Neuropathology and Applied Neurobiology (2015), 41, e56–e67
doi: 10.1111/nan.12135
Tumour necrosis factor receptor superfamily member 9 (TNFRSF9) is up-regulated in reactive astrocytes in human gliomas A.-E. Blank*, P. Baumgarten*, P. Zeiner*, C. Zachskorn*†‡, C. Löffler§, J. Schittenhelm§, C. J. Czupalla*, D. Capper¶**, K. H. Plate*†‡, P. N. Harter*†‡ and M. Mittelbronn*†‡ *Edinger Institute, Institute of Neurology, Goethe University Frankfurt, Frankfurt am Main, †German Cancer Consortium (DKTK), ‡German Cancer Research Center (DKFZ), ¶Institute of Neuropathology, University of Heidelberg, **Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, and §Department of Neuropathology, Institute of Pathology and Neuropathology, University of Tuebingen, Tuebingen, Germany
A.-E. Blank, P. Baumgarten, P. Zeiner, C. Zachskorn, C. Löffler, J. Schittenhelm, C. J. Czupalla, D. Capper, K. H. Plate, P. N. Harter and M. Mittelbronn (2015) Neuropathology and Applied Neurobiology 41, e56–e67 Tumour necrosis factor receptor superfamily member 9 (TNFRSF9) is up-regulated in reactive astrocytes in human gliomas Aims: The prognosis of patients with malignant gliomas is still dismal despite maximum treatment. Novel therapeutic alternatives targeting tumorigenic pathways are, therefore, demanded. In murine glioma models, targeting of tumour necrosis factor receptor superfamily (TNFRSF) 9 led to complete tumour eradication. Thus, TNFRSF9 might also constitute a promising target in human diffuse gliomas. As there is a lack of data, we aimed to define the expression pattern and cellular source of TNFRSF9 in human gliomas. Methods: We investigated TNFRSF9 expression in normal human central nervous system (CNS) tissue and glioma specimens using immunohistochemistry, immunofluorescence and Western blotting techniques. Results: Our results show
that TNFRSF9 is considerably up-regulated in human gliomas when compared with normal brain tissue. In addition, our data provides evidence for an immune cellindependent de novo expression pattern of TNFRSF9 in mainly non-neoplastic reactive astrocytes and excludes classic immunological cell types, namely lymphocytes and microglia as the source of TNFRSF9. Moreover, TNFRSF9 is predominantly expressed in a perivascular and peritumoural distribution with significantly higher expression in IDH-1 mutant gliomas. Conclusions: Our findings provide a novel, TNFRSF9-positive, reactive astrocytic phenotype and challenge the therapeutic suitability of TNFRSF9 as a promising target for human gliomas.
Keywords: astrocytes, CD137, glioma, gliosis, TNFRSF9
Introduction Human central nervous system (CNS) tumours consist of heterogeneous cell populations including neoplastic, as Correspondence: Michel Mittelbronn, Edinger Institute, Institute of Neurology, Goethe University Frankfurt, D-60528 Frankfurt, Germany. Tel: +49 (0) 69 6301 84169; Fax: +49 (0) 69 6301 84150; E-mail: [email protected]
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well as reactive, glial, immune and vessel-associated cells [1]. As patient prognosis is still dismal even with maximum treatment, which includes surgical resection in combination with radio- and chemotherapy, more specific anti-tumoural therapy strategies are urgently needed. Among other approaches, immunotherapy showed promising results in cell culture and animal studies and has already entered clinical investigation. In animal experi© 2014 British Neuropathological Society
TNFRSF9 expression in gliomas
ments, tumour necrosis factor receptor superfamily member 9 (TNFRSF9) has been described as up-regulated in tumour-infiltrating lymphocytes (TILs). These cells can be stimulated by anti-TNFRSF9 monoclonal antibodies resulting in anti-tumoural effects [2]. TNFRSF9-activating agonistic treatment approaches are currently in clinical trials for treatment of melanoma, renal cell carcinoma and ovarian cancer [3]. In preclinical transplantable murine tumour models, agonistic anti-TNFRSF9 treatment prevented recurrences as well as metastases and even showed curative effects [4,5]. The anti-tumoural effects targeting TNFRSF9 have been attributed to enhanced cellular proliferation, survival and cytokine production of TILs in both animal and human studies [6,7]. These pro-survival, anti-tumoural capacities were mainly conferred to CD8 cytotoxic T-cells [8]. Other immune cells, such as monocytes, were also activated by TNFRSF9 stimulation. This resulted in increased proliferation, viability, morphological changes and increased adhesion capacity; TNFRSF9-related differentiation into dendritic cells was present in human but absent in murine monocytes [9]. Moreover, it has been reported that TNFRSF9-activating strategies further enhanced antibody-dependent cellular cytotoxicity (ADCC) in breast cancer patients, pointing to an additive effect of tumour-targeting antibodies such as trastuzumab (monoclonal anti-HER2 antibody) and secondary antibodies boosting the immune system [10,11]. In addition, TNFRSF9-activatory approaches increased anti-tumoural effects exerted by oncolytic viruses [12]. Most interestingly, anti-TNFRSF9 treatment revealed the best results in intracranial animal glioma models compared with subcutaneous or metastatic tumours, with animals surviving significantly longer and even achieving curative effects [13]. In murine glioma models, the combination of irradiation and an anti-TNFRSF9-based immunotherapy led to complete tumour eradication in over 60% of the glioma-harbouring animals; this was most likely related to increased TILs and production of interferon-gamma [14]. Apart from its immune modulatory anti-tumour functions, activation of TNFRSF9 on hypoxic intratumoural endothelial cells leads to increased expression of various cell adhesion molecules including: intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin, thereby facilitating the entry of activated T-lymphocytes into the tumour tissue [15]. As TNFRSF9 and its ligand TNFSF9 act in a bidirectional way, © 2014 British Neuropathological Society
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immune cells expressing TNFSF9 can be reversely stimulated after interaction with TNFRSF9 on endothelial cells [16]. A potential shortcoming of the promising preclinical studies is related to an opposite effect of the TNFRSF9TNFSF9 system in murine models and humans [17]. To date, the expression and functional role of TNFRSF9 in the normal and pathological human CNS is not entirely clear. While at first the data suggested that TNFRSF9 is mainly expressed on neurones and astrocytes, its ligand TNFSF9 is mainly found on microglial cells [18,19]. In our study, we aimed to define the expression pattern and cellular source of TNFRSF9 in human gliomas.
Materials and methods
Patient material The study is based on an analysis of 1094 formalin-fixed paraffin-embedded tissue samples from 416 patients suffering from a glial brain tumour (see Table 1). Tissue samples were retrieved from the archives of the Neurological Institute (Edinger Institute) of the Goethe University Frankfurt, Germany. For statistical analyses, tissue micro arrays were used with cores of representative tumour centre, infiltration zone and surrounding normal appearing brain tissue. In addition, exemplary whole mount sections, including five CNS tissue samples from normal CNS autopsy cases, were stained. Specimens were cut with a microtome (3 μm thickness) and placed on SuperFrostPlus slides (Microm International, Walldorf, Germany). The use of patient material was endorsed by the local ethical committee of the Goethe University Frankfurt, Germany (GS 04/09; NP 03/11).
Immunohistochemistry For immunohistochemistry a mouse monoclonal antihuman TNFRSF9 antibody (dilution 1:40; clone S16 Novocastra/Leica Microsystems, Wetzlar, Germany) was used. Tissue labelling was performed using the DiscoveryXT immunohistochemistry system (Ventana Medical Systems, Illkirch, France). A cell-conditioning pretreatment was performed for 68 min followed by a 4-min blocking step with Inhibitor D. The primary antibody was applied for 32 min, followed by a secondary antibody (Discovery Universal Secondary Antibody) for 32 min. After washing steps, a blocking step with Blocker D for 4 min and a 16-min incubation with one drop of SA-HRP D were NAN 2015; 41: e56–e67
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Table 1. Patient data of pilocytic and diffuse gliomas
Pilocytic astrocytoma Diffuse astrocytoma Oligodendroglioma Oligoastrocytoma Anaplastic astrocytoma Anaplastic oligodendroglioma Anaplastic oligoastrocytoma Glioblastoma
WHO grade
Male/ female
Median age (range)
Patients (n)
Tumour localization (supratentorial/ infratentorial)
I II II II III III III IV
17/30 10/6 3/5 5/11 18/15 9/8 20/11 140/108
14 31 34.5 42.5 43 49 43 61
47 16 8 16 33 17 31 248
16/31 16/0 8/0 16/0 32/1 17/0 31/0 246/2
(0–75) (5–52) (4–65) (21–67) (22–66) (19–67) (23–71) (7–80)
mIDH-1 (R132H, mut/wt)
Ki-67 index (in %, 95% CI)
0/46 8/6 4/4 13/3 18/15 9/8 22/9 9/239
2.7 0.9 0.8 0.6 6.2 4.6 4.0 13.0
(1.7–3.8) (0.2–1.5) (0.0–2.3) (0.2–1.0) (3.6–8.7) (1.1–8.1) (1.4–6.7) (11.6–14.3)
mut/wt, mutant/wild-type; CI, confidence interval.
