Thomsen-Friedenreich Antigen Research Articles

Fully Synthetic TF-Based Self-Adjuvanting Vaccine Simultaneously Triggers iNKT Cells and Mincle and Protects Mice against Tumor Development.

The Thomsen-Friedenreich (TF) antigen has proven to be a promising target for developing novel therapeutic cancer vaccines. Here, a new strategy that TF antigen covalently coupled with KRN7000 and vizantin was developed. The resulting three-component vaccine KRN7000-TF-vizantin simultaneously triggers invariant natural killer T (iNKT) cells and macrophage-inducible C-type lectin (Mincle) signaling pathways, eliciting much stronger TF-specific immune responses than glycoprotein vaccine TF-KLH/alum and the corresponding two-component conjugate vaccines TF-KRN7000 and TF-vizantin. The analysis of IgG isotypes and the secretion of cytokines revealed that KRN7000-TF-vizantin induced Th1/Th2 mixed immune responses, where Th1 was dominant. In vivo experiments demonstrated that KRN7000-TF-vizantin increased the survival rate and survival time of tumor-challenged mice, and surviving mice rejected further tumor attacks without any additional treatment. This work demonstrates that covalently coupled KRN7000 and vizantin could serve as a promising TF-based vaccine carrier for antitumor immune therapy, and KRN7000-TF-vizantin features great potential to be a vaccine candidate.

Jacalin-Curcumin Complex Sensitizes the Breast Cancer MDA-MB-231 Cell Line.

Protein-drug interactions are crucial for understanding drug delivery and cell functions. Jacalin is a suitable molecule for such targeting, as it specifically recognizes the tumor-associated Thomsen-Friedenreich (TF) antigen that is expressed on the glycosylated proteins in cancer cells. The present paper describes the interaction of curcumin and jacalin, a possible carrier molecule for the delivery of antitumor drugs due to its ability to recognize tumor cells. Our results have shown that both steady-state fluorescence and fluorescent labelling of jacalin are two reliable methods to determine jacalin-curcumin interactions. The affinity of jacalin for curcumin is consistently within the micromolar range (using fluorescence and microscale thermophoresis) showing high-affinity binding of the complex. In vitro experiments on triple-negative breast cancer MDA-MB-231 cells indicated inhibition of cell growth after treating with the jacalin-curcumin complex for 48 h. The cell survival fraction was significantly reduced to 50% after combined treatment. In this paper, we report for the first time about the jacalin-curcumin interaction. We quantified this unique biomolecular interaction and gathered additional information on the binding event. We observed that the jacalin-curcumin complex inhibits the proliferation of the triple-negative breast cancer MDA-MB-231 cells.

CAR-Ts redirected against the Thomsen-Friedenreich antigen CD176 mediate specific elimination of malignant cells from leukemia and solid tumors.

