Glycosphingolipids (GSLs), composed of a hydrophilic carbohydrate chain and a lipophilic ceramide anchor, play pivotal roles in countless biological processes, including the development of cancer. As part of the investigation of the vertebrate glycome, GSL analysis is undergoing rapid expansion owing to the application of modern mass spectrometry. Here we introduce direct coupling of IR-MALDI-o-TOF mass spectrometry with the TLC overlay binding assay for the structural characterization of GSLs. We matched three complementary methods including (i) TLC separation of GSLs, (ii) their detection with oligosaccharide-specific proteins, and (iii) in situ MS analysis of protein-detected GSLs. The high specificity and sensitivity is demonstrated by use of antibodies, bacterial toxins, and a plant lectin. The procedure works on a nanogram scale, and detection limits of less than 1 ng at its best of immunostained GSLs were obtained. Furthermore, only crude lipid extracts of biological sources are required for TLC-IR-MALDI-MS, omitting any laborious GSL downstream purification procedures. This strategy was successfully applied to the identification of cancer-associated GSLs in human hepatocellular and pancreatic tumors. Thus, the in situ TLC-IR-MALDI-MS of immunolabeled GSLs opens new doors by delivering specific structural information of trace quantities of GSLs with only a limited investment in sample preparation.

BackgroundDespite progress in adjuvant chemotherapy in the recent decades, pancreatic and colon cancers remain common causes of death worldwide. Bacterial toxins, which specifically bind to cell surface-exposed glycosphingolipids, are a potential novel therapy. We determined the expression of globotriaosylceramide (Gb3Cer/CD77), the Shiga toxin receptor, in human pancreatic and colon adenocarcinomas.Methodology/Principal FindingsTissue lipid extracts of matched pairs of cancerous and adjacent normal tissue from 21 pancreatic and 16 colon cancer patients were investigated with thin-layer chromatography overlay assay combined with a novel mass spectrometry approach. Gb3Cer/CD77 was localized by immunofluorescence microscopy of cryosections from malignant and corresponding healthy tissue samples. 62% of pancreatic and 81% of colon adenocarcinomas showed increased Gb3Cer/CD77 expression, whereas 38% and 19% of malignant pancreas and colon tissue, respectively, did not, indicating an association of this marker with neoplastic transformation. Also, Gb3Cer/CD77 was associated with poor differentiation (G>2) in pancreatic cancer (P = 0.039). Mass spectrometric analysis evidenced enhanced expression of Gb3Cer/CD77 with long (C24) and short chain fatty acids (C16) in malignant tissues and pointed to the presence of hydroxylated fatty acid lipoforms, which are proposed to be important for receptor targeting. They could be detected in 86% of pancreatic and about 19% of colon adenocarcinomas. Immunohistology of tissue cryosections indicated tumor-association of these receptors.Conclusions/SignificanceEnhanced expression of Gb3Cer/CD77 in most pancreatic and colon adenocarcinomas prompts consideration of Shiga toxin, its B-subunit or B-subunit-derivatives as novel therapeutic strategies for the treatment of these challenging malignancies.

Pancreatic adenocarcinoma confers one of the highest mortality rates in malignant human tumors with very poor prognosis. Because as yet no treatments are available that produce a substantial survival benefit for this fatal neoplasia, new therapeutic concepts are urgently required to support cancer standard treatment. In search of tumorassociated gangliosides with therapeutic background, we probed a random collection of cancerous and adjacent normal postoperative tissue samples from 38 patients for the expression of CD75s-and iso-CD75s-gangliosides. We exhaustively analyzed the expression of CD75s-1-ganglioside (IV 6 Neu5Ac-nLc4Cer) and structurally closely related iso-CD75s-1-ganglioside (IV 3 Neu5Ac-nLc4Cer) by means of immunohistology of cryosections and semiquantitative TLC of tissue lipid extracts combined with mass spectrometry. CD75s-1-and iso-CD75s-1-ganglioside showed an elevated expression in 42% and 66% of the tumors, respectively, indicating a significant association with neoplastic transformation (P = 0.001). Thus, increased expression of CD75s-1-and iso-CD75s-1-gangliosides renders these cell surface molecules promising candidates for oncologic applications. Further statistical analysis revealed a significant enhancement of CD75s-1-ganglioside in the group of less differentiated tumors (grade >2) suggesting this ganglioside as a potential marker for poor differentiation. The CD75s-specific antitumor drug rViscumin, which represents the recombinant counterpart of the ribosome-inactivating lectin viscumin, has successfully passed clinical phase I trials and provides an opportunity for treating pancreatic cancer. Consequently, if an enhanced expression is existent in malignant tissues, we propose the targeting of CD75s-gangliosides with rViscumin as a novel potential strategy in adjuvant treatment of pancreatic malignancies. [Mol Cancer Ther 2008;7(8):2464 -75]

