Fucosylation Enhances CD34⁺ Hematopoietic Stem Cell Homing and Longevity via E-Selectin–Mediated Adhesion and Signaling

Fucosylation Enhances CD34⁺ Hematopoietic Stem Cell Homing and Longevity via E-Selectin–Mediated Adhesion and Signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fucosylation Enhances CD34⁺ Hematopoietic Stem Cell Homing and Longevity via E-Selectin–Mediated Adhesion and Signaling Asma S. Al-Amoodi, Arwa A. Alghuneim, Jana S. Malki, Shuho Nozue, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7961408/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jan, 2026 Read the published version in BMC Cancer → Version 1 posted 12 You are reading this latest preprint version Abstract Background Hematopoietic stem cell transplantation (HSCT) is a cornerstone treatment for blood disorders and hematological malignancies, although its efficacy is limited by inefficient stem cell homing to the bone marrow. We previously demonstrated that fucosylated HSC ligands interact with endothelial E-selectin to facilitate homing. However, the downstream consequences of modulating fucosylation in HSCs remain unclear. Here, we systematically characterized how enhancing or inhibiting fucosylation—via recombinant human fucosyltransferase 6 (FTVI) or 2-fluoro-L-fucose (2FF), respectively—affects migration, signaling, and engraftment of human granulocyte-colony stimulating factor-mobilized peripheral blood CD34⁺ (mPB-CD34⁺) cells. Methods Live-cell imaging under flow, phosphoproteomics, and transcriptomics were used to characterize rolling dynamics and intracellular signaling, and in vivo homing was assessed in immunodeficient xenograft mouse models. Results Fucosylation enhanced tether and sling formation, improved E-selectin binding, and increased homing to the bone marrow and spleen. FTVI-treated cells activated MAPK and PI3K/AKT/mTOR pathways and showed enriched Rho-GTPase signaling, associated with proliferation and migration. In contrast, 2FF-treated cells had impaired migration and reduced rolling efficiency. Long-term studies confirmed enhanced repopulation and self-renewal capacity of fucosylated cells. Conclusion Fucosylation critically modulates E-selectin interactions, migration, and intracellular signaling in HSCs. These findings highlight glycoengineering as a promising strategy to enhance HSC transplantation outcomes in cancer therapy. E-selectin fucosylation glycoengineering HSCT HSC homing sialyl Lewis X Rho-GTPase PI3K/AKT/mTOR MAPK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction HSCT can stop or even reverse the progression of various types of diseases from hematological malignancies to autoimmune disorders [ 1 ]. Understanding the mechanisms that facilitate successful transplantation is crucial to improving outcomes and preventing relapse. Since HSCT is administered intravenously, its success depends on the ability of circulating HSCs to locate the bone marrow niche and home [ 2 ] – and this is directly related to the interaction between the glycan ligand Sialyl Lewis X (sLe x ) on the cell surface [ 3 , 4 ] and the E-selectin adhesion molecule that is stably expressed on marrow endothelial cells [ 5 ]. This interaction starts a multi-step process also involving chemokine receptors and integrins that guides HSCs into the bone marrow niche. Studies have shown that sLe x levels have a direct impact on the physiological behavior of HSCs [ 6 – 9 ]. In cancer cells, enhanced sLe x expression is associated with increased metastasis [ 10 , 11 ], which happens because key glycosyltransferases are upregulated [ 12 , 13 ], such as fucosyltransferases (FUTs) that are directly involved in fucosylation and have been linked to cancer progression [ 14 ]. Consequently, several studies have investigated ways to obstruct fucosylation based on the biochemistry of FUTs, such as the prominent inhibitor 2-Fluoro-fucose (2FF) [ 15 ], an analog of GDP-fucose, which interferes with the salvage pathway in GDP-fucose synthesis [ 16 ] and has been shown to effectively block leukocyte adherence and rolling interactions with selectins in a sickle cell mouse model [ 17 ] and oncogenic mouse models [ 18 ]. Alternatively, in other clinical contexts such as HSCT, promoting fucosylation can be useful for HSCs whose surface expression of the sLe x epitope is low [ 19 ], which inhibits bone marrow homing and limits transplantation success [ 20 ]. In human CD34 + HSCs, reduced sLe x expression is caused by a lack of α-1,3-FUT enzymes [ 19 ] responsible for adding the fucose sugar to the GlcNAc terminal, thereby impairing sLe x formation and inhibiting the interactions between HSCs and bone marrow endothelium [ 19 , 21 , 22 ]. Recombinant human fucosyltransferase 6 (FTVI) has been shown to effectively promote HSC homing of both mouse [ 23 ] and human cells in mouse models [ 21 , 24 , 25 ]. A 2015 clinical trial of cord blood-derived HSCs showed that ex vivo fucosylation using FTVI promotes engraftment [ 26 ]. We have previously demonstrated that ex vivo fucosylation promoted sLe x epitope levels in murine and human HSCs, thereby enhancing their migration, engraftment, and self-renewal capacities, effectively increasing their stemness [ 23 , 27 ]. However, the precise phenotypic changes and molecular pathways associated with improved engraftment in fucosylated HSCs remain unclear. This study sought to shed light on the cellular processes resulting from fucosylation treatment by manipulating fucose’s formation on human granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood CD34 + (mPB-CD34 + ) HSCs through two treatments, FTVI to enhance sLe x expression and 2FF to inhibit it. We then analyzed the associated intrinsic parameters of cell cycling, signaling, viability, and physiological flow. Our results indicate that fucosylation enhances the adhesive and migratory capacity of mPB-CD34⁺ HSCs by improving E-selectin interactions, while also activating Rho-GTPase and PI3K/AKT/mTOR signaling pathways that promote early entry into the cell cycle. Notably, these changes appear to preserve long-term stemness, suggesting that fucosylation plays a multifaceted role in regulating both short-term engraftment and durable hematopoietic reconstitution. Methods Human CD34 + stem cell culture and fucosylation treatment Human CD34 + progenitor cells (1 × 10 6 ; Lonza). Thawed cells were centrifuged, resuspended in stem cell media (Serum-Free Expansion Medium II, Stem Cell Technologies), and treated with 10× StemSpan CD34 + Expansion Supplement (Stem Cell Technologies). 2FF (400 µM; Biosynth Carbosynth) was added to the culture for several days as described in previously published work [ 15 ]. FTVI treatment was performed as previously established [ 23 ]. Briefly, rhFTVI enzyme (KAICO Ltd.) was added in a buffer using 0.5 mM GDP L-Fucose (Sigma–Aldrich), 25 mM HEPES (Gibco), 5 mM magnesium chloride, 0.1% human serum albumin (Sigma–Aldrich) in HBSS media (Gibco), and incubation was performed at 37°C for 30 min. After incubation, cells were washed twice in a sterile buffer containing 5% FBS and 2 mM EDTA (Thermo Fisher Scientific). Flow-based and microfluidic rolling assays Fluorescence multiplex cell rolling (FMCR) was conducted as previously established [ 28 ]. Briefly, the control/untreated cells were stained with green cell mask membrane (522/535), 2FF-treated cells with orange cell mask membrane (554/567), and FTVI-treated cells with Vybrant DiD (644/665). Confocal laser scanning microscopy (Zeiss) was performed on a parallel plate flow chamber (Bioptechs) including a coverslip with E-selectin-expressing Chinese hamster ovary (CHO-E) cells. Microfluidic-based rolling assay Wide-field fluorescence imaging experiments were conducted using a home-built fluorescence microscopy setup [ 29 , 30 ]. E-selectin-deposited microfluidic chambers were connected to the syringe pump (PHD Ultra, Harvard Apparatus). The stained cells were injected into the E-selectin-deposited microfluidic chambers through silicon tubing (inner diameter of 0.8 mm, ibidi GmbH) connected to the syringe pump and perfused on the stage of an inverted fluorescence microscope at a flow rate of 2 dyn/cm 2 . Real-time fluorescence imaging of the live cells was performed using an EMCCD camera with a 60× objective lens at 0.92 mV laser power (see Extended Methods in the Online Supplementary Appendix). In vivo transplantation and imaging mPB-CD34 + HSCs were stained with Xeno Light DiR dye, washed, and injected into NSG mice. Fluorescence imaging was conducted at various time points. E-selectin blocking experiments utilized anti-CD62E antibodies. Bone marrow engraftment and BrdU incorporation were analyzed via flow cytometry. All procedures adhered to ethical guidelines and approvals (see Extended methods in the Online Supplementary Appendix). Phosphopeptide enrichment and data processing Following treatment with FTVI and E-selectin, cells lysates were centrifuged and the proteins were digested via the S-Trap protocol, and peptides were desalted. Approximately 5% of the peptides were used for global proteome profiling, while the remainder underwent phosphopeptide enrichment using TiO2 microspheres. After washing and elution, phosphopeptides were desalted and analyzed by mass spectrometry. Data were processed using MSFragger against a comprehensive protein database, identifying differentially expressed proteins based on specified criteria (see Extended methods in the Online Supplementary Appendix). Single-cell RNA sequencing mPB-CD34 + cells treated with FTVI and/or E-selectin were used to prepare a single-cell sequencing library using a Chromium Next GEM Single cell 3' kit (10× Genomics) according to the manufacturer’s instructions. Library size distribution and concentration was validated using an Agilent 2100 Bioanalyzer (Agilent Technologies) and Invitrogen Qubit 2.0 Fluorometer (Life Technologies-Invitrogen), respectively. The libraries were sequenced on a NovaSeq 6000 SP flow cell (Illumina) by Novogene. We used 10× Genomics Cell Ranger software (version 7.0.0) to perform reference alignment, unique molecular identifier (UMI) collapsing and counting, and initial filtering. We identified highly variable genes (FindVariableGenes; Seurat) and performed subpopulation clustering (FindCluster; Seurat). Cell cycle phase was determined (CellCycleScoring) and the gene ontology (GO) term, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and Reactome enrichment analyses were performed using clusterProfiler. Statistical analysis Data analysis was performed using GraphPad Prism (version 9 for Mac, GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard deviation (SD). Unless otherwise indicated, Student’s t-test was used to compare two treatment groups; one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test was used for multiple comparisons for more than two treatment groups. Statistical significance was set at P ≤ 0.05. Graphs were constructed using GraphPad Prism. Results 2FF treatment dramatically inhibits core fucosylation, reducing E-selectin binding sLe x can be detected by anti-sLe x antibodies such as HECA-452 [ 23 , 27 , 31 – 35 ]. We examined the impact of removing fucose through 2FF treatment on sLe x expression and E-selectin binding in human mPB-CD34 + HSCs. Cells were treated with 2FF for 12–96 h, then analyzed using HECA-452 and recombinant E-selectin by flow cytometry. Both sLe x expression and E-selectin binding were decreased in a time-dependent manner (Fig. 1 A), with sLe x levels reduced to only ~ 8% of the original level at 72 h. Western blot confirmed this decrease (Fig. 1 B), and in sLe x levels and Aleuria aurantia lectin (AAL) blotting further supported reduced cell-surface fucosylation ( Figure S1 A). Consistent with earlier findings [ 23 , 24 ], FTVI treatment increased sLe x to ~ 99%, while 2FF dramatically reduced it to < 10%, as shown by HECA-452 staining (Fig. 1 C). AAL blots of 2FF-treated cell lysates showed markedly reduced intensity, while FTVI enhanced it ( Figure S1 B ). We also examined how 2FF and FTVI modulate E-selectin’s interaction with its ligands—CD43, CD44, and PSGL-1 [ 33 ]—via immunoprecipitation followed by E-selectin blotting. As expected, 2FF abrogated ligand binding, whereas FTVI reinforced this interaction (Fig. 1 D ) . These findings highlight how altering sLe x levels via fucosylation directly influences E-selectin binding capacity in mPB-CD34 + cells. Inhibiting fucosylation in mPB-CD34 + cells reduces SDF-1-mediated transmigration but does not impact clonogenicity Given the importance of core fucosylation in pathophysiology, we evaluated how 2FF affects the colony-forming ability of mPB-CD34 + cells. After a 3-day 2FF treatment, no significant difference in colony formation was observed compared to control cells (Fig. 2 A, 2 B). This aligns with our previous findings that FTVI treatment does not markedly alter HSC clonogenicity [ 23 , 24 ]. Since several FUTs (FUT3–7 and FUT9) [ 36 ] participate in the biosynthesis of sLe x , we examined how 2FF and FTVI influence FUT expression. qRT-PCR showed that neither treatment significantly changed α1,3-FUT transcript levels (Fig. 2 C). Although 2FF reduced FUT9 expression, its role in sLe x synthesis is limited. As HSCs use CXCR4 to migrate toward the chemokine SDF-1α during homing to the bone marrow, we assessed treatment effects on SDF-1α-directed chemotaxis. 