E-selectin Ligand Research Articles

E-selectin affinity glycoproteomics reveals neuroendocrine proteins and the secretin receptor as a poor-prognosis signature in colorectal cancer.

Colorectal cancer (CRC) cells express sialylated Lewis antigens (sLe), crucial for metastasis via E-selectin binding. However, these glycoepitopes lack cancer specificity, and E-selectin-targeted glycoproteins remain largely unknown. Here, we established a framework for identifying metastasis-linked glycoproteoforms. More than 70% of CRC tumors exhibited overexpression of sLeA/X, yet without discernible associations with metastasis or survival. However, The Cancer Genome Atlas (TCGA) analysis unveiled differing expression patterns of sLeA/X-related glycogenes correlating with disease severity, indicating context-dependent regulation by distinct glycosyltransferases. Deeper exploration of metastatic tumor sialoglycoproteome identified nearly 600 glycoproteins, greatly expanding our understanding of the metastasis-related glycoproteome. These glycoproteins were linked to cell adhesion, oncogenic pathways, and neuroendocrine functions. Using an in-house algorithm, the secretin receptor (SCTR) emerged as a top-ranked targetable glycoprotein. Tumor screening confirmed SCTR's association with poor prognosis and metastasis, with N-glycosylation adding cancer specificity to this glycoprotein. Prognostic links were reinforced by TCGA-based investigations. In summary, SCTR, a relatively unknown CRC glycoprotein, holds potential as a biomarker of poor prognosis and as an E-selectin ligand, suggesting an unforeseen role in disease dissemination. Future investigations should focus on this glycoprotein's biological implications for clinical applications.

Glycoengineered extracellular vesicles released from antibacterial hydrogel facilitate diabetic wound healing by promoting angiogenesis.

Diabetic wounds have become a global healthcare burden owing to impaired angiogenesis and persistent infections. Extracellular vesicles (EVs) can improve diabetic wounds, though their targeting ability is limited. In this study, we investigated the performance of a novel hydrogel dressing comprised of gelatin methacryloyl, glycoengineered EVs, and polylysine in treating infected diabetic wounds. High-throughput single-cell RNA sequencing (scRNA-seq) and immunofluorescence staining revealed that E-selectin (SELE) levels were higher in diabetic wounds than in non-diabetic wounds. Mesenchymal stromal cells (MSCs) were transfected with a lentivirus containing fucosyltransferase VII (FUT7) and a CD63-P19-Nluc vector to enhance the expression of sialyl Lewis X (sLeX), the ligand of E-selectin, on the surface of EVs (s-EVs) derived from transfected MSCs (s-MSCs). s-EVs can target human umbilical vein endothelial cells (HUVECs) under lipopolysaccharide stimulation and promote the function of stimulated HUVECs in vitro. To promote and sustain the release of s-EVs, we fabricated a gelatin methacryloyl (Gel)/poly-L-lysine methacryloyl (PL)-5 hydrogel with good antibacterial ability, biocompatibility and mechanical properties. In a mouse experiment, s-EV@Gel/PL-5 exhibited excellent angiogenesis and anti-inflammatory abilities and further promoted the healing of infected diabetic wounds. Our findings demonstrated the potential of the s-EV@Gel/PL-5 hydrogel in the clinical treatment of diabetic infectious wounds.

Association of circulating cytokine levels and tissue-infiltrating myeloid cells with achalasia: results from Mendelian randomization and validation through clinical characteristics and single-cell RNA sequencing.

