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BackgroundImmunotherapy is currently part of the standard of care for patients with advanced-stage non-small cell lung cancer (NSCLC). However, many patients do not respond to this treatment, therefore combination strategies are being explored to increase clinical benefit. The PEMBRO-RT trial combined the therapeutic programmed cell death 1 (PD-1) antibody pembrolizumab with stereotactic body radiation therapy (SBRT) to increase the overall response rate and study the effects on the tumor microenvironment (TME).MethodsHere, immune infiltrates in the TME of patients included in the PEMBRO-RT trial were investigated. Tumor biopsies of patients treated with pembrolizumab alone or combined with SBRT (a biopsy of the non-irradiated site) at baseline and during treatment were stained with multiplex immunofluorescence for CD3, CD8, CD20, CD103 and FoxP3 for lymphocytes, pan-cytokeratin for tumors, and HLA-ABC expression was determined.ResultsThe total number of lymphocytes increased significantly after 6 weeks of treatment in the anti-PD-1 group (fold change: 1.87, 95% CI: 1.06 to 3.29) and the anti-PD-1+SBRT group (fold change: 2.29, 95% CI: 1.46 to 3.60). The combination of SBRT and anti-PD-1 induced a 4.87-fold increase (95% CI: 2.45 to 9.68) in CD103+cytotoxic T-cells 6 weeks on treatment and a 2.56-fold increase (95% CI: 1.03 to 6.36) after anti-PD-1 therapy alone. Responders had a significantly higher number of lymphocytes at baseline than non-responders (fold difference 1.85, 95% CI: 1.04 to 3.29 for anti-PD-1 and fold change 1.93, 95% CI: 1.08 to 3.44 for anti-PD-1+SBRT).ConclusionThis explorative study shows that that lymphocyte infiltration in general, instead of the infiltration of a specific lymphocyte subset, is associated with response to therapy in patients with NSCLC.Furthermore, anti-PD-1+SBRT combination therapy induces an immunological abscopal effect in the TME represented by a superior infiltration of cytotoxic T cells as compared with anti-PD-1 monotherapy.

BackgroundImmune checkpoint inhibitors (ICI) can lead to long-term responses in patients with metastatic melanoma. Still many patients with melanoma are intrinsically resistant or acquire secondary resistance. Previous studies have used primary or metastatic tumor tissue for biomarker assessment. Especially in melanoma, metastatic lesions are often present at different anatomical sites such as skin, lymph nodes, and visceral organs. The anatomical site may directly affect the tumor microenvironment (TME). To evaluate the impact of tumor evolution on the TME and on ICI treatment outcome, we directly compared paired primary and metastatic melanoma lesions for tumor mutational burden (TMB), HLA-ABC status, and tumor infiltrating lymphocytes (TILs) of patients that received ipilimumab.MethodsTMB was analyzed by sequencing primary and metastatic melanoma lesions using the TruSight Oncology 500 assay. Tumor tissues were subjected to multiplex immunohistochemistry to assess HLA-ABC status and for the detection of TIL subsets (B cells, cytotoxic T cells, helper T cells, and regulatory T cells), by using a machine-learning algorithm.ResultsWhile we observed a very good agreement between TMB of matched primary and metastatic melanoma lesions (intraclass coefficient=0.921), such association was absent for HLA-ABC status, TIL density, and subsets thereof. Interestingly, analyses of different metastatic melanoma lesions within a single patient revealed that TIL density and composition agreed remarkably well, rejecting the hypothesis that the TME of different anatomical sites affects TIL infiltration. Similarly, the HLA-ABC status between different metastatic lesions within patients was also comparable. Furthermore, high TMB, of either primary or metastatic melanoma tissue, directly correlated with response to ipilimumab, whereas lymphocyte density or composition did not. Loss of HLA-ABC in the metastatic lesion correlated to a shorter progression-free survival on ipilimumab.ConclusionsWe confirm the link between TMB and HLA-ABC status and the response to ipilimumab-based immunotherapy in melanoma, but no correlation was found for TIL density, neither in primary nor metastatic lesions. Our finding that TMB between paired primary and metastatic melanoma lesions is highly stable, demonstrates its independency of the time point and location of acquisition. TIL and HLA-ABC status in metastatic lesions of different anatomical sites are highly similar within an individual patient.

Angiosarcomas (AS) are extremely rare and aggressive vascular malignancies subdivided in de novo primary AS (pAS) and secondary AS (sAS). We hypothesize that the combination of immunological and genomic profiles significantly differs between primary and secondary AS, with potential impact on treatment strategies and a role for immunotherapy. Tumor-infiltrating lymphocytes were analyzed using multiplex immunohistochemistry from 79 pAS and 178 sAS. Median cell density was significantly higher in sAS for CD3+ T-cells (p < 0.001), CD8+ cytotoxic T-cells (p = 0.033), CD4+ T-helper cells (p < 0.001) and FoxP3+ T-regulatory cells (p < 0.001). CD20+ B-cell density was comparable (p = 0.417). Comprehensive genomic profiling was performed in 25 pAS and 25 sAS. A (likely) pathogenic mutation was detected in 80% of pAS vs. 88% of sAS (p = 0.702). Amplifications were found in 15% of pAS vs. 84% of sAS (p < 0.001). DNA damage response (DDR) pathway mutations (p = 0.021) and MYC amplifications (p < 0.001) were predominantly seen in sAS. In conclusion we observed a clear and clinical relevant distinction in immune infiltration and genomic profiles between pAS and sAS. The T-cell infiltrated tumor microenvironment and frequent DDR gene mutations, especially in sAS, warrant clinical trials with immunotherapy.

Tissue specimens taken from primary tumors or metastases contain important information for diagnosis and treatment of cancer patients. Multispectral imaging allows in situ visualization of heterogeneous cell subsets, such as lymphocytes, in tissue samples. Many image processing pipelines first segment cell boundaries and then measure marker expression to assign cell phenotypes. In dense tissue environments such as solid tumors, segmentation-based phenotyping can be inaccurate due to segmentation errors or overlapping cell boundaries. Here we introduce a machine learning pipeline design called ImmuNet that directly identifies the positions and phenotypes of immune cells without determining their exact boundaries. ImmuNet is easy to train: human annotators only need to click on immune cells and rank their expression of each marker; full annotation of tissue regions is not necessary. We demonstrate that ImmuNet is a suitable approach for immune cell detection and phenotyping in multiplex im-munohistochemistry: it compares favourably to segmentation-based methods, especially in dense tissues, and we externally validate ImmuNet results by comparing them to flow cytometric measurements from the same tissue. In summary, ImmuNet performs well on diverse tissue specimens, takes relatively little effort to train and implement, and is a simpler alternative to segmentation-based approaches when only cell positions and phenotypes, but not their shapes are required for downstream analyses. We hope that ImmuNet will help cancer researchers to analyze multichannel tissue images more easily and accurately.

