Increased expression of SSEA-4 on TKI-resistant non-small cell lung cancer with EGFR-T790M mutation.

Non-small cell lung cancer (NSCLC), a major life-threatening disease accounting for 85% of all lung cancer cases, has been treated with tyrosine kinase inhibitors (TKIs), but often resulted in drug resistance, and approximately 60% of TKI-resistant cases are due to acquired secondary (epithelial growth factor receptor) EGFR-T790M mutation. To identify alternative targets for TKI-resistant NSCLC with EGFR-T790M mutation, we found that the three globo-series glycosphingolipids are increasingly expressed on this type of NSCLC cell lines, and among them, the increase of stage-specific embryonic antigen-4 (SSEA-4) expression is the most significant. Compared to TKI-sensitive cell lines, SSEA-4 and the key enzyme ß3GalT5 responsible for the synthesis of SSEA3 are more expressed in TKI-resistant NSCLC cell lines with EGFR-T790M mutation, and the expression levels strongly correlate with poor survival in patients with EGFR mutation. In addition, we demonstrated that a SSEA-4 targeted monoclonal antibody, especially the homogeneous glycoform with well-defined Fc glycan designed to improve effective functions, is highly effective against this subpopulation of NSCLC in cell-based and animal studies. These findings provide a direction for the prediction of tumor recurrence and treatment of TKI-resistant NSCLC with EGFR-T790M mutation.

Homogeneous antibody and CAR-T cells with improved effector functions targeting SSEA-4 glycan on pancreatic cancer.

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.

Mucins Shed from the Laminated Layer in Cystic Echinococcosis Are Captured by Kupffer Cells via the Lectin Receptor Clec4F.

Cystic echinococcosis is caused by the larval stages (hydatids) of cestode parasites belonging to the species cluster Echinococcus granulosus sensu lato, with E. granulosus sensu stricto being the main infecting species. Hydatids are bladderlike structures that attain large sizes within various internal organs of livestock ungulates and humans. Hydatids are protected by the massive acellular laminated layer (LL), composed mainly of mucins. Parasite growth requires LL turnover, and abundant LL-derived particles are found at infection sites in infected humans, raising the question of how LL materials are dealt with by the hosts. In this article, we show that E. granulosus sensu stricto LL mucins injected into mice are taken up by Kupffer cells, the liver macrophages exposed to the vascular space. This uptake is largely dependent on the intact mucin glycans and on Clec4F, a C-type lectin receptor which, in rodents, is selectively expressed in Kupffer cells. This uptake mechanism operates on mucins injected both in soluble form intravenously (i.v.) and in particulate form intraperitoneally (i.p.). In mice harboring intraperitoneal infections by the same species, LL mucins were found essentially only at the infection site and in the liver, where they were taken up by Kupffer cells via Clec4F. Therefore, shed LL materials circulate in the host, and Kupffer cells can act as a sink for these materials, even when the parasite grows in sites other than the liver.

Mechanism of Antigen Presentation and Specificity of Antibody Cross-Reactivity Elicited by an Oligosaccharide-Conjugate Cancer Vaccine.

Polysaccharides have been successfully used as immunogens for the development of vaccines against bacterial infection; however, there are no oligosaccharide-based vaccines available to date and no previous studies of their processing and presentation. We reported here the intracellular enzymatic processing and antigen presentation of an oligosaccharide-conjugate cancer vaccine prepared from the glycan of Globo-H (GH), a globo-series glycosphingolipid (GSL). This oligosaccharide-conjugate vaccine was shown to elicit antibodies against the glycan moieties of all three globo-series GSLs that are exclusively expressed on many types of cancer and their stem cells. To understand the specificity and origin of cross-reactivity of the antibodies elicited by the vaccine, we found that the vaccine is first processed by fucosidase 1 in the early endosome of dendritic cells to generate a common glycan antigen of the GSLs along with GH for MHC class II presentation. This work represents the first study of oligosaccharide processing and presentation and is expected to facilitate the design and development of glycoconjugate vaccines based on oligosaccharide antigens.

Signaling pathway of globo-series glycosphingolipids and ß1,3-galactosyltransferase V (ß3GalT5) in breast cancer.

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme ß1,3-galactosyltransferase V (ß3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of ß3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of ß3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

O-GlcNAcylation regulates the stability and enzymatic activity of the histone methyltransferase EZH2.