performed. For diaminobenzidine (DAB) visualization, the sections were incubated with one drop of DAB D and one drop of DAB H2O2 D for 8 min, followed by a copper enhancer (Copper D, all Ventana Medical Systems) for 4 min. Specimens were washed and consequently counterstained with haematoxylin and bluing reagent and mounted. For double immunohistochemistry, anti-CD137 staining was followed by a secondary antibody step using the RedMap or BlueMap detection kit (all Ventana Medical Systems) and the following primary antibodies (incubation time 32 min each): rabbit polyclonal anti-human GFAP (dilution 1:10000; Z0334; Dako, Hamburg, Germany), rabbit polyclonal anti-human Olig2 (dilution 1:500; AB9610; Millipore, Darmstadt, Germany), mouse monoclonal IgG2a anti-human IDH-1 R132H (dilution 1:50; clone H09; Dianova, Hamburg, Germany), mouse monoclonal IgG1 anti-human CD68 (dilution 1:500; clone KP1; Dako), rabbit polyclonal anti-human Iba-1 (dilution 1:1000; Wako Chemicals, Richmond, VA, USA), mouse monoclonal IgG1 anti-human CD45/LCA (dilution: 1:2000; clone 2B11+PD7/26, Dako), mouse monoclonal IgG1 anti-human Ki-67 (dilution 1:200; clone MIB-1; Dako), mouse monoclonal IgG2a anti-human BRAF V600E (dilution 1:100; clone VE1, Spring Bioscience, Pleasanton, CA, USA) and rabbit monoclonal IgG anti-human phospho-S6 Ribosomal Protein (Ser240/ 244) (dilution 1:2000; clone D68F8, Cell Signaling Technology, Danvers, MA, USA). Subsequently, specimens were incubated with one drop of secondary antibody mix (Discovery Universal Secondary Antibody), followed by a blocking step for 4 min and a signal amplification via streptavidin alkaline phosphatase © 2014 British Neuropathological Society
(Blocker R1, SA-Alk Phos R) for 16 min. For visualization, chromogen was added (Fast Red for 16 min, BlueMap for 24 min), then samples were repeatedly washed and heated and finally counterstained using haematoxylin II and bluing reagent for 12 min (all Ventana Medical Systems). Images were analysed and recorded on an Olympus BX-50 microscope (Olympus, Hamburg, Germany).
Primary tumour cultures Native tumour tissue was taken directly from the surgical intervention, evaluated macroscopically by a neuropathologist and processed under sterile conditions. First, mechanical dissociation steps were performed followed by an enzymatic dissociation using a solution containing Leibovitz-L15 medium (Life technologies, Carlsbad, CA, USA), Papain suspension (Worthington Biochemical, Lakewood, NJ, USA), EDTA 0.5 M (AppliChem, Darmstadt, Germany) and Desoxyribonuclease 1 Typ II (DNAse1, Sigma-Aldrich, St. Louis, MO, USA). The shredded cell suspension was incubated for 30 min at 37°C, mixed every 10 min and afterwards washed and centrifuged several times with DMEM-F12 medium (Life technologies). Cells were cultured over two subsequent passages under either serum-containing or serum-free conditions to investigate the expression of TNFRSF9 in conditions that are known to preserve the primary tumours’ genetic pattern (DMEM, Life technologies; FCS, Provitro, Germany; Penicillin/Streptomycin, SigmaAldrich; neurosphere medium containing additional growth factors as EGF and bFGF: DMEM-F12, B27 Supplement and HEPES, Life technologies; EGF and bFGF, PeproTech; Penicillin/Streptomycin, Sigma-Aldrich). NAN 2015; 41: e56–e67
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Microglia isolation To obtain primary microglial cells from normal and glioma tissue, we used magnetic cell separation (MACS® Sorting, Miltenyi Biotec, Bergisch Gladbach, Germany). The native tissue was fully dissociated mechanically and enzymatically to gain a cell suspension that was incubated with antibody-linked magnetic particles against the microglial CD11b epitope (MicroBeads). The resulting cell suspension was subsequently separated via MACS Columns in a magnetic field, in which the antibody-linked microglial cells were collected and the non-labelled cells discarded (all Miltenyi Biotec).
Immunofluorescence Immunofluorescent stainings were performed using a mouse monoclonal anti-human TNFRSF9 antibody (dilution 1:40; clone S16 Novocastra/Leica Microsystems). After a centrifugation step, media was removed from the primary tumour spheres, followed by two washing steps with PBS (Life Technologies). Afterwards, tumour spheres were fixed for 10 min in precooled methanol at −20°C (Sigma-Aldrich). This procedure was followed by a blocking step with Roti blocking solution (1:10, Roth, Karlsruhe, Germany) for 1 h at room temperature (RT). The sphere specimens were incubated using the primary anti-human TNFRSF9 antibody overnight at 4°C and subsequently labelled with the first secondary antibody for 1 h at RT (dilution 1:500; Alexa Fluor568, human antimouse IgG, Invitrogen, Carlsbad, CA, USA). Nuclear counterstaining was performed using Topro-3 (dilution 1:500, Invitrogen). Fluorescence images were analysed and recorded on a Nikon Eclipse 80i fluorescence microscope (Nikon, Düsseldorf, Germany). After recording, digital images were adjusted with ImageJ (National Institutes of Health, Bethesda, MD, USA).
Western blot Isolated cells and primary tumour tissue were collected to generate protein lysates for Western blotting. After centrifugation, the cell suspension was transferred into liquid nitrogen and the cracked cells were stored for at least 1 h at −80°C. RIPA-Lysis buffer was used for protein retrieval. Afterwards, the protein lysates were quantified by a BCAAssay (Micro BCA™ Protein Assay Kit, Thermo Scientific, Waltham, MA, USA). Gel electrophoresis was performed © 2014 British Neuropathological Society
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using 12.5% SDS-polyacrylamide gels with 20 μg protein samples resuspended in sample buffer according to standard procedures. The fractionated cells were transferred to a nitrocellulose membrane (Amersham Hybond ECL Membrane, GE Healthcare, Little Chalfont, Buckinghamshire, UK) using a blotting chamber for 30 min according to the manufacturer’s protocols (Bio-Rad Laboratories, Hercules, CA, USA). To avoid unspecific antibody signals, the membrane was blocked by blocking solution for at least 1 h at RT (Rotiblock in PBS, Roth). Several initial washing steps using PBS/T (Tween 20, Sigma-Aldrich) were followed by incubation with the primary mouse monoclonal antihuman TNFRSF9 antibody as previously described (clone S16 Novocastra/Leica Microsystems). A rabbit polyclonal IgG anti-human β-Actin antibody (Abcam, Cambridge, UK) was used serving as loading control. Primary antibodies were applied for 2 h and after additional washing steps the secondary antibody was added for 1 h. After removal of nonbinding antibodies by five washing steps with PBS/T and PBS, respectively, immunodetection was performed using horseradish peroxidase substrate Luminol reagent (Santa Cruz Biotechnology, Dallas, TX, USA) for 3 min. The blots were analysed on a X-ray film (Super RX, Fujifilm, Tokio, Japan) and developed with a X-ray film processor (Curix 60, AGFA, Mortsel, Belgium).
Scoring TNFRSF9 expression was assessed by taking both staining intensity and frequency into account using a previously established protocol [20]. The semiquantitative scores consist of a frequency score ranging from 0 to 4: 0: 0–1%, 1: 1–10%, 2: 10–25%, 3: 25–50% and 4: >50% of all cells showing a positive nuclear staining. Likewise, intensity was recorded in a similar semiquantitative approach as follows: 0: no staining, 1: weak staining, 2: moderate staining, 3: strong staining. The results for staining intensity and frequency were multiplied, such that the final expression cell score reflected both. The evaluation and photographic documentation of the immunohistochemical staining was performed using an Olympus BX50 light microscope.
TCGA and REMBRANDT platform analyses Gene expression signatures were analysed in 424 primary gliomas and 11 normal brain samples by assessing the TCGA data portal which applied the Agilent 244 K NAN 2015; 41: e56–e67
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G4502A microarray to determine mRNA profiles (http:// tcga.cancer.gov/) [21]. Data were assessed in November 2011.
Statistical analysis The semiquantitative TNFRSF9 scores were assigned as an ordinal scale response variable and statistical differences among WHO grades were assessed using the non-parametric Wilcoxon test. A significance level of alpha = 0.05 was selected for all tests. Statistical analysis was performed using JMP 8.0 software (SAS, Cary, NC, USA).
