ganglioside--gd2 and Glioma

The objective of this study was to determine the feasibility, toxicity, and potential therapeutic benefits of an adjuvant active immunotherapy using a tumour specific ganglioside (GD2) conjugate for the adjuvant treatment of recurrent or progressive gliomas. Seven patients with proven GD2 expression in surgical specimens underwent a vaccination course with GD2-KLH/MPL-A conjugate. The follow-up was performed according to WHO guidelines regarding common toxicity criteria. Antibody titres against the ganglioside and the adjuvants were analysed. All patients developed a local type 4 reaction. Anti-GD2-antibody titres could not be detected, despite high titres against the immunoadjuvants. No tumour regression was observed. The disease remained stable for a median of 21.5 weeks (6-34 weeks). The median survival time after the first immunization was 47 weeks. The medial total survival time was 76 weeks. Adverse effects have not been observed. Active GD2-KLH/MPL-A immunization was technically feasible, but did not elicit anti-GD2 antibody generation.

Topics: Adjuvants, Immunologic; Adult; Aged; Animals; Brain Neoplasms; Enzyme-Linked Immunosorbent Assay; Feasibility Studies; Female; Gangliosides; Glioma; Humans; Immunoglobulin G; Immunoglobulin M; Immunotherapy, Active; Male; Mice; Mice, Inbred BALB C; Middle Aged; Neoplasm Recurrence, Local; Radiography; Vaccination

High expression of gangliosides GD3 and GD2 is observed in human gliomas. The functions of GD3 and GD2 in malignant properties have been reported in glioma cells in vitro, but those functions have not yet been investigated in vivo. In this study, we showed that deficiency of GD3 synthase (GD3S, St8sia1) attenuated glioma progression and clinical and pathological features in a platelet-derived growth factor B-driven murine glioma model. Lack of GD3S resulted in the prolonged lifespan of glioma-bearing mice and low-grade pathology in generated gliomas. Correspondingly, they showed reduced phosphorylation levels of Akt, Erks, and Src family kinases in glioma tissues. A DNA microarray study revealed marked alteration in the expression of various genes, particularly in MMP family genes, in GD3S-deficient gliomas. Re-expression of GD3S restored expression of MMP9 in primary-cultured glioma cells. We also identified a transcription factor, Ap2α, expressed in parallel with GD3S expression, and showed that Ap2α was critical for the induction of MMP9 by transfection of its cDNA and luciferase reporter genes, and a ChIP assay. These findings suggest that GD3S enhances the progression of gliomas by enhancement of the Ap2α-MMP9 axis. This is the first report to describe the tumor-enhancing functions of GD3S in vivo.

Topics: Animals; Astrocytes; Brain Neoplasms; Cells, Cultured; Disease Models, Animal; Disease Progression; Gangliosides; Gene Expression Regulation, Neoplastic; Glioma; Longevity; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; Sialyltransferases; Transfection

Sialic acid-containing glycosphingolipids, gangliosides, are considered as cancer associated antigens in neuro-ectoderm-derived tumors such as melanomas and neuroblastomas. In particular, gangliosides GD3 and GD2 are expressed in human gliomas. It has been reported that their expression levels increase along with increased malignant properties. However, the implication of GD3/GD2 in human glioma cells has never been clarified, at least to the best of our knowledge. In this study, we introduced the cDNA of GD3 synthase (GD3S)(ST8SIA1) into a glioma cell line, U-251MG, that expresses neither GD3 nor GD2, thereby establishing transfectant cells U-251MG-GD3S(+) expressing high levels of GD3 and GD2 on the cell surface. In these U-251MG‑GD3S(+) cell lines, signaling molecules such as Erk1/2, Akt, p130Cas, paxillin and focal adhesion kinase were activated, leading to the enhancement of invasion activity and motility. It was then demonstrated that the U-251MG-GD3S(+) cells could proliferate under culture conditions with low or no serum concentrations without undergoing cell cycle arrest by escaping the accumulation of p16 and p21. All these results suggested that GD3 and GD2 highly expressed in gliomas confer increased invasion and mobility, cell growth abilities under low serum conditions, and increased ratios of the S-G2/M phase in the cell cycle.

