Major Cell Surface Research Articles

CD44 and its implication in neoplastic diseases.

CD44, a nonkinase single span transmembrane glycoprotein, is a major cell surface receptor for many other extracellular matrix components as well as classic markers of cancer stem cells and immune cells. Through alternative splicing of CD44 gene, CD44 is divided into two isoforms, the standard isoform of CD44 (CD44s) and the variant isoform of CD44 (CD44v). Different isoforms of CD44 participate in regulating various signaling pathways, modulating cancer proliferation, invasion, metastasis, and drug resistance, with its aberrant expression and dysregulation contributing to tumor initiation and progression. However, CD44s and CD44v play overlapping or contradictory roles in tumor initiation and progression, which is not fully understood. Herein, we discuss the present understanding of the functional and structural roles of CD44 in the pathogenic mechanism of multiple cancers. The regulation functions of CD44 in cancers-associated signaling pathways is summarized. Moreover, we provide an overview of the anticancer therapeutic strategies that targeting CD44 and preclinical and clinical trials evaluating the pharmacokinetics, efficacy, and drug-related toxicity about CD44-targeted therapies. This review provides up-to-date information about the roles of CD44 in neoplastic diseases, which may open new perspectives in the field of cancer treatment through targeting CD44.

MAdCAM-1 co-stimulation combined with retinoic acid and TGF-β induces blood CD8+ T cells to adopt a gut CD101+ TRM phenotype

Resident memory T cells (TRMs) help control local immune homeostasis and contribute to tissue protective immune responses. The local cues that guide their differentiation and localization are poorly defined. We demonstrate that MAdCAM-1, a ligand for the gut homing receptor α4β7 integrin, in the presence of retinoic acid and TGF-β provide a costimulatory signal that induces blood CD8+ T cells to adopt a TRM -like phenotype. These cells express CD103 (integrin αE) and CD69, the two major TRM cell surface markers, along with CD101. They also express CCR5, CCR9 and α4β7, three receptors associated with gut homing. A subset also express E-cadherin, a ligand for αEβ7. Fluorescent lifetime imaging indicated an αEβ7 and E-cadherin cis interaction on the plasma membrane. This report advances our understanding of the signals that drive the differentiation of CD8+ T cells into TRMs and provides a means to expand these cells in vitro, thereby affording an avenue to generate more effective tissue specific immunotherapies.

Abstract 335: BD®OMICS-Guard Sample Preservation Buffer preserves mRNA and cell surface epitopes for single-cell sequencing applications

