A Versatile One-Step Enzymatic Strategy for Efficient Imaging and Mapping of Tumor-Associated Tn Antigen.

Tn antigen (CD175), recognized as the precursor monosaccharide (α-GalNAc) of mucin O-glycan, is a well-known tumor-associated carbohydrate antigen (TACA). It has emerged as a potential biomarker for cancer diagnosis and prognosis. However, the role it plays in cancer biology remains elusive due to the absence of a sensitive and selective detection method. In this study, we synthesized two new probes based on a unique uridine-5'-diphospho-α-d-galactose (UDP-Gal) derivative, each functionalized with either a fluorescence or a cleavable biotin tag, to develop an innovative one-step enzymatic labeling strategy, enabling the visualization, enrichment, and site-specific mapping of the Tn antigen with unparalleled sensitivity and specificity. Our versatile strategy has been successfully applied to detect and image Tn antigen across various samples, including the complex cell lysates, live cells, serum, and tissue samples. Compared to the traditional lectin method, this one-step enzymatic method is simpler and more efficient (>10/100-fold in sensitivity). Furthermore, it allowed us to map 454 Tn-glycoproteins and 624 Tn-glycosylation sites from HEK293FTn+ and Jurkat cells. Therefore, our strategy provides an exceptionally promising tool for revealing the biological functions of the Tn antigen and advancing cancer diagnostics.

Spectrally separated dual-label upconversion luminescence lateral flow assay for cancer-specific STn-glycosylation in CA125 and CA15-3.

Multiplexed lateral flow assays (LFAs) offer efficient on-site testing by simultaneously detecting multiple biomarkers from a single sample, reducing costs. In cancer diagnostics, where biomarkers can lack specificity, multiparameter detection provides more information at the point-of-care. Our research focuses on epithelial ovarian cancer (EOC), where STn-glycosylated forms of CA125 and CA15-3 antigens can better discriminate cancer from benign conditions. We have developed a dual-label LFA that detects both CA125-STn and CA15-3-STn within a single anti-STn antibody test line. This utilizes spectral separation of green (540 nm) and blue (450 nm) emitting erbium (NaYF4:Yb3+, Er3+)- and thulium (NaYF4: Yb3+, Tm3+)-doped upconverting nanoparticle (UCNP) reporters conjugated with antibodies against the protein epitopes in CA125 or CA15-3. This technology allows the simultaneous detection of different antigen variants from a single test line. The developed proof-of-concept dual-label LFA was able to distinguish between the ascites fluid samples from diagnosed ovarian cancer patients (n = 10) and liver cirrhosis ascites fluid samples (n = 3) used as a negative control. The analytical sensitivity of CA125-STn for the dual-label LFA was 1.8 U/ml in buffer and 3.6 U/ml in ascites fluid matrix. Here we demonstrate a novel approach of spectrally separated measurement of STn-glycosylated forms of two different cancer-associated protein biomarkers by using UCNP reporter technology.

Immune cells surveil aberrantly sialylated O-glycans on megakaryocytes to regulate platelet count.

Immune thrombocytopenia (ITP) is a platelet disorder. Pediatric and adult ITP have been associated with sialic acid alterations, but the pathophysiology of ITP remains elusive, and ITP is often a diagnosis of exclusion. Our analysis of pediatric ITP plasma samples showed increased anti-Thomsen-Friedenreich antigen (TF antigen) antibody representation, suggesting increased exposure of the typically sialylated and cryptic TF antigen in these patients. The O-glycan sialyltransferase St3gal1 adds sialic acid specifically on the TF antigen. To understand if TF antigen exposure associates with thrombocytopenia, we generated a mouse model with targeted deletion of St3gal1 in megakaryocytes (MK) (St3gal1MK-/-). TF antigen exposure was restricted to MKs and resulted in thrombocytopenia. Deletion of Jak3 in St3gal1MK-/- mice normalized platelet counts implicating involvement of immune cells. Interferon-producing Siglec H-positive bone marrow (BM) immune cells engaged with O-glycan sialic acid moieties to regulate type I interferon secretion and platelet release (thrombopoiesis), as evidenced by partially normalized platelet count following inhibition of interferon and Siglec H receptors. Single-cell RNA-sequencing determined that TF antigen exposure by MKs primed St3gal1MK-/- BM immune cells to release type I interferon. Single-cell RNA-sequencing further revealed a new population of immune cells with a plasmacytoid dendritic cell-like signature and concomitant upregulation of the immunoglobulin rearrangement gene transcripts Igkc and Ighm, suggesting additional immune regulatory mechanisms. Thus, aberrant TF antigen moieties, often found in pathological conditions, regulate immune cells and thrombopoiesis in the BM, leading to reduced platelet count.