Background: Angiosarcomas (AS) are a rare heterogenous group of soft tissue sarcomas (STS) that form in the lining of blood vessels or lymphatic vessels. They comprise of primary (de novo) AS and secondary AS. The etiology of primary AS is unknown. Secondary AS arise due to DNA damaging factors like prior radiotherapy (RT), ultraviolet (UV) light exposure or chronic lymphedema (Stewart Treves syndrome). Treatment options are limited and their prognosis is poor. Development of new treatment strategies is difficult due to the rarity of these subgroups of STS. Genomic profiling of primary and secondary AS may provide a rationale for targeted treatment strategies. Method: Tumor samples were retrospectively collected from patients diagnosed with AS in the Netherlands. Patients were categorized as primary/secondary AS. Genomic profiles were analyzed using “TruSight Oncology 500”, a Next Generation Sequencing panel that analyzes variants in 523 cancer relevant genes. Results: Tumor DNA from 51 treatment naive AS patients was analyzed. The cohort comprised of 26 patients with a primary AS, divided in 5 subgroups: Heart (n=5, 10%), primary breast (n=5, 10%), skin not UV associated (n=4, 8%), soft tissue (n=5, 10%) and visceral (n=7, 14%) AS. The other 25 patients had a secondary AS, subdivided in RT-associated (n=13, 25%), Stewart Treves (n=5, 10%) and UV-associated (n=7, 14%) AS. Mean Tumor Mutational Burden (TMB) was 7.1 mutations per Mb for all patients (4.2 mut/Mb in primary AS vs 10.1 in secondary AS, p = 0.91). High TMB (≥10 mut/Mb) was found in 6 patients (12%) divided over 3 subgroups: UV associated AS (n=3/7 (43%)), visceral AS (n=2/7 (28%)) and skin not UV associated AS (n=1/4 (25%)). No patients were microsatellite instable. A pathogenic mutation, amplification or deletion was identified in 82% of all patients (n=42, 70% of primary AS vs 100% of secondary AS (p= Conclusion: We showed a clear distinction in genomic profiles of AS subgroups with specific pathogenic alterations. Especially secondary AS may benefit from treatment with ICI based on frequent MYC amplifications, DDR mutations, and high TMB. These data show clear evidence for the development of future treatment strategies with targeted therapy and ICI for this rare heterogeneous group of STS. Citation Format: Stefan G. van Ravensteijn, Yvonne M. Versleijen-Jonkers, Melissa H. Hillebrandt-Roeffen, Maikel Nederkoorn, Mark A. Gorris, Kiek Verrijp, Leonie I. Kroeze, Tessa J. de Bitter, Richarda M. de Voer, Uta E. Flucke, Ingrid M. Desar. The genomic landscape of primary and secondary angiosarcomas, a rare heterogenous group of soft tissue sarcomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB140.

The tumour microenvironment (TME) forms a major obstacle in effective cancer treatment and for clinical success of immunotherapy. Conventional co-cultures have shed light onto multiple aspects of cancer immunobiology, but they are limited by the lack of physiological complexity. We develop a human organotypic skin melanoma culture (OMC) that allows real-time study of host-malignant cell interactions within a multicellular tissue architecture. By co-culturing decellularized dermis with keratinocytes, fibroblasts and immune cells in the presence of melanoma cells, we generate a reconstructed TME that closely resembles tumour growth as observed in human lesions and supports cell survival and function. We demonstrate that the OMC is suitable and outperforms conventional 2D co-cultures for the study of TME-imprinting mechanisms. Within the OMC, we observe the tumour-driven conversion of cDC2s into CD14+ DCs, characterized by an immunosuppressive phenotype. The OMC provides a valuable approach to study how a TME affects the immune system., Conventional co-culture systems often lack physiological complexity of the tumor microenvironment. Here, the authors report an organotypic skin melanoma culture and use this model to investigate the tumor induced suppression on dendritic cells.

Contains fulltext : 202248.pdf (Publisher’s version ) (Closed access) Diffuse gliomas often carry point mutations in isocitrate dehydrogenase ( IDH1(mut)), resulting in metabolic stress. Although IDH(mut) gliomas are difficult to culture in vitro, they thrive in the brain via diffuse infiltration, suggesting brain-specific tumor-stroma interactions that can compensate for IDH-1 deficits. To elucidate the metabolic adjustments in clinical IDH(mut) gliomas that contribute to their malignancy, we applied a recently developed method of targeted quantitative RNA next-generation sequencing to 66 clinical gliomas and relevant orthotopic glioma xenografts, with and without the endogenous IDH-1(R132H) mutation. Datasets were analyzed in R using Manhattan plots to calculate distance between expression profiles, Ward's method to perform unsupervised agglomerative clustering, and the Mann Whitney U test and Fisher's exact tests for supervised group analyses. The significance of transcriptome data was investigated by protein analysis, in situ enzymatic activity mapping, and in vivo magnetic resonance spectroscopy of orthotopic IDH1(mut)- and IDH(wt)-glioma xenografts. Gene set enrichment analyses of clinical IDH1(mut) gliomas strongly suggest a role for catabolism of lactate and the neurotransmitter glutamate, whereas, in IDH(wt) gliomas, processing of glucose and glutamine are the predominant metabolic pathways. Further evidence of the differential metabolic activity in these cancers comes from in situ enzymatic mapping studies and preclinical in vivo magnetic resonance spectroscopy imaging. Our data support an evolutionary model in which IDH(mut) glioma cells exist in symbiosis with supportive neuronal cells and astrocytes as suppliers of glutamate and lactate, possibly explaining the diffuse nature of these cancers. The dependency on glutamate and lactate opens the way for novel approaches in the treatment of IDH(mut) gliomas.-Lenting, K., Khurshed, M., Peeters, T. H., van den Heuvel, C. N. A. M., van Lith, S. A. M., de Bitter, T., Hendriks, W., Span, P. N., Molenaar, R. J., Botman, D., Verrijp, K., Heerschap, A., ter Laan, M., Kusters, B., van Ewijk, A., Huynen, M. A., van Noorden, C. J. F., Leenders, W. P. J. Isocitrate dehydrogenase 1-mutated human gliomas depend on lactate and glutamate to alleviate metabolic stress.

BACKGROUND: It is assumed that high pore densities in flow diverters (FDs) are beneficial for intracranial aneurysm (IA) healing. However, various animal studies are not conclusive on the issue, suggesting that other factors are in play. One important factor might be wall apposition. OBJECTIVE: To (1) determine the relationship between FD pore density and aneurysm occlusion, and (2) determine the relationship between FD wall apposition and aneurysm occlusion. METHODS: Saccular aneurysms were microsurgically created in the aorta of 36 Wistar rats. Twelve rats received a low pore density FD (10 pores/mm2), 12 rats received a high pore density FD (23 pores/mm2), and the remaining 12 rats served as a control group. Six animals from each group were sacrificed 1 and 3 mo after surgery. We determined aneurysm occlusion, the number of struts not in contact with the aorta wall, and the average distance from malapposed struts to aorta wall through histology. RESULTS: No significant differences were found in aneurysm occlusion between the low pore density and high pore density groups (P > .05) after 1 and 3 mo of follow-up. The average number of malapposed struts was lower for the occluded aneurysm group (4.4 +/- 1.9) compared to the nonoccluded aneurysm group (7.7 +/- 2.6, P < .01). The average distance between malapposed struts and parent artery wall was lower for the occluded aneurysm group (33.9 mum +/- 11.5 mum) than for the nonoccluded aneurysm group (48.7 mum +/- 18.8 mum, P < .05). CONCLUSION: Wall apposition is more important than pore density for aneurysm occlusion.