Shiga toxin (Stx, synonymous to verotoxin, VT) binds with high and low affinity to the globo-series neutral glycosphingolipids (GSLs), globotriaosylceramide (Gb3Cer or Galalpha4Galbeta4Glcbeta1Cer, also known as CD77) and globotetraosylceramide (Gb4Cer or GalNAcbeta3Galalpha4Galbeta4Glcbeta1Cer), respectively, which represent the targets of Stxs on many different cell types. B-cell-derived Raji cells and THP-1 cells of monocytic origin are widely used for the investigation of Stx-mediated cellular response, because Stx is known to cause cell death in both cell lines. Despite their functional importance, the Stx receptors of Raji and THP-1 cells have so far not been investigated. This prompted us to explore the structures of their GSL receptors in detail by means of nanoelectrospray ionization quadrupole time-of-flight mass spectrometry (nanoESI-QTOF-MS) with collision-induced dissociation (CID) in conjunction with Stx1 as well as anti-Gb3Cer and anti-Gb4Cer antibodies. Using the combination of a thin-layer chromatography (TLC) overlay assay and MS(1) and MS(2) analysis we identified Gb3Cer (d18:1, C24:1/C24:0) as the prevalent Stx1-receptor accompanied by less abundant Gb3Cer (d18:1, C16:0) in the neutral GSL fraction of Raji cells. The same Gb3Cer species but with almost equal proportions of the C24:1/C24:0 and C16:0 variants were found in THP-1 cells. In addition, unusual hydroxylated Gb3Cer (d18:1, C24:1/C24:0) and Gb3Cer (d18:1, C26:1) could be identified in trace quantities in both cell lines. As the most obvious difference between Raji and THP-1 cells we observed the expression of Gb4Cer in THP-1 cells, whereas Raji cells failed to express this elongation product of Gb3Cer. Both short- and long-chain fatty acid carrying Gb4Cer (d18:1, C16:0) and Gb4Cer (d18:1, C24:1/C24:0), respectively, were the prevalent Gb4Cer variants. This first report on the differential expression of Gb3Cer and Gb4Cer and their structural diversity in lymphoid and myeloid cell lines supports the hypothesis that such heterogeneities might play a functional role in the molecular assembly of GSLs in membrane organization and cellular signaling of Stx-susceptible cells.

The sialic acid-specific cytotoxic lectin viscumin and its recombinant equivalent rViscumin specifically bind to CD75s-gangliosides with terminal Neu5Acα6Galβ4GlcNAc sequence. We, therefore, comparatively analyzed the content of CD75s-gangliosides and closely related iso-CD75s-gangliosides (terminated by Neu5Acα3Galβ4GlcNAc sequence) and the gene expression of associated β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) and β-galactoside α-2,3-sialyltransferase 6 (ST3GAL6), respectively, in 35 hepatocellular carcinoma (HCC) patients. Ganglioside structures were identified in lipid extracts of matched pairs of malignant and nonmalignant liver tissues by thin-layer chromatography immunodetection coupled with infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry. CD75s- and iso-CD75s-gangliosides were found to be deregulated in tumor tissues and showed an elevated occurrence in 35 and 41% of HCCs, respectively, compared with nontumoral liver tissues. Statistical analysis revealed a correlation between enhanced iso-CD75s-ganglioside amount and a poor histopathological differentiation (τ = 0.317, P = 0.045) and a significant association of CD75s- and iso-CD75s-ganglioside levels in nontumorous (τ = 0.392, P = 0.003) and in tumorous tissues (τ = 0.650, P < 0.001). Quantitative real-time polymerase chain reaction gene expression analysis of sialyltransferases exhibited no difference in ST6GAL1 expression in cancerous and adjacent noncancerous tissues. Interestingly, the ST3GAL6 expression was significantly diminished in HCCs (P = 0.003). The results indicate that the occurrence of CD75s- and iso-CD75s-gangliosides in tumor tissues is largely independent of the transcriptional expression of ST6GAL1 and ST3GAL6, respectively. Thus, further experiments are required to explore the rationale behind the differential ganglioside level and to validate the applicability of CD75s- and iso-CD75s-gangliosides as targets for individual HCC therapies.