Chimeric antigen receptor-engineered T cells (CAR-Ts) are investigated in various clinical trials for the treatment of cancer entities beyond hematologic malignancies. A major hurdle is the identification of a target antigen with high expression on the tumor but no expression on healthy cells, since "on-target/off-tumor" cytotoxicity is usually intolerable. Approximately 90% of carcinomas and leukemias are positive for the Thomsen-Friedenreich carbohydrate antigen CD176, which is associated with tumor progression, metastasis and therapy resistance. In contrast, CD176 is not accessible for ligand binding on healthy cells due to prolongation by carbohydrate chains or sialylation. Thus, no "on-target/off-tumor" cytotoxicity and low probability of antigen escape is expected for corresponding CD176-CAR-Ts. Using the anti-CD176 monoclonal antibody (mAb) Nemod-TF2, the presence of CD176 was evaluated on multiple healthy or cancerous tissues and cells. To target CD176, we generated two different 2nd generation CD176-CAR constructs differing in spacer length. Their specificity for CD176 was tested in reporter cells as well as primary CD8+ T cells upon co-cultivation with CD176+ tumor cell lines as models for CD176+ blood and solid cancer entities, as well as after unmasking CD176 on healthy cells by vibrio cholerae neuraminidase (VCN) treatment. Following that, both CD176-CARs were thoroughly examined for their ability to initiate target-specific T-cell signaling and activation, cytokine release, as well as cytotoxicity. Specific expression of CD176 was detected on primary tumor tissues as well as on cell lines from corresponding blood and solid cancer entities. CD176-CARs mediated T-cell signaling (NF-κB activation) and T-cell activation (CD69, CD137 expression) upon recognition of CD176+ cancer cell lines and unmasked CD176, whereby a short spacer enabled superior target recognition. Importantly, they also released effector molecules (e.g. interferon-γ, granzyme B and perforin), mediated cytotoxicity against CD176+ cancer cells, and maintained functionality upon repetitive antigen stimulation. Here, CD176L-CAR-Ts exhibited slightly higher proliferation and mediator-release capacities. Since both CD176-CAR-Ts did not react towards CD176- control cells, their response proved to be target-specific. Genetically engineered CD176-CAR-Ts specifically recognize CD176 which is widely expressed on cancer cells. Since CD176 is masked on most healthy cells, this antigen and the corresponding CAR-Ts represent a promising approach for the treatment of various blood and solid cancers while avoiding "on-target/off-tumor" cytotoxicity.

Chemoenzymatic Tagging of Tn/TF/STF Antigens in Living Systems.

Truncated mucin-type O-glycans, such as Tn-associated antigens, are aberrantly expressed biomarkers of cancer, but remain challenging to target. Reactive antibodies to these antigens either lack high-affinity or are prone to antigen escape. Here, we have developed a robust chemoenzymatic strategy for the global labeling of Tn-associated antigens, i.e. Tn (GalNAcα-O-Ser/Thr), Thomsen-Friedenreich (Galβ1-3GalNAcα-O-Ser/Thr, TF) and STF (Neu5Acα2-3Galβ1-3GalNAcα-O-Ser/Thr, STF) antigens, in human whole blood with high efficiency and selectivity. This method relies on the use of the O-glycan sialyltransferase ST6GalNAc1 to transfer a sialic acid-functionalized adaptor to the GalNAc residue of these antigens. By tagging, the adaptor functionalized antigens can be easily targeted by customized strategies such as, but not limited to, chimeric antigen receptor T-Cells (CAR-T). We expect this tagging system to find broad applications in cancer diagnostics and targeting in combination with established strategies.

Engineered Glycan-Binding Proteins for Recognition of the Thomsen–Friedenreich Antigen and Structurally Related Disaccharides

Glycan-binding proteins (GBPs) are widely used reagents for basic research and clinical applications. These reagents allow for the identification and manipulation of glycan determinants without specialized equipment or time-consuming experimental methods. Existing GBPs, mainly antibodies and lectins, are limited, and discovery or creation of reagents with novel specificities is time consuming and difficult. Here, we detail the generation of GBPs from a small, hyper-thermostable DNA-binding protein by directed evolution. Yeast surface display of a variable library of rcSso7d proteins was screened to find variants with selectivity toward the cancer-associated glycan Galβ1-3GalNAcα or Thomsen-Friedenreich antigen and various relevant disaccharides. Characterization of these proteins shows them to have specificities and affinities on par with currently available lectins. The proteins can be readily functionalized with fluorophores or biotin using sortase-mediated ligation to create reagents that prove useful for glycoprotein blotting and cell staining applications. The presented methods for the development of GBPs toward specific saccharides of interest will have great impact on both biomedical and glycobiological research.