Abstract Multiomics has emerged as a powerful tool to derive insights on the transcriptomic and proteomic data of human cancer samples and preclinical models of blood and tumor malignancies. Due to the sensitivity of sequencing techniques, sample quality and stability can potentially interfere with results. BD® OMICS-Guard Sample Preservation Buffer (SPB), a novel preservation solution, allows cells and bulk tissues to be stored for up to 72h at 4 °C, while reliably capturing the true biology of cells. This reagent gently preserves mRNA and protein integrity without the traditional cross-linking and harsh fixatives, increasing flexibility and expanding options for study designs. Here, we showcase the preservation properties ofthis reagent for multiomic readouts in human cells and mouse tissue across three different timepoints.BD® OMICS-Guard SPB’s preservation of mRNA from human PBMC and bulk mouse spleen tissues wasevidenced by WTA for differentially expressed genes in samples preserved for 24, 48 and 72h comparedto fresh/unpreserved controls. Protein epitope preservation was demonstrated via CITE-Seq assay withthe BD® AbSeq Human Immune Discovery Panel (human PBMC) or a 30-plex panel of anti-mouse BD®AbSeq Antibody-Conjugated Oligonucleotides (mouse splenocytes) and confirmed with multicolor flowcytometry. For both sample types, high correlation R2 values (>0.87) were calculated for the preservation time points versus fresh controls. Concomitant flow cytometry analysis of major cell types and surface protein expression was consistent with sequencing data for all samples. Specificity and sensitivity of major cell type markers and cell surface proteins across cell types and time is visualized in the BD®AbSeq Antibody-generated heatmap for each sample. Additionally, BD® OMICS-Guard SPB maintains mRNA integrity in targeted gene assays and VDJ full-length assays, suggesting robust preservation of select transcripts readily interrogated in single-cell profiling. Batch effect across time and technical replicates for both sample types was limited with the BD Rhapsody™ HT Xpress System and delineated by donor/sample specific tSNEs across time. We demonstrate that the BD® OMICS-Guard Sample Preservation Buffer effectively preserves both mRNA and surface proteins for single-cell experiments. BD® OMICS-Guard SPB allows the convenient preservation of samples such as tumor biopsies to be collected and stored for up to 72h at 4 °C while maintaining mRNA and protein integrity, allowing the flexibility for precious samples to be potentially shipped to a central sequencing site to ensure standardized sequencing workflows. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD and BD Rhapsody are trademarks of Becton, Dickinson and Company or its affiliates. © 2023 BD. All rights reserved. Citation Format: Samuel X. Shi, Cynthia Sakofsky, Zhiqi Zhang, Samatha Vadrevu, Xiaoshan Shi, Hye-Won Song, Aruna Ayer. BD®OMICS-Guard Sample Preservation Buffer preserves mRNA and cell surface epitopes for single-cell sequencing applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 335.

Increased CD44 expression in primary meningioma: its clinical significance and association with peritumoral brain edema.

CD44 is a major cell surface receptor involved in cell adhesion and migration. The overexpression of CD44 is a poor prognostic factor in many neoplasms, including meningiomas. The aim of this study was to investigate the association between CD44 gene expression and clinical signatures of primary meningiomas. CD44 gene expression was quantitatively evaluated by snap freezing tumor tissues obtained from 106 patients with primary meningioma. The relationships between CD44 expression and clinical signatures of meningiomas, including histological malignancy, tumor volume, and peritumoral brain edema (PTBE), were analyzed. PTBE was assessed using the Steinhoff classification (SC) system (from SC 0 to SC III). CD44 gene expression in WHO grade 2 and 3 meningiomas was significantly higher than that in grade 1 meningiomas. In addition, CD44 expression increased with the severity of PTBE. Particularly, among the grade 1 meningiomas or small-sized tumors (maximum tumor diameter < 43 mm), CD44 expression in tumors with severe PTBE (SC II or III) was significantly higher than that in tumors without or with mild PTBE (SC 0 or I). Multivariate logistic regression analysis also revealed that overexpression of CD44 was an independent significant factor of severe PTBE development in primary meningiomas. In addition to tumor cell aggressiveness, CD44 expression promotes the development of PTBE in meningioma. Since PTBE is a strong factor of tumor-related epilepsy or cognitive dysfunction in patients with meningioma, CD44 is thus a potential therapeutic target in meningioma with PTBE.

Mifepristone Modulates the Polarization of Th1/Th2 by Inducing the Mature of Monocyte‐Derived Dendritic Cells

AbstractAs an anti‐implantation contraception medicine, mifepristone can alter the development of the endometrium. In contrast, little research is performed to decipher the effect of mifepristone on Th1/Th2 immune homeostasis in the endometrium. CD14+ monocytes derived from healthy donors are cultured with interleukin (IL)‐4 and granulocyte macrophage‐colony stimulating factor to induce monocyte‐derived dendritic cells (DCs), which are further incubated with mifepristone. Mifepristone incubation could induce up‐regulated CD83 and major histocompatibility complex cell surface receptor staining on monocyte‐derived DCs. Functionally, mifepristone incubation could enhance the allostimulatory capacity of DCs to promote the proliferation of allogeneic T cells with up‐regulated Th1 cell proportion and down‐regulated Th2 cell proportion. On the other side, increased Th1 cytokines release (interferon‐γ, tumor necrosis factor α), inhibited Th2 cytokines release (IL‐10, IL‐4), promoted T‐Box transcription factor 21 transcription, and inhibited GATA binding protein 3 transcription are also identified. Mifepristone‐stimulated DCs could alter the Th1/Th2 transcription balance to favor Th1 skewing inflammatory environment.