// Sanne A.M. van Lith 1, * , Ilse Roodink 1, 5, * , Joost J.C. Verhoeff 2 , Petri I. Makinen 3 , Jari P. Lappalainen 3 , Seppo Yla-Herttuala 3, 4 , Jos Raats 5 , Erwin van Wijk 6 , Ronald Roepman 7 , Stef J. Letteboer 7 , Kiek Verrijp 1 , William P.J. Leenders 1 1 Department of Pathology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands 2 Department of Radiotherapy, Amsterdam Medical Center, 1100 DD, Amsterdam, The Netherlands 3 Department of Biotechnology and Molecular Medicine, University of Eastern Finland, FI-70211, Kuopio, Finland 4 Science Service Center and Gene Therapy Unit, Kuopio University Hospital, 70210 Kuopio, Finland 5 Modiquest BV, LSP, Molenstraat 110, 5342 CC, Oss, The Netherlands 6 Department of Otorhinolaryngology, RadboudUMC, 6500 HB, Nijmegen, The Netherlands 7 Department of Genetics, RadboudUMC, 6500 HB, Nijmegen,The Netherlands * These authors contributed equally to this work Correspondence to: William Leenders, email: william.leenders@radboudumc.nl Keywords: glioma, stroma, targeting, nanobody, macrophages Received: June 30, 2016 Accepted: September 19, 2016 Published: September 26, 2016 ABSTRACT Diffuse gliomas are primary brain cancers that are characterised by infiltrative growth. Whereas high-grade glioma characteristically presents with perinecrotic neovascularisation, large tumor areas thrive on pre-existent vasculature as well. Clinical studies have revealed that pharmacological inhibition of the angiogenic process does not improve survival of glioblastoma patients. Direct targeting of tumor vessels may however still be an interesting therapeutic approach as it allows pinching off the blood supply to tumor cells. Such tumor vessel targeting requires the identification of tumor-specific vascular targeting agents (TVTAs). Here we describe a novel TVTA, C-C7, which we identified via in vivo biopanning of a llama nanobody phage display library in an orthotopic mouse model of diffuse glioma. We show that C-C7 recognizes a subpopulation of tumor blood vessels in glioma xenografts and clinical glioma samples. Additionally, C-C7 recognizes macrophages and activated endothelial cells in atherosclerotic lesions. By using C-C7 as bait in yeast-2-hybrid (Y2H) screens we identified dynactin-1-p150Glued as its binding partner. The interaction was confirmed by co-immunostainings with C-C7 and a commercial anti-dynactin-1-p150Glued antibody, and via co-immunoprecipitation/western blot studies. Normal brain vessels do not express dynactin-1-p150 Glued and its expression is reduced under anti-VEGF therapy, suggesting that dynactin-1-p150 Glued is a marker for activated endothelial cells. In conclusion, we show that in vivo phage display combined with Y2H screenings provides a powerful approach to identify tumor-targeting nanobodies and their binding partners. Using this combination of methods we identify dynactin-1-p150 Glued as a novel targetable protein on activated endothelial cells and macrophages.

Contains fulltext : 181795.pdf (Publisher’s version ) (Closed access) The receptor tyrosine kinase (RTK) MET represents a promising tumor target in a subset of glioblastomas. Most RTK inhibitors available in the clinic today, including those inhibiting MET, affect multiple targets simultaneously. Previously, it was demonstrated that treatment with cabozantinib (MET/VEGFR2/RET inhibitor) prolonged survival of mice carrying orthotopic patient-derived xenografts (PDX) of the MET-addicted glioblastoma model E98, yet did not prevent development of recurrent and cabozantinib-resistant tumors. To exclude VEGFR2 inhibition-inflicted blood-brain barrier normalization and diminished tumor distribution of the drug, we have now investigated the effects of the novel MET-selective inhibitor Compound A in the orthotopic E98 xenograft model. In vitro, Compound A proved a highly potent inhibitor of proliferation of MET-addicted cell lines. In line with its target selectivity, Compound A did not restore the leaky blood-brain barrier and was more effective than cabozantinib in inhibiting MET phosphorylation in vivo Compound A treatment significantly prolonged survival of mice carrying E98 tumor xenografts, but did not prevent eventual progression. Contrasting in vitro results, the Compound A-treated xenografts displayed high levels of AKT phosphorylation despite the absence of phosphorylated MET. Profiling by RNA sequencing showed that in vivo transcriptomes differed significantly from those in control xenografts.Implications: Collectively, these findings demonstrate the plasticity of paracrine growth factor receptor signaling in vivo and urge for prudency with in vitro drug-testing strategies to validate monotherapies. Mol Cancer Res; 15(11); 1587-97. (c)2017 AACR.

Targeted carrier systems (e.g., liposomes or nanoparticles) are used to specifically deliver drugs to a site of interest. Site-direction can be achieved by attachment of targeting molecules, such as peptides, DNA/RNA, or antibodies, to the surface of the carrier. Here, the formation of polymersomes with tumor-targeting potential is described. A single-domain antibody (A12) that specifically targets PlexinD1 (a transmembrane protein overexpressed in tumor vasculature) is equipped with an azide-functionality using expressed protein ligation. This azide-containing A12 can subsequently be attached to BCN-functionalized polymersomes using a strain-promoted azide alkyne cycloaddition, thereby forming polymersomes with tumor-targeting potential.

The majority of low-grade and secondary high-grade gliomas carry heterozygous hotspot mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) or the mitochondrial variant IDH2. These mutations mostly involve Arg132 in IDH1 and Arg172 or Arg140 in IDH2. Whereas IDHs convert isocitrate to alpha-ketoglutarate (α-KG) with simultaneous reduction of NADP+ to NADPH, these IDH mutants reduce α-KG to D-2-hydroxyglutarate (D-2-HG) while oxidizing NADPH. D-2-HG is a proposed oncometabolite, acting via competitive inhibition of α-KG-dependent enzymes that are involved in metabolism and epigenetic regulation. However, much less is known about the implications of the metabolic stress, imposed by decreased α-KG and NADPH production, for tumor biology. We here present a novel heterozygous IDH1 mutation, IDH1R314C, which was identified by targeted next generation sequencing of a high grade glioma from which a mouse xenograft model and a cell line were generated. IDH1R314C lacks isocitrate-to-α-KG conversion activity due to reduced affinity for NADP+ and differs from the IDH1R132 mutants in that it does not produce D-2-HG. Because IDH1R314C is defective in producing α-KG and NADPH, without concomitant production of the D-2-HG, it represents a valuable tool to study the effects of IDH1-dysfunction on cellular metabolism in the absence of this oncometabolite.

The infiltrative behavior of diffuse gliomas severely reduces therapeutic potential of surgical resection and radiotherapy, and urges for the identification of new drug-targets affecting glioma growth and migration. To address the potential role of protein tyrosine phosphatases (PTPs), we performed mRNA expression profiling for 91 of the 109 known human PTP genes on a series of clinical diffuse glioma samples of different grades and compared our findings with in silico knowledge from REMBRANDT and TCGA databases. Overall PTP family expression levels appeared independent of characteristic genetic aberrations associated with lower grade or high grade gliomas. Notably, seven PTP genes (DUSP26, MTMR4, PTEN, PTPRM, PTPRN2, PTPRT and PTPRZ1) were differentially expressed between grade II-III gliomas and (grade IV) glioblastomas. For DUSP26, PTEN, PTPRM and PTPRT, lower expression levels correlated with poor prognosis, and overexpression of DUSP26 or PTPRT in E98 glioblastoma cells reduced tumorigenicity. Our study represents the first in-depth analysis of PTP family expression in diffuse glioma subtypes and warrants further investigations into PTP-dependent signaling events as new entry points for improved therapy. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0372-x) contains supplementary material, which is available to authorized users.

Contains fulltext : 167769.pdf (Publisher’s version ) (Closed access) BACKGROUND: Gliomas are primary brain tumors that are associated with a poor prognosis. The introduction of new treatment modalities (including immunotherapy) for these neoplasms in the last 3 decades has resulted in only limited improvement in survival. Gliomas are known to create an immunosuppressive microenvironment that hampers the efficacy of (immuno)therapy. One component of this immunosuppressive environment is the myeloid-derived suppressor cell (MDSC). METHODS: We set out to analyze the presence and activation state of MDSCs in blood (n = 41) and tumor (n = 20) of glioma patients by measuring S100A8/9 and arginase using flow cytometry and qPCR. Inhibition of T cell proliferation and cytokine production after stimulation with anti-CD3/anti-CD28 coated beads was used to measure in vitro MDSC suppression capacity. RESULTS: We report a trend toward a tumor grade-dependent increase of both monocytic (M-) and polymorphonuclear (PMN-) MDSC subpopulations in the blood of patients with glioma. M-MDSCs of glioma patients have increased levels of intracellular S100A8/9 compared with M-MDSCs in healthy controls (HCs). Glioma patients also have increased S100A8/9 serum levels, which correlates with increased arginase activity in serum. PMN-MDSCs in both blood and tumor tissue demonstrated high expression of arginase. Furthermore, we assessed blood-derived PMN-MDSC function and showed that these cells have potent T cell suppressive function in vitro. CONCLUSIONS: These data indicate a tumor grade-dependent increase of MDSCs in the blood of patients with a glioma. These MDSCs exhibit an increased activation state compared with MDSCs in HCs, independent of tumor grade.

