Effective suppression of tumor growth and hepatic metastasis of neuroblastoma by NKT-stimulatory phenyl glycolipid.

Invariant natural killer T cell (iNKT) cells produce large amounts of cytokines in response to α-Galactosylceramide (α-GalCer) stimulation. An analog containing two phenyl rings on the acyl chain, C34, was previously found to be more Th1-biased than α-GalCer and triggered greater anticancer activities against breast cancer, melanoma and lung cancer in mice. Since liver is enriched in iNKT cells, we investigated anticancer efficacy of C34 on neuroblastoma with hepatic metastasis. C34 induced Th1-biased cytokine secretions in the liver, significantly suppressed neuroblastoma growth/metastasis and prolonged mouse survival. The anti-tumor efficacy might be attributed to greater expansions of hepatic NKT, NK, CD4+ T, and CD8+ T cells as well as reduction of the number of SSCloGr1intCD11b+ subset of myeloid-derived suppressor cells (MDSCs) in the liver of tumor-bearing mice, as compared to DMSO control group. C34 also upregulated expression of CD1d and CD11c, especially in the SSCloGr1intCD11b+ subset of MDSCs, which might be killed by C34-activated NKT cells, attributing to their reduced number. In addition, C34 also induced expansion of CD4+ T, CD8+ T, and NK cells, which might eliminate neuroblastoma cells. These immune-modulating effects of C34 might act in concert in the local milieu of liver to suppress the tumor growth. Further analysis of database of neuroblastoma revealed that patients with high CD11c expression in the monocytic MDSCs in the tumor had longer survival, suggesting the potential clinical application of C34 for treatment of neuroblastoma.

Globo H ceramide is an independent prognostic marker for gallbladder cancer.

In recent studies, there has been growing interest in developing cancer therapeutics targeting Globo H ceramide, which is considered as the most prevalent tumor-associated carbohydrate antigen in epithelial cancers. In this study, we aimed to evaluate the expression of Globo H and investigate its prognostic significance in gallbladder cancer (GBC). The tumor specimens and clinical characteristics of GBC patients were collected from the tumor bank and database of Chang Gung Memorial Hospital. Globo H in tumor specimens was detected by immunohistochemistry (IHC) and mass spectrometry analysis. Through data mining, it was discovered that FUT1 and FUT2, which are key enzymes involved in the biosynthesis of Globo H, were significantly up-regulated in human gallbladder cancer (GBC). Consistent with this finding, Globo H expression was detected in 86% (128 out of 149) of GBC specimens using immunohistochemical (IHC) staining. This was the highest frequency among Globo H expressing cancers. Patients with tumors exhibiting higher Globo H expression (H-score ≥ 80) demonstrated significantly shorter disease-free survival (DFS) and overall survival (OS) (P = 0.0001 and P = 0.0004, respectively). In a multivariable Cox regression analysis, elevated Globo H expression was identified as an independent unfavorable predictor for DFS and OS (hazard ratio: 2.29 and 2.32, respectively, P = 0.008 and 0.001) in primary GBC. Globo H is an independent prognostic marker for GBC.

Priming of macrophage by glycosphingolipids from extracellular vesicles facilitates immune tolerance for embryo-maternal crosstalk.

Glycosphingolipids (GSLs) display diverse functions during embryonic development. Here, we examined the GSL profiles of extracellular vesicles (EVs) secreted from human embryonic stem cells (hESCs) and investigated their functions in priming macrophages to enhance immune tolerance of embryo implantation. When peripheral blood mononuclear cells were incubated with ESC-secreted EVs, globo-series GSLs (GHCer, SSEA3Cer, and SSEA4Cer) were transferred via EVs into monocytes/macrophages. Incubation of monocytes during their differentiation into macrophages with either EVs or synthetic globo-series GSLs induced macrophages to exhibit phenotypic features that imitate immune receptivity, i.e., macrophage polarization, augmented phagocytic activity, suppression of T cell proliferation, and the increased trophoblast invasion. It was also demonstrated that decidual macrophages in first-trimester tissues expressed globo-series GSLs. These findings highlight the role of globo-series GSLs via transfer from EVs in priming macrophages to display decidual macrophage phenotypes, which may facilitate healthy pregnancy.

High expression of embryonic stem cell marker SSEA3 confers poor prognosis and promotes epithelial mesenchymal transition in hepatocellular carcinoma.

