In Situ Heparan Sulfate-Induced Peptide Self-Assembly to Overcome the Cell Surface Glycocalyx Barrier for Cancer Treatment.

Heparan sulfate (HS) is a major component of cell surface glycocalyx with extensive negative charges and plays a protective role by preventing toxins, including small molecule drugs and anticancer cationic lytic peptides (ACLPs), from cells. However, this effect may compromise the treatment efficiency of anticancer drugs. To overcome the impedance of cancer cell glycocalyx, an HS-targeting ACLP PTP-7z was designed by fusion of an ACLP and a Zn2+-binding HS-targeting peptide. Upon Zn2+ ion binding, PTP-7z could self-assemble into uniform nanoparticles and show improved serum stability and reduced hemolysis, which enable it to self-deliver to tumor sites. The peptide PTP-7z showed a pH- and Zn2+ ion-dependent HS-binding ability, which triggers the HS-induced in situ self-assembling on the cancer cell surface in the acidic tumor microenvironment (TME). The self-assembled PTP-7z can overcome the impedance of cell glycocalyx by either disrupting cell membranes or translocating into cells through endocytosis and inducing cell apoptosis. Moreover, PTP-7z can also inhibit cancer cell migration. These results proved that HS-responsive in situ self-assembling is a practical strategy to overcome the cancer cell glycocalyx barrier for ACLPs and could be extended to the design of other peptide drugs to promote their in vivo application.

Glycomacromolecules to Tailor Crowded and Heteromultivalent Glycocalyx Mimetics.

The glycocalyx, a complex carbohydrate layer on cell surfaces, plays a crucial role in various biological processes. Understanding native glycocalyces' complexity is challenging due to their intricate and dynamic nature. Simplified mimics of native glycocalyces offer insights into glycocalyx functions but often lack molecular precision and fail to replicate key features of the natural analogues like molecular crowding and heteromultivalency. We introduce membrane-anchoring precision glycomacromolecules synthesized via solid-phase polymer synthesis (SPPoS) and thiol-induced, light-activated controlled radical polymerization (TIRP), enabling the construction of crowded and heteromultivalent glycocalyx mimetics with varying molecular weights and densities in giant unilamellar vesicles (GUVs). The incorporation and dynamics of glycomacromolecules in the GUVs are examined via microscopy and fluorescence correlation spectroscopy (FCS) and studies on lectin-carbohydrate-mediated adhesion of GUVs reveal inhibitory and promotional adhesion effects corresponding to different glycocalyx mimetic compositions, bridging the gap between synthetic models and native analogues.

Polyphenol-Reinforced Glycocalyx-Like Hydrogel Coating Induced Myocardial Regeneration and Immunomodulation.

Although minimally invasive interventional occluders can effectively seal heart defect tissue, they still have some limitations, including poor endothelial healing, intense inflammatory response, and thrombosis formation. Herein, a polyphenol-reinforced medicine/peptide glycocalyx-like coating was prepared on cardiac occluders. A coating consisting of carboxylated chitosan, epigallocatechin-3-gallate (EGCG), tanshinone IIA sulfonic sodium (TSS), and hyaluronic acid grafted with 3-aminophenylboronic acid was prepared. Subsequently, the mercaptopropionic acid-GGGGG-Arg-Glu-Asp-Val peptide was grafted by the thiol-ene "click" reaction. The coating showed good hydrophilicity and free radical-scavenging ability and could release EGCG-TSS. The results of biological experiments suggested that the coating could reduce thrombosis by promoting endothelialization, and promote myocardial repair by regulating the inflammatory response. The functions of regulating cardiomyocyte apoptosis and metabolism were confirmed, and the inflammatory regulatory functions of the coating were mainly dependent on the NF-kappa B and TNF signaling pathway.

Adhesion dynamics of Janus nanocarriers to endothelial cells: A dissipative particle dynamics study.