Utilization of novel lectin-conjugated Au nanoparticles as Thomsen-Friedenreich onco-antigen target for in vitro cytotoxicity and apoptosis induction in leukemic cell line

Leukemia is a tumor of blood-forming tissues including bone marrow and lymphatic nodes, which comprise biologically distinct subgroups. In the present study, Au NPs-PEG-Lectin was prepared as a drug targeting system for potential Thomsen-Friedenreich antigen (TF-Ag) presented on the surface of leukemic cells to induce cytotoxicity. Gold nanoparticles were prepared using citrate reduction method and conjugated with lectin via SH-PEG-COOH. The conjugate was characterized using UV/Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Zeta potential, and Scanning electron microscopy (SEM) with subsequent applications for cytotoxicity, cell cycle analysis, and apoptosis. Immunophenotypically blood samples from patients with acute lymphoblastic leukemia (ALL) were positively expressed CD45, CD95 dim expression, and low CD176 (TF-Ag) expression. Samples of acute myeloid leukemia (AML) confirmed the expression of all markers. Au NPs-PEG-Lectin conjugate showed an average size of 35.82 nm with zeta potential of −27.33 with accelerated lectin release from the conjugate at acidic pH. Au NPs-PEG-Lectin demonstrated the highest and most significant cytotoxic activity against HL-60 and K562 with IC50 of 132.5 and 314.8 μg mL−1, respectively. Flow cytometric analysis revealed induction of HL-60 cell apoptosis upon conjugate treatment in a dose-dependent pattern up to 51.03 % with no sign of necrosis with cell cycle arrest at G0/G1 phase. HL-60 cells treated with Au NPs-PEG-Lectin exhibited inter-nucleosomal DNA fragmentation. Morphologically, Phospho-Histone/BrdU dual staining indicated that Au NPs-PEG-Lectin initiated HL-60 arrest at G0/G1 phase. Taken together, molecular docking verified the possible interaction between lectin amino acids and different hydroxy groups within TF-Ag forming hydrogen bonds.

EXPRESSION OF ANTIGEN THOMSEN–FRIEDENREICH RECOGNIZED BY Amaranthus leucocarpus LECTIN IN MCF CELLS STIMULATED WITH LIPOPOLYSACCHARIDE

Altered glycosylation is a characteristic of cancer cells, where the overexpression of truncated antigens such as the Thomsen–Friedenreich (TF) antigen (Galβ1, 3GalNAcα1, O-Ser / Thr) The TF antigen is specifically recognized by the Amaranthus leucocarpus (ALL). The MCF-7 breast cancer cell line maintains characteristics similar to differentiated mammary epithelial cells. It has been linked to stimulation of breast cancer cells with lipopolysaccharide, with a higher risk of metastasis in this type of cancer. In this work the expression of TF antigen and moesin in MCF-7 stimulated with LPS at concentration of 20 ng / ml cells was determined, by flow cytometry and fluorescence microscopy. MCF-7 cells express 45% of TF antigen without stimulus with LPS, the expression of the TF antigen decreases with the time of LPS stimulation. Moesin expression increases with LPS stimulation time. A double-positive MCF-7 population for moesin and a TF-antigen of 6.34% were identified by cytometry at 6 hours of LPS stimulation, compared to cells with no stimulus, whose double positivity was 2.86%. Our results suggests that moesin could be Amaranthus leucocarpus receptor in MCF-7 cells.

Therapeutic efficacy of the humanized JAA-F11 anti-Thomsen-Friedenreich antibody constructs H2aL2a and H3L3 in human breast and lung cancer xenograft models.

The Thomsen-Friedenreich antigen (TF-Ag-α) is found on ~85% of human carcinomas but is cryptic on normal tissue. The humanized highly specific hJAA-F11-H2aL2a and -H3L3 antibodies target TF-Ag-α without binding to TF-Ag-beta (found on surface glycolipids of some normal cells). The relative affinity of H3L3 is 17 times that of H2aL2a, which would seem to favor superior efficacy, however, increased affinity can result in less tumor penetration. To assess the potential therapeutic efficacy of these antibodies, four human cancer- mouse xenograft models were treated with H2aL2a and H3L3.The tumor xenograft models used were human non-small cell lung cancer, H520, and small cell lung cancer, HTB171 in nude mice and human triple negative breast cancer, MDA-MB-231 and HCC1806 in SCID mice. H2aL2a significantly decreased tumor growth in both breast and both lung cancer models. H2aL2a showed statistically equal or better efficacy than H3L3 and has superior production capabilities. These results suggest that H2aL2a may be superior as a naked antibody, as an antibody drug conjugate or as a radiolabeled antibody, however the higher affinity of H3L3 may lead to better efficacy in bi-specific therapies in which the binding is decreased due to the presence of only one TF-Ag-α binding site.