Background Neoadjuvant chemoradiotherapy (nCRT) followed by resection of the tumor with two field lymphadenectomy is a standard treatment for esophageal cancer. After nCRT, however, in more than 70% of patients no lymph node metastases are found, suggesting extensive overtreatment. Tumor-targeted fluorescence imaging is a promising technique to detect lymph node metastases intra-operatively and guide personalized resection. The aim of this study is to identify potential viable tumor markers for fluorescence imaging of lymph node metastases in patients with esophageal adenocarcinoma (EAC). Methods Immunohistochemistry (IHC) was performed on tissue microarrays from EAC’s patients that underwent surgical resection between 2007 and 2016. Patients were subdivided in five groups, non-pretreated patients with and without metastatic lymph nodes, complete responders, partial responders and non-responders after nCRT. Five membranous markers, c-MET, CAIX, EGFR, EpCAM, HER2, and two cytoplasmic markers, VEGF-A and VEGF-A receptor were included. Tumor marker expression was scored on intensity (none (0), slight (1), moderate (2), strong (3)) and the percentage of positive cells (estimation). Threshold for positive detection rate was defined as an intensity of ≥ 2 in more than 10% the cells. Results EpCAM showed the highest expression in metastastic lymph nodes, with a median intensity of 3 (range 2–3) in > 70% of the tumor cells. Expression was found in 37 out of 39 EAC’s (95%). VEGF-A and CAIX expression was observed in 28 of 33 (85%) and 10 of 33 (30%) of metastatic lymph nodes and 34 of 39 (87%) and 17 of 39 (44%) in the primary EAC’s, respectively. For the other tumor biomarkers the detection rate ranged between 0 and 11% for metastatic lymph nodes and primary EAC’s. Only EpCAM and VEGF-A showed weak, non-specific staining in the fibrotic tissue. Conclusion High expression rates in primary EAC and metastatic lymph nodes were observed using immunohistochemical antibodies for EpCAM, VEGF-A and CA-IX, making these clinically relevant viable EAC tumor markers. A phase 1 dose finding study targeting VEGF-A by Bevacizumab-800-CW in patient with EAC is in preparation. Disclosure All authors have declared no conflicts of interest.

5036Background: Predictive biomarkers are needed to improve the proportion of pts with mCRPC that may benefit from anti-PD-1/PD-L1 ICB. A comprehensive characterization of both genomic alterations ...

Tumour metastasis is the result of a complex sequence of events, including migration of tumour cells through stroma, proteolytic degradation of stromal and vessel wall elements, intravasation, transport through the circulation, extravasation and outgrowth at compatible sites in the body (the 'seed and soil' hypothesis). However, the high incidence of metastasis from various tumour types in liver and lung may be explained by a stochastic process as well, based on the anatomical relationship of the primary tumour with the circulation and mechanical entrapment of metastatic tumour cells in capillary beds. We previously reported that constitutive VEGF-A expression in tumour xenografts facilitates this type of metastatic seeding by promoting shedding of multicellular tumour tissue fragments, surrounded by vessel wall elements, into the circulation. After transport through the vena cava, such fragments may be trapped in pulmonary arteries, allowing them to expand to symptomatic lesions. Here we tested whether this process has clinical relevance for clear cell renal cell carcinoma (ccRCC), a prototype tumour in the sense of high constitutive VEGF-A expression. To this end we collected and analysed outflow samples from the renal vein, directly after tumour nephrectomy, in 42 patients diagnosed with ccRCC. Tumour fragments in venous outflow were observed in 33% of ccRCC patients and correlated with the synchronous presence or metachronous development of pulmonary metastases (p < 0.001, Fisher's exact test). In patients with tumours that, in retrospect, were not of the VEGF-A-expressing clear cell type, tumour fragments were never observed in the renal outflow. These data suggest that, in ccRCC, a VEGF-A-induced phenotype promotes a release of tumour cell clusters into the circulation that may contribute to pulmonary metastasis.

Contains fulltext : 69878.pdf (Publisher’s version ) (Closed access) AIM OF THE STUDY: To investigate the correlation between tumour accumulation of In-111-bevacizumab and VEGF-A expression in patients with colorectal liver metastases. METHODS: Two weeks before resection of the liver metastases 12 patients were intravenously injected with In-111-labelled bevacizumab. Ten minutes and 7 d after injection a whole body scan was acquired. Seven days after the injection, 3D acquisition SPECT of the liver was performed. RESULTS: Enhanced uptake of In-111-bevacizumab in the liver metastases was observed in 9 of the 12 patients. The level of antibody accumulation in these lesions varied considerably. There was no correlation between the level of In-111-antibody accumulation and the level of VEGF-A expression in the tissue as determined by in situ hybridisation and ELISA. CONCLUSIONS: In this study, we investigated the correlation between tumour accumulation of radiolabelled bevacizumab and VEGF-A expression in patients with colorectal liver metastases. No clear-cut correlation between the level of antibody accumulation and expression of VEGF-A was found.

Contains fulltext : 153143.pdf (Publisher’s version ) (Closed access) Myeloid-derived suppressor cells (MDSCs), defined as CD33-positive major histocompatibility complex class II-negative cells, are increased in a variety of human tumors and are associated with immunosuppression. Myeloid-derived suppressor cells can be further subdivided into CD14-positive monocytic MDSC and CD15-positive granulocytic MDSC (polymorphonuclear MDSC) subpopulations. Here we analyzed MDSC subsets in the blood and tumor tissue of patients with glioma, including the most malignant variant, glioblastoma multiforme (GBM). CD33-positive major histocompatibility complex class II-negative MDSCs in blood from 21 patients with glioma and 12 healthy individuals were phenotyped and quantified by flow cytometry. Myeloid populations of the monocytic MDSC and polymorphonuclear MDSC phenotypes were both significantly increased in the blood of patients with GBM versus healthy controls. The myeloid activation markers CD80 and PD-L1 could not be detected on either of these MDSC subsets; CD124, CD86, and CD40 were detected at similar levels on MDSCs in patients with glioma and healthy donors. By contrast, in tumor cell suspensions, the MDSC population consisted almost exclusively of CD15-positive cells. Immunohistochemistry confirmed infiltration of CD15-positive major histocompatibility complex class II-negative cells in glioma tissue samples. These data support a role for cells with an MDSC phenotype in the blood and tumor microenvironment of patients with GBM.

Tumors arise initially as avascular masses in which central hypoxia induces expression of vascular endothelial growth factor-A (VEGF-A) and subsequently tumor vascularization. However, VEGF-A can also be constitutively expressed as a result of genetic events. VEGF-A is alternatively spliced to yield at least 6 different isoforms. Of these, VEGF-A(121) is freely diffusible whereas basically charged domains in the larger isoforms confer affinity for cell surface or extracellular matrix components. We previously reported that in a mouse brain metastasis model of human melanoma, VEGF-A(121) induced a qualitatively different tumor vascular phenotype than VEGF-A(165) and VEGF-A(189): in contrast to the latter ones, and VEGF-A(121) did not induce a neovascular bed but rather led to leakage and dilatation of preexistent brain vessels. Here, we correlate vascular phenotypes with spatial VEGF-A expression profiles in clinical brain tumors (low grade gliomas; n = 6, melanoma metastases; n = 4, adenocarcinoma metastases; n = 4, glioblastoma multiforme; n = 3, sarcoma metastasis; n = 1, renal cell carcinoma metastasis; n = 1). We show that tumors that constitutively express VEGF-A present with different vascular beds than tumors in which VEGF-A is expressed as a response to central hypoxia. This phenotypic difference is consistent with a model where in tumors with constitutive VEGF-A expression, all isoforms exert their effects on vasculature, resulting in a classical angiogenic phenotype. In tumors where only central parts express hypoxia-induced VEGF-A, the larger angiogenic isoforms are retained by extracellular matrix, leaving only freely diffusible VEGF-A(121) to exert its dilatation effects on distant vessels.