Porcine extraintestinal pathogenic Escherichia coli delivers two serine protease autotransporters coordinately optimizing the bloodstream infection.

Extraintestinal pathogenic Escherichia coli (ExPEC) is one of the leading causes of bloodstream infections in a broad spectrum of birds and mammals, thus poses a great threat to public health, while its underlying mechanism causing sepsis is not fully understood. Here we reported a high virulent ExPEC strain PU-1, which has a robust ability to colonize within host bloodstream, while induced a low level of leukocytic activation. Two serine protease autotransporters of Enterobacteriaceae (SPATEs), VatPU-1 and TshPU-1, were found to play critical roles for the urgent blood infection of strain PU-1. Although the Vat and Tsh homologues have been identified as virulence factors of ExPEC, their contributions to bloodstream infection are still unclear. In this study, VatPU-1 and TshPU-1 were verified to interact with the hemoglobin (a well-known mucin-like glycoprotein in red blood cell), degrade the mucins of host respiratory tract, and cleave the CD43 (a major cell surface component sharing similar O-glycosylated modifications with other glycoprotein expressed on leukocytes), suggesting that these two SPATEs have the common activity to cleave a broad array of mucin-like O-glycoproteins. These cleavages significantly impaired the chemotaxis and transmigration of leukocytes, and then inhibited the activation of diverse immune responses coordinately, especially downregulated the leukocytic and inflammatory activation during bloodstream infection, thus might mediate the evasion of ExPEC from immune clearance of blood leukocytes. Taken together, these two SPATEs play critical roles to cause a heavy bacterial load within bloodstream via immunomodulation of leukocytes, which provides a more comprehensive understanding how ExPEC colonize within host bloodstream and cause severe sepsis.

The lipidome of Crithidia fasiculataand its plasticity.

Crithidia fasiculata belongs to the trypanosomatidae order of protozoan parasites, bearing close relation to other kinetoplastid parasites such as Trypanosoma brucei and Leishmania spp. As an early diverging lineage of eukaryotes, the study of kinetoplastid parasites has provided unique insights into alternative mechanisms to traditional eukaryotic metabolic pathways. Crithidia are a monogenetic parasite for mosquito species and have two distinct lifecycle stages both taking place in the mosquito gut. These consist of a motile choanomastigote form and an immotile amastigote form morphologically similar to amastigotes in Leishmania. Owing to their close relation to Leishmania, Crithidia are a growing research tool, with continuing interest in its use as a model organism for kinetoplastid research with the added benefit that they are non-pathogenic to humans and can be grown with no special equipment or requirements for biological containment. Although comparatively little research has taken place on Crithidia, similarities to other kinetoplast species has been shown in terms of energy metabolism and genetics. Crithidia also show similarities to kinetoplastids in their production of the monosaccharide D-arabinopyranose similar to Leishmania, which is incorporated into a lipoarabinogalactan a major cell surface GPI-anchored molecule. Additionally, Crithidia have been used as a eukaryotic expression system to express proteins from other kinetoplastids and potentially other eukaryotes including human proteins allowing various co- and post-translational protein modifications to the recombinant proteins. Despite the obvious usefulness and potential of this organism very little is known about its lipid metabolism. Here we describe a detailed lipidomic analyses and demonstrate the possible placidity of Crithidia's lipid metabolis. This could have important implications for biotechnology approaches and how other kinetoplastids interact with, and scavenge nutrients from their hosts.

