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, 111 (8), 1634-45

The Glycosphingolipid P₁ Is an Ovarian Cancer-Associated Carbohydrate Antigen Involved in Migration

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The Glycosphingolipid P₁ Is an Ovarian Cancer-Associated Carbohydrate Antigen Involved in Migration

F Jacob et al. Br J Cancer.

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.

Figures

Figure 1
Figure 1
Significantly lower levels of anti-P1 IgM in cancer patients in the Australian validation cohort (n=155). (A) Box-and-whisker plots showing distribution of AGA levels of IgM and IgG to covalently attached P1 trisaccharide using suspension array. Decreased AGA levels in cancer patients were observed for IgM. (B) Kaplan–Meier curve on relapse-free survival showing that patients with low antibody levels (n=33; lower than median fluorescence signal) have slightly earlier relapse (P=0.055) than those with high antibody levels (n=25, higher than median fluorescence signal).
Figure 2
Figure 2
P1 is expressed on cancer tissue cells. (A, B) Base peak chromatograms shown for serous ovarian cancer (A (a)) and endometrioid peritoneal cancer tissue (B (a)). Selected mass peaks (red arrow) corresponding to the composition of the Pk trisaccharide (Hex3) (A and B b(i); m/z 505.31−), P tetrasaccharide (Hex3HexNAc1) (A and B c (ii); m/z 708.31−), and P1 pentasaccharide (Hex4HexNAc1) (A and B d(iii); m/z 870.31−) are represented as an extracted ion chromatogram (EIC). (C) MS2 spectrum of Pk trisaccharide (Galα1-4Galβ1-4Glcβ1) (i), P tetrasaccharide (GalNAcβ1-3Galα1-4Galβ1-4Glcβ1) (ii) and P1 pentasaccharide (Galα1-4Galβ1-4GlcNAcβ1-3Galβ1-4Glcβ1) (iii). (D) Representative flow cytometry results shown as contour plots with outliers demonstrate P1 and Pk negative cell lines HOSE6-3 and SKOV3. IGROV1 was detected positive with IgMs for both P1 and Pk. Representative contour plots showing P1 and Pk expression (FITC; ordinate) and forward scatter (FSC; abscissa). Given percentage corresponds to P1/Pk-positive cells.
Figure 3
Figure 3
Monoclonal anti-P1 IgM bind specifically to P1. Monoclonal anti-P1 antibody (P3NIL100, positive control) was incubated with different glycan amounts (0.015–0.06 μmol) prior to flow cytometric measurement of binding to IGROV1 cells. Glycoconjugates Sepharose-PAA (-Lactose, -LacNAc, -P1, -Pk) were applied to test specificity of antibodies to P1 by testing the inhibition of binding of the anti-P1 antibody. Representative contour plots out of three independent experiments showing P1 expression (FITC; ordinate) and forward scatter (FSC; abscissa). Complete inhibition is shown by increasing Seph-PAA-P1 amount with only minor inhibition of anti-P1 antibodies using Seph-PAA-Pk.
Figure 4
Figure 4
Ascites and blood plasma from cancer patients contain comparable levels of anti-glycan antibodies independent of immunoglobulin class and volume of ascites. Matched ascites and blood plasma samples (n=11) were profiled for binding of IgA+IgG+IgM (n=22 printed glycan array slides) and independently for IgA (n=22), IgG (n=22) and IgM (n=22) to printed glycan array slides. One scatterplot represents signals for 11 patients each with ascites and blood plasma. AGA to P1 trisaccharide are highlighted in black. Median signal intensity over all signals calculated for ascites and plasma are shown by horizontal and vertical solid lines, respectively. Antibody signals (RFU × 106) are shown on ascites and blood plasma axis. Cutoff (5%) separating background signals from real AGA binding are indicated by a solid line for ascites and plasma in the same scatterplot.
Figure 5
Figure 5
Human ascites-derived affinity-purified anti-P1 antibodies bind to IGROV1 cells. (A) Flow cytometry-based contour plots demonstrate the presence of IgM antibodies in ascites (1 : 3 diluted in PBS) bound to IGROV1 cells (ascites IgM, 24.3%) compared with unstained sample (control). Affinity-purified anti-P1 IgM (1 : 20) bound to IGROV1 cells (15.1% of P1-positive cells). (B) Representative ELISA demonstrating the cross-reactivity of affinity-purified anti-P1 antibodies of class IgM (dark grey) and IgG (light grey) to investigated glycans linked to PAA. (C) Printed glycan array data (scatterplot for 50 μM vs 10 μM saccharide prints) showing cross-reactivity to glycans sharing related carbohydrate structures. Grey area indicates unspecific binding of AGA as determined by 5% threshold. Inter-quartile range is shown for each glycan horizontally and vertically for 50 μM and 10 μM, respectively.
Figure 6
Figure 6
Elevated P1 expression results in increased migration rate. (A) FACS-sorted subpopulations of P1-low- and -high-expressing IGROV1 cells. Representative histograms showing P1 expression (abscissa) on cell-sorted IGROV1 cells to normalised cell count (ordinate). P1 distribution for unstained controls (red), P1-positivity P3NIL100 antibody (green) and OSK17 antibody (blue). (B) Colorimetric cell migration assay showing enhanced migratory ability of IGROV1 cells expressing high compared with low levels of P1. Stained cells counted in five fields and averaged. Representative image of cell sorted subpopulations of migrated cells (stained violet) after 18 h. (C) RTCA assay for P1-sorted IGROV1 cells showing the migrated cells (cell index) in bar graphs at the time points 0 h, 10 h, 20 h and 30 h. Left bar graph shows RTCA experiment without araC, right bar graph with araC as proliferation inhibitor. Representative figure out of two independent experiments. (D) Bar chart showing the inhibition of cell migration of human IGROV1 cells incubated with anti-P1 IgM compared with corresponding incubation with IgM isotype control. Number of migrated cells was normalised to control. Not significant (NS); *P<0.05, **P<0.01, ***P<0.001.

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