC phenotype, using an in vitro phosphatome-wide RNA interference screen. We identified a set of genes, the knockdown of which induces a significant decrease in the glioma stem cell marker CD133, indicating a role in the glioblastoma stem-like phenotype. Among these genes, the ecto-nucleotidase ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) was found to be highly expressed in GSCs compared with normal brain and neural stem cells. Knockdown of ENPP1 in cultured GSCs resulted in an overall downregulation of stem cell-associated genes, induction of differentiation into astrocytic cell lineage, impairment of sphere formation, in addition to increased cell death, accumulation of cells in G1/G0 cell cycle phase and sensitisation to chemotherapeutic treatment. Genome-wide gene expression analysis and nucleoside and nucleotide profiling revealed that knockdown of ENPP1 affects purine and pyrimidine metabolism, suggesting a link between ENPP1 expression and a balanced nucleoside-nucleotide pool in GSCs. The phenotypic changes in E-NPP1-deficient GSCs are assumed to be a consequence of decreased transcriptional function of E2F1. Together, these results reveal that E-NPP1, by acting upstream of E2F1, is indispensable for the maintenance of GSCs in vitro and hence required to keep GSCs in an undifferentiated, proliferative state.

Antiglioma activity of GoPI-sugar, a novel gold(I)-phosphole inhibitor: chemical synthesis, mechanistic studies, and effectiveness in vivo.

Glioblastoma, an aggressive brain tumor, has a poor prognosis and a high risk of recurrence. An improved chemotherapeutic approach is required to complement radiation therapy. Gold(I) complexes bearing phosphole ligands are promising agents in the treatment of cancer and disturb the redox balance and proliferation of cancer cells by inhibiting disulfide reductases. Here, we report on the antitumor properties of the gold(I) complex 1-phenyl-bis(2-pyridyl)phosphole gold chloride thio-ß-d-glucose tetraacetate (GoPI-sugar), which exhibits antiproliferative effects on human (NCH82, NCH89) and rat (C6) glioma cell lines. Compared to carmustine (BCNU), an established nitrosourea compound for the treatment of glioblastomas that inhibits the proliferation of these glioma cell lines with an IC50 of 430µM, GoPI-sugar is more effective by two orders of magnitude. Moreover, GoPI-sugar inhibits malignant glioma growth in vivo in a C6 glioma rat model and significantly reduces tumor volume while being well tolerated. Both the gold(I) chloro- and thiosugar-substituted phospholes interact with DNA albeit more weakly for the latter. Furthermore, GoPI-sugar irreversibly and potently inhibits thioredoxin reductase (IC50 4.3nM) and human glutathione reductase (IC50 88.5nM). However, treatment with GoPI-sugar did not significantly alter redox parameters in the brain tissue of treated animals. This might be due to compensatory upregulation of redox-related enzymes but might also indicate that the antiproliferative effects of GoPI-sugar in vivo are rather based on DNA interaction and inhibition of topoisomerase I than on the disturbance of redox equilibrium. Since GoPI-sugar is highly effective against glioblastomas and well tolerated, it represents a most promising lead for drug development. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.

Analysis of CIC-associated CpG island methylation in oligoastrocytoma.

AIMS: Combined deletion of the whole chromosomal arms 1p and 19q is a frequent event in oligodendroglial tumours. Recent identification of recurrent mutations in CIC on 19q and FUBP1 on 1p and their mutational patterns suggest a loss of function of the respective proteins. Surprisingly, oligoastrocytomas harbouring identical genetic characteristics regarding 1p/19q codeletion and frequent IDH1/2 mutations have been shown to carry CIC mutations in a significantly lower number of cases. The present study investigates whether epigenetic modification may result in silencing of CIC. METHODS: As IDH1/2 mutation-mediated DNA hypermethylation is a prominent feature of these tumours, we analysed a set of CIC wild-type oligoastrocytomas and other diffuse gliomas with regard to 1p/19q status for presence of CIC-associated CpG island methylation by methylation-specific PCR. RESULTS: Both methylation-specific PCR and subsequent bisulphite-sequencing of selected cases revealed an unmethylated status in all samples. CONCLUSION: Despite the hypermethylator phenotype in IDH1/2 mutant tumours and recent detection of gene silencing particularly on retained alleles in oligodendroglial tumours, hypermethylation of CIC-associated CpG islands does not provide an alternative mechanism of functional CIC protein abrogation.