Achalasia is a rare motility disorder of the esophagus often accompanied by immune dysregulation, yet specific underlying mechanisms remain poorly understood. We utilized Mendelian randomization (MR) to explore the causal effects of cytokine levels on achalasia, with cis-expression/protein quantitative trait loci (cis-eQTLs/pQTLs) for 47 cytokines selected from a genome-wide association study (GWAS) meta-analysis and GWAS data for achalasia obtained from FinnGen. For cytokines significantly linked to achalasia, we analyzed their plasma concentrations and expression differences in the lower esophageal sphincter (LES) using enzyme-linked immunosorbent assay and single-cell RNA sequencing (scRNA-seq) profiling, respectively. We further employed bioinformatics approaches to investigate underlying mechanisms. We revealed positive associations of circulating Eotaxin, macrophage inflammatory protein-1b (MIP1b), soluble E-selectin (SeSelectin) and TNF-related apoptosis-inducing ligand (TRAIL) with achalasia. When combining MR findings with scRNA-seq data, we observed upregulation of TRAIL (OR = 2.70, 95% CI, 1.20-6.07), encoded by TNFSF10, in monocytes and downregulation of interleukin-1 receptor antagonist (IL-1ra) (OR = 0.70, 95% CI 0.59-0.84), encoded by IL1RN, in FOS_macrophages in achalasia. TNFSF10high monocytes in achalasia displayed activated type I interferon signaling, and IL1RNlow FOS_macrophages exhibited increased intercellular communications with various lymphocytes, together shaping the proinflammatory microenvironment of achalasia. We identified circulating Eotaxin, MIP1b, SeSelectin and TRAIL as potential drug targets for achalasia. TNFSF10high monocytes and IL1RNlow macrophages may play a role in the pathogenesis of achalasia.

Enforced HCELL Expression Enhances Bone Marrow Stromal Cells Homing and Amelioration of Cerebral Ischemia-Reperfusion Injury via induction of PGE2.

Ischemic stroke (IS) is a significant and potentially life-threatening disease with limited treatment options, often resulting in severe disability. Bone marrow stromal cells (BMSCs) transplantation has exhibited promising neuroprotection following cerebral ischemia-reperfusion injury (CIRI). However, the effectiveness is hindered by their low homing rate when administered through the vein. In this study, we aimed to enhance the homing ability of BMSCs through lentivirus transfection to express fucosyltransferase 7. This glycosylation engineered CD44 on BMSCs to express hematopoietic cell E-selectin/L-selectin ligand (HCELL), which is the most potent E-selectin ligand. Following enforced HCELL expression, the transplantation of BMSCs was then evaluated in a middle cerebral artery occlusion (MCAO) model. Results showed that HCELL+BMSCs significantly ameliorated neurological deficits and reduced the volume of cerebral infarction. Furthermore, the transplantation led to a decrease in apoptosis by up-regulating BCL-2 and down-regulating BAX, also reduced the mRNA levels of inflammatory factors, such as interleukin-1β (IL-1β), IL-2, IL-6 and tumor necrosis factor-alpha (TNF-α) in the ischemic brain tissue. Notably, enforced HCELL expression facilitated the migration of BMSCs towards cerebral ischemic lesions and their subsequent transendothelial migration through the up-regulation of PTGS-2, increased production of PGE2 and activation of VLA-4. In summary, our study demonstrates that transplantation of HCELL+BMSCs effectively alleviates CIRI, and that enforced HCELL expression enhances the homing of BMSCs to cerebral ischemic lesions and their transendothelial migration via PTGS-2/PGE2/VLA-4. These findings indicate that enforced expression of HCELL on BMSCs could serve as a promising therapeutic strategy for the treatment of ischemic stroke.

Strengthening an Intramolecular Non-Classical Hydrogen Bond to Get in Shape for Binding.

In this research article, we report on the strengthening of a non-classical hydrogen bond (C-H⋅⋅⋅O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E-selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre-organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre-organization, the stabilization by a non-classical hydrogen bond between the H-C5 of l-fucose and the ring oxygen O5 of the neighboring d-galactose moiety is essential and yields 7.4 kJ mol-1. This effect could be further strengthened by replacing l-fucose by 6,6,6-trifluoro-l-fucose leading to an improved non-classical H-bond of 14.9 kJ mol-1, i.e., an improved pre-organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR-shifts of the H-C5Fuc, by X-ray diffraction analysis of glycomimetics co-crystallized with E-selectin as well as by isothermal titration calorimetry. Furthermore, the electron-withdrawing character of the CF3-group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid-stability, a prerequisite for gastrointestinal stability, could be substantially improved.

Chemoenzymatic Synthesis of Tri-antennary N-Glycans Terminating in Sialyl-Lewisx Reveals the Importance of Glycan Complexity for Influenza A Virus Receptor Binding.