Janus nanocarriers (NCs) provide promising features in interfacial applications such as targeted drug delivery. Herein, we use dissipative particle dynamics simulations to study the adhesion dynamics of NCs with Janus ligand compositions to the endothelial cell as a function of a series of effects, such as the initial orientation, ligand density, shape, and size of Janus NCs. The Janus NCs, with its long axis parallel to the endothelial glycocalyx (EG) layer, has the best penetration depth due to its lower potential energy and the lowest shell entropy loss. Among different shapes of Janus NCs, both the potential energy and the EG entropy loss control the penetration. In fact, at the parallel orientations, Janus shapes with a robust mechanical strength and larger surface area at the EG/water interface can rotate and penetrate more efficiently. An increase in the ligand density of Janus NCs increases entropy losses of both the hydrophilic and the hydrophobic ligands and decreases the potential energy. Thus, for a specific Janus NCs, functionalizing with an appropriate ligand density would help driving forces prevail over barriers of penetration into the EG layer. For a particular ligand density, once the radius of the Janus NCs exceeds the appropriate size, barriers such as hydrophobic ligands and shell entropy losses are also reinforced significantly and surpass driving forces. Our observations reveal that entropy losses for hydrophobic ligands of Janus NCs and for the shell of NCs are decisive for the adhesion and penetration of Janus NCs to endothelial cells.

Organ-on-a-Chip Approach for Accelerating Blood-Brain Barrier Nanoshuttle Discovery.

Organ-on-a-chip, which recapitulates the dynamics of in vivo vasculature, has emerged as a promising platform for studying organ-specific vascular beds. However, its practical advantages in identifying vascular-targeted drug delivery systems (DDS) over traditional in vitro models remain underexplored. This study demonstrates the reliability and efficacy of the organ-on-a-chip in screening efficient DDS by comparing its performance with that of a conventional transwell, both designed to simulate the blood-brain barrier (BBB). The BBB nanoshuttles discovered through BBB Chip-based screening demonstrated superior functionality in vivo compared to those identified using transwell methods. This enhanced effectiveness is attributed to the BBB Chip's accurate replication of the structure and dynamics of the endothelial glycocalyx, a crucial protective layer within blood vessels, especially under shear stress. This capability of the BBB Chip has enabled the identification of molecular shuttles that efficiently exploit the endothelial glycocalyx, thereby enhancing transendothelial transport efficacy. Our findings suggest that organ-on-a-chip technology holds considerable promise for advancing research in vascular-targeted DDS due to its accurate simulation of molecular transport within endothelial systems.

Cationic Polysaccharides Bind to the Endothelial Cell Surface Extracellular Matrix Involving Heparan Sulfate.

Cationic polysaccharides have been extensively studied for drug delivery via the bloodstream, yet few have progressed to clinical use. Endothelial cells lining the blood vessel wall are coated in an anionic extracellular matrix called the glycocalyx. However, we do not fully comprehend the charged polysaccharide interactions with the glycocalyx. We reveal that the cationic polysaccharide poly(acetyl, arginyl) glucosamine (PAAG) exhibits the highest association with the endothelial glycocalyx, followed by dextran (neutral) and hyaluronan (anionic). Furthermore, we demonstrate that PAAG binds heparan sulfate (HS) within the glycocalyx, leading to intracellular accumulation. Using an in vitro glycocalyx model, we demonstrate a charge-based extent of association of polysaccharides with HS. Mechanistically, we observe that PAAG binding to HS occurs via a condensation reaction and functionally protects HS from degradation. Together, this study reveals the interplay between polysaccharide charge properties and interactions with the endothelial cell glycocalyx toward improved delivery system design and application.

Glycocalyx-Mimicking Nanoparticles with Differential Organ Selectivity for Drug Delivery and Therapy.

Organ-selective drug delivery is expected to maximize the efficacy of various therapeutic modalities while minimizing their systemic toxicity. Lipid nanoparticles and polymersomes can direct the organ-selective delivery of mRNAs or gene editing machineries, but their delivery is limited to mostly liver, spleen, and lung. A platform that enables delivery to these and other target organs is urgently needed. Here, a library of glycocalyx-mimicking nanoparticles (GlyNPs) comprising five randomly combined sugar moieties is generated, and direct in vivo library screening is used to identify GlyNPs with preferential biodistribution in liver, spleen, lung, kidneys, heart, and brain. Each organ-targeting GlyNP hit show cellular tropism within the organ. Liver, kidney, and spleen-targeting GlyNP hits equipped with therapeutics effectively can alleviate the symptoms of acetaminophen-induced liver injury, cisplatin-induced kidney injury, and immune thrombocytopenia in mice, respectively. Furthermore, the differential organ targeting of GlyNP hits is influenced not by the protein corona but by the sugar moieties displayed on their surface. It is envisioned that the GlyNP-based platform may enable the organ- and cell-targeted delivery of therapeutic cargoes.

