Search

Characterization of tissue surrounding glioblastoma (GBM) is a focus for translational research because tumor recurrence invariably occurs in this area. We investigated the expression of the progenitor/ stem cell markers GD3 ganglioside and NG2 proteoglycan in GBM, peritumor tissue (brain adjacent to tumor, BAT) and cancer stem-like cells (CSCs) isolated from GBM (GCSCs) and BAT (PCSCs). GD3 and NG2 immunohistochemistry was performed in paired GBM and BAT specimens from 40 patients. Double-immunofluorescence was carried out to characterize NG2-positive cells of vessel walls. GD3 and NG2 expression was investigated in GCSCs and PCSCs whose tumorigenicity was also evaluated in Scid/bg mice. GD3 and NG2 expression was higher in tumor tissue than in BAT. NG2 decreased as the distance from tumor margin increased, regardless of the tumor cell presence, whereas GD3 correlated with neoplastic infiltration. In BAT, NG2 was coexpressed with a-smooth muscle actin (α-SMA) in pericytes and with nestin in the endothelium. Higher levels of NG2 mRNA and protein were found in GCSCs while GD3 synthase was expressed at similar levels in the 2 CSC populations. PCSCs had lower tumorigenicity than GCSCs. These data suggest the possible involvement of GD3 and NG2 in pre/pro-tumorigenic events occurring in the complex microenvironment of the tissue surrounding GBM.

Gangliosides are sialic acid-containing glycosphingolipids located mainly in outlet membrane plasma. Tumors usually accumulate gangliosides as disialoganglioside GD3 which is widely expressed in melanomas and neuroblastomas. GD3 has been associated with tumor progression, conferring tumorigenic characteristics to cells such as increased proliferation and migration. In order to investigate the role of GD3 in cell migration, we have transfected the GD3 synthase gene (ST8Sia I) in a melanocyte cell line. In this work we have demonstrated that overexpression of GD3 upon transfection of GD3 synthase was associated with an increased migratory response towards laminin-1. GD3 and 9-O-acetyl-GD3 gangliosides, which colocalize with integrins in cell lamellipodia, are positive modulators of integrin function in the migratory response while GD3 was left behind as a molecular footprint. Indeed, addition of exogenous GD3 to the cells stimulated cell migration whereas exogenously added GM3 inhibited it. However, depletion of gangliosides using a glucosylceramide synthase inhibitor led cells more migratory. Glycosphingolipids content modulates FGF-2-induced cell migration as we demonstrated that FGF-2 induces cell migration. However, this effect was abolished after using glucosylceramide synthase inhibitor, suggesting that GD3 may modulate the motogenic activity of FGF-2. Finally, we showed that GD3 synthase transfected melanocytes release extracellular vesicles (EVs) enriched in GD3. These EVs are responsible for modulating cell migration response due to increase average velocity of GD3 negative cells as we showed in a real-time wound-healing assay. This study shows that EVs from a subset of GD3 positive cells may contribute to the horizontal signaling propagation to a different subset of GD3 negative cells. Thus, data present here led us to an important insight into how EVs carrying GD3 ganglioside could participate in intercellular communication modulating cellular migration. Citation Format: Andreia H. Otake, Ana Paula M. Duarte, Renata de Freitas Saito, Alexandre F. Ramos, Roger Chammas. GD3 ganglioside-enriched extracellular vesicles stimulate melanocyte migration [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A73.

We have analyzed sequence homologies between nonimmunogenic phage displayed peptides mimicking GD3 ganglioside and human/mouse self-proteins. The GD3 ganglioside phagotopes showed homology to proteins involved in carbohydrate metabolism/transport. Besides this expected homology, molecular mimicry of critical regulatory proteins was found. These data contribute to our understanding of the structural relatedness of antigenic determinants defined by specific anti-GD3 monoclonal antibodies and, in addition, suggest that molecular mimicry might explain the nonimmunogenicity of these peptides otherwise characterized by specificity to the mAb counterpart. We conclude that construction of peptides harboring motifs absent or scarcely represented in endogenous self-proteins might be a useful approach in melanoma immunotherapy.

