Identifying Proteins Involved in Glioma Angiogenesis: a Proteomics Approach

Approximately half of tumors encountered in the brain represent metastases from neoplasms located elsewhere in the body. A large part of the other half represents meningiomas arising from the membranes covering the brain. The remaining group consists of so-called primary brain tumors, which are tumors arising from the cellular components of brain tissue itself. Most of these tumors are called gliomas because they putatively arise from glial cells. Gliomas are the most frequently encountered subtypes of primary brain tumors. Glial tumor cells still display signs of glial differentiation to some extent [1]. Relative to the major cancers affecting humans like lung-, breast -, colonic - and prostate cancer, glial neoplasms are only a minority. The morbidity and mortality of this group is, however, highest of all. Hence, the impact of these tumors on the well-being of the patients and the economic consequences thereof warrants research efforts comparable to those undertaken in the major cancer groups. In several respects gliomas differ from other tumors. They hardly ever metastasize. Further, neoplastic glial cells infiltrate brain tissue diffusely by mechanisms of migration. Because of the diffuse infiltrative character there are no clear-cut tumor borders and therefore, radical surgery is never possible. Another peculiarity of gliomas is their continuous metamorphosis: over time the tumors change their histological appearance. This goes along with increasing genetic instability. The tumors become more cellular; the cells more pleomorphic and finally necrotic areas appear. The metamorphosis goes along with an increase in blood vessels and a change in the structure of the blood vessel walls. Although gliomas are among the most vascularized tumors, it is surprising that anti-angiogenesis therapies have been relatively unsuccessful so far. To improve this situation it is necessary to increase the specificity of the therapeutic targets in the glioma vascularization

Expression site of P2RY12 in residential microglial cells in astrocytomas correlates with M1 and M2 marker expression and tumor grade

The role of resident microglial cells in the pathogenesis and progression of glial tumors is still obscure mainly due to a lack of specific markers. Recently P2RY12, a P2 purinergic receptor, was introduced as a specific marker for microglial cells under normal and pathologic conditions. Here we analyzed the expression of P2RY12 in astrocytomas of various malignancy grades in relation to markers for M1 and M2 macrophage activation profiles by using two web-based glioma datasets and confocal immunohistochemistry to 28 astrocytoma samples grades II-IV. In the gliomas, P2RY12 immunoreactivity delineated CD68 negative cells with otherwise microglial features from CD68 positive tumor associated macrophages (TAMs). The presence of P2RY12 positive cells correlated positively with overall survival. P2RY12 mRNA levels and membrane-bound localization of P2RY12 were inversely correlated with increasing malignancy grade, and the expression site of P2RY12 shifted from cytoplasmic in low-grade gliomas, to nuclear in high-grade tumors. The cytoplasmic expression of P2RY12 was associated with the expression of M1 markers, characteristic of the pro-inflammatory macrophage response. In contrast, the nuclear localization of P2RY12 was predominant in the higher graded tumors and associated with the expression of the M2 marker CD163. We conclude that P2RY12 is a specific marker for resident microglia in glioma and its expression and localization correspond to tumor grade and predominant stage of M1/M2 immune response

RNA from stabilized whole blood enables more comprehensive immune gene expression profiling compared to RNA from peripheral blood mononuclear cells

Monitoring changes in the immune profile in blood samples can help identifying changes in tumor biology and therapy responsiveness over time. Immune-related gene expression profiles offer a highly reproducible method to monitor changes of the immune system. However, measuring gene expression profiles in whole blood samples can be complicated because of the high protein and enzyme abundancy that affect the stability and quality of the RNA. Peripheral blood mononuclear cells (PBMCs) are one the most commonly used source for immune cell RNA extraction, though, this method does not reflect all components of the peripheral blood. The aim of this study was to determine the differences in immune-related gene expression between RNA isolated from stabilized whole blood and RNA isolated from PBMCs. Whole blood samples from 12 pancreatic cancer patients were collected before and after chemotherapy (n = 24). Blood samples were collected in both EDTA tubes, and Tempus tubes containing an RNA stabilizer (total n = 48). PBMCs were isolated from EDTA samples using Ficoll and were snap frozen. Subsequently, immune-related gene expression was profiled using the PanCancer Immune Profiling Panel of NanoString technology. Gene expression profiles of PBMCs were compared to that of Tempus tubes using the Advanced Analysis module of nSolver software. Both types of samples provided good quality RNA and gene expression measurements. However, RNA isolated from Tempus tubes resulted in significantly higher gene counts than PBMCs; 107/730 genes were exclusively detected in Tempus samples, while under the detection limit in PBMCs. In addition, 192/730 genes showed significantly higher gene counts in Tempus samples, 157/730 genes showed higher gene counts in PBMCs. Thus, RNA isolated from whole blood stabilizing blood tubes, such as Tempus tubes, enable higher gene counts and more comprehensive measurements of gene expression profiles compared to RNA isolated from PBMCs