Protein O-glycosylation by attachment of ß-N-acetylglucosamine (GlcNAc) to the Ser or Thr residue is a major posttranslational glycosylation event and is often associated with protein folding, stability, and activity. The methylation of histone H3 at Lys-27 catalyzed by the methyltransferase EZH2 was known to suppress gene expression and cancer development, and we previously reported that the O-GlcNAcylation of EZH2 at S76 stabilized EZH2 and facilitated the formation of H3K27me3 to inhibit tumor suppression. In this study, we employed a fluorescence-based method of sugar labeling combined with mass spectrometry to investigate EZH2 glycosylation and identified five O-GlcNAcylation sites. We also find that mutation of one or more of the O-GlcNAcylation sites S73A, S76A, S84A, and T313A in the N-terminal region decreases the stability of EZH2, but does not affect its association with the PRC2 components SUZ12 and EED. Mutation of the C-terminal O-GlcNAcylation site (S729A) in the catalytic domain of EZH2 abolishes the di- and trimethylation activities, but not the monomethylation of H3K27, nor the integrity of the PRC2/EZH2 core complex. Our results show the effect of individual O-GlcNAcylation sites on the function of EZH2 and suggest an alternative approach to tumor suppression through selective inhibition of EZH2 O-GlcNAcylation.

Glycolipid GD3 and GD3 synthase are key drivers for glioblastoma stem cells and tumorigenicity.

The cancer stem cells (CSCs) of glioblastoma multiforme (GBM), a grade IV astrocytoma, have been enriched by the expressed marker CD133. However, recent studies have shown that CD133(-) cells also possess tumor-initiating potential. By analysis of gangliosides on various cells, we show that ganglioside D3 (GD3) is overexpressed on eight neurospheres and tumor cells; in combination with CD133, the sorted cells exhibit a higher expression of stemness genes and self-renewal potential; and as few as six cells will form neurospheres and 20-30 cells will grow tumor in mice. Furthermore, GD3 synthase (GD3S) is increased in neurospheres and human GBM tissues, but not in normal brain tissues, and suppression of GD3S results in decreased GBM stem cell (GSC)-associated properties. In addition, a GD3 antibody is shown to induce complement-dependent cytotoxicity against cells expressing GD3 and inhibition of GBM tumor growth in vivo. Our results demonstrate that GD3 and GD3S are highly expressed in GSCs, play a key role in glioblastoma tumorigenicity, and are potential therapeutic targets against GBM.

Stage-specific embryonic antigen-3 (SSEA-3) and ß3GalT5 are cancer specific and significant markers for breast cancer stem cells.

The discovery of cancer stem cells (CSCs), which are responsible for self-renewal and tumor growth in heterogeneous cancer tissues, has stimulated interests in developing new cancer therapies and early diagnosis. However, the markers currently used for isolation of CSCs are often not selective enough to enrich CSCs for the study of this special cell population. Here we show that the breast CSCs isolated with CD44(+)CD24(-/lo)SSEA-3(+) or ESA(hi)PROCR(hi)SSEA-3(+) markers had higher tumorigenicity than those with conventional markers in vitro and in vivo. As few as 10 cells with CD44(+)CD24(-/lo)SSEA-3(+) formed tumor in mice, compared with more than 100 cells with CD44(+)CD24(-/lo). Suppression of SSEA-3 expression by knockdown of the gene encoding ß-1,3-galactosyltransferase 5 (ß3GalT5) in the globo-series pathway, led to apoptosis in cancer cells specifically but had no effect on normal cells. This finding is further supported by the analysis of SSEA-3 and the two related globo-series epitopes SSEA4 and globo-H in stem cells (embryonic stem cells and induced pluripotent stem cells) and various normal and cancer cells, and by the antibody approach to target the globo-series glycans and the late-stage clinical trials of a breast cancer vaccine.

CLEC5A-Mediated Enhancement of the Inflammatory Response in Myeloid Cells Contributes to Influenza Virus Pathogenicity In Vivo.

