Graphical abstract graphic file with name fx1.jpg [65]Open in a new tab Highlights * • BLCA cells resist low oxygen and glucose, stopping proliferation and enhancing invasion * • Stressed cells rely on mitophagy and fatty acid β-oxidation for energy * • BLCA cells and tumors display immature glycophenotypes in response to stress * • Stress-induced immature glycosylation drives resistance to anoikis and invasion __________________________________________________________________ Health sciences; Medicine; Medical specialty; Internal medicine; Oncology; Natural sciences; Biological sciences; Systems biology; Cancer systems biology; Cancer Introduction Bladder cancer (BLCA) remains one of the deadliest malignancies of the genitourinary tract due to high intra and inter-tumoral molecular heterogeneity.[66]^1 This has delayed a more comprehensive understanding of tumor spatiotemporal status and affected the efficiency of precise clinical interventions. Although genetic alterations are considered primary causes of cancer development, downstream phenotypic changes induced by the tumor microenvironment are among the driving forces of progression and dissemination. The generation of hypoxic niches characterized by decreased oxygen availability (≤2% O[2]) is a microenvironment hallmark of solid tumors.[67]^2 Not surprisingly, the presence of hypoxic regions is a pivotal independent poor prognosis factor in several cancers, including urothelial carcinomas.[68]^3 Uncontrolled tumor cell proliferation supported by avid glucose consumption and glycolysis in the presence of oxygen and fully functioning mitochondria (the Warburg effect) is a common feature of solid tumors.[69]^4 Rapid proliferation is frequently accompanied by flawed neoangiogenesis, resulting in suboptimal oxygen and nutrient supply to cancer cells in the periphery of blood vessels. Poor vascularization and competition for nutrients require constant metabolic remodeling and exploitation of alternative survival strategies by cancer cells. Although many tumor cells faced with suboptimal growth conditions undergo programmed cell death and necrosis, some subpopulations show tremendous molecular plasticity to adapt to hypoxic and nutrient-deprived microenvironments.[70]^5 Low oxygen levels induce metabolic rewiring toward anaerobic glycolysis and microenvironment acidification, while contributing to the maintenance of cancer stem cells and acquisition of epithelial-to-mesenchymal transition (EMT) traits, decisively dictating tumor fate.[71]^6^,[72]^7 Moreover, slow-dividing cancer cells in hypoxic regions can escape many cytotoxic drugs targeting rapidly dividing cells, also being sufficiently shielded from many other therapeutic agents compared to the tumor bulk.[73]^8 Although the mechanisms of cellular adaptation to hypoxia are already well known, few studies have interrogated how cancer cells can also withstand low levels of glucose. Furthermore, there is little knowledge of the underlying molecular alterations occurring at the cell surface, which dictate poor prognosis and may be easily targeted in theragnostic interventions. The cell surface is densely covered by a layer of complex glycans (glycocalyx), which result from the concerted activity of a wide variety of glycosyltransferases, glycosidases, and sugar nucleotide transporters across the secretory pathways.[74]^9^,[75]^10 Moreover, glycosylation reflects microenvironmental stimuli in response to alterations in glycogenes expression and metabolic imbalances.[76]^11^,[77]^12 While aberrant glycosylation, manifested by O-glycans shortening, is a common feature of most epithelial tumors,[78]^13 there is still a significant gap in identifying the common macroenvironmental drivers responsible for these molecular changes in solid tumors. Nevertheless, seminal observations have linked the Warburg effect with increased flux of glucose and other nutrients into the hexosamine biosynthetic pathway (HBP), resulting in higher mucin-type O-glycosylation and sialylation in cancer cells, directly impacting on glycocalyx composition.[79]^14^,[80]^15 These alterations influence the activation of oncogenic signaling, inducing immune tolerance, migration, cell-cell, and cell-matrix adhesion.[81]^11 Furthermore, they suggest that metabolic imbalances may play a role in supporting cancer progression by influencing glycocalyx remodeling. Expanding on these findings, a prior explorative study from our group has provided initial evidence that hypoxia augments the malignancy of BLCA cells while concurrently inhibiting protein O-glycosylation extension.