Endothelial Glycocalyx Degradation Patterns in Sepsis-Associated Pediatric Acute Respiratory Distress Syndrome: A Single Center Retrospective Observational Study.

BACKGROUND: Sepsis-associated destruction of the pulmonary microvascular endothelial glycocalyx (EGCX) creates a vulnerable endothelial surface, contributing to the development of acute respiratory distress syndrome (ARDS). Constituents of the EGCX shed into circulation, glycosaminoglycans and proteoglycans, may serve as biomarkers of endothelial dysfunction. We sought to define the patterns of plasma EGCX degradation products in children with sepsis-associated pediatric ARDS (PARDS), and test their association with clinical outcomes. METHODS: We retrospectively analyzed a prospective cohort (2018-2020) of children (≥1 month to <18 years of age) receiving invasive mechanical ventilation for acute respiratory failure for ≥72 h. Children with and without sepsis-associated PARDS were selected from the parent cohort and compared. Blood was collected at time of enrollment. Plasma glycosaminoglycan disaccharide class (heparan sulfate, chondroitin sulfate, and hyaluronan) and sulfation subtypes (heparan sulfate and chondroitin sulfate) were quantified using liquid chromatography tandem mass spectrometry. Plasma proteoglycans (syndecan-1) were measured through an immunoassay. RESULTS: Among the 39 mechanically ventilated children (29 with and 10 without sepsis-associated PARDS), sepsis-associated PARDS patients demonstrated higher levels of heparan sulfate (median 639 ng/mL [interquartile range, IQR 421-902] vs 311 [IQR 228-461]) and syndecan-1 (median 146 ng/mL [IQR 32-315] vs 8 [IQR 8-50]), both p = 0.01. Heparan sulfate subtype analysis demonstrated greater proportions of N-sulfated disaccharide levels among children with sepsis-associated PARDS (p = 0.01). Increasing N-sulfated disaccharide levels by quartile were associated with severe PARDS (n = 9/29) with the highest quartile including >60% of the severe PARDS patients (test for trend, p = 0.04). Higher total heparan sulfate and N-sulfated disaccharide levels were independently associated with fewer 28-day ventilator-free days in children with sepsis-associated PARDS (all p < 0.05). CONCLUSIONS: Children with sepsis-associated PARDS exhibited higher plasma levels of heparan sulfate disaccharides and syndecan-1, suggesting that EGCX degradation biomarkers may provide insights into endothelial dysfunction and PARDS pathobiology.

Chronic cranial window implantation for high-resolution intravital imaging of the endothelial glycocalyx in mouse cortex.

The endothelial glycocalyx is an integral component of the brain vascular barrier. Visualizing its structure in vivo is essential to understand its physiological and pathophysiological mechanisms. Here, we present a surgical protocol for chronic cranial window implantation in mice, alongside the use of multiphoton microscopy tools to image the cortical vasculature. We describe steps for cranial window implantation, intravenous injection of fluorescent markers, and intravital imaging. We then detail a technique to quantify glycocalyx thickness using Imaris image analysis software. For complete details on the use and execution of this protocol, please refer to Gray et al. (2023).1.

Endothelial glycocalyx-associated molecules as potential serological markers for sepsis-associated encephalopathy: A systematic review and meta-analysis.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is characterized by a diffuse cerebral dysfunction that accompanies sepsis in the absence of direct central nervous system infection. The endothelial glycocalyx is a dynamic mesh containing heparan sulfate linked to proteoglycans and glycoproteins, including selectins and vascular/intercellular adhesion molecules (V/I-CAMs), which protects the endothelium while mediating mechano-signal transduction between the blood and vascular wall. During severe inflammatory states, components of the glycocalyx are shed into the circulation and can be detected in soluble forms. Currently, SAE remains a diagnosis of exclusion and limited information is available on the utility of glycocalyx-associated molecules as biomarkers for SAE. We set out to synthesize all available evidence on the association between circulating molecules released from the endothelial glycocalyx surface during sepsis and sepsis-associated encephalopathy. METHODS: MEDLINE (PubMed) and EMBASE were searched since inception until May 2, 2022 to identify eligible studies. Any comparative observational study: i) evaluating the association between sepsis and cognitive decline and ii) providing information on level of circulating glycocalyx-associated molecules was eligible for inclusion. RESULTS: Four case-control studies with 160 patients met the inclusion criteria. Meta-analysis of biomarkers ICAM-1 (SMD 0.41; 95% CI 0.05-0.76; p = 0.03; I2 = 50%) and VCAM-1 (SMD 0.55; 95% CI 0.12-0.98; p = 0.01; I2 = 82%) revealed higher pooled mean concentration in patients with SAE compared to the patients with sepsis alone. Single studies reported elevated levels of P-selectin (MD 0.80; 95% CI -17.77-19.37), E-selectin (MD 96.40; 95% Cl 37.90-154.90), heparan sulfate NS2S (MD 19.41; 95% CI 13.37-25.46), and heparan sulfate NS+NS2S+NS6S (MD 67.00; 95% CI 31.00-103.00) in patients with SAE compared to the patients with sepsis alone. CONCLUSION: Plasma glycocalyx-associated molecules are elevated in SAE and may be useful for early identification of cognitive decline in sepsis patients.

