lewis-x-antigen and Brain-Neoplasms

CD15-containing glycoconjugates have a common trisaccharide residue, 3-fucosyl-N-acetyllactosamine, which can be recognized by a panel of monoclonal antibodies. Immunohistochemical studies revealed a widespread distribution of CD15 in several epithelial non-neural tissues as well as in the CNS. In the mature mammalian brain CD15-containing glycolipids and glycoproteins are constantly present in astrocytes, whereas oligodendrocytes and particular subpopulations of neurons are variably immunostained. CD15 immunoreactive astrocytes are spatially distributed in some brain regions, which points to specialized functions of astroglial subpopulations. The expression of CD15 follows a timely ordered pattern during the development of glial cells and neurons of certain brain areas, such as the human and rat cerebellum and the mouse visual system. During morphogenesis, CD15 may exert either growth-promoting or growth-repulsive activities to guide cell migration. In CNS lesions altered expression patterns of CD15 may occur. For example, in human gliomas the staining intensity for CD15 inversely correlates with the grade of malignancy. In degenerative brain diseases reactive astrocytes may reveal an increased labelling intensity on their cell surface as well as an abnormal cytosolic accumulation of the epitope. The functional significance of CD15 in the CNS is not exactly known yet. The carbohydrate could be involved in cellular adhesion and/or as receptor molecule in signal transduction pathways, as has recently been demonstrated for leukocyte-platelet or leukocyte-endothelial cell interactions.

Topics: Adult; Animals; Astrocytes; Brain; Brain Diseases; Brain Neoplasms; Glioma; Glycoconjugates; Humans; Lewis X Antigen; Mammals; Mice; Morphogenesis; Neuroglia; Neurons; Oligodendroglia; Organ Specificity; Rats

The phenotype of glioma-initiating cells (GIC) is modulated by cell-intrinsic and cell-extrinsic factors. Phenotypic heterogeneity and plasticity of GIC is an important limitation to therapeutic approaches targeting cancer stem cells. Plasticity also presents a challenge to the identification, isolation, and propagation of purified cancer stem cells. Here we use a barcode labelling approach of GIC to generate clonal populations over a number of passages, in combination with phenotyping using the established stem cell markers CD133, CD15, CD44, and A2B5. Using two cell lines derived from isocitrate dehydrogenase (IDH)-wildtype glioblastoma, we identify a remarkable heterogeneity of the phenotypes between the cell lines. During passaging, clonal expansion manifests as the emergence of a limited number of barcoded clones and a decrease in the overall number of clones. Dual-labelled GIC are capable of forming traceable clonal populations which emerge after as few as two passages from mixed cultures and through analyses of similarity of relative proportions of 16 surface markers we were able to pinpoint the fate of such populations. By generating tumour organoids we observed a remarkable persistence of dominant clones but also a significant plasticity of stemness marker expression. Our study presents an experimental approach to simultaneously barcode and phenotype glioma-initiating cells to assess their functional properties, for example to screen newly established GIC for tumour-specific therapeutic vulnerabilities.

Topics: AC133 Antigen; Antigens, CD; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Cells, Cultured; Clone Cells; Flow Cytometry; Glioma; Humans; Hyaluronan Receptors; Immunophenotyping; Lewis X Antigen; Microscopy, Confocal; Neoplastic Stem Cells; Tumor Microenvironment

Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.

Topics: AC133 Antigen; Brain Neoplasms; Cell Line, Tumor; Culture Media, Serum-Free; Flow Cytometry; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Intercellular Signaling Peptides and Proteins; Lewis X Antigen; Neoplastic Stem Cells; Neural Stem Cells; Reverse Transcriptase Polymerase Chain Reaction

Most patients with glioblastoma (GBM) die within 2 years. A major therapeutic goal is to target GBM stem cells (GSCs), a subpopulation of cells that contribute to treatment resistance and recurrence. Since their discovery in 2003, GSCs have been isolated using single-surface markers, such as CD15, CD44, CD133, and α6 integrin. It remains unknown how these single-surface marker-defined GSC populations compare with each other in terms of signaling and function and whether expression of different combinations of these markers is associated with different functional capacity. Using mass cytometry and fresh operating room specimens, we found 15 distinct GSC subpopulations in patients, and they differed in their MEK/ERK, WNT, and AKT pathway activation status. Once in culture, some subpopulations were lost and previously undetectable ones materialized. GSCs that highly expressed all 4 surface markers had the greatest self-renewal capacity, WNT inhibitor sensitivity, and in vivo tumorigenicity. This work highlights the potential signaling and phenotypic diversity of GSCs. Larger patient sample sizes and antibody panels are required to confirm these findings.