Large-scale preparation of sialyl-Tn antigen-containing peptides from mucin-like glycoproteins in boar seminal gel.

Sialyl-Tn antigen, a tumor antigen, is a valuable ligand for the purification of proteins that specifically bind to it. Here, we developed a new method for the preparation of large amounts of sialyl-Tn antigen-containing peptides from an unused resource, boar seminal gel. The glycopeptides were prepared from the actinase E digests by a combination of gel filtration and hydrophilic partitioning.

Expression of Thomsen-Friedenreich Antigen in Colorectal Cancer and Association with Microsatellite Instability.

Microsatellite instability (MSI) is a molecular phenotype due to a deficient DNA mismatch repair (dMMR). In colorectal cancer (CRC), dMMR/MSI is associated with several clinical and histopathological features, influences prognosis, and is a predictive factor of response to therapy. In daily practice, dMMR/MSI profiles are identified by immunohistochemistry and/or multiplex PCR. The Thomsen-Friedenreich (TF) antigen was previously found to be a potential single marker to identify MSI-high gastric cancers. Therefore, in this study, we aimed to disclose a possible association between TF expression and MSI status in CRC. Furthermore, we evaluated the relationship between TF expression and other clinicopathological features, including patient survival. We evaluated the expression of the TF antigen in a cohort of 25 MSI-high and 71 microsatellite stable (MSS) CRCs. No association was observed between the expression of the TF antigen and MSI-high status in CRC. The survival analysis revealed that patients with MSI-high CRC showed improved survival when the TF antigen was expressed. This finding holds promise as it indicates the potential use of the TF antigen as a biomarker of better prognosis in MSI-high CRCs that should be validated in an independent and larger CRC cohort.

Identification of Tn antigen O-GalNAc-expressing glycoproteins in human carcinomas using novel anti-Tn recombinant antibodies.

The Tn antigen is a neoantigen abnormally expressed in many human carcinomas and expression correlates with metastasis and poor survival. To explore its biomarker potential, new antibodies are needed that specifically recognize this antigen in tumors. Here we generated two recombinant antibodies to the Tn antigen, Remab6 as a chimeric human IgG1 antibody and ReBaGs6 as a murine IgM antibody and characterized their specificities using multiple biochemical and biological approaches. Both Remab6 and ReBaGs6 recognize clustered Tn structures, but most importantly do not recognize glycoforms of human IgA1 that contain potential cross-reactive Tn antigen structures. In flow cytometry and immunofluorescence analyses, Remab6 recognizes human cancer cell lines expressing the Tn antigen, but not their Tn-negative counterparts. In immunohistochemistry (IHC), Remab6 stains many human cancers in tissue array format but rarely stains normal tissues and then mostly intracellularly. We used these antibodies to identify several unique Tn-containing glycoproteins in Tn-positive Colo205 cells, indicating their utility for glycoproteomics in future biomarker studies. Thus, recombinant Remab6 and ReBaGs6 are useful for biochemical characterization of cancer cells and IHC of tumors and represent promising tools for Tn biomarker discovery independently of recognition of IgA1.