Epigenetic screen of human DNA repair genes identifies aberrant promoter methylation of NEIL1 in head and neck squamous cell carcinoma.

Aberrant promoter methylation of different DNA repair genes has a critical role in the development and progression of various cancer types, including head and neck squamous cell carcinomas (HNSCCs). A systematic analysis of known human repair genes for promoter methylation is however missing. We generated quantitative promoter methylation profiles in single CpG units of 160 human DNA repair genes in a set of DNAs isolated from fresh frozen HNSCC and normal tissues using MassARRAY technology. Ninety-eight percent of these genes contained CpG islands (CGIs) in their promoter region; thus, DNA methylation is a potential regulatory mechanism. Methylation data were obtained for 145 genes, from which 15 genes exhibited more than a 20% difference in methylation levels between tumor and normal tissues, manifested either as hypermethylation or as hypomethylation. Analyses of promoter methylation with mRNA expression identified the DNA glycosylase NEIL1 (nei endonuclease VIII-like 1) as the most prominent candidate gene. NEIL1 promoter hypermethylation was confirmed in additional fresh frozen HNSCC samples, normal mucosa, HNSCC cell lines and primary human skin keratinocytes. The investigation of laser-microdissected tissues further substantiated increased methylation levels in tumor versus matched non-tumor cells. Immunohistological analysis revealed significantly less NEIL1 protein expression in tumor tissues. 5-Aza-2'-deoxycytidine treatment and DNMT1 knockdown resulted in the re-expression of NEIL1 in HNSCC cell lines, which initially carried hypermethylated promoter regions. In conclusion, our results suggest that DNA methylation contributes to the downregulation of NEIL1 expression and might thus have a role in modulating the response to therapies of HNSCC.

Notch1 signaling promotes survival of glioblastoma cells via EGFR-mediated induction of anti-apoptotic Mcl-1.

The Notch1-mediated signaling pathway has a central role in the maintenance of neural stem cells and contributes to growth and progression of glioblastomas, the most frequent malignant brain tumors in adults. Here, we demonstrate that the Notch1 receptor promotes survival of glioblastoma cells by regulation of the anti-apoptotic Mcl-1 protein. Notch1-dependent regulation of Mcl-1 occurs cell type dependent at a transcriptional or post-translational level and is mediated by the induction of epidermal growth factor receptor (EGFR). Inhibition of the Notch1 pathway overcomes apoptosis resistance and sensitizes glioblastoma cells to apoptosis induced by ionizing radiation, the death ligand TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) or the Bcl-2/Bcl-XL inhibitor ABT-737. In conclusion, targeting Notch1 might represent a promising novel strategy in the treatment of glioblastomas.

RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival.

The concept of cancer stem-like cells (CSCs) has gained considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. This sub-population of tumor cells has been intensively investigated and their role in therapy resistance as well as tumor recurrence has been demonstrated. In that respect, development of therapeutic strategies that target CSCs (and possibly also the tumor bulk) appears a promising approach in patients suffering from primary brain tumors. In the present study, we utilized RNA interference (RNAi) to screen the complete human kinome and phosphatome (682 and 180 targets, respectively) in order to identify genes and pathways relevant for the survival of brain CSCs and thereby potential therapeutical targets for glioblastoma. We report of 46 putative candidates including known survival-related kinases and phosphatases. Interestingly, a number of genes identified are involved in metabolism, especially glycolysis, such as PDK1 and PKM2 and, most prominently PFKFB4. In vitro studies confirmed an essential role of PFKFB4 in the maintenance of brain CSCs. Furthermore, high PFKFB4 expression was associated with shorter survival of primary glioblastoma patients. Our findings support the importance of the glycolytic pathway in the maintenance of malignant glioma cells and brain CSCs and imply tumor metabolism as a promising therapeutic target in glioblastoma.