[82]^16 However, how hypoxia and glucose restriction shape the cancer glycome and its underlying functional implications remains mostly unaddressed. In this study, we employed a multi-omics approach combining transcriptomics, metabolomics, glycomics, and phosphoproteomics to characterize in-depth the molecular plasticity of BLCA cells under hypoxia and low glucose and its contribution to aggressiveness. Having observed significant glycome remodeling in response to these stressors, we have conducted functional glycomics studies supported by well-characterized glycoengineered cell models to determine how altered glycosylation impacts BLCA progression. Important insights were generated to understand how BLCA adapts to oxygen and nutrient deprivation, aiming to identify more aggressive BLCA subpopulations envisaging future clinical interventions. Results Hypoxia and low glucose induce BLCA aggressiveness To better understand the molecular adaptability of BLCA cells to hypoxia and low glucose, four BLCA cell models reflecting different grades of the disease (RT4-grade 1, 5637-grade 2, T24-grade 3, HT1197-grade 4) were cultured under low oxygen (0.1% O[2]) and reduced glucose (≤10%) levels. This was expected to mimic microenvironmental conditions encountered by cells growing far apart from blood vessels.[83]^16 Cells responded rapidly to these challenges, stabilizing the hypoxia-inducible factor HIF-1α, which became more pronounced in the absence of glucose, supporting HIF-1α pivotal role in adaptive responses to microenvironmental stress ([84]Figure 1A). Notably, HIF-1α was also increased in 5637 cells grown under atmospheric oxygen tension but with very low glucose (normoxia-Glc), reinforcing the existence of a non-canonical regulation of HIF-1α stabilization regardless of oxygen availability.[85]^17 The exception was the T24 cell line, which showed a modest HIF-1α increase under stress, even though nuclear accumulation was notorious in subsequent experiments. This was accompanied by an increase in lactate levels, which were rapidly extruded to the extracellular space under low oxygen (Hypoxia), suggesting the adoption of anaerobic glycolysis as main bioenergetic pathway and capacity to maintain intracellular homeostasis[86]^7 ([87]Figure 1B). Low glucose (normoxia-Glc) also increased lactate as a result of aerobic glycolysis, except for HT1197 cells. However, it remained in the intracellular compartment, suggesting that low oxygen may be critical for activating extrusion mechanisms. Under these conditions, lactate may be converted into pyruvate to fuel the Krebs cycle. The combined effect of hypoxia and low glucose (Hypoxia-Glc) lowered lactate close to vestigial levels, strongly supporting the activation of an alternative energy-producing pathway to glycolysis. Figure 1. [88]Figure 1 [89]Open in a new tab BLCA cells exhibit remarkable tolerance to hypoxia and low glucose, adopting a quasi-quiescent and more aggressive invasive behavior (A) Hypoxia or low glucose (normoxia-Glc) significantly upregulates HIF-1α expression in BLCA cell lines, which is further enhanced when combined (hypoxia-Glc). (B) BLCA cells cultured in hypoxia and low glucose produce residual levels of lactate. Individually, these stressors induce the production of lactate. (C) Hypoxia and low glucose significantly suppress cell proliferation. Individually, low oxygen or low glucose inhibits cell proliferation. The combination of these stressors further exacerbates this effect in all cell lines. (D) BLCA cells maintain their viability under hypoxia and low glucose. The combined environmental stress from hypoxia and low glucose does not significantly impact the viability of 5637 and T24 cells. RT4 and HT1197 cells exhibit a 30%–45% reduction in viability under these conditions, suggesting a limited adaptive capacity. (E) BLCA cells display increased invasiveness under hypoxia or low glucose. This is significantly potentiated when both stimuli are combined. (F) BLCA cells demonstrate remarkable adaptability to microenvironmental changes with minimal impact on cell viability. Restoring oxygen and glucose levels does not affect cell viability, underscoring the high plasticity of these cells to endure drastic microenvironmental changes. (G) BLCA cells restore basal proliferation after 48 h of reoxygenation with glucose restoration. Both 5637 and T24 cells regain proliferative capacity, fully reinstating proliferation after 48 h, highlighting their plasticity in responding to