BACKGROUND: Malignant cells may arise from dedifferentiation of mature cells and acquire features of the progenitor cells. Definitive endoderm from which liver is derived, expresses glycosphingolipids (GSLs) such as stage-specific embryonic antigen 3 (SSEA3), Globo H, and stage-specific embryonic antigen 4 (SSEA4). Herein, we evaluated the potential prognosis value of the three GSLs and biological functions of SSEA3 in hepatocellular carcinoma (HCC). METHODS: The expression of SSEA3, Globo H, and SSEA4 in tumor tissues obtained from 328 patients with resectable HCC was examined by immunohistochemistry staining. Epithelial mesenchymal transition (EMT) and their related genes were analyzed by transwell assay and qRT-PCR, respectively. RESULTS: Kaplan Meier survival analysis showed significantly shorter relapse-free survival (RFS) for those with higher expression of SSEA3 (p < 0.001), Globo H (p < 0.001), and SSEA4 (p = 0.005) and worse overall survival (OS) for those with high expression of either SSEA3 (p < 0.001) or SSEA4 (p = 0.01). Furthermore, multivariable Cox regression analysis identified the SSEA3 as an independent predictor for RFS (HR: 2.68, 95% CI: 1.93-3.72, p < 0.001) and OS (HR: 2.99, 95% CI: 1.81-4.96, p < 0.001) in HCC. Additionally, SSEA3-ceramide enhanced the EMT of HCC cells, as reflected by its ability to increase migration, invasion and upregulate the expression of CDH2, vimentin, fibronectin, and MMP2, along with ZEB1. Moreover, ZEB1 silencing abrogated the EMT-enhancing effects of SSEA3-ceramide. CONCLUSIONS: Higher expression of SSEA3 was an independent predictor for RFS and OS in HCC and promoted EMT of HCC via upregulation of ZEB1.

KIR/KIR-ligand genotypes and clinical outcomes following chemoimmunotherapy in patients with relapsed or refractory neuroblastoma: a report from the Children's Oncology Group.

BACKGROUND: In the Children's Oncology Group ANBL1221 phase 2 trial for patients with first relapse/first declaration of refractory high-risk neuroblastoma, irinotecan and temozolomide (I/T) combined with either temsirolimus (TEMS) or immunotherapy (the anti-GD2 antibody dinutuximab (DIN) and granulocyte macrophage colony stimulating factory (GM-CSF)) was administered. The response rate among patients treated with I/T/DIN/GM-CSF in the initial cohort (n=17) was 53%; additional patients were enrolled to permit further evaluation of this chemoimmunotherapy regimen. Potential associations between immune-related biomarkers and clinical outcomes including response and survival were evaluated. METHODS: Patients were evaluated for specific immunogenotypes that influence natural killer (NK) cell activity, including killer immunoglobulin-like receptors (KIRs) and their ligands, Fc gamma receptors, and NCR3. Total white cells and leucocyte subsets were assessed via complete blood counts, and flow cytometry of peripheral blood mononuclear cells was performed to assess the potential association between immune cell subpopulations and surface marker expression and clinical outcomes. Appropriate statistical tests of association were performed. The Bonferroni correction for multiple comparisons was performed where indicated. RESULTS: Of the immunogenotypes assessed, the presence or absence of certain KIR and their ligands was associated with clinical outcomes in patients treated with chemoimmunotherapy rather than I/T/TEMS. While median values of CD161, CD56, and KIR differed in responders and non-responders, statistical significance was not maintained in logistic regression models. White cell and neutrophil counts were associated with differences in survival outcomes, however, increases in risk of event in patients assigned to chemoimmunotherapy were not clinically significant. CONCLUSIONS: These findings are consistent with those of prior studies showing that KIR/KIR-ligand genotypes are associated with clinical outcomes following anti-GD2 immunotherapy in children with neuroblastoma. The current study confirms the importance of KIR/KIR-ligand genotype in the context of I/T/DIN/GM-CSF chemoimmunotherapy administered to patients with relapsed or refractory disease in a clinical trial. These results are important because this regimen is now widely used for treatment of patients at time of first relapse/first declaration of refractory disease. Efforts to assess the role of NK cells and genes that influence their function in response to immunotherapy are ongoing. TRIAL REGISTRATION NUMBER: NCT01767194.

Conformational alteration in glycan induces phospholipase Cß1 activation and angiogenesis.