Contains fulltext : 36095.pdf (Publisher’s version ) (Closed access) BACKGROUND: Thy-1.1 transgenic mice develop hypercellular focal and segmental glomerulosclerosis (FSGS) lesions that mimic human collapsing FSGS, in 7 days after injection with anti-Thy-1.1 antibodies. These lesions consist of proliferating parietal epithelial cells (PECs). We questioned whether the angiotensin converting enzyme inhibitor (ACE), captopril, could prevent the development of FSGS and if protection is related to the timing of drug administration. METHODS: First, we compared the effect of captopril treatment with angiotensin II-(ANGII) independent antihypertensive therapy (triple therapy). Second, we tested the effects of captopril administered over four different time intervals: days -7 to 0 (Ca-7>0), days -7 to 7 (Ca-7>7), days 0-7 (Ca0>7) and days 3-7 (Ca3>7) (day 0 being the day of injection of the antibody). RESULTS: In anti-Thy-1.1 injected control (C) mice we observed dedifferentiation and activation of podocytes, reflected by loss of ASD33 and increased expression of desmin, followed by a marked accumulation of PECs forming hypercellular lesions. PECs showed an increased expression of connective tissue growth factor (CTGF). Triple therapy or captorpil pre-treatment (Ca-7>0) had no significant effect on albuminuria or FSGS. In contrast, Ca0>7 and Ca3>7 treatment significantly lowered albuminuria and attenuated development of FSGS. The latter two treatments attenuated loss of ASD33 expression by podocytes but could not prevent increased desmin expression. In addition, these treatments reduced CTGF expression by PECs and prevented PEC proliferation. CONCLUSIONS: ACE inhibition, but not triple therapy, prevents the development of FSGS, suggesting an important role for ANGII. ACE inhibition has a protective effect even when started 3 days after the initial podocyte insult, which is probably related to the ability of ACE-inhibition to block PEC activation and proliferation.

Background - Grade IV diffuse gliomas (glioblastomas) are notoriously difficult to treat. Many studies aim at targeting tumor-specific aberrations, such as mutations in genes encoding oncogenic receptor tyrosine kinases (RTKs). Of high interest as a tumor target in diffuse glioma is the RTK MET, which is amplified in a significant proportion of glioblastomas, and a number of MET inhibitors have been developed. However, most RTK inhibitors available in the clinic today, including those inhibiting MET, are not entirely selective and inhibit additional kinases at the doses used. They therefore may induce potentially undesired off-target effects, such as blood-brain barrier normalization in the case of concomitant VEGFR2 inhibition. Methods – We studied the effects of the novel, selective MET-kinase inhibitor (Compound A) and the combined VEGFR2/RET/MET inhibitor cabozantinib on MET activation and proliferation in the MET-amplified E98 astrocytoma cell line in vitro, using western blot analysis and MTT proliferation assays. Effects of compound A were also studied in mice carrying orthotopic xenografts of the same E98 cell line. Survival was monitored and effects of MET inhibition were investigated by immunohistochemistry using phospho-specific antibodies. Results – E98 cells were highly sensitive to treatment with Compound A in vitro (IC50~9.4 nM). Furthermore, Compound A effectively inhibited phosphorylation of MET in orthotopic E98 xenografts. In contrast, we have previously shown that after treatment with cabozantinib in the same model the high levels of phosphorylated MET were maintained. While treatment with Compound A significantly prolonged the survival of E98-xenograft bearing mice, tumors still developed and showed extensive AKT phosphorylation in the absence of MET phopshorylation, suggesting in vivo therapy resistance mechanisms. Interestingly, resistance to Compound A was not seen in vitro in an E98 cell line derived from Compound A-resistant tumor xenografts. Conclusions – Compound A is a promising, highly selective MET kinase inhibitor with activity against gliomas with constitutive MET signaling. Selective MET kinase inhibitors may be more suited for treatment of glioma than combined VEGFR2/MET inhibitors, as the latter may induce vessel normalization resulting in poor tumor penetration. Similar to clinical experience, E98 tumor-bearing mice ultimately develop resistance to TKIs. Combination therapies targeting both MET and potential resistance pathways may be required to allow long-term tumor treatment. Citation Format: Corina van den Heuvel, Anna Navis, Houshang Amiri, Kiek Verrijp, Arend Heerschap, Karen Rex, Isabelle Dussault, Sean Caenepeel, Angela Coxon, Pieter Wesseling, William Leenders. Selective MET kinase inhibition in MET-dependent glioma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2077. doi:10.1158/1538-7445.AM2017-2077

// Annika M. Bourgonje 1 , Anna C. Navis 2 , Jan T.G. Schepens 1 , Kiek Verrijp 2 , Liesbeth Hovestad 3 , Riet Hilhorst 3 , Sheila Harroch 4 , Pieter Wesseling 2,5 , William P.J. Leenders 2 and Wiljan J.A.J. Hendriks 1 1 Department of Cell Biology , Radboud University Medical Center, Nijmegen, The Netherlands 2 Department of Pathology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, The Netherlands 3 PamGene International BV, ’s-Hertogenbosch, The Netherlands 4 Department of Neuroscience, Institut Pasteur, Paris, France 5 Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands Correspondence: Wiljan J.A.J. Hendriks, email: // Keywords : PTPRZ1, diffuse infiltrative growth, signal transduction, cell migration, tyrosine phosphorylation, PDZ Received : June 07, 2014 Accepted : August 18, 2014 Published : August 19, 2014 Abstract Gliomas are primary brain tumors for which surgical resection and radiotherapy is difficult because of the diffuse infiltrative growth of the tumor into the brain parenchyma. For development of alternative, drug-based, therapies more insight in the molecular processes that steer this typical growth and morphodynamic behavior of glioma cells is needed. Protein tyrosine phosphatase PTPRZ-B is a transmembrane signaling molecule that is found to be strongly up-regulated in glioma specimens. We assessed the contribution of PTPRZ-B protein domains to tumor cell growth and migration, via lentiviral knock-down and over-expression using clinically relevant glioma xenografts and their derived cell models. PTPRZ-B knock-down resulted in reduced migration and proliferation of glioma cells in vitro and also inhibited tumor growth in vivo . Interestingly, expression of only the PTPRZ-B extracellular segment was sufficient to rescue the in vitro migratory phenotype that resulted from PTPRZ-B knock-down. In contrast, PTPRZ-B knock-down effects on proliferation could be reverted only after re-expression of PTPRZ-B variants that contained its C-terminal PDZ binding domain. Thus, distinct domains of PTPRZ-B are differentially required for migration and proliferation of glioma cells, respectively. PTPRZ-B signaling pathways therefore represent attractive therapeutic entry points to combat these tumors.

Contains fulltext : 118357.pdf (Publisher’s version ) (Open Access) Anti-angiogenic treatment of glioblastoma with Vascular Endothelial Growth Factor (VEGF)- or VEGF Receptor 2 (VEGFR2) inhibitors normalizes tumor vessels, resulting in a profound radiologic response and improved quality of life. This approach however does not halt tumor progression by diffuse infiltration, as this phenotype is less angiogenesis dependent. Combined inhibition of angiogenesis and diffuse infiltrative growth would therefore be a more effective treatment approach in these tumors. The HGF/c-MET axis is important in both angiogenesis and cell migration in several tumor types including glioma. We therefore analyzed the effects of the c-MET- and VEGFR2 tyrosine kinase inhibitor cabozantinib (XL184, Exelixis) on c-MET positive orthotopic E98 glioblastoma xenografts, which routinely present with angiogenesis-dependent areas of tumor growth, as well as diffuse infiltrative growth. In cultures of E98 cells, cabozantinib effectively inhibited c-MET phosphorylation, concomitant with inhibitory effects on AKT and ERK1/2 phosphorylation, and cell proliferation and migration. VEGFR2 activation in endothelial cells was also effectively inhibited . Treatment of BALB/c nu/nu mice carrying orthotopic E98 xenografts resulted in a significant increase in overall survival. Cabozantinib effectively inhibited angiogenesis, resulting in increased hypoxia in angiogenesis-dependent tumor areas, and induced vessel normalization. Yet, tumors ultimately escaped cabozantinib therapy by diffuse infiltrative outgrowth via vessel co-option. Of importance, in contrast to the results from experiments, blockade of c-MET activation was incomplete, possibly due to multiple factors including restoration of the blood-brain barrier resulting from cabozantinib-induced VEGFR2 inhibition. In conclusion, cabozantinib is a promising therapy for c-MET positive glioma, but improving delivery of the drug to the tumor and/or the surrounding tissue may be needed for full activity.

Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5–13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations., Author Summary Idiopathic dilated cardiomyopathy (DCM) is estimated to be of genetic origin in 20%–48% of the patients. Almost all currently known genetic defects show dominant inheritance, although especially in younger children recessive causes have been proposed to contribute considerably to DCM. Knowledge of the genetic causes and pathophysiological mechanisms is essential for prognosis and treatment. Here, we studied several individual young patients (5–13 years old) with idiopathic and sometimes asymptomatic dilated cardiomyopathy. The key to identification of the gene was the finding of abnormal protein N-glycosylation. Via homozygosity mapping and functional knowledge of the N-glycosylation pathway, the causative gene could be identified as dolichol kinase (DOLK). Since DCM is very rare in N-glycosylation disorders (Congenital Disorders of Glycosylation, CDG) and most patients with CDG present with a multisystem involvement, we studied the underlying pathophysiological cause of this life-threatening disease. Biochemical experiments in affected heart tissue showed deficient O-mannosylation of alpha-dystroglycan, which could be correlated with the dilated cardiomyopathy. Our results thus highlight nonsyndromic DCM as a novel presentation of DOLK-CDG, via deficient O-mannosylation of alpha-dystroglycan.

Contains fulltext : 97113.pdf (Publisher’s version ) (Open Access) INTRODUCTION: Tenascin-X (TNX) is an extracellular matrix (ECM) glycoprotein, the absence of which in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS), a group of inherited connective tissue disorders characterized by joint hypermobility, skin hyperextensibility, and tissue fragility. A mouse model of TNX-deficient type EDS has been used to characterize the dermatological, orthopedic, and obstetrical features. The growing insight in the clinical overlap between myopathies and inherited connective tissue disorders asks for a study of the muscular characteristics of inherited connective tissue diseases. Therefore, this study aims to define the muscular phenotype of TNX knockout (KO) mice. MATERIALS AND METHODS: We performed a comprehensive study on the muscular phenotype of these TNX KO mice, consisting of standardized clinical assessment, muscle histology, and gene expression profiling of muscle tissue. Furthermore, peripheral nerve composition was studied by histology and electron microscopy. RESULTS: The main findings are the presence of mild muscle weakness, mild myopathic features on histology, and functional upregulation of genes encoding proteins involved in ECM degradation and synthesis. Additionally, sciatic nerve samples showed mildly reduced collagen fibril density of endoneurium. DISCUSSION: The muscular phenotype of TNX KO mice consists of mild muscle weakness with histological signs of myopathy and of increased turnover of the ECM in muscle. Furthermore, mildly reduced diameter of myelinated fibers and reduction of collagen fibril density of endoneurium may correspond with polyneuropathy in TNX-deficient EDS patients. This comprehensive assessment can serve as a starting point for further investigations on neuromuscular function in TNX KO mice.

Contains fulltext : 87540.pdf (Publisher’s version ) (Closed access) INTRODUCTION: In aromatic L-amino acid decarboxylase (AADC) deficiency, a neurotransmitter biosynthesis defect, paradoxical normal or increased levels of urinary dopamine have been reported. Genotype/phenotype correlations or alternative metabolic pathways may explain this remarkable finding, but were never studied systematically. METHODS: We studied the mutational spectrum and urinary dopamine levels in 20 patients with AADC-deficiency. Experimental procedures were designed to test for alternative metabolic pathways of dopamine production, which included alternative substrates (tyramine and 3-methoxytyrosine) and alternative enzymes (tyrosinase and CYP2D6). RESULTS/DISCUSSION: In 85% of the patients the finding of normal or increased urinary levels of dopamine was confirmed, but a relation with AADC genotype could not be identified. Renal microsomes containing CYP2D were able to convert tyramine into dopamine (3.0 nmol/min/g protein) but because of low plasma levels of tyramine this is an unlikely explanation for urinary dopamine excretion in AADC-deficiency. No evidence was found for the production of dopamine from 3-methoxytyrosine. Tyrosinase was not expressed in human kidney. CONCLUSION: Normal or increased levels of urinary dopamine are found in the majority of AADC-deficient patients. This finding can neither be explained by genotype/phenotype correlations nor by alternative metabolic pathways, although small amounts of dopamine may be formed via tyramine hydroxylation by renal CYP2D6. CYP2D6-mediated conversion of tyramine into dopamine might be an interesting target for the development of new therapeutic strategies in AADC-deficiency. 01 december 2010

Contains fulltext : 87890.pdf (Publisher’s version ) (Closed access) Tumor vasculature is in general highly heterogeneous. This characteristic is most prominent in high-grade gliomas, which present with areas of angiogenic growth, next to large areas of diffuse infiltrative growth in which tumor cells thrive on pre-existent brain vasculature. This limits the effectiveness of anti-angiogenic compounds as these will not affect more matured and co-opted vessels. Therefore, additional destruction of existing tumor vasculature may be a promising alternative avenue to effectively deprive tumors from blood. This approach requires the identification of novel tumor vascular targeting agents, which have broad tumor vessel specificities, ie are not restricted to newly formed vessels. Here, we describe the generation of a phage library displaying nanobodies that were cloned from lymphocytes of a Llama which had been immunized with clinical glioma tissue. In vivo biopanning with this library in the orthotopic glioma xenograft models E98 and E434 resulted in the selection of various nanobodies which specifically recognized glioma vessels in corresponding glioma xenografts. Importantly, also nanobodies were isolated which discriminated incorporated pre-existent vessels in highly infiltrative cerebral E434 xenografts from normal brain vessels. Our results suggest that the generation of nanobody-displaying immune phage libraries and subsequent in vivo biopanning in appropriate animal models is a promising approach for the identification of novel vascular targeting agents. 01 januari 2010

Tumour metastasis is the result of a complex sequence of events, including migration of tumour cells through stroma, proteolytic degradation of stromal and vessel wall elements, intravasation, transport through the circulation, extravasation and outgrowth at compatible sites in the body (the 'seed and soil' hypothesis). However, the high incidence of metastasis from various tumour types in liver and lung may be explained by a stochastic process as well, based on the anatomical relationship of the primary tumour with the circulation and mechanical entrapment of metastatic tumour cells in capillary beds. We previously reported that constitutive VEGF-A expression in tumour xenografts facilitates this type of metastatic seeding by promoting shedding of multicellular tumour tissue fragments, surrounded by vessel wall elements, into the circulation. After transport through the vena cava, such fragments may be trapped in pulmonary arteries, allowing them to expand to symptomatic lesions. Here we tested whether this process has clinical relevance for clear cell renal cell carcinoma (ccRCC), a prototype tumour in the sense of high constitutive VEGF-A expression. To this end we collected and analysed outflow samples from the renal vein, directly after tumour nephrectomy, in 42 patients diagnosed with ccRCC. Tumour fragments in venous outflow were observed in 33% of ccRCC patients and correlated with the synchronous presence or metachronous development of pulmonary metastases (p0.001, Fisher's exact test). In patients with tumours that, in retrospect, were not of the VEGF-A-expressing clear cell type, tumour fragments were never observed in the renal outflow. These data suggest that, in ccRCC, a VEGF-A-induced phenotype promotes a release of tumour cell clusters into the circulation that may contribute to pulmonary metastasis.