Topics: AC133 Antigen; Animals; Brain Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genetic Heterogeneity; Glioblastoma; Humans; Hyaluronan Receptors; Lewis X Antigen; Mice; Neoplastic Stem Cells

The identification of prognostically and therapeutically relevant molecular markers is fundamental to the further development of personalised therapies in brain tumours. Current therapeutic options for the treatment of gliomas rely mainly on surgical resection and the inhibition of tumour cell proliferation by irradiation and chemotherapy. Glioma stem cells are a subpopulation of proliferating tumour cells that have self-renewal capacity and can give rise to heterogeneous cells that comprise the tumour and are thought to play a role in the resistance of gliomas to therapy. The aim of this study was to evaluate the expression of markers of glioma stem cells and differentiated glial cells in proliferating glioma cells in comparison to the overall expression of the respective markers in the tumour tissue.. Tissue microarrays were assembled from specimen of pilocytic astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, glioblastoma, oligodendroglioma, anaplastic oligodendroglioma, ependymoma, and anaplastic ependymoma. These were immunohistochemically double stained with antibodies against the proliferation-associated antigen Ki67 and marker proteins for glioma stem cells (CD133, Nestin, Musashi, CD15, CD44), and differentiated glioma cells (GFAP, MAP2c).. The expression of both glial and glioma stem cell markers differs between proliferating and non-proliferating glioma cells. Furthermore, the proliferating cells in the different glial tumour entities show a different expression profile.. Further analysis of marker expression in proliferating glioma cells and correlation with clinical outcome and susceptibility to irradiation and chemotherapy might help establish new biomarkers and therapies for glioma.

Topics: AC133 Antigen; Biomarkers, Tumor; Brain Neoplasms; Cell Proliferation; Gene Expression Regulation, Neoplastic; Glioma; Humans; Hyaluronan Receptors; Lewis X Antigen; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; RNA-Binding Proteins

The difficulty in treatment of glioblastoma is a consequence of its natural infiltrative growth and the existence of a population of therapy-resistant glioma cells that contribute to growth and recurrence. To identify cells more likely to have these properties, we examined the expression in tumor specimens of several protein markers important for glioma progression including the intermediate filament protein, Nestin (NES), a glucose transporter (Glut1/SLC2A1), the glial lineage marker, glial fibrillary acidic protein, and the proliferative indicator, Ki-67. We also examined the expression of von Willebrand factor, a marker for endothelial cells as well as the macrophage/myeloid markers CD163 and CD15. Using a multicolor immunofluorescence and hematoxylin and eosin staining approach with archival formalin-fixed, paraffin embedded tissue from primary, recurrent, and autopsy IDH1 wildtype specimens combined with high-resolution tissue image analysis, we have identified highly proliferative NES(+)/Glut1(-) cells that are preferentially perivascular. In contrast, Glut1(+)/NES(-) cells are distant from blood vessels, show low proliferation, and are preferentially located at the borders of pseudopalisading necrosis. We hypothesize that Glut1(+)/NES(-) cells would be naturally resistant to conventional chemotherapy and radiation due to their low proliferative capacity and may act as a reservoir for tumor recurrence.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Brain Neoplasms; Glioblastoma; Glucose Transporter Type 1; Humans; Ki-67 Antigen; Lewis X Antigen; Nestin; Neuroglia; Receptors, Cell Surface; Tumor Cells, Cultured; Tumor Microenvironment; Tumor-Associated Macrophages

