乳铁蛋白在人脑胶质瘤中的表达及意义

目的：通过对乳铁蛋白生物学特性的研究,分析其在人脑胶质瘤中的表达及意义。方法：通过免疫组化方法测定10例非肿瘤脑组织和46例胶质瘤（其中低级别胶质瘤27例（I级和II级）和高级别胶质瘤19例（III级和Ⅳ级）中乳铁蛋白的表达情况并分析二者的相关性。用RT—PCR技术及Westem—blot方法测定非肿瘤脑组织、低级别组胶质瘤和高级别胶质瘤组乳铁蛋白的表达量并分析二者的相关性。结果：乳铁蛋白在非肿瘤脑组织及不同级别胶质瘤中的表达情况不同,其在低级别胶质瘤中有表达,略低于非肿瘤脑组织;而在高级别胶质瘤中表达显著减少或者表达不明显,明显低于低级别胶质瘤和非肿瘤脑组织（免疫组化、RT．PCR及Western．Blot的P值分别为P=0．001、P=0．003、P=0．004）。结论：乳铁蛋白在非肿瘤脑组织和不同级别胶质瘤中的表达有差异,提示乳铁蛋白参与了人脑胶质瘤发生与发展的过程。高级别胶质瘤的高度恶性和高侵袭性可能与乳铁蛋白表达的下调从而丧失对肿瘤细胞的抑制作用相关。     

Prognostic implication of histological oligodendroglial tumor component: clinicopathological analysis of 111 cases of malignant gliomas

The favorable prognosis of high-grade oligodendroglial tumor such as glioblastoma (GBM) with oligodendroglioma component (GBMO) has been suggested; however, the studies which examine the prognostic significance of oligodendroglial tumor were limited. In this study, we performed a histopathology-based reevaluation of 111 cases of high grade gliomas according to the latest World Health Organization (WHO), and compared the clinical outcomes between oligodendroglial tumors and pure astrocytic tumors. The survival analysis revealed that the patients with high grade oligodendroglial tumor including GBMO significantly indicated better prognosis compared to the patients with high grade pure astrocytic tumors (GBM and AA, anaplastic astrocytoma) as expected, and the obtained survival curves were almost identical to those from the patients with conventional Grade III or Grade IV tumors, respectively. Moreover, if the cases of oligodendroglial tumor were histopathologically excluded, the patients with AA exhibited extremely poor prognosis which was similar to that of GBM, suggesting that the histological identification of oligodendroglial tumor component, even partially, prescribe the prognosis of high grade glioma patients. This is the prominent report of retrospective clinicopathological analysis for high-grade gliomas throughout Grade III and IV, especially referring to the prognostic value of histological oligodendroglial tumor component; in addition, our results might offer an alternative aspect for the grading of high-grade astrocytic/oligodendroglial tumors.     

Recognition and lysis by natural killers of tumor cells with participation of laminin

According to the data obtained in the present work, the receptor complex of mouse natural killers (NK) includes laminin, antibody to which blocks EK-activity (NKA regardless of the presence of complement. Preincubation of mouse splenocytes with anti-laminin serum led to a decrease in their NKA towards tumor cells-targets (CT), the NKA activity decreasing 2 times with respect to cultivated cells of rat hepatoma HTC, while 10 times - to cultivated cells of human erythroblastosis K562. Pretreatment of aplenocytes with noraml nonimmune serum did not lead to a change of NKA. Quite different was the pattern after the tumor cell preincubation with anti-laminin serum: pretreatment of CT K562 led to a twofold decrease in sensitivity of these cells to NK-lysis, whereas the pretreatment of CT K562, on the contrary, made them twice sensitive to NK-lysis. Electrophoretic separation of protein of CT plasma membranes with subsequent immunoblotting with anti-laminin immune serum revealed the presence oflaminin on HTC cell plasma membrane, which was identified as laminin 8/9 by the mass-spectrometry method, while no laminin was detected on K562 cells. Preincubation of splenocytes with laminin did nor affect NKA with respect to CT K562 and HTC. Pretreatment of CT K562 and HTC with laminin decreased the NKA to zero. The obtained data allow suggesting a doubtless participation of laminin and its receptors in CT cytolysis by NK.     