Glycosyltransferases EXTL2 and EXTL3 cellular balance dictates heparan sulfate biosynthesis and shapes gastric cancer cell motility and invasion

Heparan sulfate (HS) proteoglycans (HSPGs) are abundant glycoconjugates in cells' glycocalyx and extracellular matrix. By acting as scaffolds for protein-protein interactions, HSPGs modulate extracellular ligand gradients, cell signaling networks, and cell-extracellular matrix crosstalk. Aberrant expression of HSPGs and enzymes involved in HSPG biosynthesis and processing has been reported in tumors, with impact in cancer cell behavior and tumor microenvironment properties. However, the roles of specific glycosyltransferases in the deregulated biosynthesis of HSPGs are not fully understood. In this study, we established glycoengineered gastric cancer cell models lacking either exostosin-like glycosyltransferase 2 (EXTL2) or EXTL3 and revealed their regulatory roles in both HS and chondroitin sulfate (CS) biosynthesis and structural features. We showed that EXTL3 is key for initiating the synthesis of HS chains in detriment of CS biosynthesis, intervening in the fine-tuned balance of the HS/CS ratio in cells, while EXTL2 functions as a negative regulator of HS biosynthesis, with impact over the glycoproteome of gastric cancer cells. We demonstrated that KO of EXTL2 enhanced HS levels along with concomitant upregulation of Syndecan-4, which is a major cell surface carrier of HS. This aberrant HS expression profile promoted a more aggressive phenotype, characterized by higher cellular motility and invasion, and impaired activation of Ephrin type-A 4cell surface receptor tyrosine kinase. Our findings uncover the biosynthetic roles of EXTL2 and EXTL3 in the regulation of cancer cell GAGosylation and proteoglycans expression and unravel the functional consequences of aberrant HS/CS balance in cellular malignant features.

097 Epidermal integrin α3β1 is a regulator of cytokine, CSF-1, and of crosstalk from keratinocytes to dermal macrophages

As the major cell surface receptors for the extracellular matrix, integrins regulate adhesion and migration, and have been shown to drive tumor growth and progression. Our earlier findings have identified integrin α3β1 as a key regulator of the keratinocyte secretome and of the skin tumor microenvironment, indicating its potential efficacy as a therapeutic target. Previous mass spectrometric analysis of conditioned medium from immortalized keratinocytes indicated that α3β1 positively regulates colony-stimulating factor 1 (CSF-1), a secreted cytokine that is a primary regulator of macrophage differentiation, proliferation, and survival.

The cwp66 Gene Affects Cell Adhesion, Stress Tolerance, and Antibiotic Resistance in Clostridioides difficile.

ABSTRACTClostridioides difficile is a Gram-positive, spore-forming anaerobic bacteria that is one of the leading causes of antibiotic-associated diarrhea. The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. We constructed a cwp66 gene deletion mutant using Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 (CRISPR-Cpf1) system. The phenotypic and transcriptomic changes of the Δcwp66 mutant compared with the wild-type (WT) strain were studied. The deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the Δcwp66 mutant is more sensitive than the WT strain to clindamycin, ampicillin, and erythromycin but more resistant than the latter to vancomycin and metronidazole. Moreover, mannitol utilization capability in the Δcwp66 mutant was lost. Comparative transcriptomic analyses indicated that (i) 22.90-fold upregulation of cwpV gene and unable to express gpr gene were prominent in the Δcwp66 mutant; (ii) the cwp66 gene was involved in vancomycin resistance of C. difficile by influencing the expression of d-Alanine-d-Alanine ligase; and (iii) the mannose/fructose/sorbose IIC and IID components were upregulated in Δcwp66 mutant. The present work deepens our understanding of the contribution of the cwp66 gene to cell adhesion, stress tolerance, antibiotic resistance, and mannitol transportation of C. difficile.IMPORTANCE The cell wall protein 66 gene (cwp66) encodes a cell wall protein, which is the second major cell surface antigen of C. difficile. Although immunological approaches, such as antibodies and purified recombinant proteins, have been implemented to study the role of Cwp66 in cell adhesion, no deletion mutant of the cwp66 gene has yet been characterized. The current study provides direct evidence that the cwp66 gene serves as a major adhesion in C. difficile, and also suggested that deletion of the cwp66 gene led to the decrease of cell adhesive capacity, cell motility, and stresses tolerance (to Triton X-100, acidic environment, and oxidative stress). Interestingly, the antibiotic resistance and carbon source utilization profiles of the Δcwp66 mutant were significantly changed. These phenotypes were detrimental to the survival and pathogenesis of C. difficile in the human gut and may shed light on preventing C. difficile infection.