Live-Cell Glycocalyx Engineering.

A heavy layer of glycans forms a brush matrix bound to the outside of all the cells in our bodies; it is referred to as the "sugar forest" or glycocalyx. Beyond the increased appreciation of the glycocalyx over the past two decades, recent advances in engineering the glycocalyx on live cells have spurred the creation of cellular drugs and novel medical treatments. The development of new tools and techniques has empowered scientists to manipulate the structures and functions of cell-surface glycans on target cells and endow target cells with desired properties. Herein, we provide an overview of live-cell glycocalyx engineering strategies for controlling the cell-surface molecular repertory to suit therapeutic applications, even though the realm of this field remains young and largely unexplored.

Vascular cell behavior on glycocalyx-mimetic surfaces: Simultaneous mimicking of the chemical composition and topographical structure of the vascular endothelial glycocalyx.

The endothelial glycocalyx is a carbohydrate-rich layer overlying the outermost surface of endothelial cells. It mediates intercellular interactions by specific chemical compositions (e.g., proteoglycans containing glycosaminoglycan (GAG) side chains) and micro/nanotopography. Inspired by the endothelial glycocalyx, we fabricated a series of glycocalyx-mimetic surfaces with tunable chemical compositions (GAG-like polymers with different functional units) and topographical structures (micro/nanopatterns with pillars different in size). The combination of micro/nanopatterns and GAG-like polymers was flexibly and precisely controlled by replica molding using silicon templates (Si templates) and visible light-initiated polymerization. Human umbilical vein endothelial cells (HUVECs) and human umbilical vein smooth muscle cells (HUVSMCs) were suppressed on surfaces modified with polymers of 2-methacrylamido glucopyranose (MAG) but promoted on surfaces modified with polymers of sodium 4-vinyl-benzenesulfonate (SS) and copolymers of SS and MAG. Surface micro/nanopatterns showed highly complicated effects on surfaces grafted with different GAG-like polymers. Moreover, the spread of HUVSMCs was highly promoted on all flat/patterned surfaces containing sulfonate units, and the elongation effect was stronger on surfaces with smaller pillars. On all the flat/patterned surfaces modified with GAG-like polymers, the adsorption of human vascular endothelial growth factor (VEGF) and human basic fibroblast growth factor (bFGF) was improved, and the amount of VEGF and bFGF absorbed on patterned surfaces containing sulfonate units decreased with pattern dimensions. The decreasing trend of VEGF and bFGF adsorption was in accordance with HUVEC density, suggesting that glycocalyx-mimetic surfaces influence the adsorption of VEGF and bFGF and further influence the growth behavior of vascular cells.

Quantification and Topographical Distribution of Terminal and Linked Fucose Residues in Human Spermatozoa by Using Field Emission Scanning Electron Microscopy (FE-SEM).

The modification of sperm glycocalyx is an essential process during sperm capacitation. The presence and redistribution of terminal and linked fucose have been described during in vitro capacitation in humans. However, the influence of the capacitation time on the quantification and localization of terminal and linked fucose is still unknown. In this study, the quantitative and qualitative changes in fucosyl residues during different in vitro capacitation times (1 and 4 h), are simultaneously characterized by using Aleuria aurantia (AAA) lectin-gold labelling and high-resolution field emission scanning electron microscopy (FE-SEM) in human sperm. A significant decrease was found in the number of terminal fucose registered in the whole sperm head during the in vitro capacitation. Nevertheless, the quantification of fucose residues after 1 h of in vitro capacitation was very similar to those found after 4 h. Therefore, the changes observed in terminal and linked fucose during capacitation were not time-dependent. Furthermore, the comprehensive analysis of the topographic distribution showed the preferential fucosyl location in the acrosomal region and the presence of distinct clusters distributed over the head in all the studied conditions. Overall, these findings corroborate the validity of FE-SEM combined with gold labelling to register changes in surface molecules during in vitro sperm capacitation.