BACKGROUND: In endothelial cells, phospholipase C (PLC) ß1-activated Ca2+ is a crucial second messenger for the signaling pathways governing angiogenesis. PLCß1 is inactivated by complexing with an intracellular protein called translin-associated factor X (TRAX). This study demonstrates specific interactions between Globo H ceramide (GHCer) and TRAX, which highlight a new angiogenic control through PLCß1 activation. METHODS: Globo-series glycosphingolipids (GSLs), including GHCer and stage-specific embryonic antigen-3 ceramide (SSEA3Cer), were analyzed using enzyme-linked immunosorbent assay (ELISA) and Biacore for their binding with TRAX. Angiogenic activities of GSLs in human umbilical vein endothelial cells (HUVECs) were evaluated. Molecular dynamics (MD) simulation was used to study conformations of GSLs and their molecular interactions with TRAX. Fluorescence resonance energy transfer (FRET) analysis of HUVECs by confocal microscopy was used to validate the release of PLCß1 from TRAX. Furthermore, the in vivo angiogenic activity of extracellular vesicles (EVs) containing GHCer was confirmed using subcutaneous Matrigel plug assay in mice. RESULTS: The results of ELISA and Biacore analysis showed a stable complex between recombinant TRAX and synthetic GHCer with KD of 40.9 nM. In contrast, SSEA3Cer lacking a fucose residue of GHCer at the terminal showed ~ 1000-fold decrease in the binding affinity. These results were consistent with their angiogenic activities in HUVECs. The MD simulation indicated that TRAX interacted with the glycan moiety of GHCer at amino acid Q223, Q219, L142, S141, and E216. At equilibrium the stable complex maintained 4.6 ± 1.3 H-bonds. TRAX containing double mutations with Q223A and Q219A lost its ability to interact with GHCer in both MD simulation and Biacore assays. Removal of the terminal fucose from GHCer to become SSEA3Cer resulted in decreased H-bonding to 1.2 ± 1.0 by the MD simulation. Such specific H-bonding was due to the conformational alteration in the whole glycan which was affected by the presence or absence of the fucose moiety. In addition, ELISA, Biacore, and in-cell FRET assays confirmed the competition between GHCer and PLCß1 for binding to TRAX. Furthermore, the Matrigel plug assay showed robust vessel formation in the plug containing tumor-secreted EVs or synthetic GHCer, but not in the plug with SSEA3Cer. The FRET analysis also indicated the disruption of colocalization of TRAX and PLCß1 in cells by GHCer derived from EVs. CONCLUSIONS: Overall, the fucose residue in GHCer dictated the glycan conformation for its complexing with TRAX to release TRAX-sequestered PLCß1, leading to Ca2+ mobilization in endothelial cells and enhancing angiogenesis in tumor microenvironments.

Diversity-Oriented Synthesis of a Molecular Library of Immunomodulatory α-Galactosylceramides with Fluorous-Tag-Assisted Purification and Evaluation of Their Bioactivities in Regard to IL-2 Secretion.

Structural variants of α-galactosylceramide (α-GalCer) that stimulate invariant natural killer T (iNKT) cells constitute an emerging class of immunomodulatory agents in development for numerous biological applications. Variations in lipid chain length and/or fatty acids in these glycoceramides selectively trigger specific pro-inflammatory responses. Studies that would link a specific function to a structurally distinct α-GalCer rely heavily on the availability of homogeneous and pure materials. To address this need, we report herein a general route to the diversification of the ceramide portion of α-GalCer glycolipids. Our convergent synthesis commences from common building blocks and relies on the Julia-Kocienski olefination as a key step. A cleavable fluorous tag is introduced at the non-reducing end of the sugar that facilitates quick purification of products by standard fluorous solid-phase extraction. The strategy enabled the rapid generation of a focused library of 61 α-GalCer analogs by efficiently assembling various lipids and fatty acids. Furthermore, when compared against parent α-GalCer in murine cells, many of these glycolipid variants were found to have iNKT cell stimulating activity similar to or greater than KRN7000. ELISA assaying indicated that glycolipids carrying short fatty N-acyl chains (1fc and 1ga), an unsubstituted (1fh and 1fi) or CF3-substituted phenyl ring at the lipid tail, and a flexible, shorter fatty acyl chain with an aromatic ring (1ge, 1gf, and 1gg) strongly affected the activation of iNKT cells by the glycolipid-loaded antigen-presenting molecule, CD1d. This indicates that the method may benefit the design of structural modifications to potent iNKT cell-binding glycolipids.