A simple procedure is described for preparing GM3 ganglioside, from a few milligrams to grams, from GM1-lactone (Sonnino et al., (1985) Glycoconjugate J 2: 343-54) [1]. The synthesis was carried out under the following optimal conditions: 30 mM GM1-lactone in 0.25 M H2SO4 in DMSO, 30 min, 70 degrees C, nitrogen atmosphere, strong stirring. The yield of GM3 was 55%. The procedure applied to milligram amounts of GD1b-dilactone gave GD3 ganglioside.

Gangliosides, cell surface glycosphingolipids, are implicated in diverse biologic functions potentially important for tumor growth. Because expression of the GD3 ganglioside may have an impact on the melanoma malignancy, and therefore on the patient prognosis, we evaluated the feasibility of a retrospective immunohistochemical study of GD3 in paraffin embedded biopsies of primary melanomas. Immunoperoxidase staining of frozen and deparaffinized sections of melanoma lesions with two anti-GD3 antibodies was compared using Dako biotin-streptavidin detection kit. Residual ganglioside content was evaluated in the tissues submitted to routine histopathologic procedures using HPLC. A strong and reproducible staining was obtained with both antibodies on frozen sections of all 17 melanoma samples. However, only KM641 antibody could detect GD3 on deparaffinized sections. Biochemical quantification revealed that the Bouin fixative resulted in degradation of GD3. Additionally, most of GD3 was eluted from the tissue samples during dehydration and re-hydration steps. A subgroup of tumors particularly rich in GD3 could be detected on deparaffinized sections after standard formaldehyde fixation. Clinical evolution of such melanomas can now be compared to the group with low GD3 expression. However, any Bouin-fixed, paraffin-embedded biopsies should be excluded from such a retrospective study.

Farber's disease (FD) is a rare genetic disorder caused by ceramidase deficiency, which results in ceramide accumulation in lung, liver, colon, skeletal muscle, cartilage, and bone. Although this disease has been symptomatically characterized, little is known about its molecular pathogenetic process. Because recent studies reported that ceramide accumulation induces GD3 ganglioside formation and apoptosis, we investigated, in tissue obtained via colonoscopy from seriously involved patients, the possible involvement of ceramide in FD colonocyte destruction. Histochemical and TUNEL analyses of paraffin-embedded sections revealed that 45 ± 4.3% of FD colonocytes showed morphological signs of apoptosis compared with the 8 ± 2.3% of constitutive epithelial cell death. Importantly, immunohistochemical study for pro-apoptotic factors showed that GD3 accumulation co-localized with active caspase-3 and cleaved K18 in FD colon tissue. These findings provide evidence for a role of the apoptotic ceramide pathway in the pathogenesis of FD.

A major component of the di- and trisialoganglioside fraction of rabbit thymus was isolated. By neuraminidase treatment, methylation analysis, and CrO3 oxidation, it was identified as GD3 ganglioside with the novel disialosyl residue, NeuNGlyc(alpha, 2-8)NeuNAc(alpha, 2-3)Gal-(beta, 1-4)Glc(beta, 1-1)ceramide. Its concentration was 21.92 nmol/g of wet tissue (12.10% of the total gangliosides) and it contained 91.52% of the lipid-bound sialic acid in di- and trisialogangliosides of rabbit thymus.

Transfection of c- myc DNA into rat fibroblastic 3Y1 cell line resulted in the neosynthesis of GD3 ganglioside, as has been previously shown to occur after transfection of 3Y1 cells with adeno E1 (Nakakuma, H., Sanai, Y., Shiroki, K., and Nagai, Y. (1984) J. Biochem. 96, 1471–1480); in both cases, the products are expressed intranuclearly. Moreover, a clear correlation was identified between levels of GD3 expression and colony-forming activity (in soft agar) of myc -transformed 3Y1 cells, implying that GD3 plays some specific role in myc -induced transformation of 3Y1 cell line.