De-repression of CTGF via the miR-17-92 cluster upon differentiation of human glioblastoma spheroid cultures.

All-trans retinoic acid is a potent promoter of cellular differentiation processes, which is used in cancer therapy. Glioblastoma spheroid cultures are enriched in tumor-initiating cells, and provide a model to test new treatment options in vitro. We investigated the molecular mechanisms of response to exposure to differentiation-promoting conditions in such cultures. Microarray analyses of five independent cultures showed that after induction of differentiation, inhibitors of transforming growth factor-beta/bone morphogenetic protein, Wnt/beta-catenin and IGF signaling were upregulated, whereas expression of several microRNAs decreased, particularly that of the miR-17-92 cluster. In primary astrocytic gliomas (n=82), expression of several members of miR-17-92 was significantly higher relative to those of normal brain (n=8) and significantly increased with tumor grade progression (P<0.05). A high-level amplification of the miR-17-92 locus was detected in one glioblastoma specimen. Transfection of inhibitors of miR-17-92 induced increased apoptosis and decreased cell proliferation in glioblastoma spheroids. Mir-17-92 inhibition was also associated with increased messenger RNA (mRNA) and/or protein expression of CDKN1A, E2F1, PTEN and CTGF. The CTGF gene was shown to be a target of miR-17-92 in glioblastoma spheroids by luciferase reporter assays. Our results suggest that miR-17-92 and its target CTGF mediate effects of differentiation-promoting treatment on glioblastoma cells through multiple regulatory pathways.

Apoptosis-based treatment of glioblastomas with ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins.

Defects in the apoptotic signaling cascades contribute to the poor therapeutic response of malignant gliomas. As glioblastomas are characterized by high expression levels of anti-apoptotic Bcl-2 family proteins, we studied the effects of the novel Bcl-2 inhibitor, ABT-737, on malignant glioma cells. ABT-737 treatment released the pro-apoptotic Bax protein from its binding partner Bcl-2 and potently induced apoptotic cell death in glioblastoma cells in vitro and in vivo. The local administration of ABT-737 prolonged the survival in an intracranial glioma xenograft model. Downregulation of Mcl-1 and overexpression of Bcl-2 sensitized the cells to ABT-737-mediated apoptosis. Moreover, ABT-737 potentiated the cytotoxicity of the chemotherapeutic drugs vincristine and etoposide, and of the death ligand TRAIL. As glioma stem cells may play a crucial role for the tumor progression and the resistance to treatment in glioblastomas, we investigated the effects of ABT-737 on the subpopulation of glioma cells exhibiting stem cell characteristics. Inhibition of proliferation and induction of apoptosis by ABT-737 were less efficient in glioma stem cells than in non-stem cell-like glioma cells. As the resistance of glioma stem cells was associated with high Mcl-1 expression levels, ABT-737 treatment combined with downregulation of Mcl-1 could represent a promising novel approach in glioblastoma treatment.

The PEA-15/PED protein protects glioblastoma cells from glucose deprivation-induced apoptosis via the ERK/MAP kinase pathway.