Compartment- and malignance-dependent up-regulation of γ-glutamyltranspeptidase and dipetidylpeptidase-IV activity in human brain gliomas

γ-Glutamyltranspeptidase (GGT, syn. γ-Glutamyltransferase) and dipeptidylpeptidase-IV (DPP-IV) activity participates in metabolic and growth control of normal and tumor cells by processing biologically active peptides. Here, we report on up-regulation of these enzymes in human brain gliomas determined by catalytic enzyme histochemistry and immunocytochemistry. Higher activity of GGT was found in 50%, 68% and 81% of WHO grade II, III and IV tumors, respectively. The process started at/near the microvasculature, from where it spread to the parenchyma. On average, the enzyme activity in grade II, III and IV gliomas exceeded controls 2.0, 3.0 and 3.5-fold, respectively. Up-regulation of DPP-IV-like activity also started at the microvasculature, but mainly in pericytes and mononuclear-like cells around the vessels and dispersed in the parenchyma. Marked elevation of this enzyme activity, comprising also tumor parenchyma, occurred only in grade IV glioblastomas (65% patients; 3.6 times above controls) which can, therefore, help in their differentiation from grade III gliomas. The increase of total DPP-IV-like activity also included its two enzymatic homologs, the canonical DPP-IV/CD26 and FAP-1α. The increase in GGT is supposed to be a tumor grade dependent response of microvasculature and tumor astrocytes to stress induced by tissue hypoxia and/or the metabolic aberrancies. The increase in DPP-IV-like activity in high-grade tumors can be attributed to inflammatory/scavenging processes performed by the mononuclear-like cells and, in glioblastomas, also to regressive changes in the structure and function of the microvasculature and tumor parenchyma, including astrocyte stress response. The inverse relationship between DPP-IV-like activity and Ki67 in most glioblastomas and shorter survival time of patients with low activity of this enzyme also suggest its anti-oncogenic effects.     

Direct in vivo evidence for tumor propagation by glioblastoma cancer stem cells

High-grade gliomas (World Health Organization grade III anaplastic astrocytoma and grade IV glioblastoma multiforme), the most prevalent primary malignant brain tumors, display a cellular hierarchy with self-renewing, tumorigenic cancer stem cells (CSCs) at the apex. While the CSC hypothesis has been an attractive model to describe many aspects of tumor behavior, it remains controversial due to unresolved issues including the use of ex vivo analyses with differential growth conditions. A CSC population has been confirmed in malignant gliomas by preferential tumor formation from cells directly isolated from patient biopsy specimens. However, direct comparison of multiple tumor cell populations with analysis of the resulting phenotypes of each population within a representative tumor environment has not been clearly described. To directly test the relative tumorigenic potential of CSCs and non-stem tumor cells in the same microenvironment, we interrogated matched tumor populations purified from a primary human tumor transplanted into a xenograft mouse model and monitored competitive in vivo tumor growth studies using serial in vivo intravital microscopy. While CSCs were a small minority of the initial transplanted cancer cell population, the CSCs, not the non-stem tumor cells, drove tumor formation and yielded tumors displaying a cellular hierarchy. In the resulting tumors, a fraction of the initial transplanted CSCs maintained expression of stem cell and proliferation markers, which were significantly higher compared to the non-stem tumor cell population and demonstrated that CSCs generated cellular heterogeneity within the tumor. These head-to-head comparisons between matched CSCs and non-stem tumor cells provide the first functional evidence using live imaging that in the same microenvironment, CSCs more than non-stem tumor cells are responsible for tumor propagation, confirming the functional definition of a CSC.     