Results
TNFRSF9 is overexpressed in astrocytomas compared with normal CNS tissue Analysis of the TCGA database including 424 glioblastomas revealed that the median log2-fold mRNA
expression change for TNFRSF9 was considerably increased (by 0.54-fold) as compared with normal CNS tissue specimens (n = 11) (data not shown). To assess the in vivo expression of TNFRSF9, we first screened human tonsil tissue (Figure 1A) and the Jurkat human T-cell lymphoma line (Figure 1B), both of which were found to express membranous TNFRSF9. In contrast, normal human grey (Figure 1C) and white matter (Figure 1D) were almost completely TNFRSF9-negative. Only larger meningeal blood vessels presented with TNFRSF9-positive vessel walls, which is in line with previously published data reporting constitutive vascular TNFRSF9 expression (data not shown). In non-diffusely infiltrating human pilocytic astrocytomas WHO grade I (Figure 1E) and a large cohort of diffusely infiltrating gliomas (Figure 1F–L), prominent TNFRSF9 expression was frequently observed in a perivascular distribution pattern. In addition, TNFRSF9 was detected on large swollen cells within glial neoplasms. These cells were frequently localized in equal distances from neighbouring TNFRSF9-positive cells (Figure 1F,J) or
Figure 1. TNFRSF9 expression is up-regulated in diffuse human gliomas. TNFRSF9 immunohistochemistry in (A) normal human tonsil; (B) Jurkat cells (immortalized T lymphocyte cells); (C) normal-appearing grey matter (NAGM); (D) normal-appearing white matter (NAWM); (E) pilocytic astrocytoma, WHO grade I with vascular proliferations; (F) diffuse astrocytoma, WHO grade II; (G) anaplastic astrocytoma, WHO grade III; (H) primary glioblastoma WHO grade IV; (I) secondary glioblastoma, WHO grade IV; (J) anaplastic oligoastrocytoma, WHO grade III; (K, L) anaplastic oligodendroglioma, WHO grade III (scale bars: 100 μm in A, C–J; 50 μm in B; 200 μm in K, L). © 2014 British Neuropathological Society
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Figure 2. TNFRSF9 expression in glioma subtypes. Box-and-Whisker plots for TNFRSF9 expression in different glioma subtypes scores are depicted. Significant P-values from all pairwise comparisons of different WHO grade astrocytomas were obtained using the non-parametric Wilcoxon test. A significance level of alpha = 0.05 was selected for all comparisons. Numbers for each tumour subtype are as follows: PA: n = 47; DA: n = 16; OD: n = 8; OA: n = 18; AA: n = 33; AOD: n = 17; AOA: n = 31; GBM: n = 248.
showed very long cellular protrusions directed towards blood vessels (Figure 1H,I,K). The statistical comparison (Figure 2) of TNFRSF9 expression between the different glioma entities and malignancy grades revealed that highest scores were observed in diffuse astrocytomas WHO grade II (median expression score 2), while lowest levels were detected in WHO grade II oligodendrogliomas (median expression score 0.5). Most interestingly, in all investigated gliomas more pronounced TNFRSF9 expression was observed in cases harbouring the IDH-1 R132H mutation (see Table 1) reaching statistical significance for anaplastic astrocytomas WHO grade III (Figure 3A; P = 0.002), anaplastic oligoastrocytomas WHO grade III (Figure 3B; P = 0.025) and glioblastomas WHO grade IV (Figure 3C; P = 0.001) as compared with their wild-type counterparts.
TNFRSF9 expression is almost exclusively restricted to cells with astrocytic differentiation in human gliomas At a cellular level, TNFRSF9-positive cells within gliomas often presented with bizarre morphological features including, either thicker and shorter or longer and thinner protrusions (Figure 4A). TNFRSF9-positive cells were frequently bi- or multinucleated, typically with © 2014 British Neuropathological Society
nuclei being located at the outer border of the cell. In multiple glioma cases, these cells formed an almost complete circle surrounding prominent glioma-associated blood vessels (Figure 4A). Double immunohistochemical stainings revealed that TNFRSF9-positive cells strongly expressed GFAP (glial fibrillary acidic protein). While strongest GFAP expression was observed within cytoplasms, TNFRSF9 was most prominently expressed at the cellular membrane and on cellular protrusions (Figure 4B). Notably, GFAP expression was much stronger in TNFRSF9-positive cells as compared with surrounding TNFRSF9-negative cells. In contrast, TNFRSF9 and Olig2 expression were mutually exclusive, indicating that oligodendrocytes are most likely not a cellular source of TNFRSF9 (Figure 4C). Although Olig2 is not specific for cells with oligodendroglial differentiation, it is a reliable, sensitive tool to detect those cells. Furthermore, TNFRSF9-expressing cells were also negative for the leucocyte marker CD45 (Figure 4D) and for macrophage and microglial markers including CD68 (Figure 4E) and Iba-1 (data not shown). Finally, we assessed the proliferative potential of the TNFRSF9positive cellular fraction. Most Ki-67-positive cells lacked TNFRSF9 expression and, likewise, the TNFRSF9-positive cell fraction did not exhibit proliferative potential (Figure 4F). NAN 2015; 41: e56–e67
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Non-neoplastic, reactive astrocytes are the major cellular source of TNFRSF9 in diffuse gliomas
Figure 3. TNFRSF9 expression is associated with IDH-1 mutation status. Box-and-Whisker plots for the semiquantitative TNFRSF9 scores were assigned as ordinal scale response variables and statistical differences between IDH-1 mutated tumours in different glioma subtypes were assessed using the non-parametric Wilcoxon test. A significance level of alpha = 0.05 was selected for all comparisons.
© 2014 British Neuropathological Society
Using an antibody specific to the mutated R132H variant of the IDH-1 protein, we verified that TNFRSF9 was expressed in close proximity to the tumour bulk, while TNFRSF9-positive cells were absent in areas lacking IDH-1 R132H expression (Figure 5A,B). To address the question of whether the non-proliferating TNFRSF9-positive cell fraction is constituted of tumour cells or, more likely, of reactive glial cells, double immunostainings for TNFRSF9 and mutated IDH-1 (R132H) were performed. Double stainings revealed that TNFRSF9 was not expressed on IDH-1-mutated tumour cells (Figure 5C,D). Of note, some less frequent and non-diffuse CNS tumours (Supplemental Table S1), such as pleomorphic xanthoastrocytomas (PXA) (Supplemental Figure S1A) and subependymal giant cell astrocytomas (SEGA) (Supplemental Figure S1B), also displayed TNFRSF9 expression in tumour cells. Typically, these tumour cells harbour BRAF V600E mutation (PXA) or an alteration in the mTOR pathway (SEGA), reflected by phosphoS6-positive cells. In focal cortical dysplasias (FCD), hypertrophic or oligodendroglial-like TNFRSF9-positive cells were present (Supplemental Figure S1C). Conversely, gangliogliomas (Supplemental Figure S1D) contained very few TNFRSF9positive cells resembling those encountered in diffuse gliomas. In an additional cohort of distinct non-neoplastic CNS pathologies, strong TNFRSF9 expression was also consistently found in a subset of reactive astrocytes (Supplemental Figure S2). To corroborate the in vivo data found in diffuse gliomas, we investigated whole tumour lysates and primary glioblastoma cell cultures as well as primary glioblastoma-derived microglia cultures for TNFRSF9 expression by immunoblotting (Figure 5E). As expected, primary glioblastoma-derived microglia were completely devoid of TNFRSF9, while lysates from both whole tumour samples and primary glioblastoma cell cultures exhibited weak to moderate TNFRSF9 expression (Figure 5E). To determine if tumour spheroids express TNFRSF9 under in vitro conditions, we cultivated primary glioblastoma cells under neurosphere conditions (Figure 5F). The vast majority of neoplastic cells within the spheres remained TNFRSF9-negative, while only a small cellular fraction exhibited strong TNFRSF9 expression (Figure 5F). Similarly to the in vivo findings, the TNFRSF9-positive cells in the spheres displayed multiple long cellular processes and large cytoplasms, pointing to a reactive astrocyte morNAN 2015; 41: e56–e67
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Figure 4. TNFRSF9 is exclusively expressed on non-proliferative cells with astrocytic differentiation within diffuse gliomas. (A) TNFRSF9 expression on single multinucleated swollen cells and perivascular cell cuffs within diffuse gliomas; (B) co-expression of TNFRSF9 (brown) and glial fibrillary acid protein (GFAP, red) on swollen cell bodies with enlarged processes; no cellular co-expression of the (C) oligodendroglial marker Olig2 (red), (D) the leucocyte/lymphocyte marker CD45 (LCA, leucocyte common antigen, blue) or (E) the macrophage/microglia marker CD68 (red) with TNFRSF9 (brown) was observed; (F) TNFRSF9-positive cells (brown) lacked proliferative activity as indicated by immunopositivity for the proliferation marker Ki-67 (red). (Scale bars: 25 μm in B–F.)
phology. These findings indicate that TNFRSF9-positive reactive astrocytes might survive under cell culture conditions where they are still intermingled with neoplastic glial cells.