The clinical relevance of humoral immune responses to Globo H-KLH vaccine adagloxad simolenin (OBI-822)/OBI-821 and expression of Globo H in metastatic breast cancer.

An international randomized phase II trial of Globo H (GH) vaccine, adagloxad simolenin/OBI-821 in 349 patients with metastatic breast cancer showed longer progression-free survival (PFS) in vaccinated patients who developed anti-Globo H (anti-GH) IgG than those who did not and the placebo group. The impacts of anti-GH IgM and GH expression on peak anti-GH IgG and clinical outcome were further evaluated. The titers of anti-GH IgG and IgM were determined by ELISA. GH expression in tumor was examined by immunohistochemical staining. Immunophenotyping was conducted by flow cytometry. Adagloxad simolenin elicited anti-GH IgM which peaked at titers ≥1:80 between weeks 5 and 13. The mean anti-GH IgG titer peaked at week 41 and decreased thereafter on the completion of vaccination. One log increase in peak IgM was associated with 10.6% decrease in the HR of disease progression (HR: 0.894, 95% CI: 0.833 to 0.960, p=0.0019). Patients with anti-GH IgM ≥1:320 within first 4 weeks after vaccination had significantly higher maximum anti-GH IgM (p<0.0001) and IgG titers (p<0.0001) than those with <1:320. Moreover, the median PFS appears to be longer for patients with anti-GH IgM ≥1:320 within first 4 weeks than those with anti-GH IgM titer <1:320 (11.1 vs 7.3 months, p=0.164), but not statistically significant. Among patients with H score ≥80 for GH expression by immunohistochemistry, the vaccination group (n=42) seemed to have better PFS than the placebo group (n=23) (HR=0.59; 95% CI: 0.32 to 1.10, p=0.10), but the difference did not reach statistical significance. In addition, peak levels of anti-GH IgM were higher in patients who had lower percentage of activated regulatory T cells (Treg cells; CD4+CD45RA-Foxp3high) at baseline than those who had higher activated Treg cells (p=0.042). This study demonstrates that adagloxad simolenin induced both IgG and IgM antibodies against GH. Anti-GH IgM ≥1:320 within first 4 weeks or low activated Treg cells at baseline may help to select patients who are likely to produce a higher level of GH-specific IgM and IgG in the future.

Chemoenzymatic Synthesis of Globo-series Glycosphingolipids and Evaluation of Their Immunosuppressive Activities.

Glycosphingolipids (GSLs) play essential roles in many important biological processes, making them attractive synthetic targets. In this paper, a viable chemoenzymatic method is described for the synthesis of globo-series GSLs, namely, Gb4, Gb5, SSEA-4, and Globo H. The strategy uses a chemically synthesized lactoside acceptor equipped with a partial ceramide structure that is uniquely extended by glycosyltransferases in a highly efficient one-pot multiple enzyme (OPME) procedure. A direct and quantitative conversion of Gb4 sphingosine to Globo H sphingosine is achieved by performing two-sequential OPME glycosylations. A reduction and N-acylation protocol allows facile incorporation of various fatty acids into the lipid portions of the GSLs. The chemically well-defined lipid-modified Globo H-GSLs displayed some differences in their immunosuppressive activities, which may benefit the structural modifications of Globo H ceramides in finding new types of immunosuppressive agents. The strategy outlined in this work should be applicable to the rapid access to other complex GSLs.

Puf-A promotes cancer progression by interacting with nucleophosmin in nucleolus.