Success of tumorsphere isolation from WHO grade IV gliomas does not correlate with the weight of fresh tumor specimens: an immunohistochemical characterization of tumorsphere differentiation

Background

A trend of stage-by-stage increase in tumorsphere (TS) formation from glioma samples has been reported. Despite this trend, not all surgical specimens give rise to TSs, even World Health Organization (WHO) grade IV gliomas. Furthermore, it has been reported that differences in overall survival of primary glioblastoma patients depends on the propensity of their tumors to form TSs. However, the weights of fresh specimens vary from one surgical isolate to the next.

Methods

Accordingly, we evaluated the relationship between the weights of surgical specimens in WHO grade IV gliomas with the capacity to isolate TSs. Thirty-five fresh WHO grade IV glioma specimens were separated into two groups, based on whether they were positive or negative for TS isolation, and the relationship between TS isolation and weight of surgical specimens was assessed.

Results

We observed no significant difference in the weights of surgical samples in the two groups, and found that the optimal weight of specimens for TSs isolation was 500 mg.

Conclusion

Thus, contrary to our expectations, the ability to isolate TSs from WHO grade IV glioma specimens was not related to the weight of fresh specimens.

     

Protein interacting with C kinase 1 suppresses invasion and anchorage-independent growth of astrocytic tumor cells

Astrocytic tumors are the most common form of primary brain tumor. Astrocytic tumor cells infiltrate the surrounding CNS tissue, allowing them to evade removal upon surgical resection of the primary tumor. Dynamic changes to the actin cytoskeleton are crucial to cancer cell invasion, but the specific mechanisms that underlie the particularly invasive phenotype of astrocytic tumor cells are unclear. Protein interacting with C kinase 1 (PICK1) is a PDZ and BAR domain–containing protein that inhibits actin-related protein 2/3 (Arp2/3)-dependent actin polymerization and is involved in regulating the trafficking of a number of cell-surface receptors. Here we report that, in contrast to other cancers, PICK1 expression is down-regulated in grade IV astrocytic tumor cell lines and also in clinical cases of the disease in which grade IV tumors have progressed from lower-grade tumors. Exogenous expression of PICK1 in the grade IV astrocytic cell line U251 reduces their capacity for anchorage-independent growth, two-dimensional migration, and invasion through a three-dimensional matrix, strongly suggesting that low PICK1 expression plays an important role in astrocytic tumorigenesis. We propose that PICK1 negatively regulates neoplastic infiltration of astrocytic tumors and that manipulation of PICK1 is an attractive possibility for therapeutic intervention.     

Cyclooxygenase-2 expression in astrocytomas. Relationship with microvascular parameters, angiogenic factors expression and survival

OBJECTIVE: Cyclooxygenase-2 (COX-2) is the enzyme isoform involved in the synthesis of prostaglandins (PGs) and thromboxane from arachidonic acid. The role of the up-regulation of COX-2 in the formation and progression of gliomas has been dealt with in earlier reports, which describe increased levels of PGs within gliomas. In the present study, we examined the expression of COX-2 in diffuse gliomas of astrocytic origin in relation to microvascular parameters, angiogenic factors and survival. MATERIALS AND METHODS: A total of 83 cases of diffuse astrocytomas (grade II-IV) were analyzed by immunohistochemistry for the presence of COX-2. RESULTS: COX-2 expression was detected in 79 cases (95%) with an increased expression in grade IV as compared to grades II/III (p=0.024). A positive correlation occurred between COX-2 and angiogenic factors such as vascular endothelial growth factor (VEGF) (p<0.0001) and hypoxia inducible factor (HIF)-1alpha (p=0.005), as well as the tumours' proliferative activity (expressed as the percentage of Ki-67 positive cells) (p=0.032), and total vascular area (TVA) (p=0.040). In univariate analysis, COX-2 was associated with shortened survival (p = 0.050). Multivariate survival analysis showed that the interaction model of COX-2 with grade along with age were the only significant prognostic indicators. CONCLUSION: These results implicate COX-2 in the angiogenesis and biological aggressiveness of diffuse astrocytomas, and suggest that it would be worthwhile to examine how the inhibition of COX-2 expression may influence astrocytoma patients' survival.     