The specificity of the malarial VAR2CSA protein for chondroitin sulfate depends on 4-O-sulfation and ligand accessibility.

Placental malaria infection is mediated by the binding of the malarial VAR2CSA protein to the placental glycosaminoglycan, chondroitin sulfate. Recombinant subfragments of VAR2CSA (rVAR2) have also been shown to bind specifically and with high affinity to cancer cells and tissues, suggesting the presence of a shared type of oncofetal chondroitin sulfate (ofCS) in the placenta and in tumors. However, the exact structure of ofCS and what determines the selective tropism of VAR2CSA remains poorly understood. In this study, ofCS was purified by affinity chromatography using rVAR2 and subjected to detailed structural analysis. We found high levels of N-acetylgalactosamine 4-O-sulfation (∼80-85%) in placenta- and tumor-derived ofCS. This level of 4-O-sulfation was also found in other tissues that do not support parasite sequestration, suggesting that VAR2CSA tropism is not exclusively determined by placenta- and tumor-specific sulfation. Here, we show that both placenta and tumors contain significantly more chondroitin sulfate moieties of higher molecular weight than other tissues. In line with this, CHPF and CHPF2, which encode proteins required for chondroitin polymerization, are significantly upregulated in most cancer types. CRISPR/Cas9 targeting of CHPF and CHPF2 in tumor cells reduced the average molecular weight of cell-surface chondroitin sulfate and resulted in a marked reduction of rVAR2 binding. Finally, utilizing a cell-based glycocalyx model, we showed that rVAR2 binding correlates with the length of the chondroitin sulfate chains in the cellular glycocalyx. These data demonstrate that the total amount and cellular accessibility of chondroitin sulfate chains impact rVAR2 binding and thus malaria infection.

Comparative Characteristics of the Expression of Fucosylated Glycans and Morphometric Parameters of Terminal Placental Villi Depending on the Severity of Preeclampsia.

We performed a comparative analysis of the expression of fucosylated glycans and morphometric characteristics of the terminal villi of the placenta, depending on the severity of preeclampsia (PE). Similar patterns of the expression of fucosylated glycans in the syncytiotrophoblast glycocalyx were revealed in the placental tissue of patients with normal pregnancy and with mild and severe PE: predominance of glycans with α1,6-fucose in the core, clustered fucose residues, and LeX glycan over α1,2-fucose-containing glycans. The expression pattern of fucosylated glycans and the composition of the endothelial glycocalyx are normally close to the expression pattern and composition of the syncytiotrophoblast glycocalyx; in case of mild and severe PE, the expression pattern of fucosylated glycans was changed uniformly, and α1,2-fucose-containing glycans significantly prevailed in the endothelial glycocalyx. According to the results of Fisher's LSD test, in patients with severe PE, the total vascular area in the villus prevailed over the indices established during physiological course of pregnancy (p=0.04) and mild PE (p=0.04). Correlation analysis revealed direct and reciprocal relationships between the morphometric characteristics of the terminal villi of the placenta and the expression of fucosylated glycans in the syncytiotrophoblast and endothelium in PE. Our results indicate a changed expression of fucosylated glycans in the glycocalyx of placental barrier structures and the morphometric parameters of villi in PE of different severity, which can affect the function of the placental barrier.

Intramuscular Exposure to a Lethal Dose of Ricin Toxin Leads to Endothelial Glycocalyx Shedding and Microvascular Flow Abnormality in Mice and Swine.

Ricin toxin isolated from the castor bean (Ricinus communis) is one of the most potent and lethal molecules known. While the pathophysiology and clinical consequences of ricin poisoning by the parenteral route, i.e., intramuscular penetration, have been described recently in various animal models, the preceding mechanism underlying the clinical manifestations of systemic ricin poisoning has not been completely defined. Here, we show that following intramuscular administration, ricin bound preferentially to the vasculature in both mice and swine, leading to coagulopathy and widespread hemorrhages. Increased levels of circulating VEGF and decreased expression of vascular VE-cadherin caused blood vessel impairment, thereby promoting hyperpermeability in various organs. Elevated levels of soluble heparan sulfate, hyaluronic acid and syndecan-1 were measured in blood samples following ricin intoxication, indicating that the vascular glycocalyx of both mice and swine underwent extensive damage. Finally, by using side-stream dark field intravital microscopy imaging, we determined that ricin poisoning leads to microvasculature malfunctioning, as manifested by aberrant blood flow and a significant decrease in the number of diffused microvessels. These findings, which suggest that glycocalyx shedding and microcirculation dysfunction play a major role in the pathology of systemic ricin poisoning, may serve for the formulation of specifically tailored therapies for treating parenteral ricin intoxication.