PEA-15 (phosphoprotein enriched in astrocytes 15 kDa) is a death effector domain-containing protein, which is involved in the regulation of apoptotic cell death. Since PEA-15 is highly expressed in cells of glial origin, we studied the role of PEA-15 in human malignant brain tumors. Immunohistochemical analysis of PEA-15 expression shows strong immunoreactivity in astrocytomas and glioblastomas. Phosphorylation of PEA-15 at Ser(116) is found in vivo in perinecrotic areas in glioblastomas and in vitro after glucose deprivation of glioblastoma cells. Overexpression of PEA-15 induces a marked resistance against glucose deprivation-induced apoptosis, whereas small interfering RNA (siRNA)-mediated downregulation of endogenous PEA-15 results in the sensitization to glucose withdrawal-mediated cell death. This antiapoptotic activity of PEA-15 under low glucose conditions depends on its phosphorylation at Ser(116). Moreover, siRNA-mediated knockdown of PEA-15 abolishes the tumorigenicity of U87MG glioblastoma cells in vivo. PEA-15 regulates the level of phosphorylated extracellular-regulated kinase (ERK)1/2 in glioblastoma cells and the PEA-15-dependent protection from glucose deprivation-induced cell death requires ERK1/2 signaling. PEA-15 transcriptionally upregulates the Glucose Transporter 3, which is abrogated by the inhibition of ERK1/2 phosphorylation. Taken together, our findings suggest that Ser(116)-phosphorylated PEA-15 renders glioma cells resistant to glucose deprivation-mediated cell death as encountered in poor microenvironments, for example in perinecrotic areas of glioblastomas.

Human multidrug resistance protein 8 (MRP8/ABCC11), an apical efflux pump for steroid sulfates, is an axonal protein of the CNS and peripheral nervous system.

Dehydroepiandrosterone 3-sulfate and other neurosteroids are synthesized in the CNS and peripheral nervous system where they may modulate neuronal excitability by interacting with ligand-gated ion channels. For this modulatory activity, neurosteroids have to be locally released from either neurons or glial cells. We here identify the integral membrane protein ABCC11 (multidrug resistance protein 8) as an ATP-dependent efflux pump for steroid sulfates, including dehydroepiandrosterone 3-sulfate, and localize it to axons of the human CNS and peripheral nervous system. ABCC11 mRNA was detected in human brain by real-time polymerase chain reaction. Antibodies raised against ABCC11 served to detect the protein in brain by immunoblotting and immunofluorescence microscopy. ABCC11 was preferentially found in the white matter of the brain and co-localized with neurofilaments indicating that it is an axonal protein. Additionally, ABCC11 was localized to axons of the peripheral nervous system. For functional studies, ABCC11 was expressed in polarized Madin-Darby canine kidney cells where it was sorted to the apical membrane. This apical sorting is in accordance with the localization of ABCC11 to the axonal membrane of neurons. Inside-out plasma membrane vesicles containing recombinant ABCC11 mediated ATP-dependent transport of dehydroepiandrosterone 3-sulfate with a Km value of 21 microM. This transport function together with the localization of the ABCC11 protein in vicinity to GABAA receptors is consistent with a role of ABCC11 in dehydroepiandrosterone 3-sulfate release from neurons to sites of dehydroepiandrosterone 3-sulfate-mediated receptor modulation. Our findings may provide a basis for the characterization of mutations in the human ABCC11 gene and their linkage with neurological disorders.

Expression of mRNAs encoding for growth factors, ECM molecules, and MMP13 in mono-cultures and co-cultures of human periodontal ligament fibroblasts and alveolar bone cells.

Although the function and effects of many growth factors and extracellular matrix (ECM) molecules have been described for several periodontal tissues in vivo and in vitro, the molecular interactions involved in the communication between cells of the periodontal ligament and the alveolar bone are poorly understood. To contribute to the identification of such interactions, we have generated co-cultures (CCs) of periodontal ligament fibroblasts (PDLs) and alveolar bone cells (ABCs) and compared mRNA expression for various growth factors, ECM molecules, and matrix metalloproteinase13 (MMP13) after 1 and 2 weeks with matched mono-cultures (MCs) by reverse transcription/polymerase chain reaction. Compared with CCs of 1 week, PDLs and ABCs after 2 weeks revealed relatively high levels of all analyzed mRNAs, viz., for EGF, HGF, VEGF, TGFbeta1, collagen-I (COL1), osteonectin (ON), fibronectin (FN1), and MMP13. At week 2, when compared with MCs, CCs showed an elevation of all tested mRNAs in PDLs and ABCs, except for TGFbeta1 and FN1, which only increased in PDLs. After 1 week, when CCs were compared with MCs, mRNAs for HGF and TGFbeta1 were less abundant in PDLs and ABCs, whereas the other genes exhibited lower expression levels in only one of the cell types. Analysis of our data indicated that the expression of mRNAs for growth factors and for COL1, ON, FN1, and MMP13 was modulated in the distinct cell types with respect to culture time and culture type. The differences in the mRNA expression patterns between CCs and MCs suggest that the respective genes are involved in the molecular interactions of PDLs and ABCs.