Previously, we identified Puf-A as a novel member of Puf-family RNA-binding proteins; however, its biological functions remain obscure. Analysis of tumor samples of non-small cell lung cancer (NSCLC) showed that high Puf-A expression correlated with high histology grade and abnormal p53 status. Kaplan-Meier curve for overall survival revealed high expression of Puf-A to predict poor prognosis in stage I NSCLC. Among patients with colorectal cancer, high Puf-A expression also showed an adverse impact on overall survival. In lung cancer cell lines, downregulation of p53 increased Puf-A expression, and upregulation of p53 dampened its expression. However, luciferase reporter assays indicated that PUF-A locus harbored the p53-response element, but regulated Puf-A transcription indirectly. In vivo suppression of p53 in CCSP-rtTA/TetO-Cre/LSL-KrasG12D/p53flox/flox conditional mutant mice accelerated the progression of the KrasG12D-driven lung cancer, along with enhanced expression of Puf-A. Importantly, intranasal delivery of shPuf-A to the inducible KrasG12D/p53flox/flox mice suppressed tumor progression. Puf-A silencing led to marked decreases in the 80S ribosomes, along with decrease in S6 and L5 in the cytoplasm and accumulation in the nucleolus. Based on immunofluorescence staining and immunoprecipitation studies, Puf-A interacted with NPM1 in nucleolus. Puf-A silencing resulted in NPM1 translocation from nucleolus to nucleoplasm and this disruption of NPM1 localization was reversed by a rescue experiment. Mechanistically, Puf-A silencing altered NPM1 localization, leading to the retention of ribosomal proteins in nucleolus and diminished ribosome biogenesis, followed by cell-cycle arrest/cell death. Puf-A is a potential theranostic target for cancer therapy and an important player in cancer progression.

Globo H Is a Promising Theranostic Marker for Intrahepatic Cholangiocarcinoma.

Recent studies support the development of cancer therapeutics to target Globo H-ceramide, the most prevalent tumor-associated carbohydrate antigen in epithelial cancers. Herein, we evaluated the expression of Globo H and its prognostic significance in intrahepatic cholangiocarcinoma (ICC) and conducted preclinical studies to assess the antitumor activity of Globo H-specific antibody in thioacetamide (TAA)-induced ICC in rats. Globo H-ceramide in tumor specimens was detected by immunohistochemistry (IHC) and mass spectrometry. Antitumor efficacy of anti-Globo H mAbVK9 was evaluated in TAA-induced ICC in rat. Natural killer (NK) cells and their related genes were analyzed by IHC and quantitative real-time polymerase chain reaction. Data mining revealed that B3GALT5 and FUT2, the key enzymes for Globo H biosynthesis, were significantly up-regulated in human ICC. In addition, Globo H expression was detected in 41% (63 of 155) of ICC tumor specimens by IHC staining, and validated by mass spectrometric analysis of two IHC-positive tumors. Patients with Globo H positive tumors had significantly shorter relapse-free survival (RFS) and overall survival (P = 0.0003 and P = 0.002, respectively). Multivariable Cox regression analysis identified Globo H expression as an independent unfavorable predictor for RFS (hazard ratio: 1.66, 95% confidence interval: 1.08-2.36, P = 0.02) in ICC. Furthermore, gradual emergence of Globo H in liver tissues over 6 months in TAA-treated rats recapitulated the multistage progression of ICC in vivo. Importantly, administration of anti-Globo H mAbVK9 in rats bearing TAA-induced ICC significantly suppressed tumor growth with increased NK cells in the tumor microenvironment. Conclusion: Globo H is a theranostic marker in ICC.

Transmembrane and coiled-coil domain family 3 (TMCC3) regulates breast cancer stem cell and AKT activation.

Cancer stem cells (CSC) play a pivotal role in cancer metastasis and resistance to therapy. Previously, we compared the phosphoproteomes of breast cancer stem cells (BCSCs) enriched subpopulation and non-BCSCs sorted from breast cancer patient-derived xenograft (PDX), and identified a function unknown protein, transmembrane and coiled-coil domain family 3 (TMCC3) to be a potential enrichment marker for BCSCs. We demonstrated greater expression of TMCC3 in BCSCs than non-BCSCs and higher expression of TMCC3 in metastatic lymph nodes and lungs than in primary tumor of breast cancer PDXs. TMCC3 silencing suppressed mammosphere formation, ALDH activity and cell migration in vitro, along with reduced tumorigenicity and metastasis in vivo. Mechanistically, we found that AKT activation was reduced by TMCC3 silencing, but enhanced by TMCC3 overexpression. We further demonstrated that TMCC3 interacted directly with AKT through its 1-153 a.a. domain by cell-free biochemical assay in vitro and co-immunoprecipitation and interaction domain mapping assays in vivo. Based on domain truncation studies, we showed that the AKT-interacting domain of TMCC3 was essential for TMCC3-induced AKT activation, self-renewal, and metastasis. Clinically, TMCC3 mRNA expression in 202 breast cancer specimens as determined by qRT-PCR assay showed that higher TMCC3 expression correlated with poorer clinical outcome of breast cancer, including early-stage breast cancer. Multivariable analysis identified TMCC3 expression as an independent risk factor for survival. These findings suggest that TMCC3 is crucial for maintenance of BCSCs features through AKT regulation, and TMCC3 expression has independent prognostic significance in breast cancer. Thus, TMCC3 may serve as a new target for therapy directed against CSCs.