Discussion Several preclinical trials applying an activating antiTNFRSF9 treatment revealed curative results in different transplantable murine tumours, including gliomas [4,5,13,14]. As the exact cellular source of TNFRSF9 in the human CNS is still unclear, the aim of our study was to define the expression pattern of TNFRSF9 in human glioma and normal CNS tissue, thereby assessing its potential value for future targeted treatment approaches. Our findings indicate that TNFRSF9 is strongly overexpressed in human diffuse gliomas as compared with the normal CNS at both mRNA (data not shown) and protein levels (Figures 1 and 2). Most interestingly, high TNFRSF9 expression was significantly associated with gliomas harbouring the IDH-1 R132H mutation (Figures 2 and 3). By analysing those tumours, the cellular source of TNFRSF9 © 2014 British Neuropathological Society
expression could be linked to non-proliferative, frequently multinucleated astrocytic cells showing bizarre histomorphological features (Figures 4 and 5). This particular cellular fraction presented with a distinct distribution pattern and was most prominently observed in perivascular and peritumoural locations (Figure 5). Using double immunohistochemical staining methods, we were able to define the main cellular source of TNFRSF9 as non-proliferative, non-IDH-1-mutated, GFAP-positive, Olig2-negative astrocytes in diffuse gliomas, which, in combination with their morphological features, could be referred to as reactive astrocytes (Figure 5). Similarly, TNFRSF9-positive astrocytes with reactive changes have been detected in other neoplastic and non-neoplastic CNS pathologies (Supplemental Figure S2). However, neoplastic TNFRSF9-positive cells have also been detected in rare, non-diffuse brain tumours such as PXA and SEGA (Supplemental Figure S1). Although the anti-tumoural effects of agonistic anti-TNFRSF9 treatment in experimental glioma models are not yet fully understood, increased numbers of glioma-infiltrating lymphocytes were observed in these cases [14]. For other tumour entities, such as melanomas, NAN 2015; 41: e56–e67
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Figure 5. Non-neoplastic, reactive astrocytes constitute the cellular source of TNFRSF9 in diffuse gliomas. Immunohistochemical analyses using mutation-specific antibodies against the (A) IDH-1 R132H variant and (B) TNFRSF9 reveal a strong regional overlap between IDH-1 R132H-positive tumour cells of an anaplastic oligoastrocytoma, WHO grade III and TNFRSF9 expression, which is mainly seen in a perivascular distribution or at the tumour border. (C, D) Costainings of TNFRSF9 and mutated IDH-1 R132H excluding a co-expression of TNFRSF9 in neoplastic glial cells. (E) Immunoblotting of primary glioma spheres (lane 1), different primary glioma tissues (lanes 2–5) or primary glioma-derived microglial cells (lanes 6–7) revealing absence of TNFRSF9 in microglial cells while being present in primary glioma-derived cells or tissues. (F) Immunofluorescent analyses for TNFRSF9 were performed on primary glioma-derived spheres. Of note, as seen in primary glioma tissues, only single TNFRSF9-positive cells with long cellular protrusions were observed within the cell culture while the vast majority of cells remained TNFRSF9-negative. (Scale bars: 100 μm in A, B; 50 μm in C; 25 μm in D.) © 2014 British Neuropathological Society
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it was demonstrated that TNFRSF9 stimulation enhanced the survival and effector function of the CD8+ T-cell fraction [22]. Most recently, a novel TNFRSF9-stimulated, eomesodermin-driven CD4+ T-cell phenotype producing the cytokines IL-27, IL-15 and IL-10 capable of enhancing multipotent cytotoxic capacity has been discovered [23]. In our patient cohort, TNFRSF9 was virtually absent from glioma-infiltrating lymphocytes or microglia/ macrophages (Figure 4); a direct immune-modulatory activation of CNS intrinsic T-cells in the context of human gliomas is, therefore, highly questionable. As antitumoural effects and prolonged survival were observed in experimental intracranial sarcomas and gliomas, whereas only minimal therapeutic effects were observed in the same therapeutic approach in subcutaneous and pulmonary tumours, a general impact on the brain microenvironment was assumed [13]. Considering that the CNS is an immunoprivileged organ due to the blood-brain barrier and that only a few infiltrating lymphocytes were detected in our specimens, the finding that intracranial tumours were sensitive to systemic mAb treatment raises the question of whether or not other specialized resident immune cells, such as microglia, or alternative members of the brain microenvironment increase distinct anti-tumoural immune response. In addition, it has to be considered that a preoperative steroid treatment that avoids peritumoural oedema and is often applied in glioblastoma patients, might lead to a decreased glial reactivity and a diminished lymphocytic invasion [24–27]. In vitro studies revealed that TNFRSF9 is up-regulated by astrocytes upon stimulation with the Ca(2+)-binding protein S100B that is frequently increased during CNS pathologies [28]. The astrocytic expression of TNFRSF9 was linked to an enhanced activation status of the respective astrocytes leading to decreased neuroprotection and a modified interaction with microglia and inflammatory cells. The fact that reactive astrogliosis is potentially driven by the nuclear factor-kappa B (NF-kappaB) [29] is critical for our study, as TNFRSF9-mediated signalling mainly results in the activation of NF-kappaB [30]. Furthermore, there is an association of TNFRSF9 and the transcription factor signal transducer and activator of transcription 3 (STAT3) that contributes to the activation of astrocytes, via binding to the GFAP promoter constituting an important feature for the formation of both reactive gliosis and neoplastic astrocytes [31–33]. The question of why IDH-1-mutated specimens especially showed a prominent amount of reactive astrocytes with TNFRSF9 expression remains elusive. © 2014 British Neuropathological Society
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However, recent studies revealed that cells harbouring the mutated IDH-1 variant (R132H) produce considerably lower levels of glutathione, which is the major scavenger for radical oxygen species (ROS) [34]. The fact that ROS constitute one of the most potent inductors of reactive astrogliosis might therefore be a suitable explanation of why IDH-1-mutated gliomas exhibit significantly more TNFRSF9-associated astrogliosis [35]. Another potential explanation for significantly higher TNFRSF9 levels in IDH-1 mutant gliomas, as compared with their nonmutated counterparts, might be related to a slower growth rate in IDH-1 mutant gliomas which could allow for gliosis to develop over a longer time period. In summary, our findings reveal a novel, TNFRSF9positive, reactive astrocytic phenotype in human gliomas. One can hypothesize that most prominent TNFRSF9 signals, which are observed at the interface between glioma and CNS tissue and/or blood vessels, might be related to the fact that those areas constitute the primary site where residual astrocytes are confronted and potentially activated by glioma cells. Additionally, our study strongly challenges the applicability of successful animal glioma studies targeting TNFRSF9 to human clinical studies. However, because the role of reactive astrogliosis in gliomas is still debated, it is far too early to speculate about the suitability of therapeutically targeting TNFRSF9 in human brain tumours or other CNS disorders presenting with pronounced astrogliosis.
Acknowledgements We would like to acknowledge the support of the Deutsche Krebshilfe (A-EB) for this work. We thank Cara Laudon and Zach B. Bjornson for medical writing assistance.
Author contributions Conceived and designed the experiments: A-EB, PNH, MM. Performed the experiments: A-EB, PB, PZ, CZ, CL, CJC, DC, PNH, MM. Analysed the data: A-EB, PB, PZ, CL, DC, PNH, MM. Contributed reagents, materials, financial support: A-EB, PB, JS, KHP, MM. Wrote the paper: A-EB, PNH, MM. Supervisor of the study: PNH, MM. Corrected and approved the final version of the manuscript: all authors. NAN 2015; 41: e56–e67
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Conflict of interest The authors declare that they have no conflict of interest. Anna-Eva Blank was funded by the German Cancer Aid Foundation.