Isolipoic acid-linked gold nanoparticles bearing the thomsen friedenreich tumor-associated carbohydrate antigen: Stability and in vitro studies.

We have previously prepared gold nanoparticles (AuNPs) bearing the Thomsen-Friedenreich antigen disaccharide (TFag), a pan-carcinoma, Tumor-Associated Carbohydrate Antigen (TACA), as tools for various assays and biological applications. Conjugation to AuNPs typically involves the use of thiols due to the affinity of sulfur for the gold surface of the nanoparticle. While a use of a single thiol-containing ligand bound to the gold surface is standard practice, several studies have shown that ligands bearing multiple thiols can enhance the strength of the conjugation in a nearly linear fashion. (R)-(+)-α-Lipoic acid (LA), a naturally occurring disulfide-containing organic acid that is used as a cofactor in many enzymatic reactions, has been used as a linker to conjugate various molecules to AuNPs through its branched di-thiol system to enhance nanoparticle stability. We sought to use a similar system to increase nanoparticle stability that was devoid of the chiral center in (R)-(+)-α-lipoic acid. Isolipoic acid, an isomer of LA, where the exocyclic pentanoic acid chain is shifted by one carbon on the dithiolane ring to produce an achiral acid, was thought to act similarly as LA without the risk of any contaminating (L)-(−) isomer. We synthesized AuNPs with ligands of both serine and threonine glycoamino acids bearing the TFag linked to isolipoic acid and examined their stability under various conditions. In addition, these particles were shown to bind to Galectin-3 and inhibit the interaction of Galectin-3 with a protein displaying copies of the TFag. These agents should prove useful in the design of potential antimetastatic therapeutics that would benefit from achiral linkers that are geometrically linear and achiral.

Placental colonization by Fusobacterium nucleatum is mediated by binding of the Fap2 lectin to placentally displayed Gal-GalNAc.

While the existence of an indigenous placental microbiota remains controversial, several pathogens are known to be involved in adverse pregnancy outcomes. Fusobacterium nucleatum is an oral bacterium that is one of several bacteria associated with preterm birth. Oral fusobacteria translocate to the placenta hematogenously; however, the mechanisms localizing them to the placenta remain unclear. Here, using peanut agglutinin, we demonstrate that the level of Gal-GalNAc (Galβ1-3GalNAc; Thomsen Friedenreich antigen) found on trophoblasts facing entering maternal blood rises during gestation and is recognized by the fusobacterial Fap2 Gal-GalNAc lectin. F.nucleatum binding to human and mouse placenta correlates with Gal-GalNAc levels and is reduced upon O-glycanase treatment or with soluble Gal-GalNAc. Fap2-inactivated F.nucleatum shows reduced binding to Gal-GalNAc-displaying placental sections. In a mouse model, intravenously injected Fap2-expressing F.nucleatum, but not a Fap2 mutant, reduces mouse fetal survival by 70%.

Abstract P044: Real-time visualization of tumor cell phenotype and microenvironmental heterogeneity enabled by a hyperspectral fluorescence microendoscope