Low Expression of IL-15 and NKT in Tumor Microenvironment Predicts Poor Outcome of MYCN-Non-Amplified Neuroblastoma.

Immune tumor microenvironment (TME) in neuroblastoma (NBL) contributes to tumor behavior and treatment response. T cells and natural killer (NK) cells have been shown to play important roles in the neuroblastoma TME. However, few reports address the clinical relevance of natural killer T cells (NKTs) and interleukin-15 (IL-15), one of the crucial cytokines controlling the activation and expansion of NK/NKT cells, in NBL. In this study, we examined NKT immunoscores and IL-15 expression in both MYCN-amplified and MYCN-non-amplified NBL to correlate with clinical outcomes such as event-free survival (EFS) and overall survival (OS). From Gene Expression Omnibus (GEO) datasets GSE45480 (n = 643) and GSE49711 (n = 493), we found that NKT immunoscore and IL-15 expression were both significantly lower in MYCN-amplified NBL, and similar results were observed using our clinical NBL samples (n = 53). Moreover, NBL patients (GEO dataset GSE49711 and our clinical samples) with both lower NKT immunoscore and IL-15 expression exhibited decreased EFS and OS regardless of MYCN gene amplification status. Multivariate analysis further showed that the combination of low NKT immunoscore and low IL-15 expression level was an independent prognostic factor for poor EFS and OS in our NBL patients. These findings provide the rationale for the development of strategy to incorporate IL-15 and NKT cell therapy into the treatment regimen for neuroblastoma.

High B3GALT5 expression confers poor clinical outcome and contributes to tumor progression and metastasis in breast cancer.

BACKGROUND: Existence of breast cancer stem cells (BCSCs) is implicated in disease relapse, metastasis, and resistance of treatment. ß1,3-Galactosyltransferase 5 (B3GALT5) has been shown to be a pro-survival marker for BCSCs. However, little is known about the prognostic significance of B3GALT5 in breast cancer. METHODS: Paired tissues (tumor part and adjacent non-tumor part) from a cohort of 202 women with breast cancer were used to determine the expression levels of B3GALT5 mRNA by qRT-PCR. Kaplan-Meier and multivariable Cox proportional hazard models were used to assess survival differences in terms of relapse-free survival (RFS) and overall survival (OS). Both breast cancer cells and cancer stem cells (BCSCs) were used to see the in vitro effects of knockdown or overexpression of B3GALT5 on cell migration, invasion, and epithelial-to-mesenchymal transition (EMT). A patient-derived xenograft (PDX) model was used to see the in vivo effects of knockdown of B3GALT5 in BCSCs on tumor growth and metastasis. RESULTS: Higher expression of B3GALT5 in 202 breast cancer tissues, especially in adjacent non-tumor tissue, correlated with poor clinical outcomes including shorter OS and RFS in all patients, especially those with early stage breast cancer. In vitro studies showed B3GALT5 could enhance cell migration, invasion, mammosphere formation, and EMT. Of note, B3GALT5 upregulated the expression of ß-catenin and EMT activator zinc finger E-box binding homeobox 1 (ZEB1) pathway in BCSCs. In vivo studies showed B3GALT5 expression in BCSCs is critical for not only tumor growth but also lymph node and lung metastasis in PDX mice. CONCLUSION: Our results demonstrated the value of B3GALT5 as a prognostic marker of breast cancer, especially among the early stage patients, and its crucial roles in regulating EMT, cell migration, and stemness thereby promoting breast cancer progression.

Sialylation of CD55 by ST3GAL1 Facilitates Immune Evasion in Cancer.

Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.

O-Acetyl-GD2 as a Therapeutic Target for Breast Cancer Stem Cells.