Circulating proangiogenic cells and proteins in patients with glioma and acute myocardial infarction: Differences in neovascularization between neoplasia and tissue regeneration

Although extensive angiogenesis takes place in glial tumors, antiangiogenic therapies have remained without the expected success. In the peripheral circulation of glioma patients, increased numbers of endothelial precursor cells (EPCs) are present, potentially offering targets for antiangiogenic therapy. However, for an antiangiogenic therapy to be successful, the therapy should specifically target glioma-related EPC subsets and secreted factors only. Here, we compared the EPC subsets and plasma factors in the peripheral circulation of patients with gliomas to acute myocardial infarctions. We investigated the five most important EPC subsets and 21 angiogenesis-related plasma factors in peripheral blood samples of 29 patients with glioma, 14 patients with myocardial infarction, and 20 healthy people as controls, by FACS and Luminex assay. In GBM patients, all EPC subsets were elevated as compared to healthy subjects. In addition, HPC and KDR+ cell fractions were higher than in MI, while CD133+ and KDR+CD133+ cell fractions were lower. There were differences in relative EPC fractions between the groups: KDR+ cells were the largest fraction in GBM, while CD133+ cells were the largest fraction in MI. An increase in glioma malignancy grade coincided with an increase in the KDR+ fraction, while the CD133+ cell fraction decreased relatively. Most plasma angiogenic factors were higher in GBM than in MI patients. In both MI and GBM, the ratio of CD133+ HPCs correlated significantly with elevated levels of MMP9. In the GBM patients, MMP9 correlated strongly with levels of all HPCs. In conclusion, the data demonstrate that EPC traffic in patients with glioma, representing neoplasia, is different from that in myocardial infarction, representing tissue regeneration. Glioma patients may benefit from therapies aimed at lowering KDR+ cells and HPCs

CECR1-mediated cross talk between macrophages and vascular mural cells promotes neovascularization in malignant glioma

Glioblastomas (glioblastoma multiforme, GBM) are most malignant brain tumors characterized by profound vascularization. The activation of macrophages strongly contributes to tumor angiogenesis during GBM development. Previously, we showed that extracellular adenosine deaminase protein Cat Eye Syndrome Critical Region Protein 1 (CECR1) is highly expressed by M2-like macrophages in GBM where it defines macrophage M2 polarization and contributes to tumor expansion. In this study, the effect of CECR1 in macrophages on tumor angiogenesis was investigated. Immunohistochemical evaluation of GBM tissue samples showed that the expression of CECR1 correlates with microvascular density in the tumors, confirming data from the TCGA set. In a three-dimensional co-culture system consisting of human pericytes, human umbilical vein endothelial cells and THP1-derived macrophages, CECR1 knockdown by siRNA and CECR1 stimulation of macrophages inhibited and promoted new vessel formation, respectively. Loss and gain of function studies demonstrated that PDGFB mRNA and protein levels in macrophages are modulated by CECR1. The proangiogenic properties of CECR1 in macrophages were partially mediated via paracrine activation of pericytes by PDGFB–PDGFRβ signaling. CECR1–PDGFB–PDGFRβ cross-activation between macrophages and pericytes promoted pericyte migration, shown by transwell migration assay, and enhanced expression and deposition of periostin, a matrix component with proangiogenic properties. CECR1 function in (M2-like) macrophages mediates cross talk between macrophages and pericytes in GBM via paracrine PDGFB–PDGFRβ signaling, promoting pericyte recruitment and migration, and tumor angiogenesis. Therefore, CECR1 offers a new portent target for anti-angiogenic therapy in GBM via immune modulation.Oncogene advance online publication, 22 May 2017; doi:10.1038/onc.2017.145

