The search for tumor-associated GSL antigens has been a major focus of investigation for decades. Under normal physiological conditions, GlcCer, LacCer, Gb3 (globotriaosylceramide), Gb4 (globotetraosylceramide), GM3 (monosialodihexosylganglioside) and GD3 (disialodihexosylganglioside) have been identified as the major GSLs present in human bladder tissues [20]. However, during malignant transformation, the cellular membrane composition and organization undergo significant remodeling. As a result, cancer cells exhibit altered GSL expression, which in turn modulates transmembrane signaling pathways essential for tumor growth, invasion and metastasis [21]. These alterations in membrane composition/organization distinguish cancer cells from normal ones. Consequently, targeting such changes has been explored as a potential strategy for improving bladder cancer diagnosis and treatment.

Aberrant glycosylation, including altered GSL biosynthesis, is one of the hallmarks of malignant transformation and cancer progression [22]. Several key alterations in GSL expression patterns have been identified and extensively studied in bladder cancer (Table 1). Notably, GSL expression profiles differ between NMIBC and MIBC, suggesting their potential utility in subclassification and disease monitoring. NMIBC is predominantly characterized by a significant accumulation of GM3, whereas high-grade MIBC exhibits a shift toward higher levels of LacCer, Gb3, Gb4, and GD2 (disialotrihexosylganglioside) [23,24,25]. These differences indicate that specific GSL signatures are associated with disease progression, making them valuable markers for stratifying bladder cancer subtypes. Understanding these glycosylation patterns not only aids in bladder cancer classification and grading, but may also contribute to refining risk assessment and identifying new therapeutic targets.

The glycosyltransferase GCS, encoded by the UGCG (UDP-glucose ceramide glycosyltransferase) gene, is responsible for the initial glycosylation step leading to the synthesis of all glucose-derived GSLs. Overexpression of GCS has been observed in bladder cancer tissues, where it correlates with poor prognosis [26, 27]. In a recent study, we found that nLc4 (neolactotetraosylceramide), Gb3 and the GM3 ganglioside are significantly increased in bladder tumor tissues when compared to the normal adjacent mucosa and to normal bladder tissues from cancer-free individuals [28]. In different studies, and looking specifically into superficial bladder tumors, these exhibit a significant accumulation of GM3 [29, 30], accompanied by the upregulated expression of GM3 synthase and downregulation of Gb3 and GD3 synthases activity [23].

In the Cancer Genome Atlas Urothelial Carcinoma (TCGA-BLCA) cohort [31], high levels of B4GALNT1 (beta-1,4-N-acetyl-galactosaminyltransferase 1) expression have been strongly linked to advanced clinical stages and poor prognosis [32]. This gene encodes the glycosyltransferase responsible for the synthesis of ganglio-series GSLs, including GM2 (monosialotrihexosylganglioside), GD2, and GT2 (trisialotrihexosylganglioside), as well as GA2 (asialo-GM2, Gg3). Moreover, the genes B4GALT3 and B4GALT4 (beta-1,4-galactosyltransferases 3 and 4), involved in the biosynthesis of nLc4, have been found to be upregulated in bladder cancer compared to normal adjacent tissues [28].

The expression of Lewis antigens has been extensively studied in bladder cancer. Among type-1 fucosylated glycans, Lewis A (Lea) expression undergoes changes at early neoplastic stages, and its detection allows for the subclassification of histologically similar tumors into distinct prognostic groups. These characteristics make Lea a valuable biomarker for the diagnosis of very early premalignant urothelial alterations and assessing malignant potential in bladder cancer [33].

Among type-2 fucosylated glycans, Lewis Y (Ley) exhibits distinct expression patterns in carcinoma in situ (CIS) compared to non-CIS conditions, making it a useful marker for histological differentiation [34]. Lewis X (Lex), which is typically absent from normal urothelial cells in adults, is present in urothelial tumors regardless of tumor grade, stage, or secretor status [35].

