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Glioblastoma (GBM) is a devastating tumor and few patients survive beyond 3 years. Defining the molecular determinants underlying long-term survival is essential for insights into tumor biology and biomarker identification. We therefore investigated homogeneously treated, IDH (wt) long-term (LTS, n = 10) and short-term survivors (STS, n = 6) by microarray transcription profiling. While there was no association of clinical parameters and molecular subtypes with long-term survival, STS tumors were characterized by differential polarization of infiltrating microglia with predominance of the M2 phenotype detectable both on the mRNA and protein level. Furthermore, transcriptional signatures of LTS and STS predicted patient outcome in a large, IDH (wt) cohort (n = 468). Interrogation of overlapping genomic alterations identified concurrent gain of chromosomes 19 and 20 as a favorable prognostic marker. The strong association of this co-gain with survival was validated by aCGH in a second, independent cohort (n = 124). Finally, FISH and gene expression data revealed gains to constitute low-amplitude, clonal events with a strong impact on transcription. In conclusion, these findings provide important insights into the manipulation of the innate immune system by particularly aggressive GBM tumors. Furthermore, we genomically characterize a previously unknown, clinically relevant subgroup of glioblastoma, which can easily be identified through modern neuropathological workup.

αβ integrin is overexpressed by several carcinomas and thus considered a target for diagnostic imaging and anticancer therapies. Recently, we presented the αβ integrin-binding peptide SFITGv6 as a novel potential tracer for imaging and targeted therapy of αβ integrin-positive carcinomas. Here, we analyzed the affinity and specificity of 5 native αβ integrin-specific binders in comparison to SFITGv6. Sunflower trypsin inhibitor 1 (SFTI1)-based peptides containing arginine-glycine-aspartic acid (RGD) motif-spanning octamers of fibronectin (SFFN1), tenascin C (SFTNC), vitronectin (SFVTN), and latency-associated peptides (LAP) 1 (SFLAP1) and 3 (SFLAP3) were synthesized, and their binding potential to αβ integrin-expressing head and neck squamous cell carcinoma (HNSCC) cell lines was evaluated. Subsequently, stability, affinity, and specificity were assessed in vitro using radio-high-pressure liquid chromatography, surface plasmon resonance assay, and binding experiments including competition, kinetics, internalization, and efflux. αβ integrin binding specificity was further evaluated by peptide histochemistry. Finally, in vivo binding properties were assessed using small-animal PET imaging and biodistribution experiments in HNSCC-bearing mice, andGa-DOTA-SFLAP3 was applied for diagnostic PET/CT of an HNSCC patient. When the newly designed peptides were compared, significant binding (>20%) to several HNSCC cell lines (HNO97, HNO399, and HNO223) and a fast internalization of up to 60% and 70% were observed for SFLAP3 (GRGDLGRL) and SFITGv6 (FRGDLMQL). In contrast, the other peptides displayed binding that was moderate (SFLAP1, 4.1%-12.1%) to marginal (SFFN1, SFTNC, and SFVTN, <1%) and were therefore excluded from further analysis. Notably, SFLAP3 exhibited improved affinity for αβ integrin (mean half-maximal inhibitory concentration, 3.5 nM; dissociation constant, 7.4). Moreover, small-animal PET imaging and biodistribution studies of HNSCC xenograft mice revealed an increased tumor-specific accumulation 30-60 min after injection of Ga-labeled orLu-labeled DOTA-SFLAP3. Peptide staining further demonstrated binding specificity for SFLAP3 to HNSCC tumor cells. Finally, PET/CT scanning of an HNSCC patient showed specific SFLAP3 accumulation in the primary tumor lesion (SUV, 5.1) and in corresponding lymph node metastases (SUV, 4.1). SFLAP3 represents a promising tracer for prognostic assessment, diagnostic imaging, and possibly targeted therapy of αβ integrin-expressing tumors.

Targeted therapies are regarded as promising approaches to increase 5-year survival rate of head and neck squamous cell carcinoma (HNSCC) patients. For the selection of carcinoma-specific peptides membrane proteome of HNO97 tumor cells fractionated by the ProteomeLab PF2D system and corresponding HNO97 cells were deployed for an alternating biopanning using a sunflower trypsin inhibitor1-based phage display (SFTI8Ph) library. Stability, binding properties and affinity of novel candidates were assessed using radio-HPLC, binding experiments and surface plasmon resonance assay (SPR), respectively. Subsequently, the affinity of the peptide was verified by using peptide histochemistry, using flow cytometry, and by positron emissions tomography (PET/CT). We identified a novel ITGαβ binding peptide (SFITGv6) containing the amino acid sequence FRGDLMQL. SFITGv6 provides stability over a period of 24 hours and demonstrates high affinity ( = 14.8 nmol/L) for ITGαβ In HNO97 cells, a maximal uptake and internalization of up to 37.3% and 37.5%, respectively, was measured. Small-animal PET imaging and biodistribution studies of HNO97 xenografted Balb/c nu/nu mice showed tumor-specific accumulation of Ga- andLu-labeled DOTA-SFITGv6, respectively, 30 to 60 minutes after injection. Moreover, peptide histochemistry revealed a strong and homogenous binding of biotin-labeled SFITGv6 to HNSCC tumors and breast- and lung cancer-derived brain metastases. Finally, first PET/CT scans of HNSCC and NSCLC patients displayed SFITGv6 accumulation specifically in tumors, but not in inflammatory lesions. Thus, SFITGv6 represents a novel powerful tracer for imaging and possibly for endoradiotherapy of ITGαβ-positive carcinoma. .