2FF significantly impaired CXCR4-mediated migration of mPB-CD34 + cells (Fig. 2 D), while FTVI had no effect ( P > 0.05). These results demonstrate that inhibiting fucosylation selectively disrupts chemotactic migration of mPB-CD34 + HSCs without affecting clonogenicity. Enhanced fucosylation improves rolling and creates long, tight tethers and slings, while inhibiting fucosylation diminishes their number and size We examined treatment-induced phenotypic changes over time. Light microscopy showed that while all samples retained typical circular morphology, E-selectin treatment led to mild cell clumping, which was markedly increased in FTVI-treated samples (FTVI + E-selectin), indicating enhanced cell-cell adhesion ( Figure S2 A , S2B ). Flow-based migration assays, including FMCR assays [ 28 ] and microfluidic-based live-cell imaging [ 29 , 30 , 37 ], were used to assess the impact of fucosylation modulation on mPB-CD34 + cell rolling. Spectral overlap between fluorescence tags was minimized to distinguish samples under simultaneous excitation (405 nm, 488 nm, 633 nm; Fig. 3 A). FTVI-treatment significantly increased both the rolling duration (~ 2.2-fold, P < 0.001) and rolling cell number (~ 1.4-fold, P = 0.019), while 2FF reduced rolling cells ~ 2.7-fold ( P < 0.001) compared to controls (Fig. 3 B). Similar trends were observed under different shear stress levels (Fig. 3 C). FTVI-treatment also slowed rolling velocity, whereas 2FF accelerated it compared to control cells (Fig. 3 C). To explore the mechanism, we imaged tethers and slings via 3D epifluorescence microscopy [ 29 ]. The selectin ligand, CD44, immunolabeled using AF-647, was distributed along the entire length of these structures [ 29 ] (Fig. 3 D), consistent with prior observations in leukemic cells [ 29 ]. In untreated cells, tether and sling lengths averaged 12 µm and 19 µm, respectively. 2FF shortened them to 8 µm and 9.6 µm ( P = 0.04 and P = 0.0013), while FTVI slightly increased tether length (Fig. 3 E). Both treatments (FTVI and 2FF) reduced sling numbers ( P = 0.007 and P < 0.001, Fig. 3 F) compared untreated cells. We speculate that shorter, weaker tethers in 2FF-treated cells limit sling formation, whereas stronger tether anchoring in FTVI-treated cells may curtail sling generation. These findings suggest that fucosylation directly influences rolling behavior through changes in tether and sling dynamics: 2FF reduces rolling and increases speed, while FTVI enhances rolling via stronger, longer tethers. Boosting fucosylation improves mPB-CD34 + cell delivery to bone marrow and spleen, while inhibition restricts homing To validate our in vitro findings, we performed short- and long-term in vivo migration/homing assays in xenograft mouse models. Fucosylated mPB-CD34 + (FTVI-treated) localized prominently around the spinal cord, spleen, liver, and legs/hindlimbs (Fig. 4 A), whereas 2FF treatment reduced the overall fluorescence signal. At 48 h post-transplantation, imaging from multiple angles confirmed higher average fluorescence in FTVI-treated groups compared to controls ( P < 0.001), while 2FF had no significant effect (Fig. 4 B). Fluorescence intensity in the spleen and legs was elevated in mice that received FTVI-treated cells and reduced in mice that received 2FF-treated cells (Fig. 4 C, 4 D). Flow cytometry plots for bone marrow and blood samples are provided in Figure S3 . At 8-weeks post-transplantation, 2FF-treated cells showed impaired bone marrow engraftment ( P = 0.03), whereas FTVI-treated cells exhibited improved engraftment at 12-weeks (Fig. 4 E, Table 1 ).[ 23 ] Blocking E-selectin using anti-CD62E antibody significantly reduced bone marrow persistence of FTVI-treated cells at 12-weeks (Fig. 4 G-H), indicating that fucosylation-enhanced engraftment depends on E-selectin binding. Table 1 Engraftment efficiency of mPB-CD34 + HSCs in NSG mice. Percentages of human CD45 donor cells engrafted in recipient mouse post-transplantation of HSCs with or without the FTVI treatment. Cells Cell Injection Dose No. of mice Percentage of human cells in recipient mice (weeks) 6 8 12 Exp. 1 Buffered-CD34 pos 50,000–80,000 6 4.5, 0.05, 11.35, 0, 2.95, 16.7 (5/6) 0.55, 0.15,18.05, 0, 0.5, 8.3 (5/6) 15, 0, 35.3, 0, 6.6, 28.85 (4/6) FTVI-CD34 pos 50,000–80,000 6 0.05, 0, 6, 4.7, 12.75, 17.6 (5/6) 2.3, 0.55, 33.65, 2.6, 3, 13.55 (6/6) 6.85, 0.1, 45.8, 37.7, 54.05, 32.45 (5/5) 2FF-CD34 pos 50,000–80,000 5 11.15, 0, 3.85, 4.8, 0.85 (4/5) 3.35, 2.3, 2.6, 1.6, 0 (4/5) 15.35, 20.65, 23.5, 10.5, 0 (4/5) E-selectin function-blocking Ab-CD34 pos 50,000–80,000 5 1.9, 9.15, 0.05, 1.95, 6.45 (5/5) 0.15, 8.45, 8.85, 1.75, 0 (4/5) 0.1, 1.75, 2.6, 0.1(4/4) Exp. 2 Buffered-CD34 pos 50,000–80,000 7 6 12 16 7.1, 5.4, 8.9, 7.1, 0, 2.2, 15.7 (6/7) 70.3, 66.8, 78.7, 33.6, 0.9, 67.3, 77.4 (7/7) 74, 27.6, 85.1, 70.5, 0.4, 42.8, 91(7/7) FTVI-CD34 pos 50,000–80,000 7 15.2, 9.7, 10, 19.8, 1.2, 9.2, 2 (7/7) 61.2, 73.4, 68.9, 67.6, 3.1, 50.9 (6/6) 80.3, 30, 71.7, 78.5, 13.3, 72 (6/6) E-selectin function-blocking Ab-CD34 pos 50,000–80,000 6 1.8, 23.2, 4.2, 0, 16.3, 0.1 (5/6) 20.9, 55.6, 27.7, 3.5, 82.7, 0.9 (6/6) 12, 3.6, 24.3, 2, 89, 49.3 (6/6) We next determined lineage differentiation in peripheral blood at 8-weeks. No significant differences were observed in lymphoid (CD3 and CD19) and myeloid (CD33) lineages among groups that received untreated, 2FF-, or FTVI-treated mPB-CD34 + HSCs (Fig. 4 F). Consistent with previous findings [ 38 ], mPB-CD34 + HSCs showed a lymphoid lineage bias. Finally, we evaluated mPB-CD34 + cell viability under E-selectin stimulation. Fluorescence microscopy of calcein AM and EthD-1-stained cells showed increased cell death with FTVI + E-selectin treatment, but not with fucosylation alone ( Figure S4 A ). Annexin V-7AAD staining confirmed that this effect was transient, with cell viability recovering after 72 h ( Figure S4 B ). These results indicate that E-selectin may trigger apoptosis in FTVI-treated cells, but that fucosylation itself does not cause long-term viability loss. Together, our findings underscore the role of fucosylation in promoting mPB-CD34 + HSC engraftment, mediated through E-selectin binding, while having minimal impact on differentiation or long-term viability. Fucosylation enhances E-selectin-driven stem cell cycling via RHO and PI3K/AKT/NFκB signaling We examined whether enhanced fucosylation influences phosphoproteomic profiles and cell cycle activation in adult HSCs, which typically remain in a quiescent state to avert exhaustion and DNA replication stress. We analyzed four treatments (untreated, FTVI, E-selectin, and FTVI + E-selectin) and identified > 4,000 phosphopeptides per sample at 1% FDR (Fig. 5 A). Phosphoserine sites were most abundant across groups (> 84%). Notably, E-selectin and FTVI + E-selectin samples showed increased phosphotyrosine sites (1.23% and 1.08%, respectively). Fucosylation alone appeared to influence HSC stemness and migration pathways rather than cycling (data not shown), notably EGFR, VEGFA/VEGFR2, actin cytoskeleton regulators (Kinesin, Spectrin, and Rho GTPases), FOXO (including RBL2, USP7, MAP2K2, AGAP2, AKT1, CSNKE, STK4, MAPK14), and NFκB signaling. In fucosylated cells, dephosphorylation of MAPK14 (P-38α) may indicate suppression of HSC differentiation. We next compared E-selectin vs. FTVI + E-selectin-treated cells. Global proteomics identified 493 DEPs ( Table S2 ), predominantly nuclear (44%). GO analysis revealed enrichment in mRNA processing, DNA replication, DNA repair, mitosis, and cell cycling pathways. Phosphoproteomic profiling revealed 357 phosphopeptides (267 phosphoproteins), with 54 phosphorylated and 91 dephosphorylated DEPs (Fig. 5 B-C, Table S2 ). Key cell cycle regulators such as RB1, DYNC1LI1, NIPBL, and TP53BP1 were dephosphorylated, while LIMK2, SIK3PHKB, and TAB2 were phosphorylated, implicating MAPK (TAB2), TGFβ (LIMK2, NEDD4), and EGFR signaling (LIMK2, ABI1). Pathway enrichment identified Rho-GTPase signaling as the dominant network in FTVI + E-selectin group, involving proteins like Rac1/2 effectors, ROCK, and GTPase regulators (e.g. LIMK2, ABI1, MYO9B, ARAP1, LEMD3, and RBMX. Using a P < 0.05 threshold, we detected 446 phosphopeptides in the E-selectin group (phosphorylated: 98; dephosphorylated: 160) and 448 in the FTVI + E-selectin group (phosphorylated: 77; dephosphorylated: 199). Of these, 244 overlapped, while ~ 200 were unique to each (Fig. 5 G, Table S2 ). GO and molecular function analysis ( Figure S5 ) showed enrichment in actin/tubulin binding and MAP kinase binding. While MAPK signaling [MAPK3 (ERK), MAPK1 (ERK), MAPK11/12/13/14 (p38-β/γ/δ/α), and MAPK10 (JunK3)] was shared, only the FTVI + E-selectin group showed enrichment in small GTPase binding (e.g., HPS4, MYO9B, ARHGEF2, PAK2, MTSS2, GBF1m HPS4, ARHGEF18, and MYO9B). KEGG and Reactome pathways emphasized the importance of the Rho signaling, cytoskeleton regulation, and stemness-associated Wnt, Hedgehog, and TGFβ pathways (e.g., TRIO, HSP90AB1, FNBP1, ROCK2, DOCK8, ARHGAP1, ARHGEF18, IQGAP2, AHGAP15, SRRM1, ARFGAP2, STIP1, RBMX, and ARHGEF6; Fig. 5 E-F). To validate phosphorylation changes, flow cytometry showed PI3K/AKT/mTOR and NFκB pathway activation specifically in FTVI + E-selectin-treated cells. Reduced PTEN, an AKT inhibitor, phosphorylation supported AKT activation ( Figure S6 A ), and FOXO/EGFR pathways were induced, consistent with phosphoproteomic data. CaCl 2 , essential for E-selectin binding, did not influence phosphorylation independently (data not shown). We further confirmed NFκB activation, a transcription factor essential to stem cell cycling [ 39 ], using western blotting at 30 min and 5 h. NFkB phosphorylation was triggered by E-selectin binding, particularly in FTVI + E-selectin-treated cells ( Figure S6 B ) [ 39 ]. To explore the effects of fucosylation and E-selectin binding on cell cycle regulation, we examined the phosphorylation levels of Rb proteins, which are key regulators of cell cycle progression. We show that treatment with FTVI + E-selectin decreased the level of phosphorylated Rb proteins compared to untreated cells, indicating a cell cycle arrest ( Figure S5 D ). Collectively, these results indicate that fucosylation primes HSCs for E-selectin-mediated activation of cell cycle and stemness programs, largely via Rho GTPases, and PI3K/AKT/ NFκB signaling. Combined FTVI and E-selectin treatment drives a distinctive stem cell gene regulatory network We investigated whether enhanced E-selectin binding via fucosylation influences transcriptional networks related to cell cycling and stemness. Using single-cell RNA sequencing (scRNA-seq), we profiled 5,266 and 4,500 high-quality transcriptomes from E-selectin- and FTVI + E-selectin-treated mPB-CD34 + HSCs, respectively ( Table S3 ). UMAP visualization revealed 13 transcriptionally distinct groups, with clusters 3 and 5 enriched in FTVI + E-selectin-treated cells (Fig. 6 A). Differential gene expression analysis identified ~ 22 and ~ 33 upregulated transcripts in clusters 3 and 5, respectively ( Table S4 ). CellCycleScoring indicated a higher proportion of cells in G2/M in the FTVI + E-selectin group (Fig. 6 B), suggesting delayed reentry into G0 and prolonged cycling. Pathway enrichment analysis (GO, Reactome, and KEGG) showed that FTVI + E-selectin-treated cells were enriched in pathways related to cell cycle regulation (DNA replication, mitotic division, cyclins, G2/M transition, and MAPK4/MAPK6), homing (focal adhesion, actin cytoskeleton, Rho GTPases, cadherins, and integrins), and stemness (WNT, Hedgehog, TGF-β, and NOTCH) (Fig. 6 C). Gene set enrichment confirmed elevated Rho-GTPase-related signaling in clusters enriched with FTVI + E-selectin cells, including CDC42, Rac1, and Rac2 (Fig. 6 D). We next investigated short- and long-term cycling. Ki67-DAPI flow cytometry at 30 min revealed a ~ two-fold reduction in quiescent (G0) mPB-CD34 + cells in E-selectin and FTVI + E-selectin-treated samples (Fig. 6 E-F), indicating that fucosylation amplifies E-selectin-driven cell cycling. Notably, after 4–5 days, FTVI-treated cells returned to the G0, whereas untreated cells remained cycling (data not shown). To evaluate long-term turnover, we examined BrdU incorporation in vivo . At 12-weeks post-transplantation, BrdU-labeled hCD45 + /hCD34 + cells increased by 23% in mice receiving fucosylated HSCs compared to controls, indicating early S-phase entry (Fig. 6 G-H). However, most of these cells were in G0, supporting the idea that fucosylation enhances early self-renewal while maintaining long-term quiescence. These findings suggest that fucosylation primes HSCs to respond to E-selectin signaling by promoting early cycling and migration through Rho-GTPase pathways, while preserving long-term quiescence necessary for durable engraftment. Discussion Fucosylating stem cells is emerging as a treatment that has the potential to improve HSCT outcomes, necessitating a deeper understanding of the impact that the increased sLe x expression resulting from the treatment has on HSC function. While previous studies indicated that ex vivo fucosylation of HSCs enhances E-selectin binding and engraftment [ 21 , 23 , 26 , 40 ], this study reveals the broader implications on cellular processes including cell cycle and self-renewal. We confirmed that 