Abstract Tumor microenvironmental heterogeneity is a major driver of treatment resistance and variability in clinical response to therapy. This heterogeneity arises from variations in cellular phenotypes populating the tumor microenvironment (TME), their spatio-temporal localization and expression of surface markers, often associated with specific biological function – stemness, metabolism, proliferation, immune activation and others. Such features are usually studied through ex vivo immunofluorescence and cytometry to identify cellular phenotypes in TME; however, they cannot be applied in vivo for real time TME analysis. Moreover, current in vivo imaging techniques fail to provide real-time high-resolution visualization of the TME. Routinely employed biopsy tissue sampling through fine needle aspirates is limited by its invasive nature and inability to provide a global and dynamic overview of the TME, thus limiting our ability to study spatiotemporal TME dynamics and identify possible features leading to potentially resistant tumor phenotypes. In this study, we demonstrate the use of a hyperspectral fluorescence microendoscope (HFME) to monitor cellular phenotypes in TME with real-time visualization and high video imaging capability (~17 fps). Current fluorescence video microscopy is limited to simultaneous imaging of no more than 2 molecular markers with potential to be expanded to ~4 markers using dichroic mirrors and point detectors; the HFME demonstrated in this study can currently resolve 6 different molecular markers, simultaneously, using a multichannel linear array detector with potential to expand to 10 or more markers. Using a cocktail of near infra-red fluorophore-antibody conjugates targeted against key molecular (surface) markers of different cells in the TME, we are able to capture real-time TME dynamics at cellular resolution in two pre-clinical models; 1) a xenograft orthotopic mouse model of peritoneal carcinomatosis (disseminated metastases within the abdominal cavity) and imaging epidermal growth factor receptor (EGFR), CD44, CA125 (MUC16), transferrin receptor, Thomsen-Friedenreich carbohydrate antigen (T antigen), and CD45, 2) A syngeneic immunocompetent KPC cell line implanted mouse model of pancreatic ductal adenocarcinoma and imaging CD3, CD4, CD8a and CD45. Imaging on these tumor models was performed pre- and post-sub-therapeutic verteporfin (benzoporphyrin derivative monoacid A) photodynamic therapy (PDT, a cytotoxic light-based therapy). PDT treatment resulted in reduction of cancer cell burden, immune cell infiltration and alterations in their relative spatial localization. The results were confirmed by histopathological validation and ex vivo immunofluorescence staining of tumor tissue sections. Collectively, these results demonstrate the capability of HFME to image cancer cell phenotypes and the tumor microenvironment, in real time in live mice. With the ability to monitor cancer growth and treatment effects at a cellular level, HFME can potentially assist in customizing therapies in a patient-specific manner. Citation Format: Mohammad A. Saad, Bryan Q. Spring, Akilan Palanisami, Eric M. Kercher, Ryan T. Lang, Jason Sutin, Zhiming Mai, Tayyaba Hasan. Real-time visualization of tumor cell phenotype and microenvironmental heterogeneity enabled by a hyperspectral fluorescence microendoscope [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P044.

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

AbstractImmune 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.

3'-Sulfo-TF Antigen Determined by GAL3ST2/ST3GAL1 Is Essential for Antitumor Activity of Fungal Galectin AAL/AAGL.

Many lectins have been reported to have antitumor activities; identifying the glycan ligands in tumor cells of lectins is crucial for lectin clinical application. An edible mushroom galectin, Agrocybe aegerita lectin (AAL/AAGL), that has a high antitumor activity has been reported. In this paper, based on the glycan array data, it is showed that the Thomsen-Friedenreich antigen (TF antigen)-related O-glycans were found to be highly correlated with the antitumor activity of AAL/AAGL. Further glycosyltransferase quantification suggested that the ratio between GAL3ST2 and ST3GAL1 (GAL3ST2/ST3GAL1), which determined the 3′-sulfo-TF expression level, was highly correlated with the antitumor activity of AAL/AAGL. Overexpressing the enzyme of GAL3ST2 in HL60 and HeLa cell lines could increase the growth inhibition ratio of AAL/AAGL from 22.7 to 43.9% and 27.8 to 39.1%, respectively. However, ST3GAL1 in Jurkat cells could decrease the growth inhibition ratio from 44.7 to 35.6%. All the data suggested that the 3′-sulfo-TF antigen is one of the main glycan ligands that AAL/AAGL recognizes in tumor cells. AAL/AAGL may potentially serve as a reagent for cancer diagnosis and a targeted therapy for the 3′-sulfo-TF antigen.