Electrogenerated Chemiluminescence Immunoassays on Nanoelectrode Ensembles Platform with Magnetic Microbeads for the Determination of Carbohydrate Antigen.

This work reports a gold nanoelectrode ensembles (Au-NEE) platform taken as a disposable electrogenerated chemiluminescence (ECL) platform with immunomagnetic microbeads for ECL immunoassays for the first time. The peak-shaped voltammograms were obtained at the Au-NEE, attributed to the total diffusional overlap. The ECL intensity at Au-NEE was 12.9 folds in the Ru(bpy)32+-tri-n-propylamine (TPA) ECL system and 19.6 folds in the luminol-H2O2 system, compared with that at the Au macroelectrode using the normalized active area of the electrodes, mainly attributed to the diffusion overlap of the Au-NEE and the edge effect of the individual gold nanodisks of the Au-NEE. The ECL immunoassay on the Au-NEE platform with magnetic microbeads for the determination of cancer biomarkers was developed. Carbohydrate antigen 19-9 (CA 19-9) was chosen as a model analyte while CA 19-9 antibody on the magnetic microbeads was taken as the capture probe, and ruthenium complex-labeled CA 19-9 antibody was used as the signal probe. A "sandwich" bioconjugates on the magnetic beads were transferred onto the ECL platform, and then the ECL measurements were performed in TPA solution. The developed method showed that the ECL peak intensity was directly in proportion to the concentration of CA 19-9 in the range from 0.5 to 20 U/mL with a limit of detection of 0.4 U/mL. This work demonstrates that the Au-NEE can be employed as a useful disposable ECL platform with the merits of cheapness, low nonspecific adsorption and practical application. The proposed approach will open a new avenue in the point-of-care test for the determination of protein biomarkers.

Level of N6-Methyladenosine in Peripheral Blood RNA: A Novel Predictive Biomarker for Gastric Cancer.

BACKGROUND: Dysregulation of N6-methyladenosine (m6A) is associated with various human diseases including cancer. This study aimed to evaluate the level of m6A as a biomarker for gastric cancer (GC) diagnosis. METHODS: Peripheral blood samples were collected from 100 GC patients, 30 benign gastric disease (BGD) patients, and 75 healthy controls (HCs). Levels of m6A in total RNA and expression of m6A-related proteins were analyzed. RESULTS: The m6A levels in peripheral blood RNA were significantly increased in the GC group compared with those in the BGD or HC groups; moreover, levels increased with the progression and metastasis of GC and decreased in GC patients after surgery. The area under the curve (AUC) for m6A in the GC group was 0.929 (95% CI, 0.88-0.96), which is markedly greater than the AUCs for carcinoembryonic antigen (CEA; 0.694) and carbohydrate antigen 199 (CA199; 0.603). The combination of CEA and CA199 with m6A improved the AUC to 0.955 (95% CI, 0.91-0.98). The expressions of m6A demethylases ALKBH5 and FTO were significantly downregulated in the GC group compared with the HC group. Coculture with GC cells increased the m6A of RNA in promyelocytic (HL-60) and monocytic (THP-1) leukemia cells and nontumorigenic human peripheral blood B lymphocyte cells (PENG-EBV). Furthermore, a xenograft model enhanced the m6A in peripheral blood RNA of mice. Accordingly, expressions of ALKBH5 and FTO were decreased both in vitro and in vivo. CONCLUSIONS: Level of m6A in peripheral blood RNA is a promising noninvasive diagnostic biomarker for GC patients.

AGA Clinical Practice Update on Surveillance for Hepatobiliary Cancers in Patients With Primary Sclerosing Cholangitis: Expert Review.