Contains fulltext : 79501.pdf (Publisher’s version ) (Open Access) BACKGROUND: Plexin D1 is expressed on both tumor-associated endothelium and malignant cells in a number of clinical brain tumors. Recently we demonstrated that Plexin D1 expression is correlated with tumor invasion level and metastasis in a human melanoma progression series. The objective of this study was to examine whether Plexin D1 might be clinically useful as a pan-tumor vessel and pan-tumor cell target in solid tumors. METHODS: We examined Plexin D1 expression in clinical solid tumors (n = 77) of different origin, a selection of pre-malignant lesions (n = 29) and a variety of non-tumor related tissues (n = 52) by immunohistochemistry. Signals were verified in a selection of tissues via mRNA in situ hybridization. RESULTS: Plexin D1 is abundantly expressed on both activated established tumor vasculature and malignant cells in the majority of primary and metastatic clinical tumors, as well as on macrophages and fibroblasts. Importantly, in non-tumor related tissues Plexin D1 expression is restricted to a subset of, presumably activated, fibroblasts and macrophages. CONCLUSION: We demonstrate that Plexin D1 is in general ubiquitously expressed in tumor but not normal vasculature, as well as in malignant cells in a wide range of human tissues. This expression profile highlights Plexin D1 as a potentially valuable therapeutic target in clinical solid tumors, enabling simultaneous targeting of different tumor compartments.

Contains fulltext : 81544.pdf (Publisher’s version ) (Open Access) In this study, we examined the correlation between muscle ultrasound and muscle structure. Echo intensity (EI) of 14 muscles of two golden retriever muscular dystrophy dogs was correlated to the percentage interstitial fibrous tissue and fat in muscle biopsy. A significant correlation between interstitial fibrous tissue and EI was found (r = 0.87; p < 0.001). The separate influence of interstitial fat on muscle EI could not be established as only little fat was present. We conclude that fibrous tissue causes increased muscle EI. The high correlation between interstitial fibrous tissue and EI makes ultrasound a reliable method to determine severity of structural muscle changes.

Contains fulltext : 81037.pdf (Publisher’s version ) (Open Access) PURPOSE: Vascular endothelial growth factor-A (VEGF-A) is one of the most important factors inducing angiogenesis in tumors. Nine splice-variant isoforms of VEGF-A have been identified, each having different properties. Recently, we showed that radiolabeled anti-VEGF monoclonal antibody, bevacizumab, accumulates specifically in VEGF-A expressing tumors. In this study, we investigated in a nude mouse model which VEGF-isoforms are responsible for tumor accretion. MATERIALS AND METHODS: The humanized anti-VEGF-A antibody, A.4.6.1. (bevacizumab), was radiolabeled with In-111. The originally VEGF-negative Mel57 tumor was transfected with different VEGF isoforms (VEGF-121, VEGF-165, and VEGF-189). The obtained melanoma xenografts specifically expressing different VEGF-isoforms were used in mice. The bevacizumab uptake was examined in biodistribution studies and by gamma-camera imaging. RESULTS: The tumor cell line expressing VEGF-121 did not show specific uptake, most likely as a result of the fact that this isoform is freely diffusible. Tumors expressing VEGF-165 and -189 were clearly visualized by using gamma-camera imaging. CONCLUSION: The accumulation of radiolabeled bevacizumab in the tumor is due to interaction with VEGF-A isoforms that are associated with the tumor cell surface and/or the extracellular matrix. Scintigraphic imaging of the expression of these VEGF isoforms may thus be useful to predict response to angiogenic therapy.

Contains fulltext : 70965.pdf (Publisher’s version ) (Closed access) Vascular endothelial growth factor-A (VEGF-A) is one of the most important angiogenic factors. Here, we studied in a nude mouse model whether the expression of VEGF-A in a tumor could be imaged with a radiolabeled anti-VEGF antibody. The humanized anti-VEGF-A antibody A.4.6.1. (bevacizumab), which is reactive with all VEGF-A isoforms, was radiolabeled with In-111 or with I-125. The accumulation of the radiolabeled antibodies in VEGF-A expressing tumors (LS174T) in nude mice was examined in biodistribution studies and by gamma camera imaging. The uptake of the In-111-bevacizumab in the tumor at 3 days p.i. was significantly higher than that of I-125-bevacizumab (19.4 +/- 7.0 %ID/g vs. 9.6 +/- 3.3 %ID/g, p = 0.04). Coinjection of an excess unlabeled antibody resulted in a significant decrease in radioactivity concentration in the tumor (

Contains fulltext : 71083.pdf (Publisher’s version ) (Open Access) Plexin D1 (PLXND1) is broadly expressed on tumor vessels and tumor cells in a number of different human tumor types. Little is known, however, about the potential functional contribution of PLXND1 expression to tumor development. Expression of semaphorin 3E (Sema3E), one of the ligands for PLXND1, has previously been correlated with invasive behavior and metastasis, suggesting that the PLXND1-Sema3E interaction may play a role in tumor progression. Here we investigated PLXND1 and Sema3E expression during tumor progression in cases of melanoma. PLXND1 was not expressed by melanocytic cells in either naevi or melanomas in situ, whereas expression increased with invasion level, according to Clark's criteria. Furthermore, 89% of the metastatic melanomas examined showed membranous PLXND1-staining of tumor cells. Surprisingly, expression of Sema3E was inversely correlated with tumor progression, with no detectable staining in melanoma metastasis. To functionally assess the effects of Sema3E expression on tumor development, we overexpressed Sema3E in a xenograft model of metastatic melanoma. Sema3E expression dramatically decreased metastatic potential. These results show that PLXND1 expression during tumor development is strongly correlated with both invasive behavior and metastasis, but exclude Sema3E as an activating ligand.

Contains fulltext : 51895.pdf (Publisher’s version ) (Closed access) How and why tumors metastasize is still a matter of debate. The assumption is that mutations render tumor cells with a metastatic phenotype, enabling entrance in and transport through lymph or blood vessels. Distant outgrowth is thought to occur only in a suitable microenvironment (the seed and soil hypothesis). However, the anatomical location of most metastases in cancer patients suggests entrapment of tumor cells in the first microcapillary bed that is encountered. We here investigated how vascular endothelial growth factor-A (VEGF-A) attributes to the metastatic process. We describe here that VEGF-A enhances spontaneous metastasis by inducing intravasation of heterogeneous tumor cell clusters, surrounded by vessel wall elements, via an invasion-independent mechanism. These tumor clusters generate metastatic tissue embolisms in pulmonary arteries. Treatment of tumor-bearing mice with the antiangiogenic compound ZD6474 prevented the development of this metastatic phenotype. This work shows that tumors with high constitutive VEGF-A expression metastasize via the formation of tumor emboli and provides an alternative rationale for anti-VEGF-A therapy, namely to inhibit metastasis formation.

Tumors arise initially as avascular masses in which central hypoxia induces expression of vascular endothelial growth factor-A (VEGF-A) and subsequently tumor vascularization. However, VEGF-A can also be constitutively expressed as a result of genetic events. VEGF-A is alternatively spliced to yield at least 6 different isoforms. Of these, VEGF-A(121) is freely diffusible whereas basically charged domains in the larger isoforms confer affinity for cell surface or extracellular matrix components. We previously reported that in a mouse brain metastasis model of human melanoma, VEGF-A(121) induced a qualitatively different tumor vascular phenotype than VEGF-A(165) and VEGF-A(189): in contrast to the latter ones, and VEGF-A(121) did not induce a neovascular bed but rather led to leakage and dilatation of preexistent brain vessels. Here, we correlate vascular phenotypes with spatial VEGF-A expression profiles in clinical brain tumors (low grade gliomas; n = 6, melanoma metastases; n = 4, adenocarcinoma metastases; n = 4, glioblastoma multiforme; n = 3, sarcoma metastasis; n = 1, renal cell carcinoma metastasis; n = 1). We show that tumors that constitutively express VEGF-A present with different vascular beds than tumors in which VEGF-A is expressed as a response to central hypoxia. This phenotypic difference is consistent with a model where in tumors with constitutive VEGF-A expression, all isoforms exert their effects on vasculature, resulting in a classical angiogenic phenotype. In tumors where only central parts express hypoxia-induced VEGF-A, the larger angiogenic isoforms are retained by extracellular matrix, leaving only freely diffusible VEGF-A(121) to exert its dilatation effects on distant vessels.