Sphere formation is an indicator of tumor aggressiveness independent of the tumor grade; however, its relation to progression-free survival (PFS) is less known. This study was designed to assess the neurosphere forming ability among low grade glioma (LGG) and high-grade glioma (HGG), its stem cell marker expression, and correlation to PFS. Tumor samples of 140 patients, including (LGG; n = 67) and (HGG; n = 73) were analyzed. We used sphere forming assay, immunofluorescence, and immunohistochemistry to characterize the tumors. Our study shows that, irrespective of the pathological sub type, both LGG and HGG formed neurospheres in vitro under conventional sphere forming conditions. However, the number of neurospheres formed from tumor tissues were significantly higher in HGG compared to LGG (P < 0.0001). Different grades of glioma were further characterized for the expression of stem cell marker proteins and lineage markers. When neurospheres were analyzed, CD133 positive cells were identified in addition to CD15 and nestin positive cells in both LGG and HGG. When these neurospheres were subjected to differentiation, cells positive for GFAP and β-tubulin III were observed. Expression of stem cell markers and β-tubulin III were prominent in HGG compared to LGG, whereas GFAP expression was higher in LGG than in HGG. Kaplan-Meier survival analysis demonstrated that neurosphere forming ability was significantly associated with shorter PFS (P < 0.05) in both LGG and HGG. Our results supports earlier studies that neurosphere formation may serve as a definitive indicator of stem cell population within the tumor and thus a better predictor of PFS than the tumor grades alone. © 2018 IUBMB Life, 71(1):244-253, 2019.

Topics: AC133 Antigen; Adolescent; Adult; Aged; Biomarkers, Tumor; Brain Neoplasms; Cell Differentiation; Child; Child, Preschool; Female; Gene Expression; Glial Fibrillary Acidic Protein; Glioma; Humans; Infant; Lewis X Antigen; Male; Middle Aged; Neoplasm Grading; Neoplastic Stem Cells; Nestin; Neurons; Prognosis; Spheroids, Cellular; Survival Analysis; Tubulin

Metastatic non-small cell lung (NSCLC) cancer represents one of the most common types of brain metastasis. The mechanisms involved in how circulating cancer cells transmigrate into brain parenchyma are not fully understood. The aim of this work was to investigate the role of fucosylated carbohydrate epitopes CD15 and sialyated CD15s in cancer adhesion to brain-derived endothelial cells and determine their influence in blood-brain barrier (BBB) disruption METHODS: Three distinct, independent methods were used to measure brain endothelial integrity and include voltohmmeter (EVOM™), impedance spectroscopy (CellZscope®) and electric cell-substrate impedance sensing system (ECIS™). Two fucosyltransferases (FUT4 and 7) responsible for CD15 and CD15s synthesis were modulated in four human cancer cell lines (three lung cancer and one glioma).. Overexpression of CD15 or CD15s epitopes led to increase in adhesion of cancer cells to cerebral endothelial cells compared with wild-type and cells with silenced CD15 or CD15s (p < 0.01). This overexpression led to the disruption of cerebral endothelial cell monolayers (p < 0.01). Knockdown of FUT4 and FUT7 in metastatic cancer cells prevented disruption of an in vitro BBB model. Surprisingly, although the cells characterised as 'non-metastatic', they became 'metastatic' -like when cells were forced to over-express either FUT4 or FUT7.. Results from these studies suggest that overexpression of CD15 and CD15s could potentiate the transmigration of circulating NSCLC cells into the brain. The clinical significance of these studies includes the possible use of these epitopes as biomarkers for metastasis.

Topics: Blood-Brain Barrier; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Movement; Endothelial Cells; Fucosyltransferases; Humans; Lewis X Antigen; Lung Neoplasms; Tumor Cells, Cultured

Glioblastoma multiforme (GBM) is a highly heterogeneous and aggressive brain tumor with a dismal prognosis. Development of resistance towards cytostatic drugs like the GBM standard drug temozolomide is a severe problem in GBM treatment. One potential source of GBM relapse could be so called cancer stem like cells (CSCs). These represent an undifferentiated subpopulation of cells with high potential for tumor initiation. Furthermore, it has been shown that differentiated GBM cells can regain CSC properties when exposed to continuous temozolomide treatment in vitro. In this study, treatment of several primary GBM cell lines with clinically relevant doses of temozolomide increased their tumorigenicity as determined by colony formation assays in soft agar. Increased tumorigenicity is a known property of CSCs. Hence, therapy options that specifically target CSCs are under investigation. CSCs appear to be particularly dependent on mitochondria biogenesis which may represent a useful target for CSC elimination. Toxicity towards mitochondria is a known side effect of several antibiotics. Thus, addition of antibiotics like doxycycline may represent a useful tool to inhibit CSCs in GBM. Here, we show that combining temozolomide treatment of primary GBM cells with doxycycline could counteract the increase of tumorigenicity induced by temozolomide treatment.