microenvironmental challenges. (H) After 24 h of reoxygenation with glucose restoration, BLCA cells exhibit a significant reduction in invasion, which is fully restored under normoxia after 48 h. (I) Hypoxia and low glucose increase T24 cells' resistance to cisplatin across a wide range of concentrations, including its IC50, whereas 5637 cells remain unchanged. Error bars represent mean ± SD for three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test and the Mann-Whitney test were used for statistical analysis. Results were considered statistically significant when p < 0.05. Concomitantly, we observed a striking decline in cell proliferation (approximately 1.5-fold) in all cell lines under hypoxia, which was significantly enhanced upon glucose suppression ([90]Figure 1C). Cells were arrested in G2/M checkpoint, most likely due to depletion of the substrates required for DNA synthesis, as it was later confirmed by metabolomics ([91]Figure S1). Notably, the viability of 5637 and T24 cells was not affected after 24 h of microenvironmental stress, as highlighted by little changes in the percentage of apoptotic and pre-apoptotic cells in comparison to normoxia ([92]Figure 1D). Expanding this study, we found that 5637 and T24 were even able to withstand these stressors for at least 72 h without loss of cell viability, demonstrating their significant adaptability. In contrast, RT4 and HT1197 cells viability was decreased by approximately 30%–50% after 24 h. As such, 5637 and T24 cells were selected for more in-depth functional and molecular studies. After 24 h, 5637 and T24 cells responded to oxygen or glucose shortage by increasing invasion of Matrigel in vitro ([93]Figure 1E). Strikingly, their combination (hypoxia-Glc) enhanced further invasion, highlighting the pivotal role played by both stressors. Finally, reoxygenation and access to glucose restored proliferation after 24 h and induced a massive drop in invasion without inducing apoptosis, suggesting little oxidative stress from drastic alterations in the microenvironment ([94]Figures 1F–1H). Collectively, this demonstrated that certain BLCA cell subpopulations are well capable of accommodating hypoxia-induced stress, while concomitantly acquiring more aggressive and motile phenotypes. Overall, our results suggest that oxygen and glucose levels act as an on-off switch for proliferation and invasion. Interestingly, resistance to cancer cell death, proliferation decline, and activation of invasion traits have been closely linked to lactic acidosis as a result of either hypoxia or glucose shortage.[95]^7 Our observations support the adoption of similar behaviors in the absence of lactate. Finally, we addressed 5637 and T24 tolerance to cisplatin, generally used in the clinics against less proliferative tumor cells. Under stress, BLCA cells significantly increased tolerance to cisplatin as observed for T24 and 5637 cells ([96]Figure 1I), suggesting the adoption of cell chemoresistance mechanisms, which could be directly related to decreased proliferative potential promoted by stress conditions. Stress-induced transcriptome rewiring supports cancer aggressiveness and metabolic reprogramming The 5637 and T24 BLCA cells showed markedly different transcriptomes but common responses to hypoxia and glucose shortage ([97]Figure 2A). A total of 4,044 genes were differentially expressed in response to stress (1,722 upregulated, 2,322 downregulated; [98]Figure 2B and [99]Tables S1 and [100]S2), thus supporting significant transcriptome remodeling. Under hypoxia and glucose deprivation, cells activated stress-related genes driving more undifferentiated (KRT17),[101]^18 poorly proliferative (PPP1R15A/GADD34[102]^19), and cell-death-resistant (PPP1R15A/GADD34,[103]^20 DDIT4,[104]^21 PFKFB3[105]^22) phenotypes ([106]Figure 2C). Furthermore, upregulation of DDIT4, HK2, PFKFB3, and PPP1R15A/GADD34 supported the activation of autophagic events.[107]^23^,[108]^24^,[109]^25^,[110]^26 In particular, PPP1R15A/GADD34 plays a key role in sustaining autophagy during starvation, thus enabling lysosomal biogenesis and a sustained autophagic flux,[111]^26 whereas DDIT4 has been linked to adaptive survival mechanisms permitting cells to resist metabolic stress.[112]^27 Finally, we observed significant downregulation of genes linked to lipogenesis (ELOVL6),[113]^28 as well as both proliferation and positive regulators of differentiation (FOSB, CSF3/G-CSF).