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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Material and methods: Patient data. Figure S1. TNFRSF9 expression in primary non-diffuse tumours and malformations. Haematoxylin and eosin (HE) stainings (left A–D) and immunohistochemistry for TNFRSF9 (middle A–D and C–D right) as well as for BRAF V600E (right A) and phosphoS6 (right B) in (A) a pleomorphic xanthoastrocytoma (PXA), (B) subependymal giant cell astrocytoma (SEGA), (C) focal cortical dysplasia (FCD) and (D) ganglioglioma. (Scale bars: 200 μm in A–B; 100 μm in C–D left and middle; 50 μm in C–D right.) Figure S2. TNFRSF9 expression is associated with reactive astrogliosis also in non-neoplastic CNS lesions. HE stainings (left A–E) and immunohistochemistry for TNFRSF9 (middle A–E) of (A) ischemic stroke (tissue surrounding a pseudocyst of an old infarction > 6 months), (B) cavernoma, (C) an acute demyelinating encephalomyelitis (ADEM), (D) CNS tissue surrounding a CNS lymphoma and (E) foetal CNS tissue suffering from intrauterine hypoxia (subventricular zone) are depicted. (Scale bars: 50 μm in A right, C left and right, D right, E middle and right; 100 μm in A left and middle, B right, C middle, D left and middle and E left; 200 μm in B left and middle.) Table S1. Patient data of neoplastic and non-neoplastic CNS lesions. Received 10 October 2013 Accepted after revision 4 March 2014 Published online Article Accepted on 10 March 2014
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doi: 10.1111/nan.12135
Tumour necrosis factor receptor superfamily member 9 (TNFRSF9) is up-regulated in reactive astrocytes in human gliomas A.-E. Blank*, P. Baumgarten*, P. Zeiner*, C. Zachskorn*†‡, C. Löffler§, J. Schittenhelm§, C. J. Czupalla*, D. Capper¶**, K. H. Plate*†‡, P. N. Harter*†‡ and M. Mittelbronn*†‡ *Edinger Institute, Institute of Neurology, Goethe University Frankfurt, Frankfurt am Main, †German Cancer Consortium (DKTK), ‡German Cancer Research Center (DKFZ), ¶Institute of Neuropathology, University of Heidelberg, **Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, and §Department of Neuropathology, Institute of Pathology and Neuropathology, University of Tuebingen, Tuebingen, Germany
A.-E. Blank, P. Baumgarten, P. Zeiner, C. Zachskorn, C. Löffler, J. Schittenhelm, C. J. Czupalla, D. Capper, K. H. Plate, P. N. Harter and M. Mittelbronn (2015) Neuropathology and Applied Neurobiology 41, e56–e67 Tumour necrosis factor receptor superfamily member 9 (TNFRSF9) is up-regulated in reactive astrocytes in human gliomas Aims: The prognosis of patients with malignant gliomas is still dismal despite maximum treatment. Novel therapeutic alternatives targeting tumorigenic pathways are, therefore, demanded. In murine glioma models, targeting of tumour necrosis factor receptor superfamily (TNFRSF) 9 led to complete tumour eradication. Thus, TNFRSF9 might also constitute a promising target in human diffuse gliomas. As there is a lack of data, we aimed to define the expression pattern and cellular source of TNFRSF9 in human gliomas. Methods: We investigated TNFRSF9 expression in normal human central nervous system (CNS) tissue and glioma specimens using immunohistochemistry, immunofluorescence and Western blotting techniques. Results: Our results show
that TNFRSF9 is considerably up-regulated in human gliomas when compared with normal brain tissue. In addition, our data provides evidence for an immune cellindependent de novo expression pattern of TNFRSF9 in mainly non-neoplastic reactive astrocytes and excludes classic immunological cell types, namely lymphocytes and microglia as the source of TNFRSF9. Moreover, TNFRSF9 is predominantly expressed in a perivascular and peritumoural distribution with significantly higher expression in IDH-1 mutant gliomas. Conclusions: Our findings provide a novel, TNFRSF9-positive, reactive astrocytic phenotype and challenge the therapeutic suitability of TNFRSF9 as a promising target for human gliomas.
Keywords: astrocytes, CD137, glioma, gliosis, TNFRSF9
Introduction Human central nervous system (CNS) tumours consist of heterogeneous cell populations including neoplastic, as Correspondence: Michel Mittelbronn, Edinger Institute, Institute of Neurology, Goethe University Frankfurt, D-60528 Frankfurt, Germany. Tel: +49 (0) 69 6301 84169; Fax: +49 (0) 69 6301 84150; E-mail: [email protected]
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well as reactive, glial, immune and vessel-associated cells [1]. As patient prognosis is still dismal even with maximum treatment, which includes surgical resection in combination with radio- and chemotherapy, more specific anti-tumoural therapy strategies are urgently needed. Among other approaches, immunotherapy showed promising results in cell culture and animal studies and has already entered clinical investigation. In animal experi© 2014 British Neuropathological Society
TNFRSF9 expression in gliomas
ments, tumour necrosis factor receptor superfamily member 9 (TNFRSF9) has been described as up-regulated in tumour-infiltrating lymphocytes (TILs). These cells can be stimulated by anti-TNFRSF9 monoclonal antibodies resulting in anti-tumoural effects [2]. TNFRSF9-activating agonistic treatment approaches are currently in clinical trials for treatment of melanoma, renal cell carcinoma and ovarian cancer [3]. In preclinical transplantable murine tumour models, agonistic anti-TNFRSF9 treatment prevented recurrences as well as metastases and even showed curative effects [4,5]. The anti-tumoural effects targeting TNFRSF9 have been attributed to enhanced cellular proliferation, survival and cytokine production of TILs in both animal and human studies [6,7]. These pro-survival, anti-tumoural capacities were mainly conferred to CD8 cytotoxic T-cells [8]. Other immune cells, such as monocytes, were also activated by TNFRSF9 stimulation. This resulted in increased proliferation, viability, morphological changes and increased adhesion capacity; TNFRSF9-related differentiation into dendritic cells was present in human but absent in murine monocytes [9]. Moreover, it has been reported that TNFRSF9-activating strategies further enhanced antibody-dependent cellular cytotoxicity (ADCC) in breast cancer patients, pointing to an additive effect of tumour-targeting antibodies such as trastuzumab (monoclonal anti-HER2 antibody) and secondary antibodies boosting the immune system [10,11]. In addition, TNFRSF9-activatory approaches increased anti-tumoural effects exerted by oncolytic viruses [12]. Most interestingly, anti-TNFRSF9 treatment revealed the best results in intracranial animal glioma models compared with subcutaneous or metastatic tumours, with animals surviving significantly longer and even achieving curative effects [13]. In murine glioma models, the combination of irradiation and an anti-TNFRSF9-based immunotherapy led to complete tumour eradication in over 60% of the glioma-harbouring animals; this was most likely related to increased TILs and production of interferon-gamma [14]. Apart from its immune modulatory anti-tumour functions, activation of TNFRSF9 on hypoxic intratumoural endothelial cells leads to increased expression of various cell adhesion molecules including: intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and E-selectin, thereby facilitating the entry of activated T-lymphocytes into the tumour tissue [15]. As TNFRSF9 and its ligand TNFSF9 act in a bidirectional way, © 2014 British Neuropathological Society
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immune cells expressing TNFSF9 can be reversely stimulated after interaction with TNFRSF9 on endothelial cells [16]. A potential shortcoming of the promising preclinical studies is related to an opposite effect of the TNFRSF9TNFSF9 system in murine models and humans [17]. To date, the expression and functional role of TNFRSF9 in the normal and pathological human CNS is not entirely clear. While at first the data suggested that TNFRSF9 is mainly expressed on neurones and astrocytes, its ligand TNFSF9 is mainly found on microglial cells [18,19]. In our study, we aimed to define the expression pattern and cellular source of TNFRSF9 in human gliomas.
Materials and methods
Patient material The study is based on an analysis of 1094 formalin-fixed paraffin-embedded tissue samples from 416 patients suffering from a glial brain tumour (see Table 1). Tissue samples were retrieved from the archives of the Neurological Institute (Edinger Institute) of the Goethe University Frankfurt, Germany. For statistical analyses, tissue micro arrays were used with cores of representative tumour centre, infiltration zone and surrounding normal appearing brain tissue. In addition, exemplary whole mount sections, including five CNS tissue samples from normal CNS autopsy cases, were stained. Specimens were cut with a microtome (3 μm thickness) and placed on SuperFrostPlus slides (Microm International, Walldorf, Germany). The use of patient material was endorsed by the local ethical committee of the Goethe University Frankfurt, Germany (GS 04/09; NP 03/11).
Immunohistochemistry For immunohistochemistry a mouse monoclonal antihuman TNFRSF9 antibody (dilution 1:40; clone S16 Novocastra/Leica Microsystems, Wetzlar, Germany) was used. Tissue labelling was performed using the DiscoveryXT immunohistochemistry system (Ventana Medical Systems, Illkirch, France). A cell-conditioning pretreatment was performed for 68 min followed by a 4-min blocking step with Inhibitor D. The primary antibody was applied for 32 min, followed by a secondary antibody (Discovery Universal Secondary Antibody) for 32 min. After washing steps, a blocking step with Blocker D for 4 min and a 16-min incubation with one drop of SA-HRP D were NAN 2015; 41: e56–e67
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Table 1. Patient data of pilocytic and diffuse gliomas
Pilocytic astrocytoma Diffuse astrocytoma Oligodendroglioma Oligoastrocytoma Anaplastic astrocytoma Anaplastic oligodendroglioma Anaplastic oligoastrocytoma Glioblastoma
WHO grade
Male/ female
Median age (range)
Patients (n)
Tumour localization (supratentorial/ infratentorial)
I II II II III III III IV
17/30 10/6 3/5 5/11 18/15 9/8 20/11 140/108
14 31 34.5 42.5 43 49 43 61
47 16 8 16 33 17 31 248
16/31 16/0 8/0 16/0 32/1 17/0 31/0 246/2
(0–75) (5–52) (4–65) (21–67) (22–66) (19–67) (23–71) (7–80)
mIDH-1 (R132H, mut/wt)
Ki-67 index (in %, 95% CI)
0/46 8/6 4/4 13/3 18/15 9/8 22/9 9/239
2.7 0.9 0.8 0.6 6.2 4.6 4.0 13.0
(1.7–3.8) (0.2–1.5) (0.0–2.3) (0.2–1.0) (3.6–8.7) (1.1–8.1) (1.4–6.7) (11.6–14.3)
mut/wt, mutant/wild-type; CI, confidence interval.