Topics: Anti-Bacterial Agents; Antineoplastic Agents, Alkylating; Biomarkers, Tumor; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Dacarbazine; DNA Modification Methylases; DNA Repair Enzymes; Doxycycline; Drug Resistance, Neoplasm; Fucosyltransferases; Glioblastoma; Humans; Lewis X Antigen; Mitochondria; Neoplastic Stem Cells; Nestin; Temozolomide; Tumor Stem Cell Assay; Tumor Suppressor Proteins

Emerging evidence has emphasized the importance of cancer therapies targeting an abnormal metabolic state of tumor-initiating cells (TICs) in which they retain stem cell-like phenotypes and nicotinamide adenine dinucleotide (NAD

Topics: Aging; Animals; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Colony-Forming Units Assay; Glioblastoma; Humans; Lactic Acid; Lewis X Antigen; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; NAD; NADP Transhydrogenases; Neoplastic Stem Cells; Oxygen Consumption; RNA, Small Interfering; Sirtuin 3; Xenograft Model Antitumor Assays

Glioblastoma is the most common primary tumor of the brain and has few long-term survivors. The local and systemic immunosuppressive environment created by glioblastoma allows it to evade immunosurveillance. Myeloid-derived suppressor cells (MDSCs) are a critical component of this immunosuppression. Understanding mechanisms of MDSC formation and function are key to developing effective immunotherapies. In this study, we developed a novel model to reliably generate human MDSCs from healthy-donor CD14+ monocytes by culture in human glioma-conditioned media. Monocytic MDSC frequency was assessed by flow cytometry and confocal microscopy. The resulting MDSCs robustly inhibited T cell proliferation. A cytokine array identified multiple components of the GCM potentially contributing to MDSC generation, including Monocyte Chemoattractive Protein-1, interleukin-6, interleukin-8, and Macrophage Migration Inhibitory Factor (MIF). Of these, Macrophage Migration Inhibitory Factor is a particularly attractive therapeutic target as sulforaphane, a naturally occurring MIF inhibitor derived from broccoli sprouts, has excellent oral bioavailability. Sulforaphane inhibits the transformation of normal monocytes to MDSCs by glioma-conditioned media in vitro at pharmacologically relevant concentrations that are non-toxic to normal leukocytes. This is associated with a corresponding increase in mature dendritic cells. Interestingly, sulforaphane treatment had similar pro-inflammatory effects on normal monocytes in fresh media but specifically increased immature dendritic cells. Thus, we have used a simple in vitro model system to identify a novel contributor to glioblastoma immunosuppression for which a natural inhibitor exists that increases mature dendritic cell development at the expense of myeloid-derived suppressor cells when normal monocytes are exposed to glioma conditioned media.

Topics: Brain Neoplasms; CD11b Antigen; Cell Hypoxia; Cell Line, Tumor; Culture Media, Conditioned; Fucosyltransferases; Glioblastoma; Humans; Isothiocyanates; Lewis X Antigen; Myeloid-Derived Suppressor Cells; Sulfoxides

CD15, which is overexpressed on various cancers, has been reported as a cell adhesion molecule that plays a key role in non-CNS metastasis. However, the role of CD15 in brain metastasis is largely unexplored. This study provides a better understanding of CD15/CD62E interaction, enhanced by tumor necrosis factor-α (TNF-α), and its correlation with brain metastasis in non-small cell lung cancer (NSCLC).. CD15 and E-selectin (CD62E) expression was demonstrated in both human primary and metastatic NSCLC cells using flow cytometry, immunofluorescence, and Western blotting. The role of CD15 was investigated using an adhesion assay under static and physiological flow live-cell conditions. Human tissue sections were examined using immunohistochemistry.. CD15, which was weakly expressed on hCMEC/D3 human brain endothelial cells, was expressed at high levels on metastatic NSCLC cells (NCI-H1299, SEBTA-001, and SEBTA-005) and at lower levels on primary NSCLC (COR-L105 and A549) cells (P < .001). The highest expression of CD62E was observed on hCMEC/D3 cells activated with TNF-α, with lower levels on metastatic NSCLC cells followed by primary NSCLC cells. Metastatic NSCLC cells adhered most strongly to hCMEC/D3 compared with primary NSCLC cells. CD15 immunoblocking decreased cancer cell adhesion to brain endothelium under static and shear stress conditions (P < .0001), confirming a correlation between CD15 and cerebral metastasis. Both CD15 and CD62E expression were detected in lung metastatic brain biopsies.. This study enhances the understanding of cancer cell-brain endothelial adhesion and confirms that CD15 plays a crucial role in adhesion in concert with TNF-α activation of its binding partner, CD62E.