[114]^29^,[115]^30^,[116]^31 Furthermore, hypoxia and low glucose levels negatively regulated tumor suppressors (EGR1)[117]^32 and activated genes linked to invasive/migratory capacity (ETS1,[118]^33 DDIT4,[119]^34 TAGLN,[120]^35 TMEM158[121]^36). Collectively, BLCA cells showed remarkable transcriptomic adaptability to microenvironmental stress, supporting increased cancer aggressiveness. Genes involved in cell-cell adhesion, cell proliferation, programmed cell death, DNA damage, metabolism reprogramming, and oxidation-reduction processes ([122]Figure 2D) were triggered, in accordance with the observed functional alterations ([123]Figure 1). The transcriptome of bladder tumors (n > 400 cases) showed significant correlation with hypoxia-related genes observed in stressed cell lines ([124]Figure S2). Moreover, a distinct hypoxic phenotype (characterized by TAGLN, SLC2A3, TRIB3, and TMEM158 upregulation) was identified in more aggressive tumors and associated with a worse prognosis ([125]Figures 2E and 2F). Importantly, this hypoxic phenotype was discriminative of tumors compared to healthy tissues ([126]Figure 2G). Figure 2. [127]Figure 2 [128]Open in a new tab BLCA cell lines under hypoxia and low glucose experience profound transcriptome remodeling, linked to the acquisition of more aggressive phenotypes, which is supported by the poor prognosis observed in TCGA-BLCA patients (A) BLCA cell lines under hypoxia and low glucose display distinct transcriptomes but share common responses to these conditions. PCA for transcriptomics data reveals that PC1 (94% variance) primarily distinguishes differences between cell lines, whereas PC2 (5% variance) highlights marked changes between normoxic and stressed cells. (B) The volcano plot showcases global transcriptional changes between normoxia and hypoxia plus low glucose. Exposure to these stressors alters the expression of 4,044 genes (1,722 upregulated, 2,322 downregulated), indicating significant transcriptome remodeling. (C) Bi-clustering heatmap of the top 30 differentially expressed genes illustrates co-regulation under stress, supporting proliferation arrest, resistance to cell death, and invasion. Heatmap plots log2 transformed expression values of genes in samples. (D) Enrichment analysis of GO terms for differentially expressed genes reveals alterations in key pathways associated with cell-cell adhesion, cell proliferation, and resistance to cell death. (E) Prognostic evaluation identifies a hypoxia and glucose-deprivation-linked four-gene signature (TAGLN^high; SLC2A3^high; TRIB3^high; TMEM158^high). Univariate Cox regression analysis of the top 30 differentially expressed genes identifies seven genes associated with OS. Higher expression levels of four genes, upregulated under hypoxia and low glucose, significantly correlate with poor OS, constituting a stress signature. (F) Validation of the prognosis significance of the hypoxia-related four-gene signature in BLCA patients from TCGA. Kaplan-Meier curves of OS and PFS show significantly worse clinical outcomes for patients displaying the stress-related gene signature compared to the remaining patients in the cohort. (G) Bi-clustering heatmap showing the association between the stress-related signature and bladder tumors. Heatmap plots log2 transformed expression values of the four hypoxia-related differentially expressed genes, showing clear differentiation between cancer and healthy bladder samples. Microenvironmental stress induces catabolic metabolism To gain more insights into the metabolic reprogramming induced by hypoxia and glucose deprivation, we performed a mass-spectrometry-based untargeted metabolomics study on 5637 and T24 cells ([129]Tables S3 and [130]S4). As highlighted by [131]Figure 3A, T24 and 5637 cells present distinct metabolic fingerprints under normoxia, in line with the different molecular backgrounds observed at the transcriptome level ([132]Figure 2). However, similar responses were observed facing low oxygen and glucose, characterized by a statistically significant reduction in the levels of 85 metabolites and increments in 8 species. Increased metabolites included several fatty acid-carnitine derivatives, whereas nucleotide sugar donors (UDP-Glc and UDP-GalNAc), citric acid, and gluconic acid were substantially decreased ([133]Figures 3B and 3C). High levels of fatty acid-carnitine derivatives (pentadecanoylcarnitine, L-palmitoylcarnitine, heptadecanoyl carnitine, stearoyl carnitine, arachidyl carnitine; [134]Figures 3B and 3C) support active translocation of long-chain fatty acids across the inner mitochondrial membrane for subsequent β-oxidation. Interestingly, increased