DESCRIPTION: The purpose of this clinical practice update is to define key principles in the surveillance of hepatobiliary cancers including cholangiocarcinoma, gallbladder adenocarcinoma, and hepatocellular carcinoma in patients with primary sclerosing cholangitis (PSC). METHODS: The recommendations outlined in this expert review are based on available published evidence including observational studies and systematic reviews, and incorporates expert opinion where applicable. BEST PRACTICE ADVICE 1: Surveillance for cholangiocarcinoma and gallbladder cancer should be considered in all adult patients with PSC regardless of disease stage, especially in the first year after diagnosis and in patients with ulcerative colitis and those diagnosed at an older age. BEST PRACTICE ADVICE 2: Surveillance for cholangiocarcinoma and gallbladder cancer should include imaging by ultrasound, computed tomography, or magnetic resonance imaging, with or without serum carbohydrate antigen 19-9, every 6 to 12 months BEST PRACTICE ADVICE 3: Endoscopic retrograde cholangiopancreatography with brush cytology should not be used routinely for surveillance of cholangiocarcinomas in PSC. BEST PRACTICE ADVICE 4: Cholangiocarcinomas should be investigated by endoscopic retrograde cholangiopancreatography with brush cytology with or without fluorescence in situ hybridization analysis and/or cholangioscopy in PSC patients with worsening clinical symptoms, worsening cholestasis, or a dominant stricture. BEST PRACTICE ADVICE 5: Fine-needle aspiration of perihilar biliary strictures should be used with caution in PSC patients considered to be liver transplant candidates because of concerns for tumor seeding if the lesion is a cholangiocarcinoma. BEST PRACTICE ADVICE 6: Surveillance for cholangiocarcinoma should not be performed in PSC patients with small-duct PSCs or those younger than age 20. BEST PRACTICE ADVICE 7: The decision to perform a cholecystectomy in PSC patients with a gallbladder polyp should be based on the size and growth of the polyp, as well as the clinical status of the patient, with the knowledge of the increased risk of gallbladder cancer in polyps greater than 8 mm. BEST PRACTICE ADVICE 8: Surveillance for hepatocellular carcinoma in PSC patients with cirrhosis should include ultrasound, computed tomography, or magnetic resonance imaging, with or without α-fetoprotein every 6 months.

Application value of nomogram and prognostic factors of gastric cancer patients who underwent D2 radical lymphadenectomy.

BACKGROUND: The aim of this study was to explore the prognostic factors and establish a nomogram to predict the long-term survival of gastric cancer patients. METHODS: The clinicopathological data of 421 gastric cancer patients, who were treated with radical D2 lymphadenectomy by the same surgical team between January 2009 and March 2017, were collected. The analysis of long-term survival was performed using Cox regression analysis. Based on the multivariate analysis results, a prognostic nomogram was formulated to predict the 5-year survival rate probability. RESULTS: In the present study, the total overall 3-year and 5-year survival rates were 58.7 and 45.8%, respectively. The results of the univariate Cox regression analysis revealed that tumor staging, tumor location, Borrmann type, the number of lymph nodes dissected, the number of lymph node metastases, positive lymph nodes ratio, lymphocyte count, serum albumin, CEA, CA153, CA199, BMI, tumor size, nerve invasion, and vascular invasion were prognostic factors for gastric cancer (all, P < 0.05). However, merely tumor staging, tumor location, positive lymph node ratio, CA199, BMI, tumor size, nerve invasion, and vascular invasion were independent risk factors, based on the results of the multivariate Cox regression analysis (all, P < 0.05). The nomogram based on eight independent prognostic factors revealed a well-degree of differentiation with a concordance index of 0.76 (95% CI: 0.72-0.79, P < 0.001), which was better than the AJCC-7 staging system (concordance index = 0.68). CONCLUSION: The present study established a nomogram based on eight independent prognostic factors to predict long-term survival in gastric cancer patients. The nomogram would be beneficial for more accurately predicting the prognosis of gastric cancer, and provide important basis for making individualized treatment plans following surgery.

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.

Amplified photoelectrochemical immunoassay for the tumor marker carbohydrate antigen 724 based on dye sensitization of the semiconductor composite C3N4-MoS2.