Streptococcus Pneumoniae-Associated Hemolytic Uremic Syndrome in the Era of Pneumococcal Vaccine

Streptococcus pneumoniae-associated hemolytic uremic syndrome (Sp-HUS) is a serious complication of invasive pneumococcal disease that is associated with increased mortality in the acute phase and morbidity in the long term. Recently, Sp-HUS definition has undergone revision and cases are categorized as definite, probable, and possible, based on less invasive serological investigations that evaluate Thomsen-Friedenreich crypt antigen (T-antigen) activation. In comparison to the pre-vaccine era, Sp-HUS incidence seems to be decreasing after the introduction of 7-serotype valence and 13-serotype valence pneumococcal vaccines in 2000 and 2010, respectively. However, Sp-HUS cases continue to occur secondary to vaccine failure and emergence of non-vaccine/replacement serotypes. No single hypothesis elucidates the molecular basis for Sp-HUS occurrence, although pneumococcal neuraminidase production and formation of T-antigen antibody complexes on susceptible endothelial and red blood cells continues to remain the most acceptable explanation. Management of Sp-HUS patients remains supportive in nature and better outcomes are being reported secondary to earlier recognition, better diagnostic tools and improved medical care. Recently, the addition of eculizumab therapy in the management of Sp-HUS for control of dysregulated complement activity has demonstrated good outcomes, although randomized clinical trials are awaited. A sustained pneumococcal vaccination program and vigilance for replacement serotypes will be the key for persistent reduction in Sp-HUS cases worldwide.

Sialylation of HCV E2 Glycoprotein-Specific and Natural Anti-Glycan (TF, αGal) Antibodies as Signatures of Liver Damage

The E2 glycoprotein is the target of broadly neutralizing antibodies against Hepatitis C Virus (HCV). There is evidence that the HCV E2-specific antibody glycosylation profile is associated with hepatic fibrosis progression. The main aim of this study was to compare the sialylation of E2-specific and naturally occurring antiglycan Abs to determine whether their combination could be beneficial for the non-invasive evaluation of hepatic damage. Fifty-eight patients with various stages of hepatic fibrosis or without were tested. The sialylation of HCV E2 glycoprotein-specific antibodies (E2-Abs), the Thomsen-Friedenreich antigen- and αGal glycotope-specific antibodies (TF-Abs, αGal-Abs) was analysed using the ELISA platform. The level of IgG Abs and their reactivity to Sialospecific Sambucus Nigra Lectin (SNA) were determined and changes in Abs sialylation were analysed based on the stage of liver fibrosis, HCV genotype and antiviral therapy efficacy. The late stage of liver Fibrosis (F4) was characterized by dramatically decreased E2-Ab SNA reactivity unlike stages with no fibrosis (P=0.003) and stages F1–F3 (P=0.0007). In contrast, antiglycan Abs showed an increased sialylation. In multiple regression analysis, the combination of E2 and TF-Abs sialylation patterns gave a significant advantage in assessing liver damage. A high rate of discrimination between F0 and F4 stages of fibrosis as well as between F1–F3 and F4 was obtained (ACC=0.948 and ACC=0.90, respectively). Thus, the combined analysis of disease-specific and natural Abs sialylation can remarkably enhance the clinical value of the approach in the non-invasive evaluation of hepatic damage.

Increased ERK phosphorylation and caveolin-1 expression on K562 human chronic myelogenous leukemia cells by jacalin, a dietary plant lectin.