Additionally, the expression of fucosyltransferases (FUT) involved in the synthesis of Lewis antigens (FUT1-7 and FUT9) can also be altered in bladder cancer. FUT4 is overexpressed in multiple bladder cancer cell lines, promoting neoplastic cell proliferation and invasion [36]. FUT6 and FUT7 show increased expression in invasive cells compared to noninvasive ones [29]. A recent study further confirmed that FUT7 expression is elevated in bladder cancer tissues relative to normal tissues and is associated with poor prognosis. This upregulation was validated through immunohistochemical analysis and ELISA-based detection of FUT7 in the serum of bladder cancer patients, reinforcing its potential as a biomarker for bladder cancer detection [37].

The sialylation status of glycans in bladder cancer is directly linked to tumor progression. Sialyltransferases involved in the biosynthesis of sialylated glycans have an active role in providing clues to understand the mechanisms of tumorigenicity. Sialyl Lewis X (SLex), generated by the enzymatic transfer of sialic acid to the Lex antigen, is highly expressed in invasive urothelial tumors and cell lines but absent in noninvasive cells [29]. Similarly, Sialyl Lewis A (SLea), also known as carbohydrate antigen 19-9 (CA19-9), is detected in bladder carcinoma tissues [38] and is elevated in the urine of bladder cancer patients [39]. Notably, serum levels of SLea are significantly increased in patients with metastatic or advanced bladder cancer, suggesting its potential utility in monitoring disease progression [40].

The expression of sialyltransferases is also altered in bladder cancer. ST3GAL5 (also called GM3 synthase) is downregulated in tumor tissues compared to adjacent normal bladder tissues, with its expression levels higher in low-grade tumors than in high-grade bladder cancer [41]. Conversely, the mRNA expression levels of ST8SIA1 (the enzyme that converts GM3 to GD3) are significantly decreased in bladder cancer tumors compared to normal bladder tissues, and its low expression is associated with increased pathological grade and invasiveness [42].

These bladder cancer-specific alterations in the composition and repertoire of GSLs contribute to multiple hallmarks of cancer, including activation of invasion and metastasis, immune evasion, and resistance to cell death. These features will be explored in detail in the next subsections of this review and are summarized in Fig. 2.

Glycosphingolipid dynamics during bladder cancer progression. The repertoire and content of GSLs can influence different hallmarks of bladder cancer. Regarding invasion and metastasis, overexpression of GD2 is linked to an increased expression of mesenchymal genes, having a pro-metastatic role [24, 25]; exposure of bladder cancer cells to TGF-β decreases GM2 expression and leads to the acquisition of EMT features [46]; overexpression of NEU3 promotes invasiveness of bladder cancer through the activation of ERK and PI3K signaling [55]; high presence of GCS [26] and B4GALNT1 [32] is correlated with a metastatic phenotype; upregulation of FUT7 enhances migration and invasion capabilities of bladder cancer cells [37] and SLex and SLea antigens mediate E-selectin-dependent adhesion of some bladder tumor cells to microvascular endothelial cells, increasing the metastatic potential [49]. Regarding invasion and metastasis inhibition, GM3 mediates the interaction of α3β1 integrin with CD9 [13] and suppresses the tyrosine phosphorylation levels of EGFR, reducing cell proliferation, motility and adhesion [16]; ST8SIA1 overexpression decreases the phosphorylation of EGFR, JAK and STAT3 by increasing GM3’s sialylation, which inhibits invasiveness [42]; and the interaction of GM2 [14] or GM2-GM3 heterodimer [15] with CD82 inhibits HGF-induced activation of c-Met, impairing cell motility. GSL-related molecules can also have a role in regulating tumor cell apoptosis. Overexpression of galectin-3 promotes the activation of the PI3K/Akt pathway, inhibiting TRAIL-induced apoptosis [59]. The interaction of galectin-3 with poly-LacNAc-containing glycans on the surface of tumor cells can mask them from NK cells, facilitating immune evasion and tumor progression [56, 62]. Overall, these biological processes highlight the contribution of GSLs as promising immunotherapeutic targets or drug agents for bladder cancer treatment. This includes the administration of GSLs and GSL modulators/inhibitors, the use of antibodies or anti-cancer drugs that target GSLs, and the manipulation of genes involved in GSL biosynthesis through genetic engineering

Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose polarity, integrity and adhesion, adopting a mesenchymal phenotype that enhances their migratory capacity. The acquisition of EMT by cancer cells is characterized by the loss of E-cadherin and upregulation of mesenchymal markers such as N-cadherin, fibronectin and vimentin. This transition is driven by multiple growth factors, including epidermal growth factor (EGF), hepatocyte growth factor (HGF), and transforming growth factor-β (TGF-β) [63].