2FF treatment substantially suppressed, while FTVI markedly increased, sLe x expression and E-selectin ligand activity of mPB-CD34 + cells, consistent with previous studies [ 24 , 27 ]. Importantly, 2FF-mediated inhibition impaired E-selectin binding and chemotactic migration toward SDF-1α, whereas clonogenic potential remained unaffected, suggesting that fucosylation selectively governs migration without compromising differentiation capacity. Other studies, however, have reported that 2FF significantly affected differentiation in certain cancer types such as liver cancer, but not in others, like breast cancer [ 15 , 41 ]. This discrepancy may be context/cell type specific. The observed suppression of mPB-CD34 + cell migration in vitro by 2FF may reflect global glycan alterations that extend beyond sLe x downregulation, affecting the functionality of multiple adhesion molecules. For instance, glycosylation of proteins like Notch is critical for their transcriptional activity and regulation of hematopoiesis and stemness [ 42 – 44 ]. Thus, our findings reinforce the role of fucosylation as a key post-translational modification essential for both E-selectin recognition and broader HSC biology. We next investigated the physical interactions of HSCs with E-selectin under shear stress using flow-based rolling assays [ 28 ] and our microfluidic platform [ 29 , 30 , 37 ]. FTVI treatment significantly increased the number of rolling cells and rolling duration, likely due to improved tether and sling mediated interactions. These structures, visualized using live-cell fluorescence imaging, were longer and more stable in FTVI-treated cells compared to controls, supporting the role of fucosylation in enhancing mechanical anchorage to the vascular endothelium. The dynamic elongation, anchoring, long length, and multiplicity of the tether and sling are exploited by cells to slow their rolling and interact with other surface molecules [ 28 , 45 ]. In contrast, 2FF-treated cells displayed reduced tether formation and increased rolling velocity, consistent with impaired adhesion. In vivo homing and engraftment assays further substantiated the functional impact of fucosylation. FTVI-treated cells demonstrated enhanced localization to the bone marrow and spleen in xenograft models, while 2FF-treated cells exhibited impaired early migration. The inhibitory effect of the 2FF treatment was most pronounced at 6–8 weeks after transplantation, and engraftment efficiency was comparable to that in untreated cells at 12 weeks after injection. These results reflect the role of fucosylation during the initial stage of HSC migration and suggest that a low-but-sufficient number of HSCs can reach the bone marrow and repopulate and sustain it. Long-term engraftment analysis further confirmed the impact of fucosylation on the engraftment efficiency of mPB-CD34 + cells in the bone marrow at 12 weeks post-transplantation, consistent with our previous finding [ 23 , 27 ]. These observations align with clinical studies exploring fucosylation to improve cord blood HSC transplantation, where enhanced sLe x expression was shown to accelerate neutrophil recovery and improve early engraftment kinetics [ 26 ]. Interestingly, while FTVI alone did not trigger cell aggregation, FTVI + E-selectin-treated cells showed increased adhesion and transient apoptosis. These effects were not due to fucosylation per se , but rather the amplified interaction with E-selectin. Consistent with previous reports [ 46 ] actively proliferating cells may be more susceptible to apoptosis following strong E-selectin engagement. Notably, cell viability recovered after 72 hours, indicating a reversible effect. With regard to cell cycle regulation, we found that E-selectin binding in the context of FTVI treatment promoted the proliferation of mPB-CD34 + cells at the expense of dormant (G0) cell numbers, as validated in E-selectin knockouts in vivo [ 47 ]. HSCs are typically maintained in the G0 state near osteoblasts in the bone marrow niche to avoid depletion. To preserve homeostasis, HSCs may undergo asymmetric division, yielding one stem cell and one committed progenitor [ 48 ]. This mechanism may underlie the enhanced engraftment seen in FTVI-treated mice. Notably, this cycling effect appeared transient; at later time points, cells accumulated in the G2/M phase but did not re-enter the cycle. Future studies are needed to determine how FTVI and E-selectin together influence asymmetric division and long-term HSC maintenance. Importantly, viability returned to baseline levels within 72 hours, indicating that the pro-cycling and apoptotic responses to E-selectin are temporary and reversible. In our investigation of the molecular underpinnings of these observations, phosphoproteomic and single-cell transcriptomic analyses demonstrated that FTVI + E-selectin treatment activated EGFR, PI3K/AKT/mTOR and NFκB pathways, in which are critical regulators of HSC regeneration and cycling. The EGFR pathway promotes HSC regeneration and survival following total body irradiation, as systematic infusion of epidermal growth factor into irradiated mice dramatically improved HSC secondary engraftment. In addition to its regenerative role, EGFR is a key mediator of cell cycle regulation, acting upstream of PI3K and AKT. Activated Akt promotes cyclin expression and Rb inhibition, driving cell cycle progression. Moreover, EGFR can function as transcription factor for cell cycle regulating genes, such as CYCLIN D1 and C-MYC , further enhancing proliferation. NFκB activation occurs through PI3K, which converts phosphatidylinositol-4,5-biphosphate into phosphatidylinositol-4,5,6-triphosphate, a scaffold and activator of AKT. E-selectin binding has been found to activate NFκB as early as 30 min post-administration, and transcription factor regulates hematopoiesis. NFκB deletion in HSCs disrupts gene expression—such as through c-Met upregulation—impairing engraftment and hematopoiesis, whereas constitutive NFκB activity enhances HSC proliferation and represses quiescence [ 39 ]. Our data highlight a critical role for Rho-GTPase signaling in mediating the effects of fucosylation and E-selectin binding. Among the classically regulated Rho GTPases, Rho, Rac, Cdc42—well-studied in HSC biology—were significantly enriched in our phosphoproteomic and transcriptome analyses following FTVI and E-selectin treatment. These GTPases regulate key HSC functions, including homing, migration, cell cycle progression, and interactions with microenvironmental cues [ 38 , 49 , 50 ]. Notably, Rac is activated by cell surface receptors such as c-kit, CXCR4, and α4β1, which are likely upregulated in fucosylation-enhanced migrating HSCs. Our findings align with prior studies demonstrating that Rac and Cdc42 are essential for HSC retention in the bone marrow [ 51 ], long-term engraftment of HSCs [ 52 ], and HSC mobilization [ 53 ], while loss of their activity leads to impaired function and increased apoptosis. In addition to the previously discussed pathways, the activation of stemness-associated Wnt, Hedgehog, and TGFβ signaling pathways was observed in our datasets further underscoring a regulatory network that promotes the maintenance of stemness in FTVI and E-selectin-treated cells. In conclusion, we show that enhancing fucosylation optimizes HSC tethering and rolling on E-selectin, activating EGFR/PI3K/AKT/mTOR/NFκB signaling to drive cell cycle progression. In parallel, fucosylation promotes HSC stemness and engraftment through Rho-GTPase signaling, reinforcing their retention and function in the bone marrow niche. These findings establish a mechanistic link between glycosylation and intracellular signaling pathways that regulate the fate of HSCs and suggest that extending phosphorylation-based and transcriptomic profiling may uncover additional therapeutic targets. Abbreviations HSCT Hematopoietic stem cell transplantation mPB-CD34⁺ Human granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood CD34⁺ cells rhFTVI Recombinant human α1,3-fucosyltransferase VI sLe x Sialyl Lewis X 2FF 2-Fluoro-fucose FMCR Fluorescence multiplex cell rolling UMI Unique molecular identifier GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes AAL Aleuria aurantia lectin Declarations Ethics approval and consent to participate All in vivo experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85 − 23, revised 1996) and the Implementing Regulations of the Law of Ethics of Research on Living Creatures (Kingdom of Saudi Arabia National Committee of Bioethics, 3rd Edition). All protocols were approved by the King Abdullah University of Science and Technology (KAUST) Institutional Animal Care and Use Committee (IACUC) under protocol number 17IACUC20 . Competing interests The authors declare that they have no competing interests. Correspondence Jasmeen Merzaban, Laboratory of Cell Migration and Signaling, Bioscience Program, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955 − 6900, Kingdom of Saudi Arabia; Tel.: +96628082383; E-mail: [email protected] Funding The research reported in this publication was supported by funding from a King Abdullah University of Science and Technology (KAUST) Faculty Baseline Research Funding Program (to J.S.M.) and a King Abdullah University of Science and Technology (KAUST) – KAUST Center of Excellence for Smart Health (KCSH), under award number 5932. Author Contribution A.S.A. and A.A designed and performed the experiments, analyzed the data, and wrote the manuscript. J.M. performed cell cycling and signaling experiments and wrote the manuscript. S.N. performed the microfluidic flow-based assay and S.H. supervised this work, Y.L. conducted the in vivo mouse experiments. J.K. helped in some biocomputational analyses. H.Z. and D.B. ran the phosphoproteomic experiment while A.K.A. assisted in the in vivo experiments. J.S.M. conceived and designed the study, analyzed the data, and wrote the manuscript. Acknowledgement We acknowledge the invaluable contributions of the KAUST Animal Resource Core Lab (ARCL) at the King Abdullah University of Science and Technology (KAUST) and Dr. Simona Spinelli and Mr. Stefano Pietro for their superb instruction and support. KAUST is an AAALAC International accredited institution. We thank Dr. Ioannis Isaioglou for constructive discussions on phosphoproteomics. We acknowledge the assistance of Ms. Umm Habiba with lab administration as well as the members of the Cell Migration and Signaling Laboratory for their help and insightful comments. 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Supplementary Files TableS2.xlsx TableS4.xlsx 2FF.avi FTVI.avi untreated.avi A.AlAmoodiandA.AlguneimSupplementarydataBMCCancer.docx SupplementalMaterialFullWBs.pdf Cite Share Download PDF Status: Published Journal Publication published 29 Jan, 2026 Read the published version in BMC Cancer → Version 1 posted Editorial decision: Revision requested 16 Dec, 2025 Reviews received at journal 14 Dec, 2025 Reviews received at journal 10 Dec, 2025 Reviews received at journal 07 Dec, 2025 Reviewers agreed at journal 24 Nov, 2025 Reviewers agreed at journal 24 Nov, 2025 Reviewers agreed at journal 21 Nov, 2025 Reviewers agreed at journal 21 Nov, 2025 Reviewers invited by journal 13 Nov, 2025 Editor assigned by journal 04 Nov, 2025 Submission checks completed at journal 03 Nov, 2025 First submitted to journal 03 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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06:55:13","extension":"html","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":182096,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/d2eb3e9a0b21c417679b0e87.html"},{"id":96791321,"identity":"a7798fa9-48b7-4d57-a633-8f050a4ddb6e","added_by":"auto","created_at":"2025-11-26 06:55:08","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":600898,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFucosylation profile of human mPB-CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells is altered by FTVI and 2FF treatments and impacts binding interactions with E-selectin\u003c/strong\u003e. (A) Levels of sLe\u003csup\u003ex\u003c/sup\u003e (using the anti-sLe\u003csup\u003ex\u003c/sup\u003e antibody HECA-452, upper panel) and E-selectin binding (using recombinant chimera E-selectin [E-Ig]) in human mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells following treatment with 2FF at the indicated timepoints assessed using flow cytometry. (B) Western blot analysis of HECA-452 and E-Ig in untreated and 2FF-treated human mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells. β-actin was used as loading control. (C) sLe\u003csup\u003ex\u003c/sup\u003e expression in untreated (UNT), FTVI-treated, and 2FF-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells assessed using flow cytometry. (D) Endogenous CD44, CD43, and PSGL-1 (CD162) immunoprecipitated from untreated (UNT), 2FF-treated, and FTVI-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells. The immunoprecipitated proteins were prepared for western blot analysis and blotted for E-Ig (top) or directly for each protein (bottom).