lewis-x-antigen and Glioblastoma

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 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

Glioblastoma is a common, malignant brain tumor whose disease incidence increases with age. Glioblastoma stem-like cells (GSCs) are thought to contribute to cancer therapy resistance and to be responsible for tumor initiation, maintenance, and recurrence. This study utilizes both SNP array and gene expression profiling to better understand GSCs and their relation to malignant disease. Peripheral blood and primary glioblastoma tumor tissue were obtained from patients, the latter of which was used to generate GSCs as well as a CD133pos./CD15pos. subpopulation. The stem cell features of GSCs were confirmed via the immunofluorescent expression of Nestin, SOX2, and CD133. Both tumor tissue and the isolated primary cells shared unique abnormal genomic characteristics, including a gain of chromosome 7 as well as either a partial or complete loss of chromosome 10. Individual genomic differences were also observed, including the loss of chromosome 4 and segmental uniparental disomy of 9p24.3→p21.3 in GSCs. Gene expression profiling revealed 418 genes upregulated in tumor tissue vs. CD133pos./CD15pos. cells and 44 genes upregulated in CD133pos./CD15pos. cells vs. tumor tissue. Pathway analyses demonstrated that upregulated genes in CD133pos./CD15pos. cells are relevant to cell cycle processes and cancerogenesis. In summary, we detected previously undescribed genomic and gene expression differences when comparing tumor tissue and isolated stem-like subpopulations.

Topics: AC133 Antigen; Cell Separation; Cells, Cultured; Gene Expression Profiling; Glioblastoma; Humans; Lewis X Antigen; Neoplastic Stem Cells; Polymorphism, Single Nucleotide; Specimen Handling; Up-Regulation

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

Glioblastoma is the most common and most malignant primary brain tumor with a median survival of 15 months. Moschamine is an indole alkaloid that has a serotoninergic and cyclooxygenase inhibitory effect. In this study, we sought to determine whether moschamine could exert cytotoxic and cytostatic effects on glioma cells in vitro. Moschamine was tested for toxicity in zebrafish. We investigated the effect of moschamine on U251MG and T98G glioblastoma cell lines. Viability and proliferation of the cells were examined with trypan blue exclusion assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and the xCELLigence system. Apoptosis (annexin-propidium iodide), cell cycle, and CD24/CD44/CD56/CD15 expression were tested with flow cytometry. Treatment with moschamine significantly reduced cell viability in both cell lines tested. Induction of cell death and cell cycle arrest was confirmed with flow cytometry in both cell lines. After treatment with moschamine, there was a dose-dependent decrease in CD24 and CD44 expression, whereas there was no change in CD56 and CD15 expression in T98G cell line. The zebrafish mortality on the fifth post-fertilization day was zero even for 1 mM of moschamine concentration. The treatment of glioblastoma cell lines with moschamine may represent a novel strategy for targeting glioblastoma.

Topics: Animals; Apoptosis; CD24 Antigen; CD56 Antigen; Cell Cycle Checkpoints; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Hyaluronan Receptors; Lewis X Antigen; Neoplasm Proteins; Zebrafish

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

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

Cancer stem cells (CSCs) are the most aggressive cell type in many malignancies. Cell surface proteins are generally used to isolate and characterize CSCs. Therefore, the identification of CSC-specific cell surface markers is very important for the diagnosis and treatment of malignancies. We found that Nestin (a type VI intermediate filament protein), like the glioma stem cell (GSC) markers CD133 and CD15, exhibited different levels of expression in primary human glioblastoma specimens. Similar to our previous finding that cytoplasmic Nestin is expressed as a cell surface form in mouse GSCs, the cell surface form of Nestin was also expressed at different levels in human GSCs. We isolated cell surface Nestin-positive cell populations from human GSCs by fluorescence-activated cell sorting FACS analysis, and observed that these populations exhibited robust CSC properties, such as increased tumorsphere-forming ability and tumorsphere size. Mechanistically, we found that DAPT, a γ-secretase (a multi-subunit protease complex) inhibitor, reduced the proportion of cell surface Nestin-positive cells in human GSCs in a time- and dose-dependent manner, without significant changes in total Nestin expression, implying that a post-translational modification was involved in the generation of cell surface Nestin. Taken together, our data provides the first evidence that cell surface Nestin may serve as a promising GSC marker for the isolation and characterization of heterogeneous GSCs in glioblastomas.