Sialyl-Lewisx (SLex) is involved in immune regulation, human fertilization, cancer, and bacterial and viral diseases. The influence of the complex glycan structures, which can present SLex epitopes, on binding is largely unknown. We report here a chemoenzymatic strategy for the preparation of a panel of twenty-two isomeric asymmetrical tri-antennary N-glycans presenting SLex-Lex epitopes on either the MGAT4 or MGAT5 arm that include putative high-affinity ligands for E-selectin. The N-glycans were prepared starting from a sialoglycopeptide isolated from egg yolk powder and took advantage of inherent substrate preferences of glycosyltransferases and the use of 5'-diphospho-N-trifluoracetylglucosamine (UDP-GlcNHTFA) that can be transferred by branching N-acetylglucosaminyltransferases to give, after base treatment, GlcNH2-containing glycans that temporarily disable an antenna from enzymatic modification. Glycan microarray binding studies showed that E-selectin bound equally well to linear glycans and tri-antennary N-glycans presenting SLex-Lex. On the other hand, it was found that hemagglutinins (HA) of H5 influenza A viruses (IAV) preferentially bound the tri-antennary N-glycans. Furthermore, several H5 HAs preferentially bound to N-glycan presenting SLex on the MGAT4 arm. SLex is displayed in the respiratory tract of several avian species, demonstrating the relevance of investigating the binding of, among others IAVs, to complex N-glycans presenting SLex.

Combining 5-azacitidine with the E-selectin antagonist uproleselan is an effective strategy to augment responses in myelodysplasia and acute myeloid leukaemia.

The interaction of acute myeloid leukaemic (AML) blasts with the bone marrow (BM) microenvironment is a major determinant governing disease progression and resistance to treatment. The constitutive expression of E-selectin in the vascular compartment of BM, a key endothelial cell factor, directly mediates chemoresistance via E-selectin ligand/receptors. Despite the success of hypomethylating agent (HMA)-containing regimens to induce remissions in older AML patients, the development of primary or secondary resistance is common. We report that following treatment with 5-azacitidine, promoter regions regulating the biosynthesis of the E-selectin ligands, sialyl Lewis X, become further hypomethylated. The resultant upregulation of these gene products, in particular α(1,3)-fucosyltransferase VII (FUT7) and α(2,3)-sialyltransferase IV (ST3GAL4), likely causes functional E-selectin binding. When combined with the E-selectin antagonist uproleselan, the adhesion to E-selectin is reversed and the survival of mice transplanted with AML cells is prolonged. Finally, we present clinical evidence showing that BM myeloid cells from higher risk MDS and AML patients have the potential to bind E-selectin, and these cells are more abundant in 5-azacitidine-non-responsive patients. The collective data provide a strong rationale to evaluate 5-azacitidine in combination with the E-selectin antagonist, uproleselan, in this patient population.

Optimizing cryopreservation conditions for use of fucosylated human mesenchymal stromal cells in anti-inflammatory/immunomodulatory therapeutics.

Mesenchymal stem/stromal cells (MSCs) are being increasingly used in cell-based therapies due to their broad anti-inflammatory and immunomodulatory properties. Intravascularly-administered MSCs do not efficiently migrate to sites of inflammation/immunopathology, but this shortfall has been overcome by cell surface enzymatic fucosylation to engender expression of the potent E-selectin ligand HCELL. In applications of cell-based therapies, cryopreservation enables stability in both storage and transport of the produced cells from the manufacturing facility to the point of care. However, it has been reported that cryopreservation and thawing dampens their immunomodulatory/anti-inflammatory activity even after a reactivation/reconditioning step. To address this issue, we employed a variety of methods to cryopreserve and thaw fucosylated human MSCs derived from either bone marrow or adipose tissue sources. We then evaluated their immunosuppressive properties, cell viability, morphology, proliferation kinetics, immunophenotype, senescence, and osteogenic and adipogenic differentiation. Our studies provide new insights into the immunobiology of cryopreserved and thawed MSCs and offer a readily applicable approach to optimize the use of fucosylated human allogeneic MSCs as immunomodulatory/anti-inflammatory therapeutics.