Topics: Blotting, Western; Brain Neoplasms; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; E-Selectin; Endothelial Cells; Flow Cytometry; Fluorescent Antibody Technique; Humans; Lewis X Antigen; Lung Neoplasms; Microscopy, Confocal; Neoplasm Invasiveness; Tumor Necrosis Factor-alpha

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.

Topics: Adult; Aged; Antigens, CD; Brain Neoplasms; Female; Flow Cytometry; Glioma; Histocompatibility Antigens Class II; Humans; Lewis X Antigen; Lipopolysaccharide Receptors; Male; Middle Aged; Myeloid Cells; T-Lymphocytes, Regulatory; Young Adult

Glioma stem-like cells (GSCs) could have potential for tumorigenesis, treatment resistance, and tumor recurrence (GSC hypothesis). However, the mechanisms underlying such potential has remained elusive and few ultrastructural features of the cells have been reported in detail. We therefore undertook observations of the antigenic characteristics and ultrastructural features of GSCs isolated from human glioblastomas. Tumor spheres formed by variable numbers of cells, exhibiting a variable appearance in both their size and shape, were frequently seen in GSCs expressing the stem cell surface markers CD133 and CD15. Increased cell nucleus atypia, mitochondria, rough endoplasmic reticulum, coated vesicles, and microvilli, were noted in the GSCs. Furthermore, cells at division phases and different phases of the apoptotic process were occasionally observed. These findings could imply that GSCs have certain relations with human neural stem cells (NSCs) but are primitively different from undifferentiated NSCs. The data may provide support for the GSC hypothesis, and also facilitate the establishment of future glioblastoma treatments targeting GSCs.

Topics: AC133 Antigen; Antigens, CD; Brain Neoplasms; Cell Differentiation; Cell Line, Tumor; Fucosyltransferases; Glioblastoma; Glycoproteins; Humans; Lewis X Antigen; Neoplastic Stem Cells; Neural Stem Cells; Peptides; Spheroids, Cellular

Brain tumors are typically composed of heterogeneous cells that exhibit distinct phenotypic characteristics and proliferative potentials. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. This unit describes protocols for the culture and isolation BTICs. We applied culture conditions and assays originally used for normal neural stem cells (NSCs) in vitro to a variety of brain tumors. Using fluorescence-activated cell sorting for the neural precursor cell surface marker CD133/CD15, BTICs can be isolated and studied prospectively. Isolation of BTICs from GBM bulk tumor will enable examination of dissimilar morphologies, self-renewal capacities, tumorigenicity, and therapeutic sensitivities. As cancer is also considered a disease of unregulated self-renewal and differentiation, an understanding of BTICs is fundamental to understanding tumor growth. Ultimately, it will lead to novel drug discovery approaches that strategically target the functionally relevant BTIC population.

Topics: AC133 Antigen; Animals; Antigens, CD; Biomarkers, Tumor; Brain Neoplasms; Cell Culture Techniques; Cell Differentiation; Cell Separation; Fucosyltransferases; Glycoproteins; Humans; Lewis X Antigen; Neoplasm Proteins; Neoplastic Stem Cells; Neural Stem Cells; Peptides