levels of these metabolites have been observed in the urine of advanced-stage BLCA patients,[135]^37^,[136]^38 reinforcing the close link between this metabolic phenotype and cancer aggressiveness. In addition, stressed cells showed decreased amounts of several lysophosphatidylcholines and lysophosphatidylethanolamine, which is consistent with the mobilization of lipids for mitochondrial β-oxidation[137]^39 ([138]Figure 3C) and in perfect agreement with the lipolytic phenotype highlighted by transcriptomics. Interestingly, carnitine levels were also conserved to maintain downstream lipid β-oxidation at the expenses of L-lysine and L-methionine degradation ([139]Figures 3C–3E). On the other hand, stressed cells showed significant amounts of Krebs cycle intermediates, namely citrate, but also oxoglutarate, oxaloacetate, malate, and fumarate. Interestingly, lower citrate in cancer cells has been described to favor resistance to apoptosis and cellular dedifferentiation,[140]^40 thus in agreement with functional and transcriptomics studies. Of note, we also observed amino acids consumption (lysine, valine, leucine, isoleucine; [141]Figure 3D) to support energy requirements. A joint pathway analysis, combining transcriptomics, and metabolomics confirmed the molecular rewiring of challenged cells toward a catabolic state ([142]Figure 3E). It further highlighted changes in glycolysis and gluconeogenesis, in agreement with the adoption of a lipolytic rather than a glycolytic metabolism. Figure 3. [143]Figure 3 [144]Open in a new tab Hypoxia and low glucose shift BLCA cell metabolism from glycolytic to lipolytic, increasing lipid droplet formation and reducing the number of active mitochondria (A–C) PLS-DA analysis reveals similar metabolic responses in 5637 and T24 cells under microenvironmental stress (A). Volcano plot highlights significant metabolome alterations in response to hypoxia and low glucose (B). Downregulated metabolites include UDP-Glc, UDP-GalNAc, gluconic acid, and citric acid, whereas increased metabolites indicate active fatty acid transport and β-oxidation (C). Significant reduction in key metabolites linked to nucleotide, amino acids, Krebs cycle, and lipid metabolism was observed, consistent with catabolic metabolism. An exception is the accumulation of long fatty acid acylcarnitine for transfer across the inner mitochondrial membrane for β-oxidation. (D) Pathway enrichment analysis supports fatty acid β-oxidation as the primary bioenergetic pathway in stressed cells. Key metabolic pathways, including carnitine biosynthesis and lysine/methionine degradation, contribute to fatty acid β-oxidation. (E) Hypoxia and low glucose induce lysine and methionine degradation to support acylcarnitine biosynthesis and lipid β-oxidation. (F) Joint pathway analysis incorporating transcriptomics and metabolomics studies supports changes from glycolytic to lipolytic metabolism, impacting nucleotides and sugars biosynthesis, including O-GalNAc glycans and protein O-glycosylation. (G and H) Hypoxia and low glucose increase AMP/ATP ratio (G) and activate AMPK by phosphorylation (H), indicating impaired oxidative phosphorylation and potential catabolic processes, including mitophagy. (I) Citrate synthase activity decreases under hypoxia and low glucose, suggesting a reduction in functional mitochondria. (J) TEM analysis reveals major morphological changes, including compromised mitochondria, lipid droplets, peridroplet mitochondria, membrane vesicles, and increased shedding of vesicles, indicating membrane activity changes under stress. Error bars represent mean ± SD for three independent experiments. Mann-Whitney test was used for statistical analysis. Results were considered statistically significant when p < 0.05. Not surprisingly, we found a higher AMP/ATP ratio in stressed cells ([145]Figure 3G), mainly driven by a significant decrease in ATP. This was accompanied by higher 5-AMP-activated protein kinase (AMPK) phosphorylation ([146]Figure 3H), frequently observed in more aggressive bladder tumors.