Topics: AC133 Antigen; Amyloid Precursor Protein Secretases; Antigens, CD; Biomarkers, Tumor; Cell Shape; Cell Transformation, Neoplastic; Flow Cytometry; Fluorescent Antibody Technique; Fucosyltransferases; Gene Expression Regulation, Neoplastic; Glioblastoma; Glycoproteins; Humans; Intermediate Filament Proteins; Lewis X Antigen; Neoplastic Stem Cells; Nerve Tissue Proteins; Nestin; Peptides; Protein Processing, Post-Translational; Reproducibility of Results; Single-Cell Analysis; Tumor Cells, Cultured

Aquaporin-9 (AQP9) is a membrane protein channel that is permeable to a range of small solutes, including glycerol, urea and nucleobases. Expression of AQP9 in normal brain is limited, while widespread AQP9 expression has previously been reported in human glioblastoma. However, the specific cellular expression of AQP9 in glioblastoma remains unclear. In this study, we have examined microarrays to corroborate AQP9 mRNA expression in glioma. These analyses suggested that AQP9 mRNA expression in glioblastoma is primarily explained by tumor infiltration with AQP9 expressing leukocytes. Immunolabeling confirmed that within tumor regions, AQP9 was expressed in CD15(+) and Calgranulin B(+) leukocytes, but also in larger cells that morphologically resembled glioma cells. Specificity of immunoreagents was tested in recombinant cell lines, leukocyte preparations, and sections of normal human brain and liver tissue. Apparent AQP9(+) glioma cells were frequently observed in proximity to blood vessels, where brain tumor stem cells have been observed previously. A fraction of these larger AQP9 expressing cells co-expressed the differentiated astrocyte marker GFAP. AQP9 expressing glioma cells were negative for the brain tumor stem cell marker CD15, but were observed in proximity to CD15(+) glioma cells. AQP9 expression may therefore require signals of the perivascular tumor environment or alternatively it may be restricted to a population of glioma stem cell early progenitor cells.

Topics: Aquaporins; Astrocytes; Astrocytoma; Brain; Calgranulin B; Cell Line, Tumor; Fucosyltransferases; Glioblastoma; Humans; Leukocytes; Lewis X Antigen; Liver

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

CD133+ populations of human glioblastoma multiforme (GBM) cells are reportedly enriched for tumor stem cells (TSCs) or tumor-initiating cells (TICs). Approximately 40% of freshly isolated GBM specimens, however, do not contain CD133+ tumor cells, raising the possibility that CD133 may not be a universal enrichment marker for GBM TSCs/TICs. Here we demonstrate that stage-specific embryonic antigen 1(SSEA-1/LeX)+ GBM cells fulfill the functional criteria for TSC/TIC, since (1) SSEA-1+ cells are highly tumorigenic in vivo, unlike SSEA-1- cells; (2) SSEA-1+ cells can give rise to both SSEA-1+ and SSEA-1- cells, thereby establishing a cellular hierarchy; and (3) SSEA-1+ cells have self-renewal and multilineage differentiation potentials. A distinct subpopulation of SSEA-1+ cells was present in all but one of the primary GBMs examined (n = 24), and most CD133+ tumor cells were also SSEA-1+, suggesting that SSEA-1 may be a general TSC/TIC enrichment marker in human GBMs.

Topics: Animals; Cell Cycle; Cell Differentiation; Cell Lineage; Cell Proliferation; Female; Flow Cytometry; Glioblastoma; Humans; Lewis X Antigen; Male; Mice; Mice, SCID; Neoplastic Stem Cells; Tumor Cells, Cultured