Stathmin 在微血管内皮细胞的表达与胶质瘤恶性程度关系

目的:检测Stathmin在正常脑组织及不同级别胶质瘤微血管内皮细胞中的表达情况。方法:利用结合CD105单克隆抗体的免疫磁珠内皮细胞分选系统特异性分选出68例胶质瘤微血管内皮细胞(其中低级别胶质瘤(WHO分级Ⅰ-Ⅱ)24例,高级别胶质瘤(WHO分级Ⅲ-Ⅳ)44例)和20例正常脑组织微血管内皮细胞。应用免疫组化、RT-PCR和Western blot检测Stathmin在胶质瘤微血管内皮细胞和正常脑组织微血管内皮细胞中的表达。结果:免疫组化证实Stathmin在正常脑组织微血管内皮细胞、低级别胶质瘤微血管内皮细胞和高级别胶质瘤微血管内皮细胞的表达百分率分别是20%,66%和95%(P<0.05)。RT-PCR和Western blot法检测显示,Stathmin在胶质瘤微血管内皮细胞中的表达明显增高。低级别胶质瘤组、高级别胶质瘤组分别与正常组比较,均有显著性差异(P<0.01);且低级别胶质瘤组与高级别胶质瘤组比较,有显著性差异(P<0.01),随着胶质瘤恶性程度的增加,Stathmin表达上调,具有统计学意义。结论:Stathmin在脑胶质瘤微血管内皮细胞中表达随肿瘤恶性程度增高而增加,可能为脑胶质瘤的生物治疗提供一个新靶点。     

Prognostic significance of DNA ploidy and proliferation rate in human astrocytic gliomas

DNA ploidy and the proliferative potential in 75 gliomas were investigated using bromodeoxyuridine labelling index (BrdUrd LI), S-phase fraction (SPF) and argyrophilic nucleolar organizer regions (AgNOR) technique. There were 53 highly malignant (AIII-AIV), and 22 low-grade (AI-AII) gliomas. One fragment of the tumour was fixed in Carnoy's solution for AgNOR test, while the other fragments were used for flow cytometric determination of the labelling index, SPF and DNA ploidy. For the BrdUrdLI, tumour samples from each patient were incubated in vitro for one hour at 37 degrees C with BrdUrd using the high pressure oxygen method. The tumours showed variability in the BrdUrdLI values, SPF and AgNOR counts/cell nucleus. The same percentage of DNA aneuploidy (55%) was found in high-grade as well as in low-grade gliomas. Univariate analysis showed that patients with grade I & II gliomas had significantly higher 3-year survival rate (p = 0.0193) than those with grade III and grade IV gliomas. Also patients with lower proliferation rate of tumours (BrdUrdLI < or =2.3% and AgNOR counts < or =2.6%/cell) had higher 3-year survival rate (p<0.03), which can be helpful in prognosis. Tumour ploidy or SPF had no influence on patients' survival (p = 0.7908). Cox multivariate analysis showed that only patients' age > 45 years and high tumour grade (III and IV) were significant unfavourable prognostic factors in terms of patients' survival.     