Human infections with influenza viruses exhibit mild to severe clinical outcomes as a result of complex virus-host interactions. Induction of inflammatory mediators via pattern recognition receptors may dictate subsequent host responses for pathogen clearance and tissue damage. We identified that human C-type lectin domain family 5 member A (CLEC5A) interacts with the hemagglutinin protein of influenza viruses expressed on lentiviral pseudoparticles through lectin screening. Silencing CLEC5A gene expression, blocking influenza-CLEC5A interactions with anti-CLEC5A antibodies, or dampening CLEC5A-mediated signaling using a spleen tyrosine kinase inhibitor consistently reduced the levels of proinflammatory cytokines produced by human macrophages without affecting the replication of influenza A viruses of different subtypes. Infection of bone marrow-derived macrophages from CLEC5A-deficient mice showed reduced levels of tumor necrosis factor alpha (TNF-α) and IP-10 but elevated alpha interferon (IFN-α) compared to those of wild-type mice. The heightened type I IFN response in the macrophages of CLEC5A-deficient mice was associated with upregulated TLR3 mRNA after treatment with double-stranded RNA. Upon lethal challenges with a recombinant H5N1 virus, CLEC5A-deficient mice showed reduced levels of proinflammatory cytokines, decreased immune cell infiltration in the lungs, and improved survival compared to the wild-type mice, despite comparable viral loads noted throughout the course of infection. The survival difference was more prominent at a lower dose of inoculum. Our results suggest that CLEC5A-mediated enhancement of the inflammatory response in myeloid cells contributes to influenza pathogenicity in vivo and may be considered a therapeutic target in combination with effective antivirals. Well-orchestrated host responses together with effective viral clearance are critical for optimal clinical outcome after influenza infections. IMPORTANCE: Multiple pattern recognition receptors work in synergy to sense viral RNA or proteins synthesized during influenza replication and mediate host responses for viral control. Well-orchestrated host responses may help to maintain the inflammatory response to minimize tissue damage while inducing an effective adaptive immune response for viral clearance. We identified that CLEC5A, a C-type lectin receptor which has previously been reported to mediate flavivirus-induced inflammatory responses, enhanced induction of proinflammatory cytokines and chemokines in myeloid cells after influenza infections. CLEC5A-deficient mice infected with influenza virus showed reduced inflammation in the lungs and improved survival compared to that of the wild-type mice despite comparable viral loads. The survival difference was more prominent at a lower dose of inoculum. Collectively, our results suggest that dampening CLEC5A-mediated inflammatory responses in myeloid cells reduces immunopathogenesis after influenza infections.

Glycoprotein B7-H3 overexpression and aberrant glycosylation in oral cancer and immune response.

The incidence and mortality rate of oral cancer continue to rise, partly due to the lack of effective early diagnosis and increasing environmental exposure to cancer-causing agents. To identify new markers for oral cancer, we used a sialylation probe to investigate the glycoproteins differentially expressed on oral cancer cells. Of the glycoproteins identified, B7 Homolog 3 (B7-H3) was significantly overexpressed in oral squamous cell carcinoma (OSCC), and its overexpression correlated with larger tumor size, advanced clinical stage, and low survival rate in OSCC patients. In addition, knockdown of B7-H3 suppressed tumor cell proliferation, and restoration of B7-H3 expression enhanced tumor growth. It was also found that the N-glycans of B7-H3 from Ca9-22 oral cancer cells contain the terminal α-galactose and are more diverse with higher fucosylation and better interaction with DC-SIGN [DC-specific intercellular adhesion molecule-3 (ICAM-3)-grabbing nonintegrin] and Langerin on immune cells than that from normal cells, suggesting that the glycans on B7-H3 may also play an important role in the disease.

Effect of sialylation on EGFR phosphorylation and resistance to tyrosine kinase inhibition.

Epidermal growth factor receptor (EGFR) is a heavily glycosylated transmembrane receptor tyrosine kinase. Upon EGF-binding, EGFR undergoes conformational changes to dimerize, resulting in kinase activation and autophosphorylation and downstream signaling. Tyrosine kinase inhibitors (TKIs) have been used to treat lung cancer by inhibiting EGFR phosphorylation. Previously, we demonstrated that EGFR sialylation suppresses its dimerization and phosphorylation. In this report, we further investigated the effect of sialylation on the phosphorylation profile of EGFR in TKI-sensitive and TKI-resistant cells. Sialylation was induced in cancer progression to inhibit the association of EGFR with EGF and the subsequent autophosphorylation. In the absence of EGF the TKI-resistant EGFR mutant (L858R/T790M) had a higher degree of sialylation and phosphorylation at Y1068, Y1086, and Y1173 than the TKI-sensitive EGFR. In addition, although sialylation in the TKI-resistant mutants suppresses EGFR tyrosine phosphorylation, with the most significant effect on the Y1173 site, the sialylation effect is not strong enough to stop cancer progression by inhibiting the phosphorylation of these three sites. These findings were supported further by the observation that the L858R/T790M EGFR mutant, when treated with sialidase or sialyltransferase inhibitor, showed an increase in tyrosine phosphorylation, and the sensitivity of the corresponding resistant lung cancer cells to gefitinib was reduced by desialylation and was enhanced by sialylation.