performed. For diaminobenzidine (DAB) visualization, the sections were incubated with one drop of DAB D and one drop of DAB H2O2 D for 8 min, followed by a copper enhancer (Copper D, all Ventana Medical Systems) for 4 min. Specimens were washed and consequently counterstained with haematoxylin and bluing reagent and mounted. For double immunohistochemistry, anti-CD137 staining was followed by a secondary antibody step using the RedMap or BlueMap detection kit (all Ventana Medical Systems) and the following primary antibodies (incubation time 32 min each): rabbit polyclonal anti-human GFAP (dilution 1:10000; Z0334; Dako, Hamburg, Germany), rabbit polyclonal anti-human Olig2 (dilution 1:500; AB9610; Millipore, Darmstadt, Germany), mouse monoclonal IgG2a anti-human IDH-1 R132H (dilution 1:50; clone H09; Dianova, Hamburg, Germany), mouse monoclonal IgG1 anti-human CD68 (dilution 1:500; clone KP1; Dako), rabbit polyclonal anti-human Iba-1 (dilution 1:1000; Wako Chemicals, Richmond, VA, USA), mouse monoclonal IgG1 anti-human CD45/LCA (dilution: 1:2000; clone 2B11+PD7/26, Dako), mouse monoclonal IgG1 anti-human Ki-67 (dilution 1:200; clone MIB-1; Dako), mouse monoclonal IgG2a anti-human BRAF V600E (dilution 1:100; clone VE1, Spring Bioscience, Pleasanton, CA, USA) and rabbit monoclonal IgG anti-human phospho-S6 Ribosomal Protein (Ser240/ 244) (dilution 1:2000; clone D68F8, Cell Signaling Technology, Danvers, MA, USA). Subsequently, specimens were incubated with one drop of secondary antibody mix (Discovery Universal Secondary Antibody), followed by a blocking step for 4 min and a signal amplification via streptavidin alkaline phosphatase © 2014 British Neuropathological Society
(Blocker R1, SA-Alk Phos R) for 16 min. For visualization, chromogen was added (Fast Red for 16 min, BlueMap for 24 min), then samples were repeatedly washed and heated and finally counterstained using haematoxylin II and bluing reagent for 12 min (all Ventana Medical Systems). Images were analysed and recorded on an Olympus BX-50 microscope (Olympus, Hamburg, Germany).
Primary tumour cultures Native tumour tissue was taken directly from the surgical intervention, evaluated macroscopically by a neuropathologist and processed under sterile conditions. First, mechanical dissociation steps were performed followed by an enzymatic dissociation using a solution containing Leibovitz-L15 medium (Life technologies, Carlsbad, CA, USA), Papain suspension (Worthington Biochemical, Lakewood, NJ, USA), EDTA 0.5 M (AppliChem, Darmstadt, Germany) and Desoxyribonuclease 1 Typ II (DNAse1, Sigma-Aldrich, St. Louis, MO, USA). The shredded cell suspension was incubated for 30 min at 37°C, mixed every 10 min and afterwards washed and centrifuged several times with DMEM-F12 medium (Life technologies). Cells were cultured over two subsequent passages under either serum-containing or serum-free conditions to investigate the expression of TNFRSF9 in conditions that are known to preserve the primary tumours’ genetic pattern (DMEM, Life technologies; FCS, Provitro, Germany; Penicillin/Streptomycin, SigmaAldrich; neurosphere medium containing additional growth factors as EGF and bFGF: DMEM-F12, B27 Supplement and HEPES, Life technologies; EGF and bFGF, PeproTech; Penicillin/Streptomycin, Sigma-Aldrich). NAN 2015; 41: e56–e67
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Microglia isolation To obtain primary microglial cells from normal and glioma tissue, we used magnetic cell separation (MACS® Sorting, Miltenyi Biotec, Bergisch Gladbach, Germany). The native tissue was fully dissociated mechanically and enzymatically to gain a cell suspension that was incubated with antibody-linked magnetic particles against the microglial CD11b epitope (MicroBeads). The resulting cell suspension was subsequently separated via MACS Columns in a magnetic field, in which the antibody-linked microglial cells were collected and the non-labelled cells discarded (all Miltenyi Biotec).
Immunofluorescence Immunofluorescent stainings were performed using a mouse monoclonal anti-human TNFRSF9 antibody (dilution 1:40; clone S16 Novocastra/Leica Microsystems). After a centrifugation step, media was removed from the primary tumour spheres, followed by two washing steps with PBS (Life Technologies). Afterwards, tumour spheres were fixed for 10 min in precooled methanol at −20°C (Sigma-Aldrich). This procedure was followed by a blocking step with Roti blocking solution (1:10, Roth, Karlsruhe, Germany) for 1 h at room temperature (RT). The sphere specimens were incubated using the primary anti-human TNFRSF9 antibody overnight at 4°C and subsequently labelled with the first secondary antibody for 1 h at RT (dilution 1:500; Alexa Fluor568, human antimouse IgG, Invitrogen, Carlsbad, CA, USA). Nuclear counterstaining was performed using Topro-3 (dilution 1:500, Invitrogen). Fluorescence images were analysed and recorded on a Nikon Eclipse 80i fluorescence microscope (Nikon, Düsseldorf, Germany). After recording, digital images were adjusted with ImageJ (National Institutes of Health, Bethesda, MD, USA).
Western blot Isolated cells and primary tumour tissue were collected to generate protein lysates for Western blotting. After centrifugation, the cell suspension was transferred into liquid nitrogen and the cracked cells were stored for at least 1 h at −80°C. RIPA-Lysis buffer was used for protein retrieval. Afterwards, the protein lysates were quantified by a BCAAssay (Micro BCA™ Protein Assay Kit, Thermo Scientific, Waltham, MA, USA). Gel electrophoresis was performed © 2014 British Neuropathological Society
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using 12.5% SDS-polyacrylamide gels with 20 μg protein samples resuspended in sample buffer according to standard procedures. The fractionated cells were transferred to a nitrocellulose membrane (Amersham Hybond ECL Membrane, GE Healthcare, Little Chalfont, Buckinghamshire, UK) using a blotting chamber for 30 min according to the manufacturer’s protocols (Bio-Rad Laboratories, Hercules, CA, USA). To avoid unspecific antibody signals, the membrane was blocked by blocking solution for at least 1 h at RT (Rotiblock in PBS, Roth). Several initial washing steps using PBS/T (Tween 20, Sigma-Aldrich) were followed by incubation with the primary mouse monoclonal antihuman TNFRSF9 antibody as previously described (clone S16 Novocastra/Leica Microsystems). A rabbit polyclonal IgG anti-human β-Actin antibody (Abcam, Cambridge, UK) was used serving as loading control. Primary antibodies were applied for 2 h and after additional washing steps the secondary antibody was added for 1 h. After removal of nonbinding antibodies by five washing steps with PBS/T and PBS, respectively, immunodetection was performed using horseradish peroxidase substrate Luminol reagent (Santa Cruz Biotechnology, Dallas, TX, USA) for 3 min. The blots were analysed on a X-ray film (Super RX, Fujifilm, Tokio, Japan) and developed with a X-ray film processor (Curix 60, AGFA, Mortsel, Belgium).
Scoring TNFRSF9 expression was assessed by taking both staining intensity and frequency into account using a previously established protocol [20]. The semiquantitative scores consist of a frequency score ranging from 0 to 4: 0: 0–1%, 1: 1–10%, 2: 10–25%, 3: 25–50% and 4: >50% of all cells showing a positive nuclear staining. Likewise, intensity was recorded in a similar semiquantitative approach as follows: 0: no staining, 1: weak staining, 2: moderate staining, 3: strong staining. The results for staining intensity and frequency were multiplied, such that the final expression cell score reflected both. The evaluation and photographic documentation of the immunohistochemical staining was performed using an Olympus BX50 light microscope.
TCGA and REMBRANDT platform analyses Gene expression signatures were analysed in 424 primary gliomas and 11 normal brain samples by assessing the TCGA data portal which applied the Agilent 244 K NAN 2015; 41: e56–e67
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G4502A microarray to determine mRNA profiles (http:// tcga.cancer.gov/) [21]. Data were assessed in November 2011.
Statistical analysis The semiquantitative TNFRSF9 scores were assigned as an ordinal scale response variable and statistical differences among WHO grades were assessed using the non-parametric Wilcoxon test. A significance level of alpha = 0.05 was selected for all tests. Statistical analysis was performed using JMP 8.0 software (SAS, Cary, NC, USA).