Comparison of sialylated and fucosylated N-glycans attached to Asn 6 and Asn 41 with different roles in hyaluronan and proteoglycan link protein 1 (HAPLN1)

Hyaluronan and proteoglycan link protein 1 (HAPLN1) is an extracellular matrix protein stabilizing interactions between hyaluronan and proteoglycan. Although HAPLN1 is being investigated for various biological roles, its N-glycosylation is poorly understood. In this study, the structure of N-glycopeptides of trypsin-treated recombinant human HAPLN1 (rhHAPLN1) expressed from CHO cells were identified by nano-liquid chromatography-tandem mass spectrometry. A total of 66 N-glycopeptides were obtained, including 16 and 12 N-glycans at sites Asn 6 (located in the N-terminal region) and Asn 41 (located in the Ig-like domain, which interacts with proteoglycan), respectively. The quantities (%) of each N-glycan relative to the totals (100 %) at each site were calculated. Tri- and tetra-sialylation (to resist proteolysis and extend half-life) were more abundant at Asn 6, and di- (core- and terminal-) fucosylation (to increase binding affinity and stability) and sialyl-Lewis X/a epitope (a major ligand for E-selectin) were more abundant at Asn 41. These results indicate that N-glycans attached to Asn 6 (protecting HAPLN1) and Asn 41 (supporting molecular interactions) play different roles in HAPLN1. This is the first study of site-specific N-glycosylation in rhHAPLN1, which will be useful for understanding its molecular interactions in the extracellular matrix.

Podophyllotoxin-loaded PEGylated E-selectin peptide conjugate targeted cancer site to enhance tumor inhibition and reduce side effect

E-selectin, which is highly expressed in vascular endothelial cells near tumor and get involved in the all tumor growth steps: occurrence, proliferation and metastasis, is considered as a promise targeted protein for antitumor drug discovery. Herein, we would like to report the design, preparation and the anticancer evaluation of the peptide-PEG-podophyllotoxin conjugate(PEG-Pep-PODO), in which the short peptide (CIELLQAR) was used as the E-selectin ligand for the targeting purpose and the PEG portion the molecule got the conjugate self-assembled to form a water soluble nanoparticle. In vitro release study showed that the conjugated and entrapped PODO could be released simultaneously in the presence of GSH (highly expressed in tumor environmental conditions) and the GSH would catalyze the break of the disufur bond which linked of the PODO and the peptide-PEG portion of the conjugate. Cell adhesion test of the PEG-Pep-PODO indicated that E-selectin ligand peptide CIELLQAR could get specifically and efficiently binding to the E-selectin expressing human umbilical vein endothelial cells (HUVEC). In vitro cytotoxicity assay further revealed that PEG-Pep-PODO significantly improved the selectivity of PEG-Pep-PODO for killing the tumor cells and normal cells compared with PODO solution formulation. More importantly, the in vivo experiment demonstrated that the conjugate would accumulate of the PODO payload in tumor through targeting endothelial cells in the tumor microenvironment, which resulted in the much improved in vivo inhibition of tumor growth, intratumoral microvessel density, and decreased systemic toxicity of this nanoparticle over the free PODO. Furthermore, this water soluble conjugate greatly improved the pharmacokinetic properties of the mother molecule.

Identification of potential classes of glycoligands mediating dynamic endothelial adhesion of human tumor cells.

One critical step of metastasis formation is the extravasation of circulating tumor cells from the bloodstream. This process requires the dynamic interaction of cell adhesion molecules like E-selectin on endothelial cells with carbohydrate ligands on tumor cells. To characterize these glycans in a comprehensible approach, the rolling, tethering, and firm adhesion of nine human tumor cell lines on human umbilical vein endothelial cells was analyzed using laminar flow adhesion assays. The tumor cell lines were grouped into three subsets by their canonical E-selectin ligand status (sialyl-Lewis A and X +/+, -/+, -/-) and their adhesiveness was compared after enzymatic, pharmacologic, chemical treatment or antibody blockade of the tumor cells or endothelial cells, respectively. Tumor cells were also screened regarding their glycosyltransferase expression profile. We found that although E-selectin and terminal α2,3-sialic acid largely determined firm adhesion, adhesive events did not exclusively depend on the presence of sialyl-Lewis A and/or sialyl-Lewis X. Nevertheless, two of the three sialyl-Lewis A/X-/- tumor cells additionally or fully depended on vascular cell adhesion molecule-1 for firm adhesion. The significance of O-GalNAc- and N-glycans for adhesion varied remarkably among the tumor cells. The sialyl-Lewis A/X+/+ subset showed glycoprotein-independent adhesion, suggesting a role of glycolipids as well. All sialyl-Lewis A/X-/- tumor cells lacked FUT3 and FUT7 expression as opposed to sialyl-Lewis A/X+/+ or -/+ cell lines. In summary, the glycans on tumor cells mediating endothelial adhesion are not as much restricted to sialyl-Lewis A /X as previously assumed. The present study specifically suggests α2,3-linked sialic acid, O-GalNAc glycans, glycosphingolipids, and FUT3/FUT7 products as promising targets for future studies.