Residues Comprising the Enhanced Aromatic Sequon Influence Protein N-Glycosylation Efficiency.

N-Glycosylation is an important co- and/or post-translational modification that occurs on the vast majority of the one-third of the mammalian proteome that traverses the cellular secretory pathway, regulating glycoprotein folding and functions. Previous studies on the sequence requirements for N-glycosylation have yielded the Asn-X-Ser/Thr (NXS/T) sequon and the enhanced aromatic sequons (Phe-X-Asn-X-Thr and Phe-X-X-Asn-X-Thr), which can be efficiently N-glycosylated. To further investigate the influence of sequence variation on N-glycosylation efficiency in the context of a five-residue enhanced aromatic sequon, we used the human CD2 adhesion domain (hCD2ad) to screen the i-2, i-1, i+1, and i+2 residues flanking Asn at the i position. We found that aromatic residues, especially Trp, and sulfur-containing residues at the i-2 position improved N-glycosylation efficiency, while positively charged residues such as Arg suppressed N-glycosylation. Thiol, hydroxyl, and aliphatic-based side chains at the i-1 position had higher N-glycosylation efficiency, and Cys, in particular, compensated for the negative effect of Arg at the i-2 position. Small residues and Ser at the i+1 position increased the likelihood of N-glycosylation, and Thr is better than Ser at the i+2 position. We devised an algorithm for prediction of N-glycosylation efficiency using the SAS software, employing the 120 sequences studied as a training set. We then introduced the optimized-enhanced aromatic sequons into other glycoproteins and observed an enhancement in N-glycan occupancy that was further supported by modeling the high-affinity interaction between the optimized sequence on hCD2ad and a human oligosaccharyltransferase (OST) subunit. The findings in this study provide useful information for enhancing or suppressing N-glycosylation at a site of interest and valuable data for a better understanding of OST-catalyzed N-glycosylation.

Substrate Preference and Interplay of Fucosyltransferase 8 and N-Acetylglucosaminyltransferases.

The core fucosylation of N-glycans on glycoproteins is catalyzed by fucosyltransferase 8 (FUT8) in mammalian cells and is involved in various biological functions, such as protein function, cancer progression, and postnatal development. The substrate specificity of FUT8 toward bi-antennary N-glycans has been reported, but it is unclear with regard to tri-antennary and tetra-antennary glycans. Here, we examined the specificity and activity of human FUT8 toward tri- and tetra-antennary N-glycans in the forms of glycopeptides. We found that the tri-antennary glycan [A3(2,4,2) type] terminated with N-acetylglucosamine (GlcNAc), which is generated by N-acetylglucosaminyltransferase (GnT)-IV, is a good substrate for FUT8, but the A3(2,2,6) type of tri-antennary glycan, generated by GnT-V, is not a substrate for FUT8. We also observed that core fucosylation reduced the activity of GnT-IV toward the bi-antennary glycan. Examining the correlation between the types of N-glycans and the expression levels of FUT8, GnT-IV, and GnT-V in cells revealed that these glycosyltransferases, particularly GnT-IV, play important roles in directing the branching and core fucosylation of N-glycans in vivo. This study thus provides insights into the interplay among FUT8, GnT-IV, and GnT-V in N-linked glycosylation during the assembly of glycoproteins.

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

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.

Human CLEC18 Gene Cluster Contains C-type Lectins with Differential Glycan-binding Specificity.

The human C-type lectin 18 (clec18) gene cluster, which contains three clec18a, clec18b, and clec18c loci, is located in human chromosome 16q22. Although the amino acid sequences of CLEC18A, CLEC18B, and CLEC18C are almost identical, several amino acid residues located in the C-type lectin-like domain (CTLD) and the sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain, also known as the cysteine-rich secretory proteins/antigen 5/pathogenesis-related 1 proteins (CAP) domain, are distinct from each other. Genotyping by real-time PCR and sequencing further shows the presence of multiple alleles in clec18a/b/c loci. Flow cytometry analysis demonstrates that CLEC18 (CLEC18A, -B, and -C) are expressed abundantly in human peripheral blood cells. Moreover, CLEC18 expression is further up-regulated when monocytes differentiate into macrophages and dendritic cells. Immunofluorescence staining reveals that CLEC18 are localized in the endoplasmic reticulum, Golgi apparatus, and endosome. Interestingly, CLEC18 are also detectable in human sera and culture supernatants from primary cells and 293T cells overexpressing CLEC18. Moreover, CLEC18 bind polysaccharide in Ca(2+)-independent manner, and amino acid residues Ser/Arg(339) and Asp/Asn(421) in CTLD domain contribute to their differential binding abilities to polysaccharides isolated from Ganoderma lucidum (GLPS-F3). The Ser(339) (CLEC18A) → Arg(339) (CLEC18A-1) mutation completely abolishes CLEC18A-1 binding to GLPS-F3, and a sugar competition assay shows that CLEC18 preferentially binds to fucoidan, ß-glucans, and galactans. Because proteins with the SCP/TAPS/CAP domain are able to bind sterol and acidic glycolipid, and are involved in sterol transport and ß-amyloid aggregation, it would be interesting to investigate whether CLEC18 modulates host immunity via binding to glycolipids, and are also involved in glycolipid transportation and protein aggregation in the future.