Polymeric siRNA delivery targeting integrin-β1 could reduce interactions of leukemic cells with bone marrow microenvironment

Uncontrolled proliferation of the myeloid cells due to BCR-ABL fusion has been successfully treated with tyrosine kinase inhibitors (TKIs), which improved the survival rate of Chronic Myeloid Leukemia (CML) patients. However, due to interactions of CML cells with bone marrow microenvironment, sub-populations of CML cells could become resistant to TKI treatment. Since integrins are major cell surface molecules involved in such interactions, the potential of silencing integrin-β1 on CML cell line K562 cells was explored using short interfering RNA (siRNA) delivered through lipid-modified polyethyleneimine (PEI) polymers. Reduction of integrin-β1 in K562 cells decreased cell adhesion towards human bone marrow stromal cells and to fibronectin, a major extracellular matrix protein for which integrin-β1 is a primary receptor. Interaction of K562 cells with fibronectin decreased the sensitivity of the cells to BCR-ABL siRNA treatment, but a combinational treatment with integrin-β1 and BCR-ABL siRNAs significantly reduced colony forming ability of the cells. Moreover, integrin-β1 silencing enhanced the detachment of K562 cells from hBMSC samples (2 out of 4 samples), which could make them more susceptible to TKIs. Therefore, the polymeric-siRNA delivery targeting integrin-β1 could be beneficial to reduce interactions with bone marrow microenvironment, aiding in the response of CML cells to therapeutic treatment.

Nuclear Syndecan-1 Regulates Epithelial-Mesenchymal Plasticity in Tumor Cells.

Simple SummaryThe major cause of death in cancer is that tumor cells metastasize (i.e., spread to vital organs such as the lungs and liver). Our knowledge of how metastasis occurs is incomplete and consequently, there is a lack of therapeutic strategies that target metastasis. Recent data show that inflammatory signals may influence tumor cells to undergo an identity switch, whereby they develop metastatic properties. Understanding how this identity switch is regulated is important to develop novel therapeutics. Recently, syndecan-1, a protein found on the surface of various cells, was shown to regulate the identity switch. In this study, we report that re-localization of syndecan-1 from the cell surface to the nucleus contributes to its capacity to regulate the identity switch in tumor cells. These results identify nuclear syndecan-1 as a regulator of the identity switch and open up to further studies to elucidate the mechanisms involved.Tumor cells undergoing epithelial-mesenchymal transition (EMT) lose cell surface adhesion molecules and gain invasive and metastatic properties. EMT is a plastic process and tumor cells may shift between different epithelial-mesenchymal states during metastasis. However, how this is regulated is not fully understood. Syndecan-1 (SDC1) is the major cell surface proteoglycan in epithelial cells and has been shown to regulate carcinoma progression and EMT. Recently, it was discovered that SDC1 translocates into the cell nucleus in certain tumor cells. Nuclear SDC1 inhibits cell proliferation, but whether nuclear SDC1 contributes to the regulation of EMT is not clear. Here, we report that loss of nuclear SDC1 is associated with cellular elongation and an E-cadherin-to-N-cadherin switch during TGF-β1-induced EMT in human A549 lung adenocarcinoma cells. Further studies showed that nuclear translocation of SDC1 contributed to the repression of mesenchymal and invasive properties of human B6FS fibrosarcoma cells. The results demonstrate that nuclear translocation contributes to the capacity of SDC1 to regulate epithelial-mesenchymal plasticity in human tumor cells and opens up to mechanistic studies to elucidate the mechanisms involved.