Specific GSLs interact with different signal transducers, growth factors and integrins, playing key roles in EMT regulation and tumor cell migration [15, 45, 46]. Recent evidence further supports the role of GSLs in facilitating cancer cell plasticity, including our contribution to a study demonstrating GSL involvement in ovarian cancer EMT dynamics [64].

Bladder carcinoma cells treated with TGF-β undergo EMT, exhibiting fibroblast-like morphology, reduced epithelial marker expression, and increased motility. This transformation is accompanied by a significant reduction in GM2 levels, suggesting that specific GSLs regulate EMT-associated motility [46]. Interestingly, treatment with EtDO-P4 (GCS inhibitor) induced similar EMT effects as TGF-β stimulation, likely due to the depletion of motility-suppressing GSLs. Supporting this hypothesis, exogenous addition of GM2 reversed the motility enhancement caused by EtDO-P4, while GA1 (asialo-GM1, Gg4) inhibited TGF-β-driven motility changes [46].

Furthermore, high-grade bladder cancer tissues overexpressing GD2 also showed increased mesenchymal marker expression, including N-cadherin and vimentin, indicating an EMT-positive phenotype. GD2-positive cells also exhibited cancer stem-like characteristics, correlating with elevated CD44 expression, which reinforce the role of GD2 in tumor progression and metastasis [24, 25, 65].

Single-cell RNA sequencing data from the TCGA-BLCA cohort and immunohistochemical stainings of bladder tumor tissues showed that the above-mentioned glycosyltransferase B4GALNT1 is enriched at both the RNA and protein levels in cancer-associated fibroblasts (CAFs), which simultaneously express high levels of heat shock proteins [32]. Given that CAFs actively contribute to extracellular matrix (ECM) remodeling and tumor progression, and that heat shock proteins facilitate exosome release [66, 67], this suggests that high B4GALNT1 expression is associated with enhanced ECM reorganization and intercellular communication via exosome-mediated signaling. Consequently, B4GALNT1 is proposed to facilitate tumor-stroma interactions, ultimately driving the progression of MIBC [32].

Similarly, the role of glycosyltransferases in bladder cancer progression is further supported by studies on FUT7. Bladder cancer cell lines engineered to overexpress FUT7 exhibited significantly increased proliferation, migration, and invasion capabilities, accompanied by an induction of EMT [37].

Cell adhesion and motility are primarily regulated by integrins, which mediate interactions with ECM [63]. Integrin functions can be modulated by their interaction with tetraspanins, which in turn can be mediated by association with gangliosides. CD9, a tetraspanin known to suppress cell motility, requires GM3 as a co-factor for its inhibitory function [13]. In detail, in this glycosynaptic microdomain, GM3 enhances and stabilizes the interaction between CD9 and α3β1 integrin, resulting in a low-motility phenotype.

Beyond adhesion, GM3 levels directly impact c-Src activation, a non-receptor tyrosine kinase that regulates multiple oncogenic signaling pathways. High GM3 expression, characteristic of NMIBC, suppresses c-Src activation, whereas reduced GM3 levels in invasive cells result in c-Src hyperactivation, promoting motility and invasiveness [68]. Restoring GM3 levels in invasive bladder cancer cells enhances CD9/α3β1 interactions, suppresses c-Src activity, and reverses the invasive phenotype [45]. Additionally, GM3 modulates the activity of growth factor receptors, particularly EGFR, by reducing EGFR tyrosine phosphorylation, thereby suppressing EGF-induced signaling and inhibiting proliferation and adhesion [16].