Topics: Brain Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gangliosides; Glioma; Humans; Neoplasm Invasiveness; Tumor Cells, Cultured

Diffuse intrinsic pontine glioma (DIPG) and other diffuse midline gliomas (DMGs) with mutated histone H3 K27M (H3-K27M)

Topics: Animals; Brain Neoplasms; Gangliosides; Glioma; Histones; Humans; Immunotherapy, Adoptive; Lysine; Methylation; Mice; Receptors, Antigen, T-Cell; Xenograft Model Antitumor Assays

Neuroblastic tumors (NBT) are composed by neuroblasts and Schwannian-like stroma. The origin of these two cell subtypes remains unclear. In this study, we describe, a neuroblastic-like subpopulation in neuroblastoma (NB) coexpressing GD2 and S100A6, neuroblastic and glial lineage markers respectively. The GD2(+)/S100A6(+) neuroblastic subpopulation was found to be enriched in low risk NB, distributed around the perivascular niche. Some stromal bundles showed GD2(+)/S100A6 costaining. Metastatic bone marrow specimens also showed GD2(+)/S100A6(+) cells. During in vitro retinoic acid induced differentiation of NB cell lines, rare GD2(+)/S100A6 neuroblatic cells appeared. We conclude that GD2(+)/S100A6(+) neuroblasts may represent a tumoral glial precursor subpopulation in NBT.

Topics: Antigens, Differentiation; Cell Cycle Proteins; Cell Differentiation; Cell Lineage; Gangliosides; Glioma; Humans; Neoplastic Stem Cells; Neural Stem Cells; Neuroblastoma; Neuroglia; S100 Calcium Binding Protein A6; S100 Proteins; Schwann Cells

Antibodies against two major gangliosides detected in human brain and brain tumors--Glac2 (GD3) and Gtri2 (GD2)--were tested by immunohistochemistry in an unselected sample of intracranial tumors during the years 1986 through 1991. Two groups emerged as evaluable samples, namely gliomas of different grades and meningiomas. In a pilot series, it was shown that these gangliosides could be visualized in frozen sections of cells of gliomas and meningiomas (as well as neurinomas) and in some structures of the normal brain. It was however not possible in frozen sections to further analyze the cellular or subcellular expression pattern of the mentioned components and paraffin sections with conventional processing were only weakly and diffusely stained. A modified protocol therefore was created that replaced alcohol processing by acetone. With this protocol, interpretable results in paraffin sections were obtained. With this method, 133 single intracranial tumors were investigated as to their immunohistologically detectable ganglioside expression. The most consistent result was that the whole cytoplasm of highly fibrillary (gemistocytic) astrocytes in all grades of gliomas was stained by Gtri2 (GD2) and Glac2 (GD3) with high preponderance of Gtri2 (GD2) expression. In all meningiomas, Glac2 (GD3) had a higher expression than Gtri2. No constant pattern in the other entities emerged. By comparison with GFAP expression in gliomas and vimentin in meningiomas, the colocalisation of gangliosides and intermediary filament proteins is supposed.

Topics: Antibodies, Monoclonal; Brain Neoplasms; Gangliosides; Glioma; Humans; Immunohistochemistry; Meningioma

Two monoclonal antibodies (mabs) were raised against ganglioside GD2 (Gtri2) and tested on human intracranial tumors and normal brain by immunohistochemical methods both in frozen and paraffin embedded sections. In normal brain structures, astrocytes were visualized with both mabs (BW 625 and BW 704) almost exclusively in the subventricular and subpial layer. A minor amount of myelin sheaths in well defined localisation was only recognized in frozen sections. Consequently in astrocytic tumors of different grades of malignancy (WHO I-IV), astrocytes were depicted in their characteristic shape and arrangement around vessels. In addition, staining was observed in meningiomas and schwannomas, but not in pituitary adenomas or metastatic carcinomas. In meningioma und schwannoma the staining was restricted to the cellular periphery and was again present in frozen section material and absent in paraffin embedded tissue. In astrocytes, reactive and neoplastic, obviously fibrous processes and cytoplasm were distinctly stained both in frozen and paraffin embedded sections. It is concluded that some neuroectodermal derived cells as well as myelin of defined localisation express GD2 on the membrane surfaces and in the cytoplasm. The latter may primarily be the case in fibrous astrocytes, which were stained in reactive and pathologic conditions. The reaction can be used as diagnostic tool in astrocytic tumors; its positive therapeutic significance is hampered by the fact that (1) not all cells in heterogeneous tumor populations express the epitope and (2) there are normal structures which do so.

