The glycosphingolipid P1 is an ovarian cancer-associated carbohydrate antigen involved in migration

Abstract

Background:The level of plasma-derived naturally circulating anti-glycan antibodies (AGA) to P1 trisaccharide has previously been shown to significantly discriminate between ovarian cancer patients and healthy women. Here we aim to identify the Ig class that causes this discrimination, to identify on cancer cells the corresponding P1 antigen recognised by circulating anti-P1 antibodies and to shed light into the possible function of this glycosphingolipid.Methods:An independent Australian cohort was assessed for the presence of anti-P1 IgG and IgM class antibodies using suspension array. Monoclonal and human derived anti-glycan antibodies were verified using three independent glycan-based immunoassays and flow cytometry-based inhibition assay. The P1 antigen was detected by LC-MS/MS and flow cytometry. FACS-sorted cell lines were studied on the cellular migration by colorimetric assay and real-time measurement using xCELLigence system.Results:Here we show in a second independent cohort (n=155) that the discrimination of cancer patients is mediated by the IgM class of anti-P1 antibodies (P=0.0002). The presence of corresponding antigen P1 and structurally related epitopes in fresh tissue specimens and cultured cancer cells is demonstrated. We further link the antibody and antigen (P1) by showing that human naturally circulating and affinity-purified anti-P1 IgM isolated from patients ascites can bind to naturally expressed P1 on the cell surface of ovarian cancer cells. Cell-sorted IGROV1 was used to obtain two study subpopulations (P1-high, 66.1% and P1-low, 33.3%) and observed that cells expressing high P1-levels migrate significantly faster than those with low P1-levels.Conclusions:This is the first report showing that P1 antigen, known to be expressed on erythrocytes only, is also present on ovarian cancer cells. This suggests that P1 is a novel tumour-associated carbohydrate antigen recognised by the immune system in patients and may have a role in cell migration. The clinical value of our data may be both diagnostic and prognostic; patients with low anti-P1 IgM antibodies present with a more aggressive phenotype and earlier relapse.

Altered (neo-) lacto series glycolipid biosynthesis impairs α2-6 sialylation on N-glycoproteins in ovarian cancer cells

“…As part of our initiative to comprehensively characterize nsGSLs, we have recently reported the presence of paragloboside (nLc4, precursor of P 1 ) and P 1 pentasaccharide in tumor specimens and immortal ovarian cancer cells using two complementary methods; PGC-LC-ESI-MS/MS and flow cytometry131415. In this study, we extended the profiling of nsGSLs into three distinct groups; Normal (HOSE17-1, FT33-Tag, FT190 and FT237 which were suggested as a potential origin of epithelial ovarian cancer1617), Ovarian (IGROV1, SKOV3, BG1, and CAOV3), and Non-ovarian cancer cell lines (Ls174T, HeLa, HCT15, and HCT116).…”

“…S3)14. The GSL pathways affected by the genetic disruption of B3GNT5 is hypothesized (according to the scheme presented in Fig.…”

“…We recently reported the presence of the P 1 glycan in tissue samples14 as well as on P 1 -enriched IGROV1 ovarian cancer cell lines13, serving as a cell-recognition molecule through its binding to naturally occurring anti-glycan antibodies1429. The presence of nLc4 and P 1 was detected on IGROV1 cells using flow cytometry, in which P 1 was detected using P3NIL100 antibody, a well-validated monoclonal IgM antibody shown to specifically recognize the chemically synthesized P 1 trisaccharide (Galα1-4Galβ1-4GlcNAcβ-), as indicated by glycan array profiling performed in this study.…”

“…Immuno staining and flow cytometry was performed as previously described1415. Biotinylated SNA lectin (1:500) containing 10 mM CaCl 2 was applied for detection of α2-6 neuraminic acid using flow cytometry (BD Accuri™ C6, BD Bioscience).…”

The comparison of glycosphingolipids isolated from an epithelial ovarian cancer cell line and a nontumorigenic epithelial ovarian cell line using MALDI-MS and MALDI-MS/MS

“…Not only sulfatides and/or gangliosides are elevated in ovarian tumors but also some other GSLs, such as neutral GSLs, can be altered in ovarian tumors. For example, Jacob et al recently reported that neutral GSL P 1 is expressed in ovarian cancer tissues and cultured cancer cells based on flow cytometry and LC-ESI-MS/ MS analysis [20]. Consequently, they have reported GSL P 1 as a novel tumor-associated carbohydrate antigen.…”