Epidermal Integrin α3β1 Regulates Tumor-Derived Proteases BMP-1, Matrix Metalloprotease-9, and Matrix Metalloprotease-3

As the major cell surface receptors for the extracellular matrix, integrins regulate adhesion and migration and have been shown to drive tumor growth and progression. Previous studies showed that mice lacking integrin α3β1 in the epidermis fail to form skin tumors during two-step chemical tumorigenesis, indicating a protumorigenic role for α3β1. Furthermore, genetic ablation of α3β1 in established skin tumors caused their rapid regression, indicating an essential role in the maintenance of tumor growth. In this study, analysis of immortalized keratinocyte lines and their conditioned media support a role for α3β1 in regulating the expression of several extracellular proteases of the keratinocyte secretome, namely BMP-1, matrix metalloprotease (MMP)-9, and MMP-3. Moreover, immunofluorescence revealed reduced levels of each protease in α3β1-deficient tumors, and RNA in situ hybridization showed that their expression was correspondingly reduced in α3β1-deficient tumor cells in vivo. Bioinformatic analysis confirmed that the expression of BMP1, MMP9, and MMP3 genes correlate with the expression of ITGA3 (gene encoding the integrin α3 subunit) in human squamous cell carcinoma and that high ITGA3 and MMP3 associate with poor survival outcome in these patients. Overall, our findings identify α3β1 as a regulator of several proteases within the secretome of epidermal tumors and as a potential therapeutic target.

Using a Phosphoproteomic Screen to Profile Early Changes During HCMV Infection of Human Monocytes.

During the binding and infection of monocytes, HCMV binds to at least two major cell surface receptors/receptor families: the epidermal growth factor receptor (EGFR) to initiate downstream signaling through the EGFR-PI3K pathway, and to β1- and β3-integrins to initiate downstream signaling through the integrin-c-Src pathway (Nogalski et al. PLoS Pathog 9:e1003463, 2013; Chan et al. Proc Natl Acad Sci U S A 106:22369-22374, 2009; Kim et al. Proc Natl Acad Sci U S A 113:8819-8824, 2016; Wang et al. Nature 424:456-461, 2003; Wang et al. Nat Med 11:515-521, 2005; Yurochko et al. Proc Natl Acad Sci U S A 89:9034-9038, 1992). Signaling through these receptors can occur rapidly with phosphorylation observed as early as 15s after EGF-EGFR interaction, for example (Alvarez-Salamero et al. Front Immunol 8:938, 2017). The ability to detect signaling and the consequences of that signaling are critical for our understanding of how HCMV-receptor engagement promotes infection and modulates the biology of different target cells. In this chapter we describe how we used an ELISA-based antibody platform to perform an assessment of the rapid phosphorylation events that occur in monocytes following infection. This assay can be adapted to other infection systems, time points and cell types as needed. Together, we examined via an ELISA-based antibody array a phosphoproteomic screen to search for potential phosphorylated proteins that might influence HCMV infection.

Prominin-1-Radixin axis controls hepatic gluconeogenesis by regulating PKA activity.

Prominin-1 (Prom1) is a major cell surface marker of cancer stem cells, but its physiological functions in the liver have not been elucidated. We analyzed the levels of mRNA transcripts in serum-starved primary WT (Prom1+/+ ) and KO (Prom1-/- ) mouse hepatocytes using RNA sequencing (RNA-seq) data, and found that CREB target genes were downregulated. This initial observation led us to determine that Prom1 deficiency inhibited cAMP response element-binding protein (CREB) activation and gluconeogenesis, but not cyclic AMP (cAMP) accumulation, in glucagon-, epinephrine-, or forskolin-treated liver tissues and primary hepatocytes, and mitigated glucagon-induced hyperglycemia. Because Prom1 interacted with radixin, Prom1 deficiency prevented radixin from localizing to the plasma membrane. Moreover, systemic adenoviral knockdown of radixin inhibited CREB activation and gluconeogenesis in glucagon-treated liver tissues and primary hepatocytes, and mitigated glucagon-elicited hyperglycemia. Based on these results, we conclude that Prom1 regulates hepatic PKA signaling via radixin functioning as an A kinase-anchored protein (AKAP).