Topics: Antibodies, Monoclonal; Brain Chemistry; Brain Neoplasms; Epitopes; Gangliosides; Glioma; Humans; Immunohistochemistry; Meningioma; Neurilemmoma

Monoclonal antibodies (mAbs) recognizing the disialoganglioside II3(NeuAc)2GgOse3Cer (GD2) were produced by immunizing mice with the GD2-expressing neuroblastoma cell line LAN-1 and a prefusion boost with purified GD2 coupled to Salmonella minnesota. Two IgM mAbs were isolated which demonstrated high levels of reactivity (binding ratios in excess of 100) with GD2 by solid-phase radioimmunoassay and positivity in high-performance thin-layer chromatography (HPTLC) immunostain; only one (DMAb-20) was subsequently shown by analysis with a panel of defined ganglioside species to be specific for the minimum epitope of GD2 GalNAc beta 1-4(NeuAc alpha 2-8-NeuAc alpha 2-3)Gal-, DMAb-20 was used to evaluate the expression of GD2 by malignant glioma and medulloblastoma cell lines using cell surface radioimmunoassay. indirect membrane immunofluorescence. HPTLC immunostain, and densitometric analysis of extracted gangliosides from selected cell lines. Sixteen of 20 (80%) malignant glioma and 5 of 5 medulloblastoma cell lines reacted with DMAb-20; in agreement with previous studies, 5 of 5 neuroblastoma and 2 of 3 melanoma cell lines also reacted with DMAb-20, GD2 was proportionally increased in the glioma and medulloblastoma cell lines relative to levels in normal brain, as determined by densitometric analysis. In a phenotypic survey of malignant glioma biopsies, tumor cells in 24 of 30 (80%) cases stained positively with DMAb-20. Reactive astrocytes, both within the adjacent to tumors, were frequently intensely stained. Among the morphological variants of glioblastoma examined, the most intense staining with DMAb-20 was observed in neoplastic gemistocytes, with the weakest or absent staining in small cell glioblastomas. As GD2 is a commonly expressed surface antigen of gliomas and medulloblastomas, expression of which is retained in tissue culture. DMAb-20 will be useful in determining the functional role of GD2 in cell-cell interaction, adhesion, and invasion, and in defining altered growth control mechanisms of central nervous system neoplasms in in vitro models.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Brain Neoplasms; Cell Line; Female; Fluorescent Antibody Technique; Gangliosides; Glioblastoma; Glioma; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred BALB C; Radioimmunoassay

Monoclonal antibodies (mAbs) BW 625 and BW 704, of the IgG3 isotype, bound to immunochemically indistinguishable epitopes on ganglioside II3(Neu-Ac)2-GgOse3-Cer. Despite this fact the mAbs showed a differential binding pattern on human glioma cell lines i.e. immunohistochemical data indicate that the detected epitopes are not identical. Furthermore, either mAb is able to mediate the antibody-dependent cellular cytotoxicity reaction (ADCC) and the human-complement-dependent cytotoxicity reaction (CDC) with epitope-expressing tumor cells. All cryopreserved tissue specimens from gliomas and neuroblastomas were immunohistochemically stained, whereas the other small round cell tumors of childhood, as well as melanomas and small-cell lung carcinomas, were essentially negative. Positive staining of normal cryopreserved tissues was restricted to amyelinic axons, Hassal's bodies and some connective tissue fibers in thymus and the tegumentary epithelium of skin. The high selectivity of mAb BW 704 for gliomas and neuroblastomas, the lack of cross-reactivity with major tissues and the strong ADCC and CDC potential argue for the use of mAb BW 704 in immunotherapy of neuroblastomas and gliomas.

Topics: Antibodies, Monoclonal; Antigens, Neoplasm; Epitopes; Gangliosides; Glioma; Humans; Immunoenzyme Techniques; Lactones; Melanoma, Experimental; Neoplasms, Nerve Tissue; Neurons; Tissue Distribution; Tumor Cells, Cultured