Neural progenitor cells (NPCs) derived from mouse embryonic stem (mES) cells can lead to tumors after transplantation. The cellular source of such tumors remains under debate. We investigated the tumor formation resulting from mES cell-derived NPCs in a rat stroke model and in nude mice. After 2 hr of ischemia and 48 hr of reperfusion, the NPCs were transplanted into the ischemic core of the xenogeneic rats. Four weeks after transplantation, the grafted cells were found to be viable at the border of the necrosis and had differentiated into neurons. Transplanted rats did not exhibit any behavioral improvement, because tumor formed in 90% of the animals. Immunosuppression facilitated tumor formation. Tumors were observed in 40% of normal rats after NPC transplantation when cyclosporin A was administered. Meanwhile, no tumor formation was observed without cyclosporin A. Ischemic damage also facilitated tumor formation, because NPCs gave rise to tumors in 90% of ischemic rats, a percentage significantly higher than that in intact rats, which was 40%. The SSEA-1-positive cells isolated from stage 4 are not exactly undifferentiated ES cells. They exhibited a marker gene transcription profile different from that of ES cells and did not form tumors in transplanted nude mice. The undifferentiated ES cells remaining after differentiation did not contribute to tumors either. First, the tumor formation rate resulting from undifferentiated ES cells in the brains of normal rats is 0%, significantly lower than that of NPCs. Second, transplanted NPCs that led to 100% tumors in nude mice contained approximately 1.5 × 10(3) Oct-4-positive cells; however, even 5 × 10(5) undifferentiated ES cells formed neoplasm only in 40% nude mice.

Topics: Animals; Brain Ischemia; Brain Neoplasms; Cell Count; Cell Differentiation; Embryonic Stem Cells; Immunosuppression Therapy; Lewis X Antigen; Male; Mice; Mice, Nude; Neural Stem Cells; Rats; Rats, Wistar; Stem Cell Transplantation

Glioblastoma multiforme (GBM), as many other solid tumours, contains a subpopulation of cells termed cancer stem-like cells responsible for the initiation and propagation of tumour growth. However, a unique immunophenotype/surface antigen composition for the clear identification of brain tumour stem cells (BTSC) has not yet been found. Here we report a novel code of cell surface markers for the identification of different cell subpopulations in neurospheres derived from a GBM with a primitive neuroectodermal tumour (PNET)-like component (GBM-PNET). These subgroups differ in their CD133/CD15 expression pattern and resemble cells with different stem-like genotype and developmental pathway activation levels. Strikingly, clonogenic analysis of cultures differentially expressing the investigated markers enabled the identification of distinct subpopulations of cells endowed with stem cell characteristics. High clonogenicity could be found in CD133(-)/CD15(-) and CD133(+)/CD15(+) but not in CD133(-)/CD15(+) cells. Moreover, cell subpopulations with pronounced clonogenic growth were characterized by high expression of stem cell-related genes. Interestingly, these observations were unique for GBM-PNET and differed from ordinary GBM cultures derived from tumours lacking a PNET component. This work elucidates the complex molecular heterogeneity of in vitro propagated glioblastoma-derived cells and potentially contributes to the development of novel diagnostic modalities aiming at the identification of the brain tumour stem-like cell population in a subgroup of GBMs.

Topics: AC133 Antigen; Aged; Antigens, CD; Biomarkers, Tumor; Brain Neoplasms; Cell Line, Tumor; Flow Cytometry; Fucosyltransferases; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glioblastoma; Glycoproteins; Humans; Lewis X Antigen; Male; Neoplastic Stem Cells; Neuroectodermal Tumors, Primitive; Peptides; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells

Glioblastoma is one of the most frequent primary brain tumors and is characterized by aggressive clinical behavior and biologic heterogeneity. To evaluate the prognostic implication of cancer stem cell markers in glioblastoma, the expression of these markers was investigated in a large series of glioblastoma patients in relation to the survival rate. This series includes 88 cases of glioblastoma that were diagnosed at the Chonnam University Hwasun Hospital from 2004 to 2009. The expression of newly established stem cell markers (nestin, CD133 and CD15) was detected using immunohistochemical analysis. The presence of immunopositive tumor cells was evaluated and interpreted in comparison with the patients' survival data. The expression of nestin was high in 60 cases (68.2%). CD133 and CD15 were positive in 52 cases (59.1%) and 40 cases (45.5%), respectively. No statistically significant difference in patient survival according to stem cell marker expression was observed (P > 0.05). However, gross total resection or combined radiation therapy and chemotherapy significantly prolonged survival (P = 0.04 and P = 0.04). Cox's proportional hazards model showed that the gross total resection and combined radiation therapy and chemotherapy were independent prognostic factors. Although the correlation of stem cell marker expression with clinical outcome in glioma is of considerable interest, the data do not support their prognostic value in glioblastoma. Identification of the key cells in the glioblastoma population in the context of clinical outcomes will provide insight into the mechanism of brain tumorigenesis and will be of paramount importance in determining therapeutically appropriate targets.