\u003c/p\u003e","description":"","filename":"fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/89ae527c5dff7a557b45babe.jpg"},{"id":96791315,"identity":"64dd7eda-de44-40b6-97e1-5e0267004e52","added_by":"auto","created_at":"2025-11-26 06:55:07","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":683136,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulating fucosylation does not alter the clonogenicity or fucosyltransferase expression in human mPB-CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e \u003cstrong\u003eprogenitors, but fucosylation inhibition affects chemotaxis. \u003c/strong\u003e(A) Colony-forming capacities of untreated and 2FF-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells; 1,000 and 500 cells were cultured in methylcellulose in the presence of cytokines (SCF, IL-3, EPO, and GM-CSF) for 12–14 days. The number and composition of the colonies are presented. BFU-E, erythroid burst-forming unit; CFU-GEMM, granulocyte-erythroid-megakaryocyte-macrophage colony-forming unit; CFU-GM, granulocyte-macrophage colony-forming unit. (B) Individual clones. (C) qRT-PCR of FUT enzyme expression in untreated, FVTI-treated, and 2FF-treated cells. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, compared to untreated cells (one-way ANOVA followed by Dunnett’s \u003cem\u003epost-hoc\u003c/em\u003e test). (D) Numbers and representative images of untreated, 2FF-treated, and FTVI-treated cells migrating toward SDF-1. **\u003cem\u003eP\u003c/em\u003e = 0.002, compared to untreated cells (one-way ANOVA followed by Dunnett’s \u003cem\u003epost-hoc\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/fa65f5ad66a553c3f958b733.jpg"},{"id":96791331,"identity":"5f7ef74a-19ed-4518-a5c7-2b729ffec335","added_by":"auto","created_at":"2025-11-26 06:55:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1226336,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAssessment of tether and sling formation in 2FF- and FTVI-treated mPB-CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cells through fluorescent imaging. \u003c/strong\u003e(A) Confocal microscope images showing minimal spectral overlap of fluorescently-stained untreated, 2FF-treated, and FTVI-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells. Representative images of cells under a flow shear stress of 2–7.5 dyn/cm\u003csup\u003e2\u003c/sup\u003e. The raw image (left) is shown along with reconstructions of cell rolling (middle) and rolling tracks (right) using Imaris for Tracking software. We confirmed that each experimental condition generated the minimum spectral overlap with the chosen fluorescence tag to capture distinguishable signals for each sample under simultaneous excitation (405 nm, 488 nm, and 633 nm) (B) Rolling duration and number of rolling fluorescently-stained untreated, 2FF-treated, and FTVI-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells at increasing shear stress intervals were acquired from three independent experiments. Data are presented as mean ± SD. *\u003cem\u003eP\u003c/em\u003e ≤ 0.01, **\u003cem\u003eP\u003c/em\u003e ≤ 0.001, ***\u003cem\u003eP\u003c/em\u003e ≤ 0.0001, compared to untreated cells (one-way ANOVA followed by Dunnett’s \u003cem\u003epost-hoc\u003c/em\u003e test). (C) Rolling velocity and numbers of rolling fluorescently-stained untreated, 2FF-treated, and FTVI-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells per shear stress increment were estimated from three independent experiments. (D) Fluorescence characterization of membrane tethers and slings transiently formed on untreated, 2FF-treated, and FTVI-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells during cell rolling over E-selectin. Fluorescence images of cells (immunolabeled against CD44 using Alexa-Fluor [AF]-647 dye-conjugated antibody) captured during cell rolling on E-selectin. (E) Lengths of individual tethers and slings on live untreated, 2FF-treated, and FTVI-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells rolled on E-selectin, captured using confocal fluorescence microscopy. (F) Quantification of tethers and slings. All images of rolling cells were captured at a shear stress of 2 dyn/cm\u003csup\u003e2\u003c/sup\u003e. Data in (E) and (F) are presented as mean ± SD. *\u003cem\u003eP\u003c/em\u003e ≤ 0.01, **\u003cem\u003eP\u003c/em\u003e ≤ 0.001, ***\u003cem\u003eP\u003c/em\u003e ≤ 0.0001, compared to untreated cells (one-way ANOVA followed by Dunnett’s \u003cem\u003epost-hoc\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/dda35f9a3d086ebb52ece4fb.jpg"},{"id":96917392,"identity":"96911b55-58d5-4259-ad76-862310618ea9","added_by":"auto","created_at":"2025-11-27 14:09:40","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1882709,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIn vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e study of mPB-CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cell engraftment under different fucosylation-targeting treatments (2FF or FTVI) using fluorescence IVIS and flow cytometry. \u003c/strong\u003e(A) Whole-body \u003cem\u003ein vivo\u003c/em\u003e images of NSG mice obtained 1 h post-transplantation. (B) Quantified fluorescence intensity from dorsal and side images. *\u003cem\u003eP\u003c/em\u003e ≤ 0.01 and ***\u003cem\u003eP\u003c/em\u003e ≤ 0.0001, compared to untreated cells (one-way ANOVA followed by Dunnett’s \u003cem\u003epost-hoc\u003c/em\u003e test). (C) \u003cem\u003eEx vivo\u003c/em\u003e images of hematopoietic organs at 48 h post-transplantation (D) Quantified fluorescence intensities for liver, spleen, lung, and leg tissues. (E) Engraftment of human mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells in immunodeficient NSG mice at 8 and 12-weeks post-transplantation with (F) lineage differentiation of donor cells at 8-weeks post-transplantation. *\u003cem\u003eP\u003c/em\u003e ≤ 0.01, compared to untreated cells (one-way ANOVA followed by Dunnett’s \u003cem\u003epost-hoc\u003c/em\u003e test). (G) Percentage engraftment at 12-weeks post-transplantation of mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e cells. Fucosylated HSCs lose engraftment advantage when E-selectin is blocked \u003cem\u003ein vivo\u003c/em\u003e. E-selectin activity in recipient mice was blocked via injection with anti-CD62E antibody (clone 9A9) 2 h prior to intravenous injection of the FTVI-treated mPB-CD34\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e \u003c/strong\u003ecells. **\u003cem\u003eP\u003c/em\u003e ≤ 0.001, compared to untreated cells (one-way ANOVA followed by Dunnett’s \u003cem\u003epost-hoc\u003c/em\u003e test). (H) Representative flow cytometry plots of donor cell engraftment.\u003c/p\u003e","description":"","filename":"fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/b0d6cb858de7b76b3948a039.jpg"},{"id":96791326,"identity":"b8310261-a7f7-4881-8f73-a79e597ac40b","added_by":"auto","created_at":"2025-11-26 06:55:09","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":987618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModulation of mPB-CD34\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e cell fucosylation and E-selectin binding affects PI3K/AKT/NFκB and Rho-GTPase signaling activation. \u003c/strong\u003e\u0026nbsp;(A) Counts of identified phosphosites in serine (pS), threonine (pT), and tyrosine (pY) in untreated (UNT) recombinant human fucosyltransferase VI (FTVI)-treated, E-selectin (Esel)-treated, and FTVI +Esel cotreated mPB-CD34\u003csup\u003epos\u003c/sup\u003e cells following their treatment for 0.5 h. Phosphopeptides were enriched using TiO\u003csub\u003e2\u003c/sub\u003e beads before being analyzed by timsTOF Pro 2 mass spectrometry. (B) Gene Ontology of the differentially expressed phosphorylated proteins in FTVI+Esel-treated cells compared to in the Esel treatment alone. The respective top 12 enriched GO terms in the biological process, cellular compartment, and molecular function. (C) Volcano plot for phosphorylated DEPs. Red: high relative expression; blue: low relative expression. (D) Pathway enrichment analysis of DEPs. The top 14 enriched Wiki pathways and top 12 enriched Reactome pathways based on a bubble chart. Colors denote P values, and dot sizes denote the number of DEPs. (E) The pathway enrichment analyses (Reactome, Wiki and KEGG) of the unique 204 DEPs in FTVI+Esel treated cells and (F) unique 202 DEPs in Esel-treated cells. (G) Venn diagram analysis of overlapping DEPs between FTVI+Esel and Esel treated cells. (H) The percentage of phosphorylation of the indicated proteins (PI3K, AKT, mTOR, Rb and NFkB) in untreated (UNT), FTVI-treated, E-selectin (Esel)-treated, and FTVI +Esel cotreated mPB-CD34\u003csup\u003epos\u003c/sup\u003e cells following their treatment for 0.5 h was assessed using flow cytometry. Data are presented as the mean ± SD. *\u003cem\u003eP\u003c/em\u003e ≤ 0.01, **\u003cem\u003eP\u003c/em\u003e ≤ 0.001, ***\u003cem\u003eP\u003c/em\u003e ≤ 0.0001, compared to untreated cells (one-way ANOVA followed by Dunnett’s \u003cem\u003epost-hoc\u003c/em\u003e test).\u003c/p\u003e","description":"","filename":"fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/36a01ac4f6146b46cca644dc.jpg"},{"id":96791340,"identity":"7d9ae947-1985-43dd-9791-6c8fa751493c","added_by":"auto","created_at":"2025-11-26 06:55:09","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":878547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFTVI and E-selectin treatments stimulate cell cycling in the short term, enhancing bone marrow engraftment\u003c/strong\u003e. (A) UMAP visualization of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells treated with Esel and/or FTVI, with the left panel categorized by treatment and the right panel categorized by cluster. (B) Percentage of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells under different fucosylation-targeting treatments in each cell cycle phase as determined by marker gene expression. (C) Bubble plots showing Reactome, KEGG, and GO pathway enrichment for top expressed genes in FTVI+Esel cells. (D) Gene set enrichment analysis for pathways related to cell cycle regulation and HSC migration for top expressed genes in FTVI+Esel cells. (E) Representative flow cytometry plot showing Ki67-DAPI staining of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells under different fucosylation-targeting treatments. (F) Quantification of Ki67-DAPI-stained cells showing the distribution of cells in the G0, G1, and S/G2/M phases. (G) Representative plot showing the gating strategy for BrdU-stained mice injected with untreated or FTVI-treated mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells. (H) Left-hand figure shows the percentage of BrdU-stained cells while the right-hand figures show the differences in cell cycle status between treated and untreated cells.\u003c/p\u003e","description":"","filename":"fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/a772d02651f1808a3f9b2c07.jpg"},{"id":101692256,"identity":"9b9429eb-50b8-4516-a32f-3e76c9b14a85","added_by":"auto","created_at":"2026-02-02 16:17:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10211096,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/3448921f-ae63-409c-91f9-ae0eff79eafb.pdf"},{"id":96791312,"identity":"38d95a24-4377-4177-9b82-59c48413fd89","added_by":"auto","created_at":"2025-11-26 06:55:07","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1080671,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/fdb466fbe77a851ddc6e640b.xlsx"},{"id":96791317,"identity":"469539a7-04b2-46f6-b1ea-e848fa032279","added_by":"auto","created_at":"2025-11-26 06:55:08","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1126369,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/a8a45c4b30390afcc7130e22.xlsx"},{"id":96791319,"identity":"07231416-3edf-4db2-869b-835be569075c","added_by":"auto","created_at":"2025-11-26 06:55:08","extension":"avi","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2858534,"visible":true,"origin":"","legend":"","description":"","filename":"2FF.avi","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/3c436b926cd04bdf9e722f0c.avi"},{"id":96791314,"identity":"401029cf-32c4-4ae1-956b-d57658660932","added_by":"auto","created_at":"2025-11-26 06:55:07","extension":"avi","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17414988,"visible":true,"origin":"","legend":"","description":"","filename":"FTVI.avi","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/4d094abbca31af9f918aea52.avi"},{"id":96791394,"identity":"eba28474-498a-4ebd-abdf-feb776ca231d","added_by":"auto","created_at":"2025-11-26 06:55:13","extension":"avi","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":46602832,"visible":true,"origin":"","legend":"","description":"","filename":"untreated.avi","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/cdf711aa204fd0734a12c824.avi"},{"id":96916461,"identity":"d11ab6d3-8b51-4cd0-b505-8cbd95a9abca","added_by":"auto","created_at":"2025-11-27 14:08:37","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10312646,"visible":true,"origin":"","legend":"","description":"","filename":"A.AlAmoodiandA.AlguneimSupplementarydataBMCCancer.docx","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/a99264d2f7d98bf5eff66c3d.docx"},{"id":96791387,"identity":"9aa1999a-abad-4959-a3fa-b5bc02806c81","added_by":"auto","created_at":"2025-11-26 06:55:12","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":2123268,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterialFullWBs.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7961408/v1/9c04a12f365dc4a90956fbc8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Fucosylation Enhances CD34⁺ Hematopoietic Stem Cell Homing and Longevity via E-Selectin–Mediated Adhesion and Signaling","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHSCT can stop or even reverse the progression of various types of diseases from hematological malignancies to autoimmune disorders [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Understanding the mechanisms that facilitate successful transplantation is crucial to improving outcomes and preventing relapse. Since HSCT is administered intravenously, its success depends on the ability of circulating HSCs to locate the bone marrow niche and home [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e] \u0026ndash; and this is directly related to the interaction between the glycan ligand Sialyl Lewis X (sLe\u003csup\u003ex\u003c/sup\u003e) on the cell surface [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and the E-selectin adhesion molecule that is stably expressed on marrow endothelial cells [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This interaction starts a multi-step process also involving chemokine receptors and integrins that guides HSCs into the bone marrow niche. Studies have shown that sLe\u003csup\u003ex\u003c/sup\u003e levels have a direct impact on the physiological behavior of HSCs [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In cancer cells, enhanced sLe\u003csup\u003ex\u003c/sup\u003e expression is associated with increased metastasis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], which happens because key glycosyltransferases are upregulated [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], such as fucosyltransferases (FUTs) that are directly involved in fucosylation and have been linked to cancer progression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Consequently, several studies have investigated ways to obstruct fucosylation based on the biochemistry of FUTs, such as the prominent inhibitor 2-Fluoro-fucose (2FF) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], an analog of GDP-fucose, which interferes with the salvage pathway in GDP-fucose synthesis [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] and has been shown to effectively block leukocyte adherence and rolling interactions with selectins in a sickle cell mouse model [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and oncogenic mouse models [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlternatively, in other clinical contexts such as HSCT, promoting fucosylation can be useful for HSCs whose surface expression of the sLe\u003csup\u003ex\u003c/sup\u003e epitope is low [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], which inhibits bone marrow homing and limits transplantation success [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In human CD34\u003csup\u003e+\u003c/sup\u003e HSCs, reduced sLe\u003csup\u003ex\u003c/sup\u003e expression is caused by a lack of α-1,3-FUT enzymes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] responsible for adding the fucose sugar to the GlcNAc terminal, thereby impairing sLe\u003csup\u003ex\u003c/sup\u003e formation and inhibiting the interactions between HSCs and bone marrow endothelium [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Recombinant human fucosyltransferase 6 (FTVI) has been shown to effectively promote HSC homing of both mouse [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and human cells in mouse models [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A 2015 clinical trial of cord blood-derived HSCs showed that \u003cem\u003eex vivo\u003c/em\u003e fucosylation using FTVI promotes engraftment [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. We have previously demonstrated that \u003cem\u003eex vivo\u003c/em\u003e fucosylation promoted sLe\u003csup\u003ex\u003c/sup\u003e epitope levels in murine and human HSCs, thereby enhancing their migration, engraftment, and self-renewal capacities, effectively increasing their stemness [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, the precise phenotypic changes and molecular pathways associated with improved engraftment in fucosylated HSCs remain unclear. This study sought to shed light on the cellular processes resulting from fucosylation treatment by manipulating fucose\u0026rsquo;s formation on human granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood CD34\u003csup\u003e+\u003c/sup\u003e (mPB-CD34\u003csup\u003e+\u003c/sup\u003e) HSCs through two treatments, FTVI to enhance sLe\u003csup\u003ex\u003c/sup\u003e expression and 2FF to inhibit it. We then analyzed the associated intrinsic parameters of cell cycling, signaling, viability, and physiological flow. Our results indicate that fucosylation enhances the adhesive and migratory capacity of mPB-CD34⁺ HSCs by improving E-selectin interactions, while also activating Rho-GTPase and PI3K/AKT/mTOR signaling pathways that promote early entry into the cell cycle. Notably, these changes appear to preserve long-term stemness, suggesting that fucosylation plays a multifaceted role in regulating both short-term engraftment and durable hematopoietic reconstitution.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eHuman CD34\u003c/b\u003e\u003csup\u003e+\u003c/sup\u003e \u003cb\u003estem cell culture and fucosylation treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHuman CD34\u003csup\u003e+\u003c/sup\u003e progenitor cells (1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e; Lonza). Thawed cells were centrifuged, resuspended in stem cell media (Serum-Free Expansion Medium II, Stem Cell Technologies), and treated with 10\u0026times; StemSpan CD34\u003csup\u003e+\u003c/sup\u003e Expansion Supplement (Stem Cell Technologies). 2FF (400 \u0026micro;M; Biosynth Carbosynth) was added to the culture for several days as described in previously published work [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. FTVI treatment was performed as previously established [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Briefly, rhFTVI enzyme (KAICO Ltd.) was added in a buffer using 0.5 mM GDP L-Fucose (Sigma\u0026ndash;Aldrich), 25 mM HEPES (Gibco), 5 mM magnesium chloride, 0.1% human serum albumin (Sigma\u0026ndash;Aldrich) in HBSS media (Gibco), and incubation was performed at 37\u0026deg;C for 30 min. After incubation, cells were washed twice in a sterile buffer containing 5% FBS and 2 mM EDTA (Thermo Fisher Scientific).\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eFlow-based and microfluidic rolling assays\u003c/h2\u003e\u003cp\u003e\u003cb\u003eFluorescence multiplex cell rolling (FMCR)\u003c/b\u003e was conducted as previously established [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Briefly, the control/untreated cells were stained with green cell mask membrane (522/535), 2FF-treated cells with orange cell mask membrane (554/567), and FTVI-treated cells with Vybrant DiD (644/665). Confocal laser scanning microscopy (Zeiss) was performed on a parallel plate flow chamber (Bioptechs) including a coverslip with E-selectin-expressing Chinese hamster ovary (CHO-E) cells.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMicrofluidic-based rolling assay\u003c/strong\u003e\u003cp\u003eWide-field fluorescence imaging experiments were conducted using a home-built fluorescence microscopy setup [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. E-selectin-deposited microfluidic chambers were connected to the syringe pump (PHD Ultra, Harvard Apparatus). The stained cells were injected into the E-selectin-deposited microfluidic chambers through silicon tubing (inner diameter of 0.8 mm, ibidi GmbH) connected to the syringe pump and perfused on the stage of an inverted fluorescence microscope at a flow rate of 2 dyn/cm\u003csup\u003e2\u003c/sup\u003e. Real-time fluorescence imaging of the live cells was performed using an EMCCD camera with a 60\u0026times; objective lens at 0.92 mV laser power (see Extended Methods in the Online Supplementary Appendix).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003etransplantation and imaging\u003c/b\u003e\u003c/p\u003e\u003cp\u003emPB-CD34\u0026thinsp;+\u0026thinsp;HSCs were stained with Xeno Light DiR dye, washed, and injected into NSG mice. Fluorescence imaging was conducted at various time points. E-selectin blocking experiments utilized anti-CD62E antibodies. Bone marrow engraftment and BrdU incorporation were analyzed via flow cytometry. All procedures adhered to ethical guidelines and approvals (see Extended methods in the Online Supplementary Appendix).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003ePhosphopeptide enrichment and data processing\u003c/h3\u003e\n\u003cp\u003eFollowing treatment with FTVI and E-selectin, cells lysates were centrifuged and the proteins were digested via the S-Trap protocol, and peptides were desalted. Approximately 5% of the peptides were used for global proteome profiling, while the remainder underwent phosphopeptide enrichment using TiO2 microspheres. After washing and elution, phosphopeptides were desalted and analyzed by mass spectrometry. Data were processed using MSFragger against a comprehensive protein database, identifying differentially expressed proteins based on specified criteria (see Extended methods in the Online Supplementary Appendix).\u003c/p\u003e\n\u003ch3\u003eSingle-cell RNA sequencing\u003c/h3\u003e\n\u003cp\u003emPB-CD34\u003csup\u003e+\u003c/sup\u003e cells treated with FTVI and/or E-selectin were used to prepare a single-cell sequencing library using a Chromium Next GEM Single cell 3' kit (10\u0026times; Genomics) according to the manufacturer\u0026rsquo;s instructions. Library size distribution and concentration was validated using an Agilent 2100 Bioanalyzer (Agilent Technologies) and Invitrogen Qubit 2.0 Fluorometer (Life Technologies-Invitrogen), respectively. The libraries were sequenced on a NovaSeq 6000 SP flow cell (Illumina) by Novogene. We used 10\u0026times; Genomics Cell Ranger software (version 7.0.0) to perform reference alignment, unique molecular identifier (UMI) collapsing and counting, and initial filtering. We identified highly variable genes (FindVariableGenes; Seurat) and performed subpopulation clustering (FindCluster; Seurat). Cell cycle phase was determined (CellCycleScoring) and the gene ontology (GO) term, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and Reactome enrichment analyses were performed using clusterProfiler.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData analysis was performed using GraphPad Prism (version 9 for Mac, GraphPad Software, San Diego, CA, USA). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Unless otherwise indicated, Student\u0026rsquo;s t-test was used to compare two treatment groups; one-way analysis of variance (ANOVA) followed by Dunnett\u0026rsquo;s post-hoc test was used for multiple comparisons for more than two treatment groups. Statistical significance was set at P\u0026thinsp;\u0026le;\u0026thinsp;0.05. Graphs were constructed using GraphPad Prism.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003e2FF treatment dramatically inhibits core fucosylation, reducing E-selectin binding\u003c/b\u003e\u003c/p\u003e\u003cp\u003esLe\u003csup\u003ex\u003c/sup\u003e can be detected by anti-sLe\u003csup\u003ex\u003c/sup\u003e antibodies such as HECA-452 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. We examined the impact of removing fucose through 2FF treatment on sLe\u003csup\u003ex\u003c/sup\u003e expression and E-selectin binding in human mPB-CD34\u003csup\u003e+\u003c/sup\u003e HSCs. Cells were treated with 2FF for 12\u0026ndash;96 h, then analyzed using HECA-452 and recombinant E-selectin by flow cytometry. Both sLe\u003csup\u003ex\u003c/sup\u003e expression and E-selectin binding were decreased in a time-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), with sLe\u003csup\u003ex\u003c/sup\u003e levels reduced to only\u0026thinsp;~\u0026thinsp;8% of the original level at 72 h. Western blot confirmed this decrease (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), and in sLe\u003csup\u003ex\u003c/sup\u003e levels and \u003cem\u003eAleuria aurantia\u003c/em\u003e lectin (AAL) blotting further supported reduced cell-surface fucosylation (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA).\u003c/b\u003e\u003c/p\u003e\u003cp\u003eConsistent with earlier findings [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], FTVI treatment increased sLe\u003csup\u003ex\u003c/sup\u003e to ~\u0026thinsp;99%, while 2FF dramatically reduced it to \u0026lt;\u0026thinsp;10%, as shown by HECA-452 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). AAL blots of 2FF-treated cell lysates showed markedly reduced intensity, while FTVI enhanced it (\u003cb\u003eFigure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eWe also examined how 2FF and FTVI modulate E-selectin\u0026rsquo;s interaction with its ligands\u0026mdash;CD43, CD44, and PSGL-1 [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u0026mdash;via immunoprecipitation followed by E-selectin blotting. As expected, 2FF abrogated ligand binding, whereas FTVI reinforced this interaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. These findings highlight how altering sLe\u003csup\u003ex\u003c/sup\u003e levels via fucosylation directly influences E-selectin binding capacity in mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eInhibiting fucosylation in mPB-CD34\u003c/b\u003e\u003csup\u003e+\u003c/sup\u003e \u003cb\u003ecells reduces SDF-1-mediated transmigration but does not impact clonogenicity\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eGiven the importance of core fucosylation in pathophysiology, we evaluated how 2FF affects the colony-forming ability of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells. After a 3-day 2FF treatment, no significant difference in colony formation was observed compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This aligns with our previous findings that FTVI treatment does not markedly alter HSC clonogenicity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSince several FUTs (FUT3\u0026ndash;7 and FUT9) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] participate in the biosynthesis of sLe\u003csup\u003ex\u003c/sup\u003e, we examined how 2FF and FTVI influence FUT expression. qRT-PCR showed that neither treatment significantly changed α1,3-FUT transcript levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Although 2FF reduced FUT9 expression, its role in sLe\u003csup\u003ex\u003c/sup\u003e synthesis is limited.\u003c/p\u003e\u003cp\u003eAs HSCs use CXCR4 to migrate toward the chemokine SDF-1α during homing to the bone marrow, we assessed treatment effects on SDF-1α-directed chemotaxis. 2FF significantly impaired CXCR4-mediated migration of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), while FTVI had no effect (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). These results demonstrate that inhibiting fucosylation selectively disrupts chemotactic migration of mPB-CD34\u003csup\u003e+\u003c/sup\u003e HSCs without affecting clonogenicity.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eEnhanced fucosylation improves rolling and creates long, tight tethers and slings, while inhibiting fucosylation diminishes their number and size\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWe examined treatment-induced phenotypic changes over time. Light microscopy showed that while all samples retained typical circular morphology, E-selectin treatment led to mild cell clumping, which was markedly increased in FTVI-treated samples (FTVI\u0026thinsp;+\u0026thinsp;E-selectin), indicating enhanced cell-cell adhesion (\u003cb\u003eFigure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA\u003c/b\u003e, \u003cb\u003eS2B\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eFlow-based migration assays, including FMCR assays [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and microfluidic-based live-cell imaging [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], were used to assess the impact of fucosylation modulation on mPB-CD34\u003csup\u003e+\u003c/sup\u003e cell rolling. Spectral overlap between fluorescence tags was minimized to distinguish samples under simultaneous excitation (405 nm, 488 nm, 633 nm; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). FTVI-treatment significantly increased both the rolling duration (~\u0026thinsp;2.2-fold, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and rolling cell number (~\u0026thinsp;1.4-fold, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.019), while 2FF reduced rolling cells\u0026thinsp;~\u0026thinsp;2.7-fold (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similar trends were observed under different shear stress levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). FTVI-treatment also slowed rolling velocity, whereas 2FF accelerated it compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eTo explore the mechanism, we imaged tethers and slings via 3D epifluorescence microscopy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The selectin ligand, CD44, immunolabeled using AF-647, was distributed along the entire length of these structures [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), consistent with prior observations in leukemic cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In untreated cells, tether and sling lengths averaged 12 \u0026micro;m and 19 \u0026micro;m, respectively. 2FF shortened them to 8 \u0026micro;m and 9.6 \u0026micro;m (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.04 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0013), while FTVI slightly increased tether length (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Both treatments (FTVI and 2FF) reduced sling numbers (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.007 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) compared untreated cells. We speculate that shorter, weaker tethers in 2FF-treated cells limit sling formation, whereas stronger tether anchoring in FTVI-treated cells may curtail sling generation.\u003c/p\u003e\u003cp\u003eThese findings suggest that fucosylation directly influences rolling behavior through changes in tether and sling dynamics: 2FF reduces rolling and increases speed, while FTVI enhances rolling via stronger, longer tethers.\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cb\u003eBoosting fucosylation improves mPB-CD34\u003c/b\u003e\u003csup\u003e+\u003c/sup\u003e \u003cb\u003ecell delivery to bone marrow and spleen, while inhibition restricts homing\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eTo validate our \u003cem\u003ein vitro\u003c/em\u003e findings, we performed short- and long-term \u003cem\u003ein vivo\u003c/em\u003e migration/homing assays in xenograft mouse models. Fucosylated mPB-CD34\u003csup\u003e+\u003c/sup\u003e (FTVI-treated) localized prominently around the spinal cord, spleen, liver, and legs/hindlimbs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), whereas 2FF treatment reduced the overall fluorescence signal. At 48 h post-transplantation, imaging from multiple angles confirmed higher average fluorescence in FTVI-treated groups compared to controls (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while 2FF had no significant effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eFluorescence intensity in the spleen and legs was elevated in mice that received FTVI-treated cells and reduced in mice that received 2FF-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Flow cytometry plots for bone marrow and blood samples are provided in \u003cb\u003eFigure \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e. At 8-weeks post-transplantation, 2FF-treated cells showed impaired bone marrow engraftment (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.03), whereas FTVI-treated cells exhibited improved engraftment at 12-weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] Blocking E-selectin using anti-CD62E antibody significantly reduced bone marrow persistence of FTVI-treated cells at 12-weeks (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H), indicating that fucosylation-enhanced engraftment depends on E-selectin binding.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003e\u003cb\u003eEngraftment efficiency of mPB-CD34\u003c/b\u003e\u003csup\u003e+\u003c/sup\u003e \u003cb\u003eHSCs in NSG mice. Percentages of human CD45 donor cells engrafted in recipient mouse post-transplantation of HSCs with or without the FTVI treatment.\u003c/b\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCells\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCell Injection Dose\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eNo. of mice\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e\u003cp\u003ePercentage of human cells in recipient mice (weeks)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eExp. 1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBuffered-CD34\u003csup\u003epos\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50,000\u0026ndash;80,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e4.5, 0.05, 11.35, 0, 2.95, 16.7 (5/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.55, 0.15,18.05, 0, 0.5, 8.3 (5/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15, 0, 35.3, 0, 6.6, 28.85 (4/6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFTVI-CD34\u003csup\u003epos\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50,000\u0026ndash;80,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0.05, 0, 6, 4.7, 12.75, 17.6 (5/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e2.3, 0.55, 33.65, 2.6, 3, 13.55 (6/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6.85, 0.1, 45.8, 37.7, 54.05, 32.45 (5/5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e2FF-CD34\u003csup\u003epos\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50,000\u0026ndash;80,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e11.15, 0, 3.85, 4.8, 0.85 (4/5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e3.35, 2.3, 2.6, 1.6, 0 (4/5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e15.35, 20.65, 23.5, 10.5, 0 (4/5)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-selectin function-blocking Ab-CD34\u003csup\u003epos\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50,000\u0026ndash;80,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.9, 9.15, 0.05, 1.95, 6.45 (5/5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0.15, 8.45, 8.85, 1.75, 0 (4/5)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0.1, 1.75, 2.6, 0.1(4/4)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e\u003cp\u003eExp. 2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBuffered-CD34\u003csup\u003epos\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e50,000\u0026ndash;80,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7.1, 5.4, 8.9, 7.1, 0, 2.2, 15.7 (6/7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e70.3, 66.8, 78.7, 33.6, 0.9, 67.3, 77.4 (7/7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e74, 27.6, 85.1, 70.5, 0.4, 42.8, 91(7/7)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFTVI-CD34\u003csup\u003epos\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50,000\u0026ndash;80,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e15.2, 9.7, 10, 19.8, 1.2, 9.2, 2 (7/7)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e61.2, 73.4, 68.9, 67.6, 3.1, 50.9 (6/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e80.3, 30, 71.7, 78.5, 13.3, 72 (6/6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eE-selectin function-blocking Ab-CD34\u003csup\u003epos\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e50,000\u0026ndash;80,000\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1.8, 23.2, 4.2, 0, 16.3, 0.1 (5/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e20.9, 55.6, 27.7, 3.5, 82.7, 0.9 (6/6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12, 3.6, 24.3, 2, 89, 49.3 (6/6)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWe next determined lineage differentiation in peripheral blood at 8-weeks. No significant differences were observed in lymphoid (CD3 and CD19) and myeloid (CD33) lineages among groups that received untreated, 2FF-, or FTVI-treated mPB-CD34\u003csup\u003e+\u003c/sup\u003e HSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Consistent with previous findings [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], mPB-CD34\u003csup\u003e+\u003c/sup\u003e HSCs showed a lymphoid lineage bias.\u003c/p\u003e\u003cp\u003eFinally, we evaluated mPB-CD34\u003csup\u003e+\u003c/sup\u003e cell viability under E-selectin stimulation. Fluorescence microscopy of calcein AM and EthD-1-stained cells showed increased cell death with FTVI\u0026thinsp;+\u0026thinsp;E-selectin treatment, but not with fucosylation alone (\u003cb\u003eFigure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eA\u003c/b\u003e). Annexin V-7AAD staining confirmed that this effect was transient, with cell viability recovering after 72 h (\u003cb\u003eFigure \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003eB\u003c/b\u003e). These results indicate that E-selectin may trigger apoptosis in FTVI-treated cells, but that fucosylation itself does not cause long-term viability loss.\u003c/p\u003e\u003cp\u003eTogether, our findings underscore the role of fucosylation in promoting mPB-CD34\u003csup\u003e+\u003c/sup\u003e HSC engraftment, mediated through E-selectin binding, while having minimal impact on differentiation or long-term viability.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eFucosylation enhances E-selectin-driven stem cell cycling via RHO and PI3K/AKT/NFκB signaling\u003c/h2\u003e\u003cp\u003eWe examined whether enhanced fucosylation influences phosphoproteomic profiles and cell cycle activation in adult HSCs, which typically remain in a quiescent state to avert exhaustion and DNA replication stress. We analyzed four treatments (untreated, FTVI, E-selectin, and FTVI\u0026thinsp;+\u0026thinsp;E-selectin) and identified\u0026thinsp;\u0026gt;\u0026thinsp;4,000 phosphopeptides per sample at 1% FDR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Phosphoserine sites were most abundant across groups (\u0026gt;\u0026thinsp;84%). Notably, E-selectin and FTVI\u0026thinsp;+\u0026thinsp;E-selectin samples showed increased phosphotyrosine sites (1.23% and 1.08%, respectively).