FLT3 inhibitors upregulate CXCR4 and E-selectin ligands via ERK suppression in AML cells and CXCR4/E-selectin inhibition enhances anti-leukemia efficacy of FLT3-targeted therapy in AML

FLT3 inhibitors upregulate CXCR4 and E-selectin ligands via ERK suppression in AML cells and CXCR4/E-selectin inhibition enhances anti-leukemia efficacy of FLT3-targeted therapy in AML

Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop.

The lack of Tcell infiltrates is a major obstacle to effective immunotherapy in cancer. Conversely, the formation of tumor-associated tertiary-lymphoid-like structures (TA-TLLSs), which are the local site of humoral and cellular immune responses against cancers, is associated with good prognosis, and they have recently been detected in immune checkpoint blockade (ICB)-responding patients. However, how these lymphoid aggregates develop remains poorly understood. By employing single-cell transcriptomics, endothelial fate mapping, and functional multiplex immune profiling, we demonstrate that antiangiogenic immune-modulating therapies evoke transdifferentiation of postcapillary venules into inflamed high-endothelial venules (HEVs) via lymphotoxin/lymphotoxin beta receptor (LT/LTβR) signaling. In turn, tumor HEVs boost intratumoral lymphocyte influx and foster permissive lymphocyte niches for PD1- and PD1+TCF1+ CD8 Tcell progenitors that differentiate into GrzB+PD1+ CD8T effector cells. Tumor-HEVs require continuous CD8 and NK cell-derived signals revealing that tumor HEV maintenance is actively sculpted by the adaptive immune system through a feed-forward loop.

Uproleselan Added to Cladribine Plus Low Dose Cytarabine (LDAC) in Patients with Treated Secondary Acute Myeloid Leukemia (TS-AML)