The authors describe an amplified photoelectrochemical immunoassay for the tumor marker carbohydrate antigen 724 (CA724). The method employs a C3N4-MoS2 semiconductor as the photoelectric conversion layer. The nanocomposite was characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray powder diffraction, and UV-vis diffuse reflectometry. The dye eosin Y was encapsulated into CaCO3 nanospheres which then were used as labels for antibody against CA724. In addition, Fe3O4 nanospheres were employed as magnetic platform for constructing photoelectrochemical sandwich immunoassay. The CaCO3 nanospheres can be dissolved with aid of ethylene diamine tetraacetic acid (EDTA) and the carried eosin Y in CaCO3 is released. The released dyes sensitizes the C3N4-MoS2 semiconductor, which induces photocurrent amplification. Under optimal conditions and at a typical working voltage of 0 V (vs. SCE), the photocurrent increases linearly in the range of 0.05 mU mL-1 to 500 mU mL-1 of CA724, with a 0.02 mU mL-1 detection limit. Graphical abstract The C3N4-MoS2 complex, with high efficiency of electron transport, was synthesized to construct a photoelectrochemical analytical platform. A sandwich-type immunoassay was established on the surface of magnetic beads. Carbohydrate antigen 724 in sample was detected sensitively by using sensitization of released eosin Y as signal amplifiery.

Serum N-glycans outperform CA19-9 in diagnosis of extrahepatic cholangiocarcinoma.

Extensive efforts have been devoted to improve the diagnosis of extrahepatic cholangiocarcinoma (ECCA) due to its silent clinical character and lack of effective diagnostic biomarkers. Specific alterations in N-glycosylation of glycoproteins are considered a key component in cancer progression, which can serve as a distinct molecular signature for cancer detection. This study aims to find potential serum N-glycan markers for ECCA. In total, 255 serum samples from patients with ECCA (n = 106), benign bile tract disease (BBD, n = 60) and healthy controls (HC, n = 89) were recruited. Only 2 µL of serum from individual patients was used in this assay where the N-glycome of serum glycoproteins was profiled by DNA sequencer-assisted fluorophore-assisted capillary electrophoresis (DSA-FACE) technology. Multi-parameter models were constructed by combining the N-glycans and carbohydrate antigen 19-9 (CA19-9) which is currently used clinically. Quantitative analyses showed that among 13 N-glycan structures, the bifucosylated triantennary N-glycan (peak10, NA3F2) presented the best diagnostic performance for distinguishing ECCA from BBD and HC. Two diagnostic models (Glycotest1 and Glycotest2) performed better than single N-glycan or CA19-9. Additionally, two N-glycan structures (peak9, NA3Fb; peak12, NA4Fb) were tightly related to lymph node metastasis in ECCA patients. In conclusion, sera of ECCA showed relatively specific N-glycome profiling patterns. Serum N-glycan markers and models are novel, valuable and noninvasive alternatives in ECCA diagnosis and progression monitoring.

Triazole-linked fluorescent bisboronic acid capable of selective recognition of the Lewis Y antigen.

Cell surface carbohydrates of the Lewis blood group antigens, Lewis X (Lex), Lewis Y (Ley), Lewis A (Lea), and their sialylated derivatives, such as sialy Lewis X (sLex) and sialy Lewis A (sLea), play important roles in various recognition processes. These cell surface carbohydrates have also been associated with the development and progression of many types of cancers. Recently, we synthesized four anthracene-based fluorescent bisboronic acid sensors (compounds 2a-d) linked by 'click' chemistry with tethers of different lengths to match the epitope of various Lewis group of sugars. Among the four compounds, 2a appears to have both high sensitivity and selectivity for Ley among other carbohydrate antigens.

Glycan motif profiling reveals plasma sialyl-lewis x elevations in pancreatic cancers that are negative for sialyl-lewis A.