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

In Vitro Studies to Define the Cell-Surface and Intracellular Targets of Polyarginine-Conjugated Sodium Borocaptate as a Potential Delivery Agent for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) requires pharmaceutical innovations and molecular-based evidence of effectiveness to become a standard cancer therapeutic in the future. Recently, in Japan, 4-borono-L-phenylalanine (BPA) was approved as a boron agent for BNCT against head and neck (H&N) cancers. H&N cancer appears to be a suitable target for BPA-BNCT, because the expression levels of L-type amino acid transporter 1 (LAT1), one of the amino acid transporters responsible for BPA uptake, are elevated in most cases of H&N cancer. However, in other types of cancer including malignant brain tumors, LAT1 is not always highly expressed. To expand the possibility of BNCT for these cases, we previously developed poly-arginine peptide (polyR)-conjugated mercaptoundecahydrododecaborate (BSH). PolyR confers the cell membrane permeability and tumor selectivity of BSH. However, the molecular determinants for the properties are not fully understood. In this present study, we have identified the cluster of differentiation 44 (CD44) protein and translational machinery proteins as a major cell surface target and intracellular targets of BSH-polyR, respectively. CD44, also known as a stem cell-associated maker in various types of cancer, is required for the cellular uptake of polyR-conjugated molecules. We showed that BSH-polyR was predominantly delivered to a CD44High cell population of cancer cells. Once delivered, BSH-polyR interacted with the translational machinery components, including the initiation factors, termination factors, and poly(A)-biding protein (PABP). As a proof of principle, we performed BSH-polyR-based BNCT against glioma stem-like cells and revealed that BSH-polyR successfully induced BNCT-dependent cell death specifically in CD44High cells. Bioinformatics analysis indicated that BSH-polyR would be suitable for certain types of malignant tumors. Our results shed light on the biochemical properties of BSH-polyR, which may further contribute to the therapeutic optimization of BSH-BNCT in the future.

Integrin-Mediated Adhesion in the Unicellular Holozoan Capsaspora owczarzaki

In animals, cell-matrix adhesions are essential for cell migration, tissue organization, and differentiation, which have central roles in embryonic development [1-6]. Integrins are the major cell surface adhesion receptors mediating cell-matrix adhesion in animals. They are heterodimeric transmembrane proteins that bind extracellular matrix (ECM) molecules on one side and connect to the actin cytoskeleton on the other [7]. Given the importance of integrin-mediated cell-matrix adhesion in development of multicellular animals, it is of interest to discover when and how this machinery arose during evolution. Comparative genomic analyses have shown that core components of the integrin adhesome pre-date the emergence of animals [8-11]; however, whether it mediates cell adhesion in non-metazoan taxa remains unknown. Here, we investigate cell-substrate adhesion in Capsaspora owczarzaki, the closest unicellular relative of animals with the most complete integrin adhesome [11, 12]. Previous work described that the life cycle of C.owczarzaki (hereafter, Capsaspora) includes three distinct life stages: adherent; cystic; and aggregative [13]. Using an adhesion assay, we show that, during the adherent life stage, C.owczarzaki adheres to surfaces using actin-dependent filopodia. We show that integrin β2 and its associated protein vinculin localize as distinct patches in the filopodia. We also demonstrate that substrate adhesion and integrin localization are enhanced by mammalian fibronectin. Finally, using a specific antibody for integrin β2, we inhibited cell adhesion to a fibronectin-coated surface. Our results suggest that adhesion to the substrate in C.owczarzaki is mediated by integrins. We thus propose that integrin-mediated adhesion pre-dates the emergence of animals.