The potential antitumor effects of jacalin, the plant lectin that specifically recognizes the tumor-associated Thomsen-Friedenreich antigen has been extensively studied. We had earlier reported jacalin to be mitogenic to K562, the Bcr-Abl expressing erythroleukemia cell line. The dearth of studies highlighting the proliferative effects of jacalin and other lectins motivated us to unveil the mechanism underlying the mitogenic effects of jacalin. Caveolin-1 (cav-1) is an integral membrane protein, known to play a crucial role in cell signaling, lipid transport, and membrane trafficking. The role of cav-1 in tumorigenesis is considered to be controversial as it can suppress as well as promote tumor growth, depending on the cellular context. In the present study, we propose that cav-1 plays the central role in the mitogenic effects of jacalin on the K562 cells. In accordance, the mRNA, as well as protein expression of cav-1 was found to be upregulated in the jacalin-treated K562 cells as compared to the untreated control. Further, jacalin stimulation also increased the phosphorylation of ERK and Akt. The rationale that leads to the initial conjecture about cav-1 was that the sequence of jacalin possesses a cav-1-binding site.

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 and Targeting of Thomsen-Friedenreich and IL1RAP Antigens on Chronic Myeloid Leukemia Stem Cells Using Bi-Specific Antibodies.

IntroductionQuiescent leukemia stem cells (LSCs) play a major role in therapeutic resistance and disease progression of chronic myeloid leukemia (CML). LSCs belong to the primitive population; CD34+CD38-Lin-, which does not distinguish normal hematopoietic stem cells (HSC) from CML LSCs. Because Thomsen–Friedenreich/CD176 antigen is expressed on CD34+ HSC and IL1RAP is tightly correlated to BCR-ABL expression, we sought to increase the specificity towards LSC by using additional biomarkers.MethodsWe evaluated the co-expression of both antigens on CD34+ peripheral blood mononuclear cells (PBMCs) from both healthy volunteers and CML patients, using flow cytometry. Then, we used site-directed mutagenesis to induce knob-in-hole mutations in the human IgG heavy chain and the human lambda light chain to generate the bi-specific antibody (Bis-Ab) TF/RAP that binds both antigens simultaneously. We measured complement-directed cytotoxicity (CDC) in CML samples with the Bis-Ab by flow cytometry.ResultsIn contrast to healthy volunteers, CML samples displayed a highly significant co-expression of CD176 and IL1RAP. When either a double-positive cell line or CML samples were treated with increasing doses of Bis-Ab, increased binding and CDC was observed indicating co-operative binding of the Bis-Ab as compared to monoclonal antibodies.DiscussionThese results show that the bi-specific antibody is capable of targeting IL1RAP+ and CD176+ cell population among CML PBMCs, but not corresponding normal cells in CDC assay. We hereby offer a novel strategy for the depletion of CML stem cells from the bulk population in clinical hematopoietic stem cell transplantation.

Plasmacytoid Dendritic Cells Surveil Megakaryocyte Sialic Acid to Regulate Thrombopoiesis