The sialyl-Lewis A (sLeA) glycan forms the basis of the CA19-9 assay and is the current best biomarker for pancreatic cancer, but because it is not elevated in ∼25% of pancreatic cancers, it is not useful for early diagnosis. We hypothesized that sLeA-low tumors secrete glycans that are related to sLeA but not detectable by CA19-9 antibodies. We used a method called motif profiling to predict that a structural isomer of sLeA called sialyl-Lewis X (sLeX) is elevated in the plasma of some sLeA-low cancers. We corroborated this prediction in a set of 48 plasma samples and in a blinded set of 200 samples. An antibody sandwich assay formed by the capture and detection of sLeX was elevated in 13 of 69 cancers that were not elevated in sLeA, and a novel hybrid assay of sLeA capture and sLeX detected 24 of 69 sLeA-low cancers. A two-marker panel based on combined sLeA and sLeX detection differentiated 109 pancreatic cancers from 91 benign pancreatic diseases with 79% accuracy (74% sensitivity and 78% specificity), significantly better than sLeA alone, which yielded 68% accuracy (65% sensitivity and 71% specificity). Furthermore, sLeX staining was evident in tumors that do not elevate plasma sLeA, including those with poorly differentiated ductal adenocarcinoma. Thus, glycan-based biomarkers could characterize distinct subgroups of patients. In addition, the combined use of sLeA and sLeX, or related glycans, could lead to a biomarker panel that is useful in the clinical diagnosis of pancreatic cancer. Précis: This paper shows that a structural isomer of the current best biomarker for pancreatic cancer, CA19-9, is elevated in the plasma of patients who are low in CA19-9, potentially enabling more comprehensive detection and classification of pancreatic cancers.

Application of cancer-associated glycoforms and glycan-binding probes to an in vitro diagnostic multivariate index assay for precise diagnoses of cancer.

Personalized medicine has emerged as a widely accepted trend in medicine for the efficacious and safe treatment of various diseases. It covers every medical treatment tailored according to various properties of individuals. Cancer-associated glycosylation mirrors cancer states more precisely, and this "sweet side of cancer" is thus intended to spur the development of an advanced in vitro diagnostic system. The changes of glyco-codes are often subtle and thus not easy to trace, thereby making it difficult to discriminate changes from various compounding factors. Special glycan-binding probes, often lectins, can be paired with aglycosylated antibodies to enable quantitative and qualitative measurements of glycoforms. With the in vitro diagnosis multivariate index assay (IVDMIA) considered to be capable of yielding patient-specific results, the combinatorial use of multiple glycoproteins may be a good modality to ensure disease-specific, personalized diagnoses.

(18)F-Based Pretargeted PET Imaging Based on Bioorthogonal Diels-Alder Click Chemistry.

A first-of-its-kind (18)F pretargeted PET imaging approach based on the bioorthogonal inverse electron demand Diels-Alder (IEDDA) reaction between tetrazine (Tz) and trans-cyclooctene (TCO) is presented. As proof-of-principle, a TCO-bearing immunoconjugate of the anti-CA19.9 antibody 5B1 and an Al[(18)F]NOTA-labeled tetrazine radioligand were harnessed for the visualization of CA19.9-expressing BxPC3 pancreatic cancer xenografts. Biodistribution and (18)F-PET imaging data clearly demonstrate that this methodology effectively delineates tumor mass with activity concentrations up to 6.4 %ID/g at 4 h after injection of the radioligand.

A tandem enzymatic approach for detecting and imaging tumor-associated Thomsen-Friedenreich antigen disaccharide.