Genetic analysis to complement histopathological diagnosis of brain tumors

Gliomas, the most frequent tumors originating in the human nervous system, are divided into various subtypes. Currently, microscopic examination alone is insufficient for classification and grading so that genetic profiles are increasingly being emphasized in recognition of the emerging role of molecular diagnostic approaches to glioma classification. Glioblastomas (WHO grade IV) may develop de novo (primary glioblastomas) or through progression from lower-grade astrocytomas (secondary glioblastomas), while both glioblastomas show similar histological features. In contrast, they do constitute distinct disease entities that evolve through different genetic pathways, and are likely to differ in prognosis and response to therapy. Oligodendrogliomas (WHO grade II) account for 2.7% of brain tumors and 5-18% of all gliomas. Since this tumor is recognized as a particular subtype of glioma that shows remarkable responses to chemotherapy, a correct diagnosis is of prime importance. The difficulty is that histological differentiation of oligodendrogliomas from diffuse astrocytomas is highly subjective in cases without typical morphological features and there is a lack of reliable immunohistochemical markers. While histological distinction of low-grade gliomas from reactive astrocytes is also often difficult, reactive astrocytes usually lack genetic alterations. More biological and molecular approaches to glioma classification thus appear warranted to provide improved means to achieve correct diagnoses.     

siRNA抑制stathmin表达对高级别胶质瘤血管内皮细胞迁移的影响

目的：探讨siRNA抑制Stathmin表达对高级别胶质瘤微血管内皮细胞侵袭性的影响。方法：利用结合CDl05单克隆抗体的免疫磁珠内皮细胞分选系统特异性分选出22例高级别胶质瘤微血管内皮细胞（其中8例III级胶质瘤,14例Ⅳ级胶质瘤,13例男性和9例女性组成）。应用siRNA技术抑制高级别胶质瘤血管内皮细胞中Stathmin表达,Transwell侵袭实验检测Stathmin基因沉默对高级别胶质瘤血管内皮细胞迁移的影响。结果：采用RNA干扰Stathmin的高级别胶质瘤微血管内皮细胞中Stathmin的mRNA和蛋白表达均较对照组显著下降,而迁移细胞数也显著低于对照组（P〈0．01）。结论：通过siRNA抑制高级别胶质瘤血管内皮细胞中Stathmin表达可抑制其迁移。     

Identification of COL6A1 as a differentially expressed gene in human astrocytomas

Diffuse infiltrating gliomas are the most common tumors of the central nervous system. Gliomas are classified by the WHO according to their histopathological and clinical characteristics into four classes: grade I (pilocytic astrocytoma), grade II (diffuse astrocytoma), grade III (anaplastic astrocytoma), and grade IV (glioblastoma multiforme). Several genes have already been correlated with astrocytomas, but many others are yet to be uncovered. By analyzing the public SAGE data from 21 patients, comprising low malignant grade astrocytomas and glioblastomas, we found COL6A1 to be differentially expressed, confirming this finding by real time RT-PCR in 66 surgical samples. To the best of our knowledge, COL6A1 has never been described in gliomas. The expression of this gene has significantly different means when normal glia is compared with low-grade astrocytomas (grades I and II) and high-grade astrocytomas (grades III and IV), with a tendency to be greater in higher grade samples, thus rendering it a powerful tumor marker.     

Neuronatin in a subset of glioblastoma multiforme tumor progenitor cells is associated with increased cell proliferation and shorter patient survival

Glioblastoma multiforme is the most common and malignant primary brain tumor. Recent evidence indicates that a subset of glioblastoma tumor cells have a stem cell like phenotype that underlies chemotherapy resistance and tumor recurrence. We utilized a new "multidimensional" capillary isoelectric focusing nano-reversed-phase liquid chromatography platform with tandem mass spectrometry to compare the proteomes of isolated glioblastoma tumor stem cell and differentiated tumor cell populations. This proteomic analysis yielded new candidate proteins that were differentially expressed. Specifically, two isoforms of the membrane proteolipid neuronatin (NNAT) were expressed exclusively within the tumor stem cells. We surveyed the expression of NNAT across 10 WHO grade II and III gliomas and 23 glioblastoma (grade IV) human tumor samples and found NNAT was expressed in a subset of primary glioblastoma tumors. Through additional in vitro studies utilizing the U87 glioma cell line, we found that expression of NNAT is associated with significant increases in cellular proliferation. Paralleling the in vitro results, when NNAT levels were evaluated in tumor specimens from a consecutive cohort of 59 glioblastoma patients, the presence of increased levels of NNAT were found to be a an independent risk factor (P?=?0.006) for decreased patient survival through Kaplan-Meier and multivariate analysis. These findings indicate that NNAT may have utility as a prognostic biomarker, as well as a cell-surface target for chemotherapeutic agents.     