Host syndecan-1 promotes listeriosis by inhibiting intravascular neutrophil extracellular traps.

Heparan sulfate proteoglycans (HSPGs) are at the forefront of host-microbe interactions. Molecular and cell-based studies suggest that HSPG-pathogen interactions promote pathogenesis by facilitating microbial attachment and invasion of host cells. However, the specific identity of HSPGs, precise mechanisms by which HSPGs promote pathogenesis, and the in vivo relevance of HSPG-pathogen interactions remain to be determined. HSPGs also modulate host responses to tissue injury and inflammation, but functions of HSPGs other than facilitating microbial attachment and internalization are understudied in infectious disease. Here we examined the role of syndecan-1 (Sdc1), a major cell surface HSPG of epithelial cells, in mouse models of Listeria monocytogenes (Lm) infection. We show that Sdc1-/- mice are significantly less susceptible to both intragastric and intravenous Lm infection compared to wild type (Wt) mice. This phenotype is not seen in Sdc3-/- or Sdc4-/- mice, indicating that ablation of Sdc1 causes a specific gain of function that enables mice to resist listeriosis. However, Sdc1 does not support Lm attachment or invasion of host cells, indicating that Sdc1 does not promote pathogenesis as a cell surface Lm receptor. Instead, Sdc1 inhibits the clearance of Lm before the bacterium gains access to its intracellular niche. Large intravascular aggregates of neutrophils and neutrophil extracellular traps (NETs) embedded with antimicrobial compounds are formed in Sdc1-/- livers, which trap and kill Lm. Lm infection induces Sdc1 shedding from the surface of hepatocytes in Wt livers, which is directly associated with the decrease in size of intravascular aggregated NETs. Furthermore, administration of purified Sdc1 ectodomains or DNase inhibits the formation of intravascular aggregated neutrophils and NETs and significantly increases the liver bacterial burden in Sdc1-/- mice. These data indicate that Lm induces Sdc1 shedding to subvert the activity of Sdc1 ectodomains to inhibit its clearance by intravascular aggregated NETs.

Characterization of Macrophage Galactose-type Lectin (MGL) ligands in colorectal cancer cell lines

BackgroundThe Ca2+-dependent C-type lectin receptor Macrophage Galactose-type Lectin (MGL) is highly expressed by tolerogenic dendritic cells (DC) and macrophages. MGL exhibits a high binding specificity for terminal alpha- and beta-linked GalNAc residues found in Tn, sTn and LacdiNAc antigens. These glycan epitopes are often overexpressed in colorectal cancer (CRC), and, as such, MGL can be used to discriminate tumor from the corresponding healthy tissues. Moreover, the high expression of MGL ligands is associated with poor disease-free survival in stage III of CRC tumors. Nonetheless, the glycoproteins expressed by tumor cells that are recognized by MGL have hitherto remained elusive. MethodsUsing a panel of three CRC cell lines (HCT116, HT29 and LS174T), recapitulating CRC diversity, we performed FACS staining and pull-down assays using a recombinant soluble form of MGL (and a mutant MGL as control) combined with mass spectrometry-based (glyco)proteomics. ResultsHCT116 and HT29, but not LS174T, are high MGL-binding CRC cell lines. On these cells, the major cell surface binding proteins are receptors (e.g. MET, PTK7, SORL1, PTPRF) and integrins (ITGB1, ITGA3). From these proteins, several N- and/or O-glycopeptides were identified, of which some carried either a LacdiNAc or Tn epitope. ConclusionsWe have identified cell surface MGL-ligands on CRC cell lines. General significanceAdvances in (glyco)proteomics have led to identification of candidate key mediators of immune-evasion and tumor growth in CRC.