Serum miR-338-3p and miR-199b-5p are associated with the absolute neutrophil count in patients with resectable pancreatic cancer

Background: Several peripheral blood cell counts and immune ratios, such as the systemic immune-inflammation index (SII = platelet x neutrophil count/lymphocyte count) have prognostic value in patients with resectable pancreatic cancer (PDAC). Circulating microRNAs (miRNAs) are involved in various aspects of cancer and inflammation. We aimed to identify measurable circulating miRNAs in PDAC patients correlating with systemic inflammation parameters. Methods: A total of 42 PDAC patients was included in this study: twelve in the discovery (n = 6 SII low; n = 6 SII high) and 30 patients in the validation cohort (n = 19 SII low, n = 11 SII high). MiRNAs isolated from preoperative serum samples were measured with a 352 miRNA panel in the discovery cohort and individual miRNA primers in the validation cohort, using RT-qPCR (ID3EAL assays, MiRXES). Results: Only in the discovery cohort miR-328-3p, miR-338-3p, miR-1258 and miR-199b-5p were upregulated in high compared to low SII patients (fold difference ≥ 2, P<0.05). In the total cohort (n = 42) correlations were found between miR-338-3p (r = 0.48, P = 0.002) and miR-199b-5p (r = 0.44, P = 0.005) and the absolute neutrophil count. Conclusion: Circulating miR-338-3p and miR-199b-5p are correlated to the neutrophil count in the blood of PDAC patients, suggesting a potential role of circulating miRNAs in cancer immune evasion and systemic inflammation

Circulating Biomarkers for Prediction of Objective Response to Chemotherapy in Pancreatic Cancer Patients

Pancreatic cancer is a lethal disease with increasing incidence. Most patients present with advanced disease, for which palliative systemic chemotherapy is the only therapeutic option. Despite improved median survival rates with FOLFIRINOX or gemcitabine chemotherapy compared to the best supportive care, many individual patients may not benefit from chemotherapy. Biomarkers are needed to predict who will benefit from chemotherapy and to monitor a patient’s response to chemotherapy. This review summarizes current research and future perspectives on circulating biomarkers for systemic chemotherapy response

A method to correlate mRNA expression datasets obtained from fresh frozen and formalin-fixed, paraffin-embedded tissue samples: A matter of thresholds

Background: Gene expression profiling of tumors is a successful tool for the discovery of new cancer biomarkers and potential targets for the development of new therapeutic strategies. Reliable profiling is preferably performed on fresh frozen (FF) tissues in which the quality of nucleic acids is better preserved than in formalin-fixed paraffin-embedded (FFPE) material. However, since snap-freezing of biopsy materials is often not part of daily routine in pathology laboratories, one may have to rely on archival FFPE material. Procedures to retrieve the RNAs from FFPE materials have been developed and therefore, datasets obtained from FFPE and FF materials need to be made compatible to ensure reliable comparisons are possible. Aim: To develop an efficient method to compare gene expression profiles obtained from FFPE and FF samples using the same platform. Methods: Twenty-six FFPE-FF sample pairs of the same tumors representing various cancer types, and two FFPE-FF sample pairs of breast cancer cell lines, were included. Total RNA was extracted and gene expression profiling was carried out using Illumina's Whole-Genome cDNA-mediated Annealing, Selection, extension and Ligation (WG-DASL) V3 arrays, enabling the simultaneous detection of 24,526 mRNA transcripts. A sample exclusion criterion was created based on the expression of 11 stably expressed reference genes. Pearson correlation at the probe level was calculated for paired FFPE-FF, and three cut-off values were chosen. Spearman correlation coefficients between the matched FFPE and FF samples were calculated for three probe lists with varying levels of significance and compared to the correlation based on all measured probes. Unsupervised hierarchical cluster analysis was performed to verify performance of the included probe lists to compare matched FPPE-FF samples. Results: Twenty-seven FFPE-FF pairs passed the sample exclusion criterion. From the profiles of 27 FFPE and FF matched samp