Correlation between preoperative magnetic resonance spectroscopic data on high grade gliomas and morphology of Ki-67-positive tumor cell nuclei

OBJECTIVE: To investigate possible statistical correlations between metabolic data from preoperative proton magnetic resonance spectroscopy (1HMRS) and morphology of proliferating tumor cell nuclei in anaplastic gliomas and glioblastomas. STUDY DESIGN: Ki-67-positive tumor cell nuclei in paraffin sections of surgical specimens from 36 patients (7 anaplastic gliomas, World Health Organization grade 3; 29 glioblastomas, World Health Organization grade 4) were investigated by means of a digital image analysis system. Stringent inclusion criteria were formulated for all cases with respect to histologic quality and spectroscopic examination. As morphometric variables, nuclear area, shape variables (roundness factor, size-invariate Fourier amplitudes) and density of Ki-67-positive tumor cell nuclei per reference area were determined. RESULTS: Correlation analysis according to Spearman revealed a significant positive correlation between the total creatine (TCR) peak and nuclear area (P = .005). This correlation was also found within the glioblastoma group (P = .019). There was also a significant negative correlation of nuclear area with the ratio between choline and TCR in all cases (P = .014) and within the glioblastoma group (P = .046). No significant correlation of spectroscopic data was found with nuclear shape or density of Ki-67-positive tumor cell nuclei. CONCLUSION: The results demonstrate a correlation between spectroscopic data and morphology of proliferating tumor cell nuclei (nuclear size) in high grade gliomas. This study is part of a detailed investigation of the interrelationship between preoperative 1HMRS and quantitative histomorphology of gliomas.     

磁共振灌注加权成像与弥散加权成像在脑胶质瘤分级诊断中的应用

目的:探讨磁共振灌注加权成像(perfusion weighted imaging,PWI)与弥散加权成像(diffusion weighted imaging,DWI)在脑胶质瘤分级诊断中的应用价值。方法:选取2012年1月-2017年6月在我院就诊并经病理证实为脑胶质瘤患者100例,其中高、低级别胶质瘤患者各44、56例。对所有患者行PWI、DWI检查,比较肿瘤不同区域表观扩散系数(apparent diffusion coefficient,ADC)、局部脑血流量(regional cerebral blood flow,rCBF),不同级别肿瘤实质区、瘤周水肿区rADC、rrCBF,根据ROC曲线分析rADC、rrCBF对不同级别胶质瘤的诊断阈值、敏感性、特异性。结果:与对侧相应正常脑实质比较,瘤周水肿区及肿瘤实质区ADC、rCBF均显著升高(P0.05);与瘤周水肿区比较,肿瘤实质区ADC、rCBF均显著升高(P0.05)。高级别肿瘤实质区rADC显著低于低级别肿瘤实质区(P0.05),rrCBF显著高于肿瘤实质区(P0.05)。高级别瘤周水肿区与低级别瘤周水肿区rADC间无显著差异(P0.05),高级别瘤周水肿区rrCBF显著高于低级别瘤周水肿区(P0.05)。在对高、低级别脑胶质瘤的分级中,rADC、rrCBF的曲线下面积(under the receiver operating characteristic curve,AUC)分别为0.957、0.978,均0.9。rADC诊断不同分级胶质瘤的敏感度是90.12%,特异度是95.26%,诊断阈值是13.12;rrCBF诊断不同分级胶质瘤的敏感度是92.31%,特异度是98.57%,诊断阈值是2.62。rADC与rrCBF诊断不同分级胶质瘤敏感度、特异度间无显著差异(P0.05)。结论:PWI、DWI能够为脑胶质瘤的分级诊断提供参考依据。