Synopsis: A sugar-lipid molecule called OAcGD2 is a novel marker for breast cancer stem cells. Treatment with anti-OAcGD2 mAb8B6 may have superior anticancer efficacy by targeting cancer stem cells, thereby reducing metastasis and recurrence of cancer. Background: Cancer stem cells (CSCs) that drive tumor progression and disease recurrence are rare subsets of tumor cells. CSCs are relatively resistant to conventional chemotherapy and radiotherapy. Eradication of CSCs is thus essential to achieve durable responses. GD2 was reported to be a CSC marker in human triple-negative breast cancer, and anti-GD2 immunotherapy showed reduced tumor growth in cell lines. Using a specific anti-OAcGD2 antibody, mAb8D6, we set out to determine whether OAcGD2+ cells exhibit stem cell properties and mAb8D6 can inhibit tumor growth by targeting OAcGD2+CSCs. Method: OAcGD2 expression in patient-derived xenografts (PDXs) of breast cancer was determined by flow cytometric analyses using mAb8D6. The stemness of OAcGD2+ cells isolated by sorting and the effects of mAb8B6 were assessed by CSC growth and mammosphere formation in vitro and tumor growth in vivo using PDX models. Result: We found that the OAcGD2 expression levels in six PDXs of various molecular subtypes of breast cancer highly correlated with their previously defined CSC markers in these PDXs. The sorted OAcGD2+ cells displayed a greater capacity for mammosphere formation in vitro and tumor initiation in vivo than OAcGD2- cells. In addition, the majority of OAcGD2+ cells were aldehyde dehydrogenase (ALDH+) or CD44hiCD24lo, the known CSC markers in breast cancer. Treatment of PDXs-bearing mice with mAb8B6, but not doxorubicin, suppressed the tumor growth, along with reduced CSCs as assessed by CSC markers and in vivo tumorigenicity. In vitro, mAb8B6 suppressed proliferation and mammosphere formation and induced apoptosis of OAcGD2+ breast cancer cells harvested from PDXs, in a dose-dependent manner. Finally, administration of mAb8B6 in vivo dramatically suppressed tumor growth of OAcGD2+ breast CSCs (BCSCs) with complete tumor abrogation in 3/6 mice. Conclusion: OAcGD2 is a novel marker for CSC in various subtypes of breast cancer. Anti-OAcGD2 mAb8B6 directly eradicated OAcGD2+ cells and reduced tumor growth in PDX model. Our data demonstrate the potential of mAb8B6 as a promising immunotherapeutic agent to target BCSCs.

Co-Delivery of CPT-11 and Panobinostat with Anti-GD2 Antibody Conjugated Immunoliposomes for Targeted Combination Chemotherapy.

The consistent expression of disialoganglioside GD2 in neuroblastoma tumor cells and its restricted expression in normal tissues open the possibility to use it for molecularly targeted neuroblastoma therapy. On the other hand, immunoliposomes combining antibody-mediated tumor recognition with liposomal delivery of chemotherapeutics have been proved to enhance therapeutic efficacy in brain tumors. Therefore, we develop immunoliposomes (ImmuLipCP) conjugated with anti-GD2 antibody, for targeted co-delivery of CPT-11 and panobinostat in this study. U87MG human glioma cell line and its drug resistant variant (U87DR), which were confirmed to be associated with low and high expression of cell surface GD2, were employed to compare the targeting efficacy. From in vitro cytotoxicity assay, CPT-11 showed synergism drug interaction with panobinostat to support co-delivery of both drugs with ImmuLipCP for targeted synergistic combination chemotherapy. The molecular targeting mechanism was elucidated from intracellular uptake efficacy by confocal microscopy and flow cytometry analysis, where 6-fold increase in liposome and 1.8-fold increase in drug uptake efficiency was found using targeted liposomes. This enhanced intracellular trafficking for drug delivery endows ImmuLipCP with pronounced cytotoxicity toward U87DR cells in vitro, with 1.6-fold increase of apoptosis rate. Using xenograft nude mice model with subcutaneously implanted U87DR cells, we observe similar biodistribution profile but 5.1 times higher accumulation rate of ImmuLip from in vivo imaging system (IVIS) observation of Cy5.5-labelled liposomes. Taking advantage of this highly efficient GD-2 targeting, ImmuLipCP was demonstrated to be an effective cancer treatment modality to significantly enhance the anti-cancer therapeutic efficacy in U87DR tumors, shown from the significant reduced tumor size in and prolonged survival time of experiment animals as well as diminished expression of cell proliferation and enhanced expression of apoptosis marker proteins in tumor section.

Targeting glycosphingolipids for cancer immunotherapy.