Background Treated secondary AML (TS-AML), arising after prior HMA-treated MDS, is associated with very poor prognosis (Complete Remission [CR] rates 15-30% and median Overall Survival [OS] 6-8 months). E-selectin ligand is highly expressed on AML blasts in the leukemic microenvironment and may be a marker of cell survival and resistance to chemotherapy. Exposure of leukemic blasts to HMAs has been shown to increase their expression of E-selectin ligand. Uproleselan is an E-selectin antagonist that overcomes resistance to chemotherapy in AML (Barbier, Nat Commun 2020). We sought to study the combination of low-intensity chemotherapy with Cladribine + LDAC (CLAD/LDAC) with uproleselan to overcome local and microenvironmental resistance and improve outcomes in this difficult subset. Methods This is Phase Ib/II clinical trial (NCT04848974) to evaluate the safety, tolerability, and explore the efficacy of Uproleselan added to Cladribine and LDAC. A 3+3 dose-escalation approach was implemented to evaluate 2 different dose levels for Cladribine (CLAD)+ LDAC; each 4-week cycle consists of Uproleselan (at a fixed dose of 800mg intravenously [IV]) added to IV CLAD 5 days (3.75mg/m2 and 5mg/m2; level -1 and 1, respectively) and subcutaneous LDAC twice daily 10 days (15mg, and 20mg; level -1 and 1, respectively) during induction; consolidation was similar except it was with 3-days of CLAD, for up to 6 cycles. Pts aged ≥18 years with a diagnosis of TS-AML with adequate organ function, who have not received therapy for their AML were enrolled. TS-AML is defined as AML arising from a previously treated myeloid neoplasm. Presence of the E-selectin ligand was assessed using Flow Cytometry (FC). Results 10 pts have been treated, with 9 pts currently evaluable: 6 (67%) were male and the median age was 68 years (range, 58-80); at the start of therapy, the median bone marrow blasts were 33% (1-78%), median WBC was 2.2x109/L (0.6-20.1), median platelets were 18x109/L (4-305), and median creatinine was 0.98mg/dL (0.67-1.52). Pts had received a median of 1 (1-2) treatments prior to AML transformation. Prior diagnoses were: therapy-related Myelodysplastic Syndrome (t-MDS), Chronic Myelomonocytic Leukemia (CMML), MDS and MDS/MPN in 3 (33%), 3 (33%), 2 (22%) and 1 (11%) respectively; all had received HMA, 5 (56%) additionally had Ven and 3 (33%) had stem cell transplantation (SCT) prior to enrolling. All pts had unfavorable features by ELN 2017. The most frequent mutations were:SXL1, TP53 and TET2 in 4 pts each (44%), SRSF2 and NRAS in 3 patients each (33%) and SETBP1, RUNX1 and EZH2 in 2 patients each (22%). 6 pts were evaluable for E-selectin ligand expression; the median expression was 59% (42%-95%) and median MFI was 20.5 (13-262). The most common SAEs were ≥ grade 3 neutropenic fever (70%), (including 2 grade 5 events), grade 3 bleeding (10%), and grade 2 thrombosis (5%) (Table 1). There were no dose-limiting toxicities observed on dose levels -1 or 1. Two pts treated on dose level -1 die during the study follow-up due to sepsis within the first 4-weeks during induction. Median time to 0.5x109/L neutrophil and 50x109/L platelets recovery was 29 (17-39) and 38 (33-48) days respectively. The median follow-up is 4+ months. 8 pts were evaluable for response at the time of analysis. The ORR was 62% (5/8), including 2 (25%) PR, 1 (13%) CRi, 1 (13%) CRp and 1 (13%) MLFS. There was a reduction in BM blasts in 6 pts (75%) (Figure 1). 5 pts were taken off protocol due to progression, 2 for death, 1 for allogeneic SCT and 1 continued onto maintenance in remission. The one pt who achieved negative MRD, underwent SCT and is still alive. Median OS and EFS were 4.7 and 1.3 months respectively; 4-month RFS (CRi, CRp, and MLFS) was 67%. The median cycles received was 1 (1-3), median cycles at which the best response was achieved was 1 (1-2). The 4-month OS were 100% and 60% among responders vs. non-responders, respectively (p=0.27), and the 4-month EFS were 50% and 0% respectively (p=0.01). The ORR was 40% (2/5) (p=0.85) and 33% (1/3) (p=0.85) among pts who had prior Ven exposure or prior SCT, respectively. Conclusions The combination of Cladribine + LDAC with Uproleselan was overall well tolerated with few treatment-related AEs. The combination produced an ORR of 62% in a high-risk, refractory population whose prognosis is very dismal. The relationship of E-selectin ligand expression, response to treatment, and outcomes is being analyzed. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Enforced mesenchymal stem cell tissue colonization counteracts immunopathology

Mesenchymal stem/stromal cells (MSCs) are distributed within all tissues of the body. Though best known for generating connective tissue and bone, these cells also display immunoregulatory properties. A greater understanding of MSC cell biology is urgently needed because culture-expanded MSCs are increasingly being used in treatment of inflammatory conditions, especially life-threatening immune diseases. While studies in vitro provide abundant evidence of their immunomodulatory capacity, it is unknown whether tissue colonization of MSCs is critical to their ability to dampen/counteract evolving immunopathology in vivo. To address this question, we employed a murine model of fulminant immune-mediated inflammation, acute graft-versus-host disease (aGvHD), provoked by donor splenocyte-enriched full MHC-mismatched hematopoietic stem cell transplant. aGvHD induced the expression of E-selectin within lesional endothelial beds, and tissue-specific recruitment of systemically administered host-derived MSCs was achieved by enforced expression of HCELL, a CD44 glycoform that is a potent E-selectin ligand. Compared to mice receiving HCELL− MSCs, recipients of HCELL+ MSCs had increased MSC intercalation within aGvHD-affected site(s), decreased leukocyte infiltrates, lower systemic inflammatory cytokine levels, superior tissue preservation, and markedly improved survival. Mechanistic studies reveal that ligation of HCELL/CD44 on the MSC surface markedly potentiates MSC immunomodulatory activity by inducing MSC secretion of a variety of potent immunoregulatory molecules, including IL-10. These findings indicate that MSCs counteract immunopathology in situ, and highlight a role for CD44 engagement in unleashing MSC immunobiologic properties that maintain/establish tissue immunohomeostasis.