Topics: AC133 Antigen; Adolescent; Adult; Aged; Aged, 80 and over; Antigens, CD; Biomarkers, Tumor; Brain Neoplasms; Female; Follow-Up Studies; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Glioblastoma; Glycoproteins; Humans; Intermediate Filament Proteins; Lewis X Antigen; Male; Middle Aged; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; Peptides; Prognosis; Young Adult

Intracranial and intraspinal involvement is a rare complication of Hodgkin's lymphoma. Intracranial involvement is observed in 0.2 to 0.5 percent of patients with Hodgkin's lymphoma. No specific risk factors associated with intracranial involvement have been found. We report intracranial involvement of Hodgkin's lymphoma in a patient who had previously undergone thyroidectomy due to thyroid papillary carcinoma.

Topics: Adult; Brain Neoplasms; Carcinoma, Papillary; Central Nervous System Neoplasms; Female; Hodgkin Disease; Humans; Immunohistochemistry; Ki-1 Antigen; Lewis X Antigen; Magnetic Resonance Imaging; Neoplasm Metastasis; Risk Factors; Thyroidectomy; Tomography, X-Ray Computed

Tumor initiating cells (TICs) provide a new paradigm for developing original therapeutic strategies.. We screened for TICs in 47 human adult brain malignant tumors. Cells forming floating spheres in culture, and endowed with all of the features expected from tumor cells with stem-like properties were obtained from glioblastomas, medulloblastoma but not oligodendrogliomas.. A long-term self-renewal capacity was particularly observed for cells of malignant glio-neuronal tumors (MGNTs). Cell sorting, karyotyping and proteomic analysis demonstrated cell stability throughout prolonged passages. Xenografts of fewer than 500 cells in Nude mouse brains induced a progressively growing tumor. CD133, CD15/LeX/Ssea-1, CD34 expressions, or exclusion of Hoechst dye occurred in subsets of cells forming spheres, but was not predictive of their capacity to form secondary spheres or tumors, or to resist high doses of temozolomide.. Our results further highlight the specificity of a subset of high-grade gliomas, MGNT. TICs derived from these tumors represent a new tool to screen for innovative therapies.

Topics: AC133 Antigen; Animals; Antigens, CD; Antigens, CD34; Brain Neoplasms; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Glioma; Glycoproteins; Humans; Lewis X Antigen; Mice; Mice, Nude; Neoplasm Transplantation; Neoplastic Stem Cells; Neurons; Peptides; Proteomics

Cell culture and the use of cell lines are often fundamental requirements in basic scientific research. It is of the utmost importance for researchers to ensure that the cell lines in use have a well defined origin and are routinely re-analysed to highlight possible areas of contamination. In this preliminary study species specific primers were designed to easily distinguish between human, mouse and rat DNA with standard agarose gel electrophoresis. Inter-species contamination is often the most common form of contamination experienced, with the most common of cell lines in use being of human, mouse and rat derivation. A PCR-based assay was therefore developed to ensure an accurate, quick and cost effective means of determining any cell line contamination which could be easily executed on a routine basis. Furthermore, this simple PCR is able to identify the species in the inter-species mixture of DNA and therefore provides a valuable tool for the authentication of human cell lines.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Brain Neoplasms; Cell Line, Tumor; Chromatography, Agarose; DNA Primers; DNA, Neoplasm; Glioma; Humans; Immunochemistry; Lewis X Antigen; Mice; Rats; Reagent Kits, Diagnostic; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; Species Specificity

The growth of many cancers depends on self-renewing cells called cancer stem cells or tumor-propagating cells (TPCs). In human brain tumors, cells expressing the stem cell marker CD133 have been implicated as TPCs. Here we show that tumors from a model of medulloblastoma, the Patched mutant mouse, are propagated not by CD133(+) cells but by cells expressing the progenitor markers Math1 and CD15/SSEA-1. These cells have a distinct expression profile that suggests increased proliferative capacity and decreased tendency to undergo apoptosis and differentiation. CD15 is also found in a subset of human medulloblastomas, and tumors expressing genes similar to those found in murine CD15(+) cells have a poorer prognosis. Thus, CD15 may represent an important marker for TPCs in medulloblastoma.