Results
TNFRSF9 is overexpressed in astrocytomas compared with normal CNS tissue Analysis of the TCGA database including 424 glioblastomas revealed that the median log2-fold mRNA
expression change for TNFRSF9 was considerably increased (by 0.54-fold) as compared with normal CNS tissue specimens (n = 11) (data not shown). To assess the in vivo expression of TNFRSF9, we first screened human tonsil tissue (Figure 1A) and the Jurkat human T-cell lymphoma line (Figure 1B), both of which were found to express membranous TNFRSF9. In contrast, normal human grey (Figure 1C) and white matter (Figure 1D) were almost completely TNFRSF9-negative. Only larger meningeal blood vessels presented with TNFRSF9-positive vessel walls, which is in line with previously published data reporting constitutive vascular TNFRSF9 expression (data not shown). In non-diffusely infiltrating human pilocytic astrocytomas WHO grade I (Figure 1E) and a large cohort of diffusely infiltrating gliomas (Figure 1F–L), prominent TNFRSF9 expression was frequently observed in a perivascular distribution pattern. In addition, TNFRSF9 was detected on large swollen cells within glial neoplasms. These cells were frequently localized in equal distances from neighbouring TNFRSF9-positive cells (Figure 1F,J) or
Figure 1. TNFRSF9 expression is up-regulated in diffuse human gliomas. TNFRSF9 immunohistochemistry in (A) normal human tonsil; (B) Jurkat cells (immortalized T lymphocyte cells); (C) normal-appearing grey matter (NAGM); (D) normal-appearing white matter (NAWM); (E) pilocytic astrocytoma, WHO grade I with vascular proliferations; (F) diffuse astrocytoma, WHO grade II; (G) anaplastic astrocytoma, WHO grade III; (H) primary glioblastoma WHO grade IV; (I) secondary glioblastoma, WHO grade IV; (J) anaplastic oligoastrocytoma, WHO grade III; (K, L) anaplastic oligodendroglioma, WHO grade III (scale bars: 100 μm in A, C–J; 50 μm in B; 200 μm in K, L). © 2014 British Neuropathological Society
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Figure 2. TNFRSF9 expression in glioma subtypes. Box-and-Whisker plots for TNFRSF9 expression in different glioma subtypes scores are depicted. Significant P-values from all pairwise comparisons of different WHO grade astrocytomas were obtained using the non-parametric Wilcoxon test. A significance level of alpha = 0.05 was selected for all comparisons. Numbers for each tumour subtype are as follows: PA: n = 47; DA: n = 16; OD: n = 8; OA: n = 18; AA: n = 33; AOD: n = 17; AOA: n = 31; GBM: n = 248.
showed very long cellular protrusions directed towards blood vessels (Figure 1H,I,K). The statistical comparison (Figure 2) of TNFRSF9 expression between the different glioma entities and malignancy grades revealed that highest scores were observed in diffuse astrocytomas WHO grade II (median expression score 2), while lowest levels were detected in WHO grade II oligodendrogliomas (median expression score 0.5). Most interestingly, in all investigated gliomas more pronounced TNFRSF9 expression was observed in cases harbouring the IDH-1 R132H mutation (see Table 1) reaching statistical significance for anaplastic astrocytomas WHO grade III (Figure 3A; P = 0.002), anaplastic oligoastrocytomas WHO grade III (Figure 3B; P = 0.025) and glioblastomas WHO grade IV (Figure 3C; P = 0.001) as compared with their wild-type counterparts.
TNFRSF9 expression is almost exclusively restricted to cells with astrocytic differentiation in human gliomas At a cellular level, TNFRSF9-positive cells within gliomas often presented with bizarre morphological features including, either thicker and shorter or longer and thinner protrusions (Figure 4A). TNFRSF9-positive cells were frequently bi- or multinucleated, typically with © 2014 British Neuropathological Society
nuclei being located at the outer border of the cell. In multiple glioma cases, these cells formed an almost complete circle surrounding prominent glioma-associated blood vessels (Figure 4A). Double immunohistochemical stainings revealed that TNFRSF9-positive cells strongly expressed GFAP (glial fibrillary acidic protein). While strongest GFAP expression was observed within cytoplasms, TNFRSF9 was most prominently expressed at the cellular membrane and on cellular protrusions (Figure 4B). Notably, GFAP expression was much stronger in TNFRSF9-positive cells as compared with surrounding TNFRSF9-negative cells. In contrast, TNFRSF9 and Olig2 expression were mutually exclusive, indicating that oligodendrocytes are most likely not a cellular source of TNFRSF9 (Figure 4C). Although Olig2 is not specific for cells with oligodendroglial differentiation, it is a reliable, sensitive tool to detect those cells. Furthermore, TNFRSF9-expressing cells were also negative for the leucocyte marker CD45 (Figure 4D) and for macrophage and microglial markers including CD68 (Figure 4E) and Iba-1 (data not shown). Finally, we assessed the proliferative potential of the TNFRSF9positive cellular fraction. Most Ki-67-positive cells lacked TNFRSF9 expression and, likewise, the TNFRSF9-positive cell fraction did not exhibit proliferative potential (Figure 4F). NAN 2015; 41: e56–e67
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Non-neoplastic, reactive astrocytes are the major cellular source of TNFRSF9 in diffuse gliomas
Figure 3. TNFRSF9 expression is associated with IDH-1 mutation status. Box-and-Whisker plots for the semiquantitative TNFRSF9 scores were assigned as ordinal scale response variables and statistical differences between IDH-1 mutated tumours in different glioma subtypes were assessed using the non-parametric Wilcoxon test. A significance level of alpha = 0.05 was selected for all comparisons.
© 2014 British Neuropathological Society
Using an antibody specific to the mutated R132H variant of the IDH-1 protein, we verified that TNFRSF9 was expressed in close proximity to the tumour bulk, while TNFRSF9-positive cells were absent in areas lacking IDH-1 R132H expression (Figure 5A,B). To address the question of whether the non-proliferating TNFRSF9-positive cell fraction is constituted of tumour cells or, more likely, of reactive glial cells, double immunostainings for TNFRSF9 and mutated IDH-1 (R132H) were performed. Double stainings revealed that TNFRSF9 was not expressed on IDH-1-mutated tumour cells (Figure 5C,D). Of note, some less frequent and non-diffuse CNS tumours (Supplemental Table S1), such as pleomorphic xanthoastrocytomas (PXA) (Supplemental Figure S1A) and subependymal giant cell astrocytomas (SEGA) (Supplemental Figure S1B), also displayed TNFRSF9 expression in tumour cells. Typically, these tumour cells harbour BRAF V600E mutation (PXA) or an alteration in the mTOR pathway (SEGA), reflected by phosphoS6-positive cells. In focal cortical dysplasias (FCD), hypertrophic or oligodendroglial-like TNFRSF9-positive cells were present (Supplemental Figure S1C). Conversely, gangliogliomas (Supplemental Figure S1D) contained very few TNFRSF9positive cells resembling those encountered in diffuse gliomas. In an additional cohort of distinct non-neoplastic CNS pathologies, strong TNFRSF9 expression was also consistently found in a subset of reactive astrocytes (Supplemental Figure S2). To corroborate the in vivo data found in diffuse gliomas, we investigated whole tumour lysates and primary glioblastoma cell cultures as well as primary glioblastoma-derived microglia cultures for TNFRSF9 expression by immunoblotting (Figure 5E). As expected, primary glioblastoma-derived microglia were completely devoid of TNFRSF9, while lysates from both whole tumour samples and primary glioblastoma cell cultures exhibited weak to moderate TNFRSF9 expression (Figure 5E). To determine if tumour spheroids express TNFRSF9 under in vitro conditions, we cultivated primary glioblastoma cells under neurosphere conditions (Figure 5F). The vast majority of neoplastic cells within the spheres remained TNFRSF9-negative, while only a small cellular fraction exhibited strong TNFRSF9 expression (Figure 5F). Similarly to the in vivo findings, the TNFRSF9-positive cells in the spheres displayed multiple long cellular processes and large cytoplasms, pointing to a reactive astrocyte morNAN 2015; 41: e56–e67
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Figure 4. TNFRSF9 is exclusively expressed on non-proliferative cells with astrocytic differentiation within diffuse gliomas. (A) TNFRSF9 expression on single multinucleated swollen cells and perivascular cell cuffs within diffuse gliomas; (B) co-expression of TNFRSF9 (brown) and glial fibrillary acid protein (GFAP, red) on swollen cell bodies with enlarged processes; no cellular co-expression of the (C) oligodendroglial marker Olig2 (red), (D) the leucocyte/lymphocyte marker CD45 (LCA, leucocyte common antigen, blue) or (E) the macrophage/microglia marker CD68 (red) with TNFRSF9 (brown) was observed; (F) TNFRSF9-positive cells (brown) lacked proliferative activity as indicated by immunopositivity for the proliferation marker Ki-67 (red). (Scale bars: 25 μm in B–F.)
phology. These findings indicate that TNFRSF9-positive reactive astrocytes might survive under cell culture conditions where they are still intermingled with neoplastic glial cells.
