Stage-specific embryonic antigen-4 as a potential therapeutic target in glioblastoma multiforme and other cancers

Abstract

Glioblastoma multiforme (GBM), the grade IV astrocytoma, is the most common and aggressive brain tumor in adults. Despite advances in medical management, the survival rate of GBM patients remains poor, suggesting that identification of GBM-specific targets for therapeutic development is urgently needed. Analysis of several glycan antigens on GBM cell lines revealed that eight of 11 GBM cell lines are positive for stage-specific embryonic antigen-4 (SSEA-4), and immunohistochemical staining confirmed that 38/55 (69%) of human GBM specimens, but not normal brain tissue, were SSEA-4(+) and correlated with high-grade astrocytoma. In addition, an SSEA-4-specific mAb was found to induce complement-dependent cytotoxicity against SSEA-4(hi) GBM cell lines in vitro and suppressed GBM tumor growth in mice. Because SSEA-4 is expressed on GBM and many other types of cancers, but not on normal cells, it could be a target for development of therapeutic antibodies and vaccines.

Keywords: Globo H; SSEA-3; gangliosides; glycosphingolipids; targeted therapy.

Fig. 1.

The binding characteristics of mAb MC813-70 to GBM cell lines. (A) Schematic diagram of the biosynthesis of globo-series GSLs. SSEA-3, the precursor of SSEA-4 and Globo H, is synthesized from globoside. Glycosidic linkages and graphic notations are labeled. Glc, glucose; Gal, galactose; GalNAc, N-acetylgalactosamine; Fuc, fucose; NeuAc, N-acetylneuraminic acid. (B) GBM cells were stained with Alexa Fluor 488-conjugated MC813-70, and the staining intensity was analyzed with flow cytometry. All cells examined were GBM cell lines except for SVG p12, which is a normal human fetal glial cell line transformed with SV40 large T antigen. The histograms of the cells stained with MC813-70 and isotype control are shown in gray and white, respectively.

Fig. 2.

The glycan-binding profile of mAb MC813-70. The glycan microarrays on glass slides were probed with Alexa Flour 647-conjugated MC813-70 (10 μg/mL) and were read in an array scanner at 635 nm. Data are presented as mean ± SD. C5, C5H10NH2. (Inset) The binding curve of MC813-70 to SSEA-4. The dissociation constant (K_d) of mAb MC813-70 toward SSEA-4 was detected on a glass slide printed with SSEA-4 glycan.

Fig. 3.

HPTLC immunostaining and MALDI-MS profiles of gangliosides from GBM cell lines. (A) Gangliosides were separated on an HPTLC plate and detected with MC813-70 mAb. Gangliosides from 2012Ep (a human embryonal carcinoma cell line) and YAC-1 (a mouse lymphoma cell line) served as the positive controls for SSEA-4 and GM1b, respectively. SSEA-4 with different chain lengths of fatty acids migrated as two close bands. (B) The extracted gangliosides from DBTRG GBM cells were permethylated and analyzed by MALDI-MS. The major gangliosides in DBTRG cells were GM3 (m/z = 1,371.9), GM2 (m/z = 1,617.0), Neu5Ac-(n)Lc4/Gg4Cer (m/z = 1,821.1), and Neu5Ac2-(n)Lc4/Gg4Cer (m/z = 2,182.3). SSEA-4 (Neu5Ac-Hex4-HexNAc-Cer, m/z = 2025.2) was also observed, albeit in a relatively weak signal. Gangliosides with the same glycan moiety but with different fatty acyl contents are bracketed.

Fig. 4.

Expression of SSEA-4 in GBM. (A and B) Representative images of normal brain tissues (A) and GBM (B) after immunohistochemical staining with MC-813–70. The inset in B shows a magnified picture of the small boxed area. (Scale bars, 100 μm.) (C) Statistical results of SSEA-4 IHC. GBM specimens (n = 55) and normal brain tissues (n = 19) were counterstained with hematoxylin after IHC. The staining intensity of the tissues was graded as 0 (negative), 1+ (weak), 2+ (moderate), and 3+ (strong).

Fig. 5.

MC813-70 triggered CDC in GBM cells. GBM cell lines were treated with 20 µg/mL MC813-70 and rabbit complement to observe MC813-70–induced cell lysis. The CDC activity of MC813-70 was measured by the LDH release assay as described in Materials and Methods. The data are shown as mean ± SD.

Fig. 6.

Inhibition of DBTRG tumor growth by MC813-70. On day 0, male nude mice were inoculated with DBTRG cells in the right flank, and MC813-70 or mouse IgG3 isotype control (200 µg per dose) was administered i.p. at days 11, 15, and 19. Mice were killed at day 31. The tumor volume in each group (n = 3) was measured at different time points and is shown as mean ± SD. P = 0.001 was obtained by two-way ANOVA.