The Thomsen-Friedenreich antigen (TF-antigen) occurs during exposure of the underlying Core-1 disaccharide (Gal-beta(1,3)GalNAc) through the loss of its capping sialic acid (Sia). Exposure of the cryptic TF-antigen occurs during inflammation, during acute infections with influenza viruses or bacteria, in malignancies, and is associated with thrombocytopenia. Exposure of the TF-antigen on circulating blood cells, including platelets and red blood cells (RBC), can lead to severe thrombocytopenia or hemolysis in hemolytic uremic syndrome and other immune diseases. Recent data suggest that altered Sia may cause platelet destruction because treatment with the sialidase inhibitor Tamiflu increases platelet count in healthy and thrombocytopenic patients. In humans, genetic mutations involving Sia synthesis and transport, and atypical cell surface sialylation, unrelated to any genetic mutation, are associated with reduced platelet count, supporting the role of Sia in regulating platelet count. Immune cells, including classical dendritic cells (cDCs), plasmacytoid dendritic cells (pDCs), and subsets of T cells (CD8+, CD4+, and Treg cells) can also affect immune thrombocytopenia pathogenesis. Like many other immune cells, cDCs, and pDCs express Siglecs (sialic-acid-binding immunoglobulin-like lectins), which often contain immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that act as immunosuppressors. Whether BM immune cells monitor MKs via glycan-lectin receptors, including Siglecs and Sia interactions, to control platelet production is unclear. To investigate the role of the TF-antigen in thrombopoiesis, we generated St3gal1MK-KO mice (Pf4-Cre) that display increased TF-antigen specifically in megakaryocytes (MK) and platelets. St3gal1MK-KO mice developed significant thrombocytopenia, but had normal platelet half-life, suggesting that the TF-antigen affected BM thrombopoiesis. In vitro MK maturation and proplatelet production from primary ST3Gal1MK-KO mouse BM cells were also normal, pointing to extrinsic factors in the BM environment affecting thrombopoiesis. Platelet counts of St3gal1MK-KO mice were restored to wild-type levels by 1) crossing St3gal1MK-KO mice with Jak3KO mice that have impaired of lymphoid cell development, 2) by treatment with anti-inflammatory dexamethasone, and 3) treatment with a depleting anti-CD4 antibody. Immunofluorescence staining of the St3gal1MK-KO BM revealed proplatelet structures positive for GPIba+ and the TF-antigen, being infiltrated by mononuclear cells resembling lymphocytes. We speculated that immune cells surveil megakaryocytes to control thrombopoiesis. Bulk RNAseq of CD4+ cells in St3gal1MK-KO BM confirmed a population bias for Type I interferon (IFN-I)-releasing pDCs, a cell type regulated by unique sialic acid binding lectins (Siglecs). Inhibition of IFN-I activity, by a blocking receptor antibody improved platelet counts in St3gal1MK-KO mice. Co-cultures of pDCs with MKs show inhibited pro-platelet formation when TF-antigen is present on MKs with elevated IFN-I levels. Gene set enrichment analysis of BM pDCs single cell RNASeq (scRNAseq) data further confirmed that TF-antigen exposure by MKs up-regulates IFN-I transcripts. scRNAseq also reveals a new population of immune cells with pDC transcript signature and concomitant upregulation of immunoglobulin re-arrangement gene transcripts Igkc and Ighm. In conclusion, the data shows that recognition of aberrant MK sialylation by pDCs regulates thrombopoiesis through IFN-I secretion. Disclosures No relevant conflicts of interest to declare.

Crystal structures of peanut lectin in the presence of synthetic β-N- and β-S-galactosides disclose evidence for the recognition of different glycomimetic ligands.

Carbohydrate-lectin interactions are involved in important cellular recognition processes, including viral and bacterial infections, inflammation and tumor metastasis. Hence, structural studies of lectin-synthetic glycan complexes are essential for understanding lectin-recognition processes and for the further design of promising chemotherapeutics that interfere with sugar-lectin interactions. Plant lectins are excellent models for the study of the molecular-recognition process. Among them, peanut lectin (PNA) is highly relevant in the field of glycobiology because of its specificity for β-galactosides, showing high affinity towards the Thomsen-Friedenreich antigen, a well known tumor-associated carbohydrate antigen. Given this specificity, PNA is one of the most frequently used molecular probes for the recognition of tumor cell-surface O-glycans. Thus, it has been extensively used in glycobiology for inhibition studies with a variety of β-galactoside and β-lactoside ligands. Here, crystal structures of PNA are reported in complex with six novel synthetic hydrolytically stable β-N- and β-S-galactosides. These complexes disclosed key molecular-binding interactions of the different sugars with PNA at the atomic level, revealing the roles of specific water molecules in protein-ligand recognition. Furthermore, binding-affinity studies by isothermal titration calorimetry showed dissociation-constant values in the micromolar range, as well as a positive multivalency effect in terms of affinity in the case of the divalent compounds. Taken together, this work provides a qualitative structural rationale for the upcoming synthesis of optimized glycoclusters designed for the study of lectin-mediated biological processes. The understanding of the recognition of β-N- and β-S-galactosides by PNA represents a benchmark in protein-carbohydrate interactions since they are novel synthetic ligands that do not belong to the family of O-linked glycosides.