Refining the migration and engraftment of short-term and long-term HSCs by enhancing homing-specific adhesion mechanisms

AbstractIn contrast to the short-term (ST) CD34+ stem cells, studies have suggested that long-term (LT) hematopoietic stem cells (HSCs) found in the CD34− stem cell pool have trouble migrating and engrafting when introduced through IV. To understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by ST and LT-HSCs to migrate to the bone marrow(BM). Specifically focusing on murine ST-HSCs (Flk2−CD34+) and LT-HSCs (Flk2−CD34−), we observed a distinctive expression pattern of BM homing effectors necessary for the first step, namely sialyl Lewis-X (sLex) (ligand for E-selectin), and the second step, namely CXCR4 chemokine receptor (receptor for SDF-1). sLex expression was higher on Flk2−CD34+ ST-HSCs (>60%) compared with Flk2−CD34− LT-HSCs (<10%), which correlated to binding to E-selectin. Higher concentrations of CXCR4 were observed on Flk2−CD34+ ST-HSCs compared with Flk2−CD34− LT-HSCs. Interestingly, the expression of CD26, a peptidase known to deactivate chemokines (ie, SDF-1), was higher on Flk2−CD34− LT-HSCs. Given that both E-selectin–binding and CXCR4-mediated migration are compromised in Flk2−CD34− LT-HSCs, we aimed to enhance their ability to migrate using recombinant human fucosyltransferase 6 (rhFTVI) and the CD26 inhibitor, Dip A (diprotin A). To this end, we observed that although LT-HSCs expressed low concentrations of sLex, they were able to engraft when transplanted into recipient mice. Moreover, although both CD26 inhibition and fucosylation enhanced migration of both HSC populations in vitro, only pretreatment of LT-HSCs with Dip A enhanced engraftment in vivo after transplantation into recipient mice. Remarkably, fucosylation of Flk2−CD34+ ST-HSCs consistently led to their ability to transplant secondary recipients. These data suggest that using fucosylation and Dip A to overcome the molecular disparity in adhesion mechanisms among ST-HSCs and LT-HSCs differentially influences their abilities to migrate and engraft in vivo and promotes the ability of ST-HSCs to engraft secondary recipient mice, the gold standard for testing functionality of LT-HSCs.

Dual Coating of Chitosan and Albumin Negates the Protein Corona-Induced Reduced Vascular Adhesion of Targeted PLGA Microparticles in Human Blood.

Vascular-targeted carriers (VTCs) have the potential to localize therapeutics and imaging agents to inflamed, diseased sites. Poly (lactic-co-glycolic acid) (PLGA) is a negatively charged copolymer commonly used to construct VTCs due to its biodegradability and FDA approval. Unfortunately, PLGA VTCs experienced reduced adhesion to inflamed endothelium in the presence of human plasma proteins. In this study, PLGA microparticles were coated with chitosan (CS), human serum albumin (HSA), or both (HSA-CS) to improve adhesion. The binding of sialyl Lewis A (a ligand for E-selectin)-targeted PLGA, HSA-PLGA, CSPLGA, and HSA-CSPLGA to activated endothelial cells was evaluated in red blood cells in buffer or plasma flow conditions. PLGA VTCs with HSA-only coating showed improvement and experienced 35–52% adhesion in plasma compared to plasma-free buffer conditions across all shear rates. PLGA VTCs with dual coating—CS and HSA—maintained 80% of their adhesion after exposure to plasma at low and intermediate shears and ≈50% at high shear. Notably, the protein corona characterization showed increases at the 75 and 150 kDa band intensities for HSA-PLGA and HSA-CSPLGA, which could correlate to histidine-rich glycoprotein and immunoglobulin G. The changes in protein corona on HSA-coated particles seem to positively influence particle binding, emphasizing the importance of understanding plasma protein–particle interactions.

Tumor cell E-selectin ligands determine partialefficacy of bortezomib on spontaneous lung metastasis formation of solid human tumors in vivo

Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis invivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.

FLT3 Inhibitors Upregulate CXCR4 and E-Selectin Ligands and CD44 Via ERK Suppression in AML Cells, and Blockade of CXCR4 and E-Selectin Signaling with GMI-1359 Overcomes AML Resistance to Quizartinib in Vitro and In Vivo

FLT3 Inhibitors Upregulate CXCR4 and E-Selectin Ligands and CD44 Via ERK Suppression in AML Cells, and Blockade of CXCR4 and E-Selectin Signaling with GMI-1359 Overcomes AML Resistance to Quizartinib in Vitro and In Vivo