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Glioblastoma is the most common and aggressive brain tumor, with a subpopulation of stem-like cells thought to mediate its recurring behavior and therapeutic resistance. The epithelial-mesenchymal transition (EMT) inducing factor Zeb1 was linked to tumor initiation, invasion, and resistance to therapy in glioblastoma, but how Zeb1 functions at molecular level and what genes it regulates remain poorly understood. Contrary to the common view that EMT factors act as transcriptional repressors, here we show that genome-wide binding of Zeb1 associates with both activation and repression of gene expression in glioblastoma stem-like cells. Transcriptional repression requires direct DNA binding of Zeb1, while indirect recruitment to regulatory regions by the Wnt pathway effector Lef1 results in gene activation, independently of Wnt signaling. Amongst glioblastoma genes activated by Zeb1 are predicted mediators of tumor cell migration and invasion, including the guanine nucleotide exchange factor Prex1, whose elevated expression is predictive of shorter glioblastoma patient survival. Prex1 promotes invasiveness of glioblastoma cells highlighting the importance of Zeb1/Lef1 gene regulatory mechanisms in gliomagenesis.

Proteoglycans are often overexpressed in tumors and can be found on several normal and neoplastic stem cells. In this study, we analyzed in-depth the role of CSPG4 in head and neck squamous cell carcinomas (HNSCC). Analysis of CSPG4 in a homogeneous study sample of HPV-negative stage IVa HNSCCs revealed overexpression of protein and mRNA levels in a subgroup of HNSCC tumors and a significant association of high CSPG4 protein levels with poor survival. This could be validated in three publicly available microarray datasets. As a potential cause for upregulated CSPG4 expression, we identified DNA hypomethylation in a CpG-island of the promoter region. Accordingly, we found an inverse correlation of methylation and patient outcome. Finally, CSPG4 re-expression was achieved by demethylating treatment of highly methylated HNSCC cell lines establishing a direct link between methylation and CSPG4 expression. In conclusion, we identified CSPG4 as a novel biomarker in HNSCC on several biological levels and established a causative link between DNA methylation and CSPG4 protein and mRNA expression.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer and is characterized by pathological, phenotypical and biological heterogeneity. Despite the progress in surgical and radiation techniques, the 5-year survival rate is still below 50%. 1 There is increasing evidence that epigenetic changes like DNA methylation contribute significantly to malignant transformation. Down-regulation or gene silencing caused by promoter methylation has been shown to influence tumor progression in HNSCC. 2 Chondroitin sulfate proteoglycan 4 (CSPG4, also known as NG2/ MCSP/ HW-MAA) is a highly glycosylated integral membrane protein, composed of an N-linked glycoproteinand a chondroitin sulfate proteoglycan-component. 3 Although the exact function of CSPG4 is yet unknown, it seems to be implicated in angiogenesis, tissue invasion and cell spreading. 4,5 The expression level of markers of undifferentiated cells can be accurate prognostic indicators in cancer. 6 Interestingly, there are hints that CSPG4 is expressed on normal neural stem cells and cancer stem cells of gliomas and its expression is associated with a poor prognosis and resistance to treatment. 7-11 Moreover, CSPG4 is over-expressed in epithelial tumors such as breast cancer, while in melanoma and in malignant mesothelioma it is already used for antibodybased targeted therapy. 4,12,13 As a systematic analysis of the role of CSPG4 in HNSCC is still missing, we performed an in-depth analysis of CSPG4 expression in these tumors. To prevent a potential bias in our survival analyses caused by distinct life expectations of the different TNM stages, we restricted our analyses to a more homogenous study sample consisting of HPV-negative stage IVa tumors as evidenced by p16 staining. 14, 15 We found an increased CSPG4 expression in a subgroup of HNSCCs and identified promoter methylation as regulator of CSPG4 mRNA and protein expression. Moreover, high CSPG4 expression and l...