Immunization of fucose-containing polysaccharides from Reishi mushroom induces antibodies to tumor-associated Globo H-series epitopes

Abstract

Carbohydrate-based vaccines have shown therapeutic efficacy for infectious disease and cancer. The mushroom Ganoderma lucidum (Reishi) containing complex polysaccharides has been used as antitumor supplement, but the mechanism of immune response has rarely been studied. Here, we show that the mice immunized with a l-fucose (Fuc)-enriched Reishi polysaccharide fraction (designated as FMS) induce antibodies against murine Lewis lung carcinoma cells, with increased antibody-mediated cytotoxicity and reduced production of tumor-associated inflammatory mediators (in particular, monocyte chemoattractant protein-1). The mice showed a significant increase in the peritoneal B1 B-cell population, suggesting FMS-mediated anti-glycan IgM production. Furthermore, the glycan microarray analysis of FMS-induced antisera displayed a high specificity toward tumor-associated glycans, with the antigenic structure located in the nonreducing termini (i.e., Fucα1-2Galβ1-3GalNAc-R, where Gal, GalNAc, and R represent, respectively, D-galactose, D-N-acetyl galactosamine, and reducing end), typically found in Globo H and related tumor antigens. The composition of FMS contains mainly the backbone of 1,4-mannan and 1,6-α-galactan and through the Fucα1-2Gal, Fucα1-3/4Man, Fucα1-4Xyl, and Fucα1-2Fuc linkages (where Man and Xyl represent d-mannose and d-xylose, respectively), underlying the molecular basis of the FMS-induced IgM antibodies against tumor-specific glycans.

Keywords: anti-Globo H antibody; antitumor activity; mushroom polysaccharide.

Fig. 1.

Glycan-binding patterns of the serum IgM antibodies as measured by the CFG glycan microarray. Each histogram represents different sources of IgM binding to the glycan microarray, where the x axis shows the glycan number of 611 saccharides examined and the y axis is relative fluorescent units. Serum samples (tested at 1:100 dilution) from F3-treated (A), FMS-treated (B), and PBS-treated (C) mice were collected on day 14 after four dose injections and analyzed by printed array Version 5.0 of the CFG Core H. Nine of the identified glycan structures marked with glycan numbers are indicated. Dashed circles indicate the consensus glycan epitope (H-type 3/4 structure). Bars show the average RFUs [n = 4 (A and B); n = 2 (C)].

Fig. 2.

A spectrum of tumor associated-glycans highly recognized by FMS-induced antisera. Each glycan structure with chemical linker is printed on the CFG Version 5.0, which was classified into two groups. Structures of the linkers are indicated: sp0, CH₂CH₂NH₂; sp9, CH₂CH₂CH₂CH₂CH₂NH₂; sp21, N(CH₃)OCH₂CH₂NH₂. Definition of blood group determinants (H, A, or B) is annotated.

Fig. 3.

Antitumor activities of FMS. (A) Antibody-mediated cytotoxicity (CDC) of antisera from FMS-treated mice to LLC1 and TC-1 tumor cells was determined by a lactose dehydrogenase kit. The value of antisera heated at 56 °C for 30 min (HI-antisera) is indicative of the complement depletion effect. (B) Comparison of antitumor effects between preventive (Exp-1) and therapeutic (Exp-2) FMS treatment in vivo. Control is PBS-treated mice with tumor inoculation. (C) FMS treatment suppressed tumor-associated cytokines and chemokines production in vivo. Serum samples were collected at indicated time after tumor inoculation and examined by Beadlyte mouse 21-pex kits. (D and E) Distinct binding intensities of plant lectins (AAL, 2 μg/mL; UEA-I, 10 μg/mL) and anti-Globo H mAb (MBr1, 0.5mg/mL) to Globo H (GH), FMS and F3 were determined by using the fabricated glycan microarray. (F–H) DFMS (low-Fuc content of FMS) treatment reduced CDC (F) and antitumor activities in vivo, as assessed by tumor growth curves (G) and MCP-1 production levels (H). Values show the means ± SD (n ≈ 3–5 for each experiment). n.d., not detectable; NS, no statistical significant.

Fig. 4.

Correlation between anti-glycan IgM production and B1 B cells expansion in the mice immunization with our Reishi polysaccharides. (A and B) Antisera from FMS- and DFMS-treated mice were assessed by Globo H-related printed glycan microarray (A) and FMS-coated ELISA plate (B). (A) Binding of IgM to Globo H (GH) and its truncated forms (tested at 1:100 dilution) was normalized by setting the IgM anti-Gb5 as 1. (B) Binding of IgM to FMS (tested at 1:20–1:320 dilution) was measured by detecting the absorbance at 450 nm. (C–E) Expansion of peritoneal B1 B cells upon FMS immunization. FACS profiles of B1 B cells represent FMS-treated mice and control. Additional levels of B2 B cell and macrophage are shown in Fig. S4. Numbers (%) indicate the positive cells in each gate (C). FMS induced up-regulation of plasma cell surface marker (CD138) (D) and IgM production (E) in ex vivo B1 B cells culture purified from FMS-treated mice. Means ± SD (n ≈ 3–5 for each experiment). n.d., not detectable.