[147]^41 These findings were both consistent with the adoption of catabolic processes and supported the occurrence of mitophagy. Accordingly, microenvironment-challenged cells displayed decreased citrate synthase activity ([148]Figure 3I) and higher number of autophagosomes ([149]Figure S3A). Autophagy in hypoxic cells was confirmed by dedicated immunoassays ([150]Figure S3B). Mitophagy was also later confirmed by TEM ([151]Figure 3J). In fact, microscopy evidenced a drastic decrease in the number of intact mitochondria, allied to evident mitophagy events ([152]Figures 3JI and II), translated by outer mitochondrial-membrane-associated degradation and matrix sectioning. Lipid-droplet (LD)-associated mitochondria, also known as peridroplet mitochondria (PDM), were also observed under hypoxic conditions ([153]Figures 3JII). Vesicle shedding was evident ([154]Figures 3JIII and IV), highlighting cellular communication events that should be carefully investigated in future studies. Finally, stressed cells showed considerably short and disorganized endoplasmic reticulum (ER) cisternae, contrasting with typically longer ER sections presented by cancer cells in normoxia ([155]Figures 3JV and VI), demonstrating prominent disorganization of secretory pathways where glycosylation occurs ([156]Figure S4). Overall, cellular ultrastructure was significantly altered in ways that are consistent with catabolic responses leading to lipid droplets accumulation and disorganization of major organelles linked to glycosylation. Therefore, we further investigated alterations in the ER and Golgi apparatus (GA) proteomes under hypoxia and glucose restriction ([157]Figures S5A and S5B). ER-related proteins FKBP2, HYOU1, HSP90B1, ERO1A, and HSPA5 were significantly increased, and SDF4 was decreased in both cell lines ([158]Figure S5A). This highlights common unfolded protein responses intricately linked to ER stress, including organelle fragmentation and functional impairment.[159]^42^,[160]^43^,[161]^44^,[162]^45 Regarding the Golgi, we found that GOLM1 was also decreased in both cell lines ([163]Figure S5B). GOLM1 plays a pivotal role assisting in the transportation of protein cargo through the Golgi apparatus.[164]^46 Overall, ER and GA proteome remodeling supports the adoption of stress-counteracting responses and negative regulation of apoptosis, as evidenced by the functional network in [165]Figure S5C. Collectively, proteomic findings reinforce TEM evidence of impairment in secreting organelles. Cellular signaling rewiring under stress supports aggressiveness Cellular signaling rewiring was assessed by phosphoproteomics ([166]Tables S5 and [167]S6). Under stress, 5637 and T24 BLCA cells responded similarly ([168]Figure 4A), exhibiting major alterations in relevant serine/threonine kinases and substrates linked to high motility and reduced cell adhesion, reduced cell proliferation, cell senescence, and mitochondrial reorganization ([169]Figures 4B and 4C; [170]Tables S5 and [171]S6). Moreover, cells exhibit clear survival adaptations translated by induction of autophagy, apoptosis evasion, cell-cycle arrest, Krebs cycle inhibition, and cell survival ([172]Figure 4), in full agreement with functional, transcriptome, and metabolome reprogramming. Increased endocytosis was also a relevant feature of these cells, supporting major plasma membrane remodeling observed by TEM. Notably, the most significant alterations in phosphorylation (>2-fold change to normoxia) occurred in proteins involved in the AMPK, insulin, HIF-1α, EGFR tyrosine kinase, and autophagy signaling pathways ([173]Figure 4E). The main altered phosphorylation sites under hypoxic conditions are AKT1 S473, AKT3 S472, PFKFB3 S461, CAMKK2 S511, PDHA1 S232, BAD S118, and PRKAB1 S108 ([174]Figure 4D; [175]Tables S5 and [176]S6). AKT1 S473 and AKT3 S472 act synergically to protect cells from microenvironmental and cisplatin-induced apoptosis by phosphorylating downstream molecules as mTOR and glycogen synthase kinase-3. Notably, under stress, the preferable PRKCZ substrate is AKT3 whose activity was found significantly increased ([177]Figure 4E). PRKCZ has been implicated in maintaining high motility and reduced cell adhesion of metastatic cancer cells,[178]^47 in cell-cycle regulation,[179]^48 and in mitochondrial reprogramming in senescent cells toward cell survival. Also, BAD apoptotic activity is attenuated by phosphorylation at S118, precluding binding to other Bcl-2 family members to evade apoptosis.[180]^49 Furthermore, phosphorylated PDHA1 at S232 inactivates pyruvate dehydrogenase complex,[181]^50 redirecting cells toward alternative energy pathways to Krebs cycle. Together with elevated phospho-PRKAB1 S108, this is consistent with the adoption of catabolic processes such as fatty acid β-oxidation and potential mitophagy[182]^51 sustained by AMPK activation ([183]Figures 4D and 4E). A significant decrease in several forms of RPS6 (S240, S235, S244), RAF1 S621, ELAVL1 S202, and EIF4EBP1 T70 is also compatible with impaired cancer cell proliferation observed under stress.