The disaccharide galactose-ß1,3-N-acetylgalactosamine (Galß1,3-GalNAc) attached to serine and/or threonine residues of proteins, also known as the Thomsen-Friedenreich (TF) antigen, is highly expressed in various types of human carcinomas. It has been shown to contribute to tumor development, progression, and metastasis. However, current methods have limited power in detecting and imaging TF antigens among a variety of complex cell-surface glycans. Here we describe a tandem enzymatic strategy to detect and label TF antigen disaccharide with high sensitivity and selectivity. We demonstrate that this strategy enables detection of TF antigens on proteins, profiling and identification of unknown TF antigen-modified glycoproteins, and simultaneous labeling of multiple forms of complex glycan motifs on the same cell. This approach expands the capability of glycan labeling to probe the functional role of TF antigens in cancer biology.

Human breast cancer and lymph node metastases express Gb3 and can be targeted by STxB-vectorized chemotherapeutic compounds.

BACKGROUND: The B-subunit of Shiga toxin (STxB) specifically binds to the glycosphingolipid Gb3 that is highly expressed on a number of human tumors and has been shown to target tumor cells in mouse models and ex vivo on primary colon carcinoma specimen. METHODS: Using a novel ex vivo STxB labeling (ESL) method we studied Gb3 expression in cytological specimens of primary human breast tumors from 107 patients, and in synchronous lymph node metastases from 20 patients. Fluorescent STxB was incubated with fine-needle aspiration (FNA) specimens, and Gb3 expression was evaluated by fluorescence microscopy. Furthermore, 11 patient-derived human breast cancer xenografts (HBCx) were evaluated for expression of Gb3 by ESL and FACS. In addition, the biodistribution of fluorescent STxB conjugate was studied after intravenous injection in a Gb3 positive HBCx model. RESULTS: Gb3 expression was detected in 62 of 107 patients (57.9%), mainly in epithelial tumor cells. Gb3 positivity correlated with estrogen receptor expression (p≤0.01), whereas absence of Gb3 expression in primary tumors was correlated with the presence of lymph node metastases (p≤0.03). 65% of lymph node metastases were Gb3 positive and in 40% of tested patients, we observed a statistically significant increase of metastatic Gb3 expression (p≤0.04). Using concordant ESL and flow cytometry analysis, 6 out of 11 HBCx samples were scored positive. Intravenous injections of fluorescent STxB into HBC xenografted mice showed preferential STxB accumulation in epithelial cells and cells with endothelial morphology of the tumor. CONCLUSION: The enhanced expression of Gb3 in primary breast carcinomas and its lymph node metastases indicate that the development of STxB-based therapeutic strategies is of interest in this pathology. Gb3 expressing HBCx can be used as a model for preclinical studies with STxB conjugates. Finally, the ESL technique on FNA represents a rapid and cost effective method for the stratification of patients in future clinical trials.

Aberrant fucosylation of glycosphingolipids in human hepatocellular carcinoma tissues.

BACKGROUNDS & AIMS: Glycosylation promoting or inhibiting tumour cell invasion and metastasis is of crucial importance in current cancer research. Tumour-associated carbohydrate antigens are predominantly expressed on the tumour cell surface. Glycosphingolipids (GSLs) are members of the family. To perform glycosphingolipidomic assays on neutral GSLs obtained from solid hepatocellular carcinoma (HCC) tissues and paired peritumoural tissues by linear ion trap quadrupole-electrospray ionization mass spectrometry. METHODS: Qualitative and quantitative analysis of fucosylated neutral GSLs was performed in the positive ion mode on the LTQ-XL mass spectrometer and MALDI-TOF-MS. RESULTS: A group of fucosylated neutral GSLs in HCC was found to be expressed higher in the tumour tissues, as their proportion in total cellular GSLs was 3.3-fold higher in the tumour tissues than in the peritumoural tissues (P < 0.01). Moreover, qualitative analysis of the aberrant fucosylated GSLs were completed, and seven types of fucosylated GSLs that contained terminal Fuca2Gal- structure were identified by mass spectrometry. CONCLUSIONS: Our results may lead to improved immunotherapy of HCC and contribute to understanding the role of aberrant fucosylated GSLs in the development and progress of HCC in following studies.