Topics: AC133 Antigen; Animals; Antigens, CD; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Brain Neoplasms; Disease Models, Animal; Gene Expression Profiling; Glycoproteins; Hedgehog Proteins; Humans; Lewis X Antigen; Medulloblastoma; Mice; Mice, Mutant Strains; Mice, SCID; Microarray Analysis; Molecular Sequence Data; Neoplasm Transplantation; Neoplastic Stem Cells; Neurons; Patched Receptors; Peptides; Receptors, Cell Surface; Recombinant Fusion Proteins; Signal Transduction; Stem Cells; Survival Rate; Tumor Cells, Cultured

Although flow cytometry is useful for studying neural lineage relationships, the method of dissociation can potentially bias cell analysis. We compared dissociation methods on viability and antigen recognition of mouse central nervous system (CNS) tissue and human CNS tumor tissue. Although nonenzymatic dissociation yielded poor viability, papain, purified trypsin replacement (TrypLE), and two purified collagenase/neutral protease cocktails (Liberase-1 or Accutase) each efficiently dissociated fetal tissue and postnatal tissue. Mouse cells dissociated with Liberase-1 were titrated with antibodies identifying distinct CNS precursor subtypes, including CD133, CD15, CD24, A2B5, and PSA-NCAM. Of the enzymes tested, papain most aggressively reduced antigenicity for mouse and human CD24. On human CNS tumor cells, CD133 expression remained highest after Liberase-1 and was lowest after papain or Accutase treatment; Liberase-1 digestion allowed magnetic sorting for CD133 without the need for an antigen re-expression recovery period. We conclude that Liberase-1 and TrypLE provide the best balance of dissociation efficiency, viability, and antigen retention. One implication of this comparison was confirmed by dissociating E13.5 mouse cortical cells and performing prospective isolation and clonal analysis on the basis of CD133/CD24 or CD15/CD24 expression. Highest fetal expression of CD133 or CD15 occurred in a CD24(hi) population that was enriched in neuronal progenitors. Multipotent cells expressed CD133 and CD15 at lower levels than did these neuronal progenitors. We conclude that CD133 and CD15 can be used similarly as selectable markers, but CD24 coexpression helps to distinguish fetal mouse multipotent stem cells from neuronal progenitors and postmitotic neurons. This particular discrimination is not possible after papain treatment. Disclosure of potential conflicts of interest is found at the end of this article.

Topics: AC133 Antigen; Animals; Animals, Newborn; Antigens, CD; Antigens, Surface; Brain Neoplasms; Carcinoma; CD24 Antigen; Cell Separation; Cells, Cultured; Central Nervous System; Child; Flow Cytometry; Glycoproteins; Humans; Lewis X Antigen; Mice; Mice, Inbred NOD; Mice, SCID; Nerve Tissue; Peptides; Stem Cells

We report a case of myeloid sarcoma of the brain mimicking a meningioma on CT scan. The lesion was first morphologically misdiagnosed as a lymphoma, but correctly identified by using immunochemistry with anti-myeloperoxidase, anti-CD68, anti-CD15 antibodies. An acute myeloid leukemia was diagnosed 5 months later. Myeloid sarcoma is frequently mistaken for malignant lymphoma, especially when it presents without leukemic manifestation, even at immunohistochemistry, since both express some leukocyte antigens. Careful evaluation of morphology for evidence of myeloid differentiation, and immunohistochemistry using anti-myeloperoxidase, anti-lysozyme, CD15, CD68 antibodies, should be used to confirm the diagnosis and to rule out lymphoma since the treatment is different.

Topics: Adult; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Brain Neoplasms; Diagnosis, Differential; Humans; Immunohistochemistry; Leukemia, Myeloid, Acute; Lewis X Antigen; Male; Peroxidase; Sarcoma, Myeloid; Tomography, X-Ray Computed