[184]^52^,[185]^53^,[186]^54^,[187]^55 These alterations are consistent with the adoption of more aggressive phenotypes by microenvironment-challenged cells. Figure 4. [188]Figure 4 [189]Open in a new tab Hypoxia and low glucose induce major cell signaling rewiring, promoting aggressiveness via cell-cycle arrest, quiescence, apoptosis resistance, glycolytic metabolism blockage, and autophagy (A) Despite cell-related differences, common features in oncogenic signaling are elicited by hypoxia and glucose deprivation, as shown by PCA analysis of phosphoproteome signatures. PC1 (27% variance) distinguishes cell-dependent differences, whereas PC2 (17% variance) relates to marked signaling changes linked to hypoxia and glucose deprivation. (B) Kinase-substrate enrichment analysis scores each kinase based on substrate phosphorylation, highlighting major alterations in stressed cells. Red indicates significantly activated kinases, whereas blue indicates significantly inhibited ones (Z score ≥2 or ≤2). (C) KEGG pathway enrichment analysis of phosphoproteomics data shows significant alterations in cell signaling pathways supporting cell motility, cellular senescence, and autophagy under stress. (D) Volcano plot showcases global cell signaling rewiring between normoxia and hypoxia plus low glucose, indicating the most significantly hyper- or hypo-phosphorylated proteins and precise annotation of main phosphorylation sites. (E) AMPK, insulin signaling, HIF-1α signaling, EGFR-TKI resistance, and autophagy are the most significantly altered pathways in stressed cells. Top-ranked signaling pathways with 2-fold change in stressed cells are presented, highlighting the most significant hyper- or hypo-phosphorylated proteins and precise phosphorylation sites, along with main associated cellular functions. Stress induces an immature glycocalyx The generalized disorganization of secretory organelles on stressed cells strongly suggests implications for the cells’ glycocalyx. In addition, [190]Figure 3 supports major alterations in glycosylation pathways driven by changes in metabolites linked to glycosylation. This includes UDP-GalNAc, a key sugar nucleotide for the initiation of protein O-GalNAc glycosylation and Neu5Ac, a sialic acid capping different glycoconjugates ([191]Figures 3C and [192]S4). Data also suggest low flux through the hexosamine pathway, which impacts UDP-GlcNAc biosynthesis required for glycosylation, even though this metabolite could not be detected by our approach. We further observed decreased UDP-Glc, which is a key precursor of UDP-Gal and UDP-GlcA needed for glycoproteins, proteoglycans, and glycolipids glycosylation. Finally, lower levels of gluconic acid and D-ribulose 5-phosphate as well as AMP, UMP, GMP, and GDP demonstrate alterations in the pentose phosphate pathway ([193]Figures 3C and 3D) required to generate nucleotides for nucleic acid synthesis but also glycosylation. Major inhibition of mucin-type O-GalNAc glycans biosynthesis as well as fructose and mannose metabolism required for glycosylation was also evident in the joint pathway analysis ([194]Figure 3E). In addition, downregulation of several glycogenes encoding polypeptide N-acetylgalactosaminyltransferases responsible for protein O-glycosylation initiation ([195]Table S7) was evident from transcriptome analysis. Downregulation of C1GALT1C1, encoding an essential chaperone for core 1 O-glycan T-synthase activity and further O-glycans elongation, was also observed. Interestingly, stressed cells also presented downregulation of key glycosyltransferases for early N-glycans processing in the ER but also N- and O-glycans elongation in the Golgi. Finally, we observed downregulation of several glycogenes linked to proteoglycans biosynthesis ([196]Table S7). In summary, the disorganization of secretory organelles and decreased sugar nucleotides, accompanied by a net downregulation of key glycogenes linked to glycosylation initiation and elongation, drive an immature glycophenotype. Building on our previous studies linking immature glycosylation with BLCA aggressiveness, we devoted to the precise characterization of the O-GalNAc glycome of stressed cells. This was performed by exploiting the Tn mimetic benzyl-α-GalNAc as a scaffold for further O-chain elongation, providing a good assessment of O-glycosylation pathways fitness ([197]Figures 5 and [198]S4; [199]Table S8). According to [200]Figure 5A, low oxygen and glucose significantly reduced O-glycans synthesis in both