Carbohydrate-based vaccines with a glycolipid adjuvant for breast cancer.

Globo H (GH) is a hexasaccharide specifically overexpressed on a variety of cancer cells and therefore, a good candidate for cancer vaccine development. To identify the optimal carrier and adjuvant combination, we chemically synthesized and linked GH to a carrier protein, including keyhole limpet hemocyanion, diphtheria toxoid cross-reactive material (CRM) 197 (DT), tetanus toxoid, and BSA, and combined with an adjuvant, and it was administered to mice for the study of immune response. Glycan microarray analysis of the antiserum obtained indicated that the combination of GH-DT adjuvanted with the α-galactosylceramide C34 has the highest enhancement of anti-GH IgG. Compared with the phase III clinical trial vaccine, GH-keyhole limpet hemocyanion/QS21, the GH-DT/C34 vaccine elicited more IgG antibodies, which are more selective for GH and the GH-related epitopes, stage-specific embryonic antigen 3 (SSEA3) and SSEA4, all of which were specifically overexpressed on breast cancer cells and breast cancer stem cells with SSEA4 at the highest level (>90%). We, therefore, further developed SSEA4-DT/C34 as a vaccine candidate, and after immunization, it was found that the elicited antibodies are also IgG-dominant and very specific for SSEA4.

Cell-permeable probe for identification and imaging of sialidases.

Alkyne-hinged 3-fluorosialyl fluoride (DFSA) containing an alkyne group was shown to be a mechanism-based target-specific irreversible inhibitor of sialidases. The ester-protected analog DFSA (PDFSA) is a membrane-permeable precursor of DFSA designed to be used in living cells, and it was shown to form covalent adducts with virus, bacteria, and human sialidases. The fluorosialyl-enzyme adduct can be ligated with an azide-annexed biotin via click reaction and detected by the streptavidin-specific reporting signals. Liquid chromatography-mass spectrometry/mass spectrometry analysis on the tryptic peptide fragments indicates that the 3-fluorosialyl moiety modifies tyrosine residues of the sialidases. DFSA was used to demonstrate influenza infection and the diagnosis of the viral susceptibility to the anti-influenza drug oseltamivir acid, whereas PDFSA was used for in situ imaging of the changes of sialidase activity in live cells.

Fucosyltransferase 8 as a functional regulator of nonsmall cell lung cancer.

The up-regulation of fucosyltransferase 8 (FUT8), the only enzyme catalyzing α1,6-fucosylation in mammals, has been observed in several malignant cancers including liver, ovarian, thyroid, and colorectal cancers. However, the pathological role and the regulatory mechanism of FUT8 in cancers remain largely unknown. In the current study, we report that the expression of FUT8 is up-regulated in nonsmall cell lung cancer (NSCLC) and correlates with tumor metastasis, disease recurrence, and poor survival in patients with NSCLC. Knocking down FUT8 in aggressive lung cancer cell lines significantly inhibits their malignant behaviors including in vitro invasion and cell proliferation, as well as in vivo metastasis and tumor growth. The results of glycoproteomic and microarray analyses show that FUT8 globally modifies surface antigens, receptors, and adhesion molecules and is involved in the regulation of dozens of genes associated with malignancy, suggesting that FUT8 contributes to tumor progression through multiple mechanisms. Moreover, we show that FUT8 is up-regulated during epithelial-mesenchymal transition (EMT), a critical process for malignant transformation of tumor, via the transactivation of ß-catenin/lymphoid enhancer-binding factor-1 (LEF-1). These results provide a model to illustrate the relation between FUT8 expression and lung cancer progression and point to a promising direction for the prognosis and therapy of lung cancer.

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

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., Fucα1-2Galß1-3GalNAc-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.