Discussion Several preclinical trials applying an activating antiTNFRSF9 treatment revealed curative results in different transplantable murine tumours, including gliomas [4,5,13,14]. As the exact cellular source of TNFRSF9 in the human CNS is still unclear, the aim of our study was to define the expression pattern of TNFRSF9 in human glioma and normal CNS tissue, thereby assessing its potential value for future targeted treatment approaches. Our findings indicate that TNFRSF9 is strongly overexpressed in human diffuse gliomas as compared with the normal CNS at both mRNA (data not shown) and protein levels (Figures 1 and 2). Most interestingly, high TNFRSF9 expression was significantly associated with gliomas harbouring the IDH-1 R132H mutation (Figures 2 and 3). By analysing those tumours, the cellular source of TNFRSF9 © 2014 British Neuropathological Society
expression could be linked to non-proliferative, frequently multinucleated astrocytic cells showing bizarre histomorphological features (Figures 4 and 5). This particular cellular fraction presented with a distinct distribution pattern and was most prominently observed in perivascular and peritumoural locations (Figure 5). Using double immunohistochemical staining methods, we were able to define the main cellular source of TNFRSF9 as non-proliferative, non-IDH-1-mutated, GFAP-positive, Olig2-negative astrocytes in diffuse gliomas, which, in combination with their morphological features, could be referred to as reactive astrocytes (Figure 5). Similarly, TNFRSF9-positive astrocytes with reactive changes have been detected in other neoplastic and non-neoplastic CNS pathologies (Supplemental Figure S2). However, neoplastic TNFRSF9-positive cells have also been detected in rare, non-diffuse brain tumours such as PXA and SEGA (Supplemental Figure S1). Although the anti-tumoural effects of agonistic anti-TNFRSF9 treatment in experimental glioma models are not yet fully understood, increased numbers of glioma-infiltrating lymphocytes were observed in these cases [14]. For other tumour entities, such as melanomas, NAN 2015; 41: e56–e67
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Figure 5. Non-neoplastic, reactive astrocytes constitute the cellular source of TNFRSF9 in diffuse gliomas. Immunohistochemical analyses using mutation-specific antibodies against the (A) IDH-1 R132H variant and (B) TNFRSF9 reveal a strong regional overlap between IDH-1 R132H-positive tumour cells of an anaplastic oligoastrocytoma, WHO grade III and TNFRSF9 expression, which is mainly seen in a perivascular distribution or at the tumour border. (C, D) Costainings of TNFRSF9 and mutated IDH-1 R132H excluding a co-expression of TNFRSF9 in neoplastic glial cells. (E) Immunoblotting of primary glioma spheres (lane 1), different primary glioma tissues (lanes 2–5) or primary glioma-derived microglial cells (lanes 6–7) revealing absence of TNFRSF9 in microglial cells while being present in primary glioma-derived cells or tissues. (F) Immunofluorescent analyses for TNFRSF9 were performed on primary glioma-derived spheres. Of note, as seen in primary glioma tissues, only single TNFRSF9-positive cells with long cellular protrusions were observed within the cell culture while the vast majority of cells remained TNFRSF9-negative. (Scale bars: 100 μm in A, B; 50 μm in C; 25 μm in D.) © 2014 British Neuropathological Society
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it was demonstrated that TNFRSF9 stimulation enhanced the survival and effector function of the CD8+ T-cell fraction [22]. Most recently, a novel TNFRSF9-stimulated, eomesodermin-driven CD4+ T-cell phenotype producing the cytokines IL-27, IL-15 and IL-10 capable of enhancing multipotent cytotoxic capacity has been discovered [23]. In our patient cohort, TNFRSF9 was virtually absent from glioma-infiltrating lymphocytes or microglia/ macrophages (Figure 4); a direct immune-modulatory activation of CNS intrinsic T-cells in the context of human gliomas is, therefore, highly questionable. As antitumoural effects and prolonged survival were observed in experimental intracranial sarcomas and gliomas, whereas only minimal therapeutic effects were observed in the same therapeutic approach in subcutaneous and pulmonary tumours, a general impact on the brain microenvironment was assumed [13]. Considering that the CNS is an immunoprivileged organ due to the blood-brain barrier and that only a few infiltrating lymphocytes were detected in our specimens, the finding that intracranial tumours were sensitive to systemic mAb treatment raises the question of whether or not other specialized resident immune cells, such as microglia, or alternative members of the brain microenvironment increase distinct anti-tumoural immune response. In addition, it has to be considered that a preoperative steroid treatment that avoids peritumoural oedema and is often applied in glioblastoma patients, might lead to a decreased glial reactivity and a diminished lymphocytic invasion [24–27]. In vitro studies revealed that TNFRSF9 is up-regulated by astrocytes upon stimulation with the Ca(2+)-binding protein S100B that is frequently increased during CNS pathologies [28]. The astrocytic expression of TNFRSF9 was linked to an enhanced activation status of the respective astrocytes leading to decreased neuroprotection and a modified interaction with microglia and inflammatory cells. The fact that reactive astrogliosis is potentially driven by the nuclear factor-kappa B (NF-kappaB) [29] is critical for our study, as TNFRSF9-mediated signalling mainly results in the activation of NF-kappaB [30]. Furthermore, there is an association of TNFRSF9 and the transcription factor signal transducer and activator of transcription 3 (STAT3) that contributes to the activation of astrocytes, via binding to the GFAP promoter constituting an important feature for the formation of both reactive gliosis and neoplastic astrocytes [31–33]. The question of why IDH-1-mutated specimens especially showed a prominent amount of reactive astrocytes with TNFRSF9 expression remains elusive. © 2014 British Neuropathological Society
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However, recent studies revealed that cells harbouring the mutated IDH-1 variant (R132H) produce considerably lower levels of glutathione, which is the major scavenger for radical oxygen species (ROS) [34]. The fact that ROS constitute one of the most potent inductors of reactive astrogliosis might therefore be a suitable explanation of why IDH-1-mutated gliomas exhibit significantly more TNFRSF9-associated astrogliosis [35]. Another potential explanation for significantly higher TNFRSF9 levels in IDH-1 mutant gliomas, as compared with their nonmutated counterparts, might be related to a slower growth rate in IDH-1 mutant gliomas which could allow for gliosis to develop over a longer time period. In summary, our findings reveal a novel, TNFRSF9positive, reactive astrocytic phenotype in human gliomas. One can hypothesize that most prominent TNFRSF9 signals, which are observed at the interface between glioma and CNS tissue and/or blood vessels, might be related to the fact that those areas constitute the primary site where residual astrocytes are confronted and potentially activated by glioma cells. Additionally, our study strongly challenges the applicability of successful animal glioma studies targeting TNFRSF9 to human clinical studies. However, because the role of reactive astrogliosis in gliomas is still debated, it is far too early to speculate about the suitability of therapeutically targeting TNFRSF9 in human brain tumours or other CNS disorders presenting with pronounced astrogliosis.
Acknowledgements We would like to acknowledge the support of the Deutsche Krebshilfe (A-EB) for this work. We thank Cara Laudon and Zach B. Bjornson for medical writing assistance.
Author contributions Conceived and designed the experiments: A-EB, PNH, MM. Performed the experiments: A-EB, PB, PZ, CZ, CL, CJC, DC, PNH, MM. Analysed the data: A-EB, PB, PZ, CL, DC, PNH, MM. Contributed reagents, materials, financial support: A-EB, PB, JS, KHP, MM. Wrote the paper: A-EB, PNH, MM. Supervisor of the study: PNH, MM. Corrected and approved the final version of the manuscript: all authors. NAN 2015; 41: e56–e67
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Conflict of interest The authors declare that they have no conflict of interest. Anna-Eva Blank was funded by the German Cancer Aid Foundation.
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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Material and methods: Patient data. Figure S1. TNFRSF9 expression in primary non-diffuse tumours and malformations. Haematoxylin and eosin (HE) stainings (left A–D) and immunohistochemistry for TNFRSF9 (middle A–D and C–D right) as well as for BRAF V600E (right A) and phosphoS6 (right B) in (A) a pleomorphic xanthoastrocytoma (PXA), (B) subependymal giant cell astrocytoma (SEGA), (C) focal cortical dysplasia (FCD) and (D) ganglioglioma. (Scale bars: 200 μm in A–B; 100 μm in C–D left and middle; 50 μm in C–D right.) Figure S2. TNFRSF9 expression is associated with reactive astrogliosis also in non-neoplastic CNS lesions. HE stainings (left A–E) and immunohistochemistry for TNFRSF9 (middle A–E) of (A) ischemic stroke (tissue surrounding a pseudocyst of an old infarction > 6 months), (B) cavernoma, (C) an acute demyelinating encephalomyelitis (ADEM), (D) CNS tissue surrounding a CNS lymphoma and (E) foetal CNS tissue suffering from intrauterine hypoxia (subventricular zone) are depicted. (Scale bars: 50 μm in A right, C left and right, D right, E middle and right; 100 μm in A left and middle, B right, C middle, D left and middle and E left; 200 μm in B left and middle.) Table S1. Patient data of neoplastic and non-neoplastic CNS lesions. Received 10 October 2013 Accepted after revision 4 March 2014 Published online Article Accepted on 10 March 2014
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