Aberrant expression of glycosphingolipids (GSLs) is a unique feature of cancer and stromal cells in tumor microenvironments. Although the impact of GSLs on tumor progression remains largely unclear, anticancer immunotherapies directed against GSLs are attracting growing attention. Here, we focus on GD2, a disialoganglioside expressed in tumors of neuroectodermal origin, and Globo H ceramide (GHCer), the most prevalent cancer-associated GSL overexpressed in a variety of epithelial cancers. We first summarize recent advances on our understanding of GD2 and GHCer biology and then discuss the clinical development of the first immunotherapeutic agent targeting a glycolipid, the GD2-specific antibody dinutuximab, its approved indications, and new strategies to improve its efficacy for neuroblastoma. Next, we review ongoing clinical trials on Globo H-targeted immunotherapeutics. We end with highlighting how these studies provide sound scientific rationales for targeting GSLs in cancer and may facilitate a rational design of new GSL-targeted anticancer therapeutics.

Globo H-KLH vaccine adagloxad simolenin (OBI-822)/OBI-821 in patients with metastatic breast cancer: phase II randomized, placebo-controlled study.

PURPOSE: This randomized, double-blind, placebo-controlled, parallel-group, phase II trial assessed the efficacy and safety of adagloxad simolenin (OBI-822; a Globo H epitope covalently linked to keyhole limpet hemocyanin (KLH)) with adjuvant OBI-821 in metastatic breast cancer (MBC). METHODS: At 40 sites in Taiwan, USA, Korea, India, and Hong Kong, patients with MBC of any molecular subtype and ≤2 prior progressive disease events with stable/responding disease after the last anticancer regimen were randomized (2:1) to adagloxad simolenin (AS/OBI-821) or placebo, subcutaneously for nine doses with low-dose cyclophosphamide. The primary endpoint was progression-free survival (PFS). Secondary endpoints included overall survival, correlation of clinical outcome with humoral immune response and Globo H expression, and safety. RESULTS: Of 349 patients randomized, 348 received study drug. Patients with the following breast cancer subtypes were included: hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2-) (70.4%), triple negative (12.9%), and HER2+ (16.7%), similarly distributed between treatment arms. Median PFS was 7.6 months (95% CI: 6.5-10.9) with AS/OBI-821 (n=224) and 9.2 months (95% CI: 7.3-11.3) with placebo (n=124) (HR=0.96; 95% CI: 0.74-1.25; p=0.77), with no difference by breast cancer subtype. AS/OBI-821 recipients with anti-Globo H IgG titer ≥1:160 had significantly longer median PFS (11.1 months (95% CI: 9.3-17.6)) versus those with titers <1:160 (5.5 months (95% CI: 3.7-5.6); HR=0.52; p<0.0001) and placebo recipients (HR=0.71; p=0.03). Anti-KLH immune responses were similar at week 40 between AS/OBI-821 recipients with anti-Globo IgG titer ≥1:160 and those with anti-Globo IgG titer <1:160. The most common adverse events with AS/OBI-821 were grade 1 or 2 injection site reactions (56.7%; placebo, 8.9%) and fever (20.1%; placebo, 6.5%). CONCLUSION: AS/OBI-821 did not improve PFS in patients with previously treated MBC. However, humoral immune response to Globo H correlated with improved PFS in AS/OBI-821 recipients, leading the way to further marker-driven studies. Treatment was well tolerated.NCT01516307.

Paper-Based Detection Device for Alzheimer's Disease-Detecting ß-amyloid Peptides (1-42) in Human Plasma.

The diagnosis of Alzheimer's disease (AD) is frequently missed or delayed in clinical practice. To remedy this situation, we developed a screening, paper-based (P-ELISA) platform to detect ß-amyloid peptide 1-42 (Aß42) and provide rapid results using a small volume, easily accessible plasma sample instead of cerebrospinal fluid. The protocol outlined herein only requires 3 µL of sample per well and a short operating time (i.e., only 90 min). The detection limit of Aß42 is 63.04 pg/mL in a buffer system. This P-ELISA-based approach can be used for early, preclinical stage AD screening, including screening for amnestic mild cognitive impairment (MCI) due to AD. It may also be used for treatment and stage monitoring purposes. The implementation of this approach may provide tremendous impact for an afflicted population and may well prompt additional and expanded efforts in both academic and commercial communities.