Glycocalyx Curving the Membrane: Forces Emerging from the Cell Exterior.

Morphological transitions are typically attributed to the actions of proteins and lipids. Largely overlooked in membrane shape regulation is the glycocalyx, a pericellular membrane coat that resides on all cells in the human body. Comprised of complex sugar polymers known as glycans as well as glycosylated lipids and proteins, the glycocalyx is ideally positioned to impart forces on the plasma membrane. Large, unstructured polysaccharides and glycoproteins in the glycocalyx can generate crowding pressures strong enough to induce membrane curvature. Stress may also originate from glycan chains that convey curvature preference on asymmetrically distributed lipids, which are exploited by binding factors and infectious agents to induce morphological changes. Through such forces, the glycocalyx can have profound effects on the biogenesis of functional cell surface structures as well as the secretion of extracellular vesicles. In this review, we discuss recent evidence and examples of these mechanisms in normal health and disease.

Engineering the Sialome.

The surface of every eukaryotic cell is coated in a dense layer of structurally diverse glycans that together comprise the glycocalyx, a key interface between intracellular biochemistry and the external environment. Many of the glycans within the glycocalyx terminate in anionic monosaccharides belonging to the sialic acid family. Advances in our understanding of the biological processes mediated by sialic acids at the interfaces between cells have catalyzed interest in metabolic, enzymatic, and chemical strategies to edit the total complement of cellular sialic acids-the sialome. Here, we review strategies for altering the composition of the sialome with particular focus on glycan structures and state-of-the-art tools.

Dismantling the bacterial glycocalyx: Chemical tools to probe, perturb, and image bacterial glycans.

The bacterial glycocalyx is a quintessential drug target comprised of structurally distinct glycans. Bacterial glycans bear unusual monosaccharide building blocks whose proper construction is critical for bacterial fitness, survival, and colonization in the human host. Despite their appeal as therapeutic targets, bacterial glycans are difficult to study due to the presence of rare bacterial monosaccharides that are linked and modified in atypical manners. Their structural complexity ultimately hampers their analytical characterization. This review highlights recent advances in bacterial chemical glycobiology and focuses on the development of chemical tools to probe, perturb, and image bacterial glycans and their biosynthesis. Current technologies have enabled the study of bacterial glycosylation machinery even in the absence of detailed structural information.

Syndecan-1 Depletion Has a Differential Impact on Hyaluronic Acid Metabolism and Tumor Cell Behavior in Luminal and Triple-Negative Breast Cancer Cells.

Glycosaminoglycans (GAGs) and proteoglycans (PGs) are major components of the glycocalyx. The secreted GAG and CD44 ligand hyaluronic acid (HA), and the cell surface PG syndecan-1 (Sdc-1) modulate the expression and activity of cytokines, chemokines, growth factors, and adhesion molecules, acting as critical regulators of tumor cell behavior. Here, we studied the effect of Sdc-1 siRNA depletion and HA treatment on hallmark processes of cancer in breast cancer cell lines of different levels of aggressiveness. We analyzed HA synthesis, and parameters relevant to tumor progression, including the stem cell phenotype, Wnt signaling constituents, cell cycle progression and apoptosis, and angiogenic markers in luminal MCF-7 and triple-negative MDA-MB-231 cells. Sdc-1 knockdown enhanced HAS-2 synthesis and HA binding in MCF-7, but not in MDA-MB-231 cells. Sdc-1-depleted MDA-MB-231 cells showed a reduced CD24-/CD44+ population. Furthermore, Sdc-1 depletion was associated with survival signals in both cell lines, affecting cell cycle progression and apoptosis evasion. These changes were linked to the altered expression of KLF4, MSI2, and miR-10b and differential changes in Erk, Akt, and PTEN signaling. We conclude that Sdc-1 knockdown differentially affects HA metabolism in luminal and triple-negative breast cancer model cell lines and impacts the stem phenotype, cell survival, and angiogenic factors.