[Pseudonormalization of diffusion weighted images: magnetic resonance imaging in an animal model (C6-glioma)]. / Pseudonormalisierung diffusionsgewichteter Aufnahmen: Magnetresonanztomografische Untersuchungen im Tiermodell (C6-Gliom).

PURPOSE: Previous studies on intracranial tumors indicate that a high apparent diffusion coefficient (ADC) is due to low cellularity and that lower values indicate a dense, highly cellular tumor. Diffusion is affected by three major factors: cell density, existence and distribution of vasogenic edema, and hypoxic tissue. Therefore, we studied the characteristics of diffusion-weighted magnetic resonance imaging (DWI) in a rat brain C6 glioma during tumor progression. MATERIALS AND METHODS: In male Wistar rats, C6 gliomas were implanted in the caudoputamen. At day 9, 11, 13 and 15 after tumor inoculation, conventional DWI was performed on a 2.35 Tesla small bore MRI unit (Biospec 24/40, BRUKER Medizintechnik, Ettlingen, Germany). RESULTS: On conventional T2-weighted and contrast-enhanced T1-weighted images, all tumors could well be delineated from the surrounding brain tissue and showed significant progression. On DWI, the tumors were isointense or slightly hypointense compared to the surrounding brain. On the ADC maps, the tumors could be well visualized due to increasing ADC values from day 9 to 15. The mean ADC of brain tumor tissue was 0.76 +/- 0.4 x 10 ( - 3) mm (2)/s at day 9 and 0.91 +/- 0.03 x 10 ( - 3) mm (2)/s at day 15. The mean ADC of the normal contralateral caudoputamen was 0.59 +/- 0.007 x 10 ( - 3)mm (2)/s. CONCLUSION: T2 prolongation and increased water diffusion can be balanced on DWI in C6 gliomas, resulting in isointensity on DWI (T2 shine-through washout phenomenon). ADC maps are indispensable for the correct interpretation of tumor tissue diffusion behavior.

Expression and immunolocalization of the multidrug resistance proteins, MRP1-MRP6 (ABCC1-ABCC6), in human brain.

Multidrug resistance proteins (MRPs, symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of organic anions, including cytotoxic and antiviral drugs, from cells. To identify MRP family members possibly involved in the intrinsic resistance of human brain to cytotoxic and antiviral drugs, we analyzed the expression and localization of MRP1-MRP6 in rapidly frozen perilesional samples of several regions of adult human brain obtained during neurosurgery. Quantitative polymerase chain reaction analysis showed expression of MRP1, MRP2, MRP3, MRP4, and MRP5 mRNA, whereas MRP6 mRNA was below detectability. However, immunofluorescence microscopy of cryosections from human brain showed no reactivity for the MRP2 or MRP3 proteins. The proteins MRP1, MRP4, and MRP5 were clearly localized by confocal laser scanning microscopy to the luminal side of brain capillary endothelial cells. The MRP4 and MRP5 proteins were also detected in astrocytes of the subcortical white matter. Notably, MRP5 protein was present in pyramidal neurons. MRP proteins may, thus, contribute to the cellular efflux of endogenous anionic glutathione or glucuronate conjugates (substrates for MRP1), cyclic nucleotides (substrates for MRP4 and MRP5), or glutathione (co-substrate for MRP1 and MRP4); in addition, they may play an important role in the resistance of the brain to several cytotoxic and antiviral drugs.