\u003c/p\u003e\u003cp\u003eFucosylation alone appeared to influence HSC stemness and migration pathways rather than cycling (data not shown), notably EGFR, VEGFA/VEGFR2, actin cytoskeleton regulators (Kinesin, Spectrin, and Rho GTPases), FOXO (including RBL2, USP7, MAP2K2, AGAP2, AKT1, CSNKE, STK4, MAPK14), and NFκB signaling. In fucosylated cells, dephosphorylation of MAPK14 (P-38α) may indicate suppression of HSC differentiation. We next compared E-selectin vs. FTVI\u0026thinsp;+\u0026thinsp;E-selectin-treated cells. Global proteomics identified 493 DEPs (\u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e), predominantly nuclear (44%). GO analysis revealed enrichment in mRNA processing, DNA replication, DNA repair, mitosis, and cell cycling pathways. Phosphoproteomic profiling revealed 357 phosphopeptides (267 phosphoproteins), with 54 phosphorylated and 91 dephosphorylated DEPs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C, \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Key cell cycle regulators such as RB1, DYNC1LI1, NIPBL, and TP53BP1 were dephosphorylated, while LIMK2, SIK3PHKB, and TAB2 were phosphorylated, implicating MAPK (TAB2), TGFβ (LIMK2, NEDD4), and EGFR signaling (LIMK2, ABI1). Pathway enrichment identified Rho-GTPase signaling as the dominant network in FTVI\u0026thinsp;+\u0026thinsp;E-selectin group, involving proteins like Rac1/2 effectors, ROCK, and GTPase regulators (e.g. LIMK2, ABI1, MYO9B, ARAP1, LEMD3, and RBMX.\u003c/p\u003e\u003cp\u003eUsing a \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 threshold, we detected 446 phosphopeptides in the E-selectin group (phosphorylated: 98; dephosphorylated: 160) and 448 in the FTVI\u0026thinsp;+\u0026thinsp;E-selectin group (phosphorylated: 77; dephosphorylated: 199). Of these, 244 overlapped, while\u0026thinsp;~\u0026thinsp;200 were unique to each (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG, \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). GO and molecular function analysis (\u003cb\u003eFigure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e\u003c/b\u003e) showed enrichment in actin/tubulin binding and MAP kinase binding. While MAPK signaling [MAPK3 (ERK), MAPK1 (ERK), MAPK11/12/13/14 (p38-β/γ/δ/α), and MAPK10 (JunK3)] was shared, only the FTVI\u0026thinsp;+\u0026thinsp;E-selectin group showed enrichment in small GTPase binding (e.g., HPS4, MYO9B, ARHGEF2, PAK2, MTSS2, GBF1m HPS4, ARHGEF18, and MYO9B). KEGG and Reactome pathways emphasized the importance of the Rho signaling, cytoskeleton regulation, and stemness-associated Wnt, Hedgehog, and TGFβ pathways (e.g., TRIO, HSP90AB1, FNBP1, ROCK2, DOCK8, ARHGAP1, ARHGEF18, IQGAP2, AHGAP15, SRRM1, ARFGAP2, STIP1, RBMX, and ARHGEF6; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F).\u003c/p\u003e\u003cp\u003eTo validate phosphorylation changes, flow cytometry showed PI3K/AKT/mTOR and NFκB pathway activation specifically in FTVI\u0026thinsp;+\u0026thinsp;E-selectin-treated cells. Reduced PTEN, an AKT inhibitor, phosphorylation supported AKT activation (\u003cb\u003eFigure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eA\u003c/b\u003e), and FOXO/EGFR pathways were induced, consistent with phosphoproteomic data. CaCl\u003csub\u003e2\u003c/sub\u003e, essential for E-selectin binding, did not influence phosphorylation independently (data not shown).\u003c/p\u003e\u003cp\u003eWe further confirmed NFκB activation, a transcription factor essential to stem cell cycling [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], using western blotting at 30 min and 5 h. NFkB phosphorylation was triggered by E-selectin binding, particularly in FTVI\u0026thinsp;+\u0026thinsp;E-selectin-treated cells (\u003cb\u003eFigure \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003eB\u003c/b\u003e) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eTo explore the effects of fucosylation and E-selectin binding on cell cycle regulation, we examined the phosphorylation levels of Rb proteins, which are key regulators of cell cycle progression. We show that treatment with FTVI\u0026thinsp;+\u0026thinsp;E-selectin decreased the level of phosphorylated Rb proteins compared to untreated cells, indicating a cell cycle arrest (\u003cb\u003eFigure \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003eD\u003c/b\u003e).\u003c/p\u003e\u003cp\u003eCollectively, these results indicate that fucosylation primes HSCs for E-selectin-mediated activation of cell cycle and stemness programs, largely via Rho GTPases, and PI3K/AKT/ NFκB signaling.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCombined FTVI and E-selectin treatment drives a distinctive stem cell gene regulatory network\u003c/h3\u003e\n\u003cp\u003eWe investigated whether enhanced E-selectin binding via fucosylation influences transcriptional networks related to cell cycling and stemness. Using single-cell RNA sequencing (scRNA-seq), we profiled 5,266 and 4,500 high-quality transcriptomes from E-selectin- and FTVI\u0026thinsp;+\u0026thinsp;E-selectin-treated mPB-CD34\u003csup\u003e+\u003c/sup\u003e HSCs, respectively (\u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). UMAP visualization revealed 13 transcriptionally distinct groups, with clusters 3 and 5 enriched in FTVI\u0026thinsp;+\u0026thinsp;E-selectin-treated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Differential gene expression analysis identified\u0026thinsp;~\u0026thinsp;22 and ~\u0026thinsp;33 upregulated transcripts in clusters 3 and 5, respectively (\u003cb\u003eTable \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e\u003c/b\u003e). CellCycleScoring indicated a higher proportion of cells in G2/M in the FTVI\u0026thinsp;+\u0026thinsp;E-selectin group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), suggesting delayed reentry into G0 and prolonged cycling.\u003c/p\u003e\u003cp\u003ePathway enrichment analysis (GO, Reactome, and KEGG) showed that FTVI\u0026thinsp;+\u0026thinsp;E-selectin-treated cells were enriched in pathways related to cell cycle regulation (DNA replication, mitotic division, cyclins, G2/M transition, and MAPK4/MAPK6), homing (focal adhesion, actin cytoskeleton, Rho GTPases, cadherins, and integrins), and stemness (WNT, Hedgehog, TGF-β, and NOTCH) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Gene set enrichment confirmed elevated Rho-GTPase-related signaling in clusters enriched with FTVI\u0026thinsp;+\u0026thinsp;E-selectin cells, including CDC42, Rac1, and Rac2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eWe next investigated short- and long-term cycling. Ki67-DAPI flow cytometry at 30 min revealed a\u0026thinsp;~\u0026thinsp;two-fold reduction in quiescent (G0) mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells in E-selectin and FTVI\u0026thinsp;+\u0026thinsp;E-selectin-treated samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F), indicating that fucosylation amplifies E-selectin-driven cell cycling. Notably, after 4\u0026ndash;5 days, FTVI-treated cells returned to the G0, whereas untreated cells remained cycling (data not shown).\u003c/p\u003e\u003cp\u003eTo evaluate long-term turnover, we examined BrdU incorporation \u003cem\u003ein vivo\u003c/em\u003e. At 12-weeks post-transplantation, BrdU-labeled hCD45\u003csup\u003e+\u003c/sup\u003e/hCD34\u003csup\u003e+\u003c/sup\u003e cells increased by 23% in mice receiving fucosylated HSCs compared to controls, indicating early S-phase entry (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-H). However, most of these cells were in G0, supporting the idea that fucosylation enhances early self-renewal while maintaining long-term quiescence.\u003c/p\u003e\u003cp\u003eThese findings suggest that fucosylation primes HSCs to respond to E-selectin signaling by promoting early cycling and migration through Rho-GTPase pathways, while preserving long-term quiescence necessary for durable engraftment.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eFucosylating stem cells is emerging as a treatment that has the potential to improve HSCT outcomes, necessitating a deeper understanding of the impact that the increased sLe\u003csup\u003ex\u003c/sup\u003e expression resulting from the treatment has on HSC function. While previous studies indicated that \u003cem\u003eex vivo\u003c/em\u003e fucosylation of HSCs enhances E-selectin binding and engraftment [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], this study reveals the broader implications on cellular processes including cell cycle and self-renewal.\u003c/p\u003e\u003cp\u003eWe confirmed that 2FF treatment substantially suppressed, while FTVI markedly increased, sLe\u003csup\u003ex\u003c/sup\u003e expression and E-selectin ligand activity of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells, consistent with previous studies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Importantly, 2FF-mediated inhibition impaired E-selectin binding and chemotactic migration toward SDF-1α, whereas clonogenic potential remained unaffected, suggesting that fucosylation selectively governs migration without compromising differentiation capacity. Other studies, however, have reported that 2FF significantly affected differentiation in certain cancer types such as liver cancer, but not in others, like breast cancer [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This discrepancy may be context/cell type specific. The observed suppression of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cell migration \u003cem\u003ein vitro\u003c/em\u003e by 2FF may reflect global glycan alterations that extend beyond sLe\u003csup\u003ex\u003c/sup\u003e downregulation, affecting the functionality of multiple adhesion molecules. For instance, glycosylation of proteins like Notch is critical for their transcriptional activity and regulation of hematopoiesis and stemness [\u003cspan additionalcitationids=\"CR43\" citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Thus, our findings reinforce the role of fucosylation as a key post-translational modification essential for both E-selectin recognition and broader HSC biology.\u003c/p\u003e\u003cp\u003eWe next investigated the physical interactions of HSCs with E-selectin under shear stress using flow-based rolling assays [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and our microfluidic platform [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. FTVI treatment significantly increased the number of rolling cells and rolling duration, likely due to improved tether and sling mediated interactions. These structures, visualized using live-cell fluorescence imaging, were longer and more stable in FTVI-treated cells compared to controls, supporting the role of fucosylation in enhancing mechanical anchorage to the vascular endothelium. The dynamic elongation, anchoring, long length, and multiplicity of the tether and sling are exploited by cells to slow their rolling and interact with other surface molecules [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In contrast, 2FF-treated cells displayed reduced tether formation and increased rolling velocity, consistent with impaired adhesion.\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e homing and engraftment assays further substantiated the functional impact of fucosylation. FTVI-treated cells demonstrated enhanced localization to the bone marrow and spleen in xenograft models, while 2FF-treated cells exhibited impaired early migration. The inhibitory effect of the 2FF treatment was most pronounced at 6\u0026ndash;8 weeks after transplantation, and engraftment efficiency was comparable to that in untreated cells at 12 weeks after injection. These results reflect the role of fucosylation during the initial stage of HSC migration and suggest that a low-but-sufficient number of HSCs can reach the bone marrow and repopulate and sustain it. Long-term engraftment analysis further confirmed the impact of fucosylation on the engraftment efficiency of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells in the bone marrow at 12 weeks post-transplantation, consistent with our previous finding [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These observations align with clinical studies exploring fucosylation to improve cord blood HSC transplantation, where enhanced sLe\u003csup\u003ex\u003c/sup\u003e expression was shown to accelerate neutrophil recovery and improve early engraftment kinetics [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eInterestingly, while FTVI alone did not trigger cell aggregation, FTVI\u0026thinsp;+\u0026thinsp;E-selectin-treated cells showed increased adhesion and transient apoptosis. These effects were not due to fucosylation \u003cem\u003eper se\u003c/em\u003e, but rather the amplified interaction with E-selectin. Consistent with previous reports [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] actively proliferating cells may be more susceptible to apoptosis following strong E-selectin engagement. Notably, cell viability recovered after 72 hours, indicating a reversible effect. With regard to cell cycle regulation, we found that E-selectin binding in the context of FTVI treatment promoted the proliferation of mPB-CD34\u003csup\u003e+\u003c/sup\u003e cells at the expense of dormant (G0) cell numbers, as validated in E-selectin knockouts \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. HSCs are typically maintained in the G0 state near osteoblasts in the bone marrow niche to avoid depletion. To preserve homeostasis, HSCs may undergo asymmetric division, yielding one stem cell and one committed progenitor [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. This mechanism may underlie the enhanced engraftment seen in FTVI-treated mice. Notably, this cycling effect appeared transient; at later time points, cells accumulated in the G2/M phase but did not re-enter the cycle. Future studies are needed to determine how FTVI and E-selectin together influence asymmetric division and long-term HSC maintenance. Importantly, viability returned to baseline levels within 72 hours, indicating that the pro-cycling and apoptotic responses to E-selectin are temporary and reversible.