Emerging evidence highlights the role of ECM stiffness in bladder cancer progression and recurrence. Increased ECM stiffness has been observed in bladder tumors, particularly in recurrent cases, and has been linked to enhanced tumor cell proliferation, migration, and invasion [69,70,71]. ECM stiffening activates β1 integrin, triggering downstream focal adhesion kinase (FAK) and CDC42 signaling, which leads to Yes-associated protein (YAP) activation. In stiffer tumor environments, nuclear YAP drives gene expression programs that promote tumor progression and therapy resistance [69, 71]. While the role of ECM stiffness in bladder cancer is becoming clearer, little is known about how GSLs may participate in this process. Given their roles in cell adhesion, receptor clustering, and signal transduction, GSLs may influence ECM remodeling by regulating integrin dynamics and mechanosignaling pathways. Additionally, alterations in GSL composition may affect the organization of glycosynapses, potentially impacting how tumor cells sense and respond to mechanical cues within the tumor microenvironment. Investigating the crosstalk between GSLs and ECM stiffness could provide novel insights into bladder cancer progression and reveal new therapeutic opportunities.

Focusing on sialyltransferases, downregulation of ST3GAL5 (the gene that codes for the glycosyltransferase that converts LacCer into GM3) has been linked to increased proliferation, migration and invasion of J82 bladder cancer cells, whereas its overexpression suppressed tumor progression of T24 and 5637 cells [41]. In vivo, upregulation of ST3GAL5 significantly inhibited the tumorigenicity of bladder cancer cells subcutaneously inoculated into BALB/c nude mice [41].

Conversely, increased expression levels of ST8SIA1 (the gene that codes for the glycosyltransferase that converts GM3 into GD3), was associated with reduced proliferation, migration, and invasion of bladder cancer cells [42]. Mechanistically, ST8SIA1 overexpression inhibited Janus kinase 2 (JAK2) and signal transducer and activator of transcription (STAT3) phosphorylation, leading to a significant decrease in downstream effectors involved in tumor progression, including matrix metalloproteinase-2 (MMP2), proliferating cell nuclear antigen (PCNA), cyclin D1, and B-cell lymphoma 2 (BCL2) [42]. Because GM3 has been reported to suppress EGFR phosphorylation in bladder cancer cell lines [16], it was suggested that further sialylation of GM3 by ST8SIA1 may enhance this inhibitory effect by reducing EGFR, JAK, and STAT3 phosphorylation levels.

Another important glycosynaptic microdomain involves c-Met (the receptor for HGF), tetraspanin CD82, integrins, and GSLs. c-Met is a critical growth factor receptor that controls cell motility and growth. Studies have shown that GM2, rather than GM3, preferentially interacts with CD82, inhibiting HGF-induced c-Met activation and suppressing bladder cancer cell motility [14]. Further investigations revealed that a combination of GM2 and GM3 within the CD82 complex enhances c-Met suppression, leading to a stronger inhibitory effect on cell motility [15].

NEU3 (Neuraminidase 3) is a plasma membrane-associated ganglioside-specific sialidase that hydrolyzes GM3 into LacCer. NEU3 is significantly upregulated in bladder cancer tissues compared to normal ones, and its overexpression has been shown to promote tumor cell invasion [55]. Mechanistic studies using siRNA-mediated NEU3 knockdown revealed that reducing NEU3 expression suppressed extracellular signal-regulated kinase (ERK) and PI3K signaling, disrupting key pathways involved in tumor progression. These findings suggest that NEU3 functions upstream of Ras/MAPK and PI3K/Akt signaling to promote bladder cancer aggressiveness [55].

An additional study using TCGA and TCGASpliceSeq databases identified genes from the GSL biosynthesis ganglio-series pathway as key regulators of bladder cancer bone metastasis. This glyco-pathway was linked to the co-expression of integrin beta 4 (ITGB4), which is regulated by junction plakoglobin (JUP) [53]. Briefly, ITGB4 phosphorylation in bladder cancer tissues is associated with the activation of EGFR family signaling pathways involved in tumorigenesis and metastasis. In bladder cancer patients with bone metastasis, JUP downregulates ITGB4 splicing events via the ganglio-series biosynthesis pathway, further supporting the role of GSLs in metastatic progression [53].

Binding of selectins to terminal SLex and SLea antigens initiates one of the earliest steps in the metastasis cascade [72]. In bladder cancer, E-selectin-dependent cell adhesion was significantly inhibited in vitro by anti-SLex and anti-SLea antibodies [43,