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

Isocitrate dehydrogenase 1R132H mutation in microglia/macrophages in gliomas: Indication of a significant role of microglia/macrophages in glial tumorigenesis

Somatic mutation of Isocitrate dehydrogenase 1 (IDH1) at the locus of R132 (IDH1R132H) occurs in > 70% of WHO grades II-III gliomas and secondary glioblastomas. To date it remains unknown whether the mutation is restricted to glial tumor cells. Microglial cells are the resident macrophages in the central nervous system. Tumor-infiltrating microglial cells/macrophages are major stromal cellular components of malignant gliomas and substantially contribute to the tumor mass. Differential identification of the IDH1 R132H mutant cellular components is of particular importance for understanding of the mutation-associated tumor biology. Here we discovered that a significant portion of CD68+, Iba1+, CX3CR1+ microglial cells/macrophages also harbor the IDH1R132H mutation. The findings provide novel insights for understanding the mutation-associated tumor biology relevant to clinical applications as a predictive and/or prognostic marker or therapeutic target

Adhesion molecules in iris biopsy specimens from patients with uveitis

BACKGROUND/AIMS: Earlier studies on intraocular tissue have demonstrated that T lymphocytes play a major role in the pathogenesis of uveitis. Adhesion molecules are immunoregulatory molecules for the interaction between T lymphocytes and vascular endothelium and they play an important role in the recruitment of specific T lymphocytes from the circulation into inflamed tissue. In uveitis an increased expression of some of these adhesion molecules may be expected. METHODS: The presence of adhesion molecules was investigated in iris biopsy specimens from 11 patients with uveitis and eight controls (patients with primary open angle glaucoma) immunohistochemically with a panel of monoclonal antibodies: LECAM (CD 62L), ICAM-1 (CD 54), LFA-1 (CD 11a/18), VCAM-1 (CD 106), VLA-4 (CD 49d), and HECA-452, a marker for high endothelial venules. RESULTS: Positive staining for ICAM-1, LFA-1 and VCAM-1 was found in the iris in a significantly higher number of uveitis patients than in controls. The remaining adhesion molecules were also found in a higher number of uveitis patients than in controls, but this difference did not reach statistical significance. CONCLUSION: An increased expression of adhesion molecules was found in the iris of patients with uveitis, indicating an immunoregulatory function for adhesion molecules in the pathogenesis of uveitis

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

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

Periostin Is Expressed by Pericytes and Is Crucial for Angiogenesis in Glioma

The expression of the matricellular protein periostin has been associated with glioma progression. In previous work we found an association of periostin with glioma angiogenesis. Here, we screen gliomas for POSTN expression and identify the cells that express periostin in human gliomas. In addition, we study the role of periostin in an in vitro model for angiogenesis. The expression of periostin was investigated by RT-PCR and by immunohistochemistry. In addition, we used double labeling and in situ RNA techniques to identify the expre

Quantification of Calcyclin and Heat Shock Protein 90 in Sera from Women with and without Preeclampsia by Mass Spectrometry

Purpose: The objective of present study is to determine serum levels and placental distribution of two interacting proteins calcyclin and heat shock protein 90 in preeclampsia. Experimental design: Maternal serum levels of calcyclin and heat shock protein 90 are compared throughout pregnancy from the first trimester till term among women with preeclampsia (n = 43) and age-matched normotensive pregnant controls (n = 46). A serum-based 2D LC-MS assay using Parallel Reaction Monitoring is applied to quantify both calcyclin and heat shock protein 90. Results: Serum levels of calcyclin are significantly lower in patients with preeclampsia in the second trimester of pregnancy as compared to controls (p < 0.05). Serum levels of heat shock protein 90 are significantly higher in patients with preeclampsia in the third trimester as compared to controls (p < 0.001). Conclusion and clinical relevance: Both interacting proteins calcyclin and heat shock protein 90 are notably changed in preeclamptic patients compared to controls. Calcyclin is already decreased before the onset of preeclampsia in the second trimester and HSP90 is strongly increased in the third trimester. This suggests that these proteins may play a role in the pathogenesis of preeclampsia and ought to be investigated in large cohort studies as molecular biomarkers

Differential expression of boc, spock2, and gjd3 is associated with brain metastasis of er-negative breast cancers

Background: Brain metastasis is considered one of the major causes of mortality in breast cancer patients. To invade the brain, tumor cells need to pass the blood-brain barrier by mechanisms that are partially understood. In primary ER-negative breast cancers that developed brain metastases, we found that some of the differentially expressed genes play roles in the T cell response. The present study aimed to identify genes involved in the forma

Activation of CECR1 in M2-like TAMs promotes paracrine stimulation-mediated glial tumor progression

Background. The majority of glioma-associated microglia/macrophages have been identifed as M2-type macrophages with immune suppressive and tumor supportive action. Recently, the extracellular adenosine deaminase protein Cat Eye Syndrome Critical Region Protein 1 (CECR1) was shown to regulate macrophage maturation. In this study, we investigate the role of CECR1 in the regulation of the glioma-associated macrophage response. Methods. Expression of CECR1 was assessed in human glioma samples. CECR1-mediated macrophage response was studied in vitro, using donor derived CD14+ monocytes and the THP-1 monocytic cell line. The response of the human glioma cell line U87 to conditioned medium of macrophages preconditioned with recombinant human CECR1 or CECR1 silencing was also assessed. Results. CECR1 was strongly expressed in high-grade gliomas (P <.001) and correlated positively with the M2 phenotype markers in tumor-associated microglia/macrophages (TAMs) (overall, P <.05). In vitro studies confrmed the presence of a signifcantly higher level of CECR1 expression in M2-like macrophages exposed to U87 conditioned medium (P <.001). CECR1 knockdown or stimulation of macrophages affected differentiation toward the M2-like phenotype. Stimulation of U87 cells with conditioned medium of CECR1 knockdown or stimulated macrophages affected tumor cell proliferation and migration, coinciding with altered intracellular signaling of mitogen-activated protein kinase (MAPK). In glioma tissue samples, CECR1 expression correlated with Ki67 and MAPK signaling protein. Conclusions. CECR1 is a potent regulator of TAM polarization and is consistently highly expressed by M2-type TAMs, particularly in high-grade glioma. Paracrine effects induced by CECR1 in M2-like TAMs activate MAPK signaling and stimulate the proliferation and migration of glioma cells