\u003c/p\u003e\u003cp\u003eIn our investigation of the molecular underpinnings of these observations, phosphoproteomic and single-cell transcriptomic analyses demonstrated that FTVI\u0026thinsp;+\u0026thinsp;E-selectin treatment activated EGFR, PI3K/AKT/mTOR and NFκB pathways, in which are critical regulators of HSC regeneration and cycling. The EGFR pathway promotes HSC regeneration and survival following total body irradiation, as systematic infusion of epidermal growth factor into irradiated mice dramatically improved HSC secondary engraftment. In addition to its regenerative role, EGFR is a key mediator of cell cycle regulation, acting upstream of PI3K and AKT. Activated Akt promotes cyclin expression and Rb inhibition, driving cell cycle progression. Moreover, EGFR can function as transcription factor for cell cycle regulating genes, such as \u003cem\u003eCYCLIN D1\u003c/em\u003e and \u003cem\u003eC-MYC\u003c/em\u003e, further enhancing proliferation.\u003c/p\u003e\u003cp\u003eNFκB activation occurs through PI3K, which converts phosphatidylinositol-4,5-biphosphate into phosphatidylinositol-4,5,6-triphosphate, a scaffold and activator of AKT. E-selectin binding has been found to activate NFκB as early as 30 min post-administration, and transcription factor regulates hematopoiesis. NFκB deletion in HSCs disrupts gene expression\u0026mdash;such as through \u003cem\u003ec-Met\u003c/em\u003e upregulation\u0026mdash;impairing engraftment and hematopoiesis, whereas constitutive NFκB activity enhances HSC proliferation and represses quiescence [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eOur data highlight a critical role for Rho-GTPase signaling in mediating the effects of fucosylation and E-selectin binding. Among the classically regulated Rho GTPases, Rho, Rac, Cdc42\u0026mdash;well-studied in HSC biology\u0026mdash;were significantly enriched in our phosphoproteomic and transcriptome analyses following FTVI and E-selectin treatment. These GTPases regulate key HSC functions, including homing, migration, cell cycle progression, and interactions with microenvironmental cues [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Notably, Rac is activated by cell surface receptors such as c-kit, CXCR4, and α4β1, which are likely upregulated in fucosylation-enhanced migrating HSCs. Our findings align with prior studies demonstrating that Rac and Cdc42 are essential for HSC retention in the bone marrow [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], long-term engraftment of HSCs [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], and HSC mobilization [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], while loss of their activity leads to impaired function and increased apoptosis. In addition to the previously discussed pathways, the activation of stemness-associated Wnt, Hedgehog, and TGFβ signaling pathways was observed in our datasets further underscoring a regulatory network that promotes the maintenance of stemness in FTVI and E-selectin-treated cells.\u003c/p\u003e\u003cp\u003eIn conclusion, we show that enhancing fucosylation optimizes HSC tethering and rolling on E-selectin, activating EGFR/PI3K/AKT/mTOR/NFκB signaling to drive cell cycle progression. In parallel, fucosylation promotes HSC stemness and engraftment through Rho-GTPase signaling, reinforcing their retention and function in the bone marrow niche. These findings establish a mechanistic link between glycosylation and intracellular signaling pathways that regulate the fate of HSCs and suggest that extending phosphorylation-based and transcriptomic profiling may uncover additional therapeutic targets.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eHSCT\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHematopoietic stem cell transplantation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003emPB-CD34⁺\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHuman granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood CD34⁺ cells\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003erhFTVI\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRecombinant human α1,3-fucosyltransferase VI\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003esLe\u003c/b\u003e\u003csup\u003e\u003cb\u003ex\u003c/b\u003e\u003c/sup\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSialyl Lewis X\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003e2FF\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e2-Fluoro-fucose\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eFMCR\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFluorescence multiplex cell rolling\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eUMI\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eUnique molecular identifier\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eGO\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGene Ontology\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eKEGG\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eKyoto Encyclopedia of Genes and Genomes\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cb\u003eAAL\u003c/b\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAleuria aurantia lectin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eAll in vivo experimental procedures were conducted in accordance with the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e (NIH Publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996) and the \u003cem\u003eImplementing Regulations of the Law of Ethics of Research on Living Creatures\u003c/em\u003e (Kingdom of Saudi Arabia National Committee of Bioethics, 3rd Edition). All protocols were approved by the King Abdullah University of Science and Technology (KAUST) Institutional Animal Care and Use Committee (IACUC) under protocol number \u003cb\u003e17IACUC20\u003c/b\u003e.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eCorrespondence\u003c/h2\u003e\u003cp\u003eJasmeen Merzaban, Laboratory of Cell Migration and Signaling, Bioscience Program, Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955\u0026thinsp;\u0026minus;\u0026thinsp;6900, Kingdom of Saudi Arabia; Tel.: +96628082383; E-mail: [email protected]\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe research reported in this publication was supported by funding from a King Abdullah University of Science and Technology (KAUST) Faculty Baseline Research Funding Program (to J.S.M.) and a King Abdullah University of Science and Technology (KAUST) \u0026ndash; KAUST Center of Excellence for Smart Health (KCSH), under award number 5932.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.S.A. and A.A designed and performed the experiments, analyzed the data, and wrote the manuscript. J.M. performed cell cycling and signaling experiments and wrote the manuscript. S.N. performed the microfluidic flow-based assay and S.H. supervised this work, Y.L. conducted the in vivo mouse experiments. J.K. helped in some biocomputational analyses. H.Z. and D.B. ran the phosphoproteomic experiment while A.K.A. assisted in the in vivo experiments. J.S.M. conceived and designed the study, analyzed the data, and wrote the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe acknowledge the invaluable contributions of the KAUST Animal Resource Core Lab (ARCL) at the King Abdullah University of Science and Technology (KAUST) and Dr. Simona Spinelli and Mr. Stefano Pietro for their superb instruction and support. KAUST is an AAALAC International accredited institution. We thank Dr. Ioannis Isaioglou for constructive discussions on phosphoproteomics. We acknowledge the assistance of Ms. Umm Habiba with lab administration as well as the members of the Cell Migration and Signaling Laboratory for their help and insightful comments.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll analyzed datasets are included in the supplementary tables. Raw datasets generated during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHenig I, Zuckerman T: \u003cstrong\u003eHematopoietic stem cell transplantation-50 years of evolution and future perspectives\u003c/strong\u003e. \u003cem\u003eRambam Maimonides Med J \u003c/em\u003e2014, \u003cstrong\u003e5\u003c/strong\u003e(4):e0028.\u003c/li\u003e\n\u003cli\u003eLiesveld JL, Sharma N, Aljitawi OS: \u003cstrong\u003eStem cell homing: From physiology to therapeutics\u003c/strong\u003e. \u003cem\u003eStem Cells \u003c/em\u003e2020, \u003cstrong\u003e38\u003c/strong\u003e(10):1241-1253.\u003c/li\u003e\n\u003cli\u003eSackstein R: \u003cstrong\u003eEngineering cellular trafficking via glycosyltransferase-programmed stereosubstitution\u003c/strong\u003e. \u003cem\u003eAnn N Y Acad Sci \u003c/em\u003e2012, \u003cstrong\u003e1253\u003c/strong\u003e(1):193-200.\u003c/li\u003e\n\u003cli\u003eAbuelela AF, K. 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We previously demonstrated that fucosylated HSC ligands interact with endothelial E-selectin to facilitate homing. However, the downstream consequences of modulating fucosylation in HSCs remain unclear. Here, we systematically characterized how enhancing or inhibiting fucosylation\u0026mdash;via recombinant human fucosyltransferase 6 (FTVI) or 2-fluoro-L-fucose (2FF), respectively\u0026mdash;affects migration, signaling, and engraftment of human granulocyte-colony stimulating factor-mobilized peripheral blood CD34⁺ (mPB-CD34⁺) cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eLive-cell imaging under flow, phosphoproteomics, and transcriptomics were used to characterize rolling dynamics and intracellular signaling, and in vivo homing was assessed in immunodeficient xenograft mouse models.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eFucosylation enhanced tether and sling formation, improved E-selectin binding, and increased homing to the bone marrow and spleen. FTVI-treated cells activated MAPK and PI3K/AKT/mTOR pathways and showed enriched Rho-GTPase signaling, associated with proliferation and migration. In contrast, 2FF-treated cells had impaired migration and reduced rolling efficiency. Long-term studies confirmed enhanced repopulation and self-renewal capacity of fucosylated cells.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eFucosylation critically modulates E-selectin interactions, migration, and intracellular signaling in HSCs. These findings highlight glycoengineering as a promising strategy to enhance HSC transplantation outcomes in cancer therapy.\u003c/p\u003e","manuscriptTitle":"Fucosylation Enhances CD34⁺ Hematopoietic Stem Cell Homing and Longevity via E-Selectin–Mediated Adhesion and Signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-26 06:54:57","doi":"10.21203/rs.3.rs-7961408/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-16T15:20:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-15T03:25:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-10T21:26:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-07T22:47:03+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"296014489013473175152950182264890121524","date":"2025-11-24T18:15:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135041114454857289238945953654811719157","date":"2025-11-24T10:08:10+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157602849470345891042526437479209887652","date":"2025-11-21T16:17:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42209478783521163953652465609405458484","date":"2025-11-21T15:50:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-13T15:26:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-04T06:31:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-03T21:07:54+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Cancer","date":"2025-11-03T21:04:06+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-cancer","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bcan","sideBox":"Learn more about [BMC Cancer](http://bmccancer.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bcan/default.aspx","title":"BMC Cancer","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f0e19ad0-91cc-4513-b302-2e6d65e949c2","owner":[],"postedDate":"November 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:15:00+00:00","versionOfRecord":{"articleIdentity":"rs-7961408","link":"https://doi.org/10.1186/s12885-026-15614-1","journal":{"identity":"bmc-cancer","isVorOnly":false,"title":"BMC Cancer"},"publishedOn":"2026-01-29 15:58:44","publishedOnDateReadable":"January 29th, 2026"},"versionCreatedAt":"2025-11-26 06:54:57","video":"","vorDoi":"10.1186/s12885-026-15614-1","vorDoiUrl":"https://doi.org/10.1186/s12885-026-15614-1","workflowStages":[]},"version":"v1","identity":"rs-7961408","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7961408","identity":"rs-7961408","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}