The Disparity Observed Between Murine Short-term and Long-term Hematopoietic Stem Cells in Their Ability to Migrate and Engraft When Introduced IV is Due to Distinct Adhesion Molecule Expression

CD34 has traditionally been used as a marker to enrich human bone marrow stem cells for clinical bone marrow transplants for over five decades. However, several studies support that the most primitive hematopoietic stem cells (HSCs) responsible for long-term hematopoiesis-long-term HSCs (LT-HSCs) -are found within the CD34 negative (CD34 neg) fraction of bone marrow cells. LT-HSCs are challenging to study due to their scarcity and lack of specific markers to identify them. Once these stem cells enter the circulation, they must find their way, through multistep process controlled by a variety of cell surface adhesion molecules, into the bone marrow. In contrast to the CD34 pos stem cells, studies have suggested that LT-HSCs have trouble migrating and engrafting when introduced intravenously. In order to better understand why these deficiencies exist, we set out to fully elucidate the adhesion mechanisms used by various murine hematopoietic stem cell populations to migrate. To this end, we assayed the homing effectors that are either necessary in the first step, i.e., the cell surface adhesion molecule Sialyl Lewis X (sLe x) and its interaction with selectins-mainly E-selectin-on the bone marrow endothelium, or the second step, i.e. the chemokine receptor CXCR4 and its binding to the bone marrow stroma resident chemokine SDF-1α, of cell migration.Specifically focusing on murine populations of short term HSCs (ST-HSCs; Flk2 negCD34 pos) and LT-HSCs (Flk2 negCD34 neg), flow cytometric and gene expression analysis clearly illustrated distinctive expression patterns of homing effectors with the highest contrast observed in sLe x where Flk2 negCD34 neg HSCs exhibited low expression (< 10%) and Flk2 negCD34 pos HSCs exhibited much higher expression (> 60%) which correlated to higher binding to E-selectin. Of the common E-selectin ligands identified, both Flk2 negCD34 pos and Flk2 negCD34 neg HSCs expressed CD44 and PSGL-1 to equivalent levels while CD43 was expressed at significantly lower levels on Flk2 negCD34 neg HSCs. Moreover, our data indicates that the variation observed in sLe x expression/E-selectin binding correlates with differences in the expression of key glycosyltransferase genes involved in the creation of the sLe x epitope on these E-selectin ligands. mRNA expression analysis of glycosyltransferases revealed that, in general, sialyltransferase (ST3Gals) genes were expressed at higher levels in Flk2 negCD34 neg HSCs while only one fructosyltransferase (FT4) gene was expressed at higher levels in this HSC population. Thus, these data suggest that α2,3 sialylation is intact in Flk2 negCD34 neg HSCs but α1,3 fucosylation is lacking. The expression levels of homing molecules involved in the stages of cell migration after E-selectin binding were also assessed. No differences in the expression of the integrins CD49d, CD49e and CD29 were observed between Flk2 negCD34 neg and Flk2 negCD34 pos HSCs. Interestingly, higher levels of the chemokine receptor CXCR4 were measured on the surface of Flk2 negCD34 pos HSCs compared to Flk2 negCD34 neg HSCs. Likewise, this was reflected in a much lower ability of the Flk2 negCD34 neg HSCs to migrate towards the CXCR4 ligand, SDF-1, in a transwell assay.Based on these findings that show that both E-selectin binding and CXCR4 mediated migration are compromised in Flk2 negCD34 neg HSCs, we sought to enhance this LT-HSC populations ability to migrate using well-known treatments including recombinant human fucosyltransferase 6 (rhFVI) to create the glycans necessary for selectin binding, as well as the CD26 inhibitor (Diprotin A; Dip A) to enhance the binding of chemokines to their receptors.Interestingly, although both CD26 inhibition and cell surface fucosylation remarkably enhanced the expression of homing molecules in both HSC populations in vitro, only pretreatment of Flk2 negCD34 neg HSCs with Dip A significantly enhanced engraftment in vivo after transplantation into C57BL/6N mice.This study represents the first direct analysis of E-selectin ligand expression on murine LT-HSC populations and strongly support that by temporarily enhancing the expression of migration-associated molecules on the surface of these cells, prolonged and efficient HSC engraftment can be achieved. We hope our finding will potentially shed light on methods to optimally utilize these very valuable long-term HSCs in clinical bone marrow and cord blood transplants worldwide. DisclosuresNo relevant conflicts of interest to declare.