Contains fulltext : 49831.pdf (Publisher’s version ) (Closed access) Collapsing focal segmental glomerulosclerosis (cFSGS) is characterized by hyperplasia of glomerular epithelial cells. In a mouse model of FSGS and in a patient with recurrent idiopathic FSGS, we identified the proliferating cells as parietal epithelial cells (PECs). In the present study, we have evaluated the origin of the proliferating cells in cFSGS associated with human immunodeficiency virus (HIV) and pamidronate. We performed a detailed study of glomerular lesions in biopsies of two patients with HIV-associated cFSGS and a nephrectomy specimen of a patient with pamidronate-associated cFSGS. Glomeruli were studied by serial sectioning using light and electron microscopy and immunohistochemistry to determine the epithelial cell phenotype. We used Synaptopodin, vascular endothelial growth factor, and CD10 as podocyte markers, CK8 and PAX2 as PEC markers and Ki-67 as marker of cell proliferation. The newly deposited extracellular matrix was characterized using antiheparan sulfate single-chain antibodies. The proliferating cells were negative for the podocyte markers, but stained positive for the PEC markers and the cell proliferation marker Ki-67. The proliferating PAX-2 and CK8 positive cells that covered the capillary tuft were always in continuity with PAX-2/CK8 positive cells lining Bowman's capsule. The matrix deposited by these proliferating cells stained identically to Bowman's capsule. Our study demonstrates that PECs proliferate in HIV and pamidronate-associated cFSGS. Our data do not support the concept of the proliferating, dedifferentiated podocyte.

Contains fulltext : 32854.pdf (Publisher’s version ) (Closed access) We previously reported that during mouse embryogenesis, plexin D1 (plxnD1) is expressed on neuronal and endothelial cells. Endothelial cells gradually loose plxnD1 expression during development. Here we describe, using in situ hybridization, that endothelial plxnD1 expression is regained during tumor angiogenesis in a mouse model of brain metastasis. Importantly, we found PLXND1 expression also in a number of human brain tumors, both of primary and metastatic origin. Apart from the tumor vasculature, abundant expression was also found on tumor cells. Via panning of a phage display library, we isolated two phages that carry single-domain antibodies with specific affinity towards a PLXND1-specific peptide. Immunohistochemistry with these single-domain antibodies on the same tumors that were used for in situ hybridization confirmed PLXND1 expression on the protein level. Furthermore, both these phages and the derived antibodies specifically homed to vessels in brain lesions of angiogenic melanoma in mice after i.v. injection. These results show that PLXND1 is a clinically relevant marker of tumor vasculature that can be targeted via i.v. injections.

We reported previously that vascular endothelial growth factor isoform A (VEGF-A) expression by Mel57 human melanoma cells led to tumor progression in a murine brain metastasis model in an angiogenesis-independent fashion by dilation of co-opted, pre-existing vessels and concomitant enhanced blood supply (B. Kusters et al., Cancer Res., 62: 341-345, 2002). Here, we compare the activities of the 121, 165, and 189 VEGF-A isoforms in this model by transfecting Mel57 cells with the respective cDNAs and by injecting the resulting stably transfected cell lines in the internal carotid arteries of nude mice (n = 10 for each isoform). Although the three isoforms had similar potency to induce endothelial cell proliferation, VEGF(121) expression did not result in sprouting angiogenesis but rather led to extensive vasodilation and increased permeability of pre-existing, predominantly peritumoral vessels. Sometimes, proliferating endothelial cells accumulated in vessel lumina, giving these a microvascular, glomeruloid, proliferation-like appearance. Expression of VEGF(165) or VEGF(189) was associated with induction of an intratumoral neovascular bed. In VEGF(165)-expressing tumors, daughter endothelial cells were distributed among newly formed vessels that were extensively dilated. This also occurred in VEGF(189) tumors, but there, vasodilation was less pronounced. Using contrast-enhanced magnetic resonance imaging, the different vascular phenotypes were visualized on characteristic radiological images. VEGF(165) expression was the most unfavorable of the three. Mice carrying VEGF(165) tumors became moribund earlier than those carrying VEGF(121)-expressing tumors (16 +/- 4 days versus 22 +/- 3 days). Our data demonstrate that VEGF-A isoforms differ in angiogenic properties that can be visualized by contrast-enhanced magnetic resonance imaging.

Contains fulltext : 108424.pdf (Publisher’s version ) (Open Access) 01 maart 2012

We investigated the mechanisms of vascularization in a brain metastases model of malignant melanoma. Parenchymal metastases expressing little vascular endothelial growth factor-A (VEGF-A) co-opted the preexistent brain vasculature, leading to an infiltrative phenotype. Metastases of the human melanoma cell line Mel57, engineered to express recombinant VEGF-A(165), showed accelerated growth in a combined expansive and infiltrative pattern with marked central necrosis. This difference in growth profile was accompanied by dilation of co-opted intra- and peritumoral vessels with concomitant induction of vascular permeability. Our data show that modulation of preexistent vasculature can contribute to malignant progression without induction of sprouting angiogenesis.

Contains fulltext : 80044.pdf (Publisher’s version ) (Closed access) Alpha-dystroglycanopathies such as Walker Warburg syndrome represent an important subgroup of the muscular dystrophies that have been related to defective O-mannosylation of alpha-dystroglycan. In many patients, the underlying genetic etiology remains unsolved. Isolated muscular dystrophy has not been described in the congenital disorders of glycosylation (CDG) caused by N-linked protein glycosylation defects. Here, we present a genetic N-glycosylation disorder with muscular dystrophy in the group of CDG type I. Extensive biochemical investigations revealed a strongly reduced dolichol-phosphate-mannose (Dol-P-Man) synthase activity. Sequencing of the three DPM subunits and complementation of DPM3-deficient CHO2.38 cells showed a pathogenic p.L85S missense mutation in the strongly conserved coiled-coil domain of DPM3 that tethers catalytic DPM1 to the ER membrane. Cotransfection experiments in CHO cells showed a reduced binding capacity of DPM3(L85S) for DPM1. Investigation of the four Dol-P-Man-dependent glycosylation pathways in the ER revealed strongly reduced O-mannosylation of alpha-dystroglycan in a muscle biopsy, thereby explaining the clinical phenotype of muscular dystrophy. This mild Dol-P-Man biosynthesis defect due to DPM3 mutations is a cause for alpha-dystroglycanopathy, thereby bridging the congenital disorders of glycosylation with the dystroglycanopathies.

This paper describes a new automated system to prepare slides of cytological material from suspension. The system collects material on a filter tape by filtration and transfers it to glass slides by means of pressure-fixation. Using cervical cells as a model, results show that a well-defined cell number is evenly deposited over a standardized area, while a small number of cells is retained on the tape and a negligible number lost in the filtrate. Contamination is very small. Application of the system to other cytological material (fine needle aspirations, monolayer and cell suspension cultures, agar cultures, and isolated nuclei) is shown. In general, more than one slide can be made from one sample. Several histological staining procedures as well as immunofluorescence labeling protocols can be applied to the preparations obtained in this way. This system thus introduces a method that will standardize specimen preparation, is quick, saves operator time, and can be used for both diagnostic and research applications.