cell lines ([201]Figure S6 and [202]Table S8). More detailed glycomic characterization in [203]Figure 5C showed that both cell lines abundantly express fucosylated (m/z 746.40; H1N1F1; type 3 H-antigen) and sialylated T (m/z 933.48; H1N1S1) antigens, also exhibiting several extended core 2 O-glycans of variable lengths and degrees of fucosylation and sialylation. Low amounts of shorter O-glycans such as core 3 (m/z 613.33; N2) and STn (m/z 729.38; N1S1) antigens could also be observed. Complementary flow cytometry analysis using lectins (VVA for Tn; VVA after NeuAse for STn; PNA for T; PNA after NeuAse for STn; and GSL II after PNGAse F for terminal GlcNAc) was employed to gather semi-quantitative data on most relevant glycan structures ([204]Figure 5B). This was particularly relevant for estimating the Tn antigen, which could not be directly assessed by the adopted MS methodology. Accordingly, 60%–80% of the cells originally expressed Tn, whereas only 15%–30% expressed STn. The reduction of oxygen and glucose significantly impacted the glycome of cells, inducing a more homogeneous simple cell glycophenotype characterized by few short-chain O-glycans without chain extension beyond core 2, mainly mono- (m/z 933.48; H1N1S1) and, to less extent, di-sialylated (m/z 1294.65; H1N1S2) T antigens and core 3 (N2) ([205]Figures 5A–5C). Under hypoxia and glucose deprivation, STn (N1S1) expression remained negligible as few cells express this glycan (>10% of the cells) ([206]Figure 5B). Interestingly, no extension of pre-existing core 3 was observed, reinforcing the inexorable expression of shorter structures by stressed cells. Furthermore, we found that the main driver for the lack of glycan extension was the reduction in glucose ([207]Figure 5C). Notably, BLCA cells regained the capacity to extend glycans after reoxygenation and reintroduction of glucose ([208]Figure 5C), demonstrating significant plasticity and that microenvironmental stressors act as an on-off switch for O-glycosylation. Complementary flow cytometry studies showed that the percentage of cells expressing T antigen decreased under hypoxia and glucose deprivation in comparison to normoxia, whereas the abundance of sialylated T antigens was significantly increased ([209]Figure 5B). Also, the percentage of Tn-expressing cells increased under stress, whereas STn levels remained residual, as most Tn antigen is maintained in its non-sialylated form. Overall, these findings highlight that O-glycosylation extension is antagonized under hypoxia and glucose deprivation, leading to more homogeneous glycophenotypes with accumulation of shorter O-glycans. Interestingly, we found similar patterns in a wider panel of cell lines, including RT4 and HT1197, but also esophageal, gastric, and colorectal cancer cells and human monocyte-derived macrophages ([210]Figure S7 and [211]Table S9), suggesting common response patterns to stress by cancer cells and normal immune cells. Notably, in these cells, core 2 was significantly decreased but not completely lost. Finally, we exposed BLCA cells to deferoxamine (DFX), an ion chelator known to stabilize HIF-1α expression, and confirmed nuclear accumulation of HIF-1α ([212]Figure 5A). Upon exposure to DFX, both cell lines exhibited a significant decrease in the expression of C1GALT1C1, a crucial glycosyltransferase responsible for O-chain elongation beyond the Tn antigen. Additionally, in 5637 cells, GCNT1, which regulates core 2 biosynthesis, is downregulated, whereas T24 cells upregulate ST3GAL1 ([213]Figure S8). These alterations could potentially contribute to a premature stop in glycans elongation. Interestingly, DFX did not induce significant changes in the glycosylation of both cell lines, indicating that the HIF-1α transcription factor might not exert a major influence on glycome remodeling, despite its role in regulating crucial O-glycogenes expression. Our observations also highlight the need for careful interpretation of the glycome based on glycogenes expression. Finally, seeking deeper insights on the relation between glycogenes expression and the glycophenotype, we re-analyzed by RT-PCR the expression of a wide array of glycosyltransferases involved in the initial steps of O-glycans biosynthesis ([214]Figure 5D). Glucose suppression impacted more significantly glycogenes expression than hypoxia, inducing significant overexpression of glycosyltransferases linked to O-glycans elongation, namely C1GALT1, C1GALT1C1 (for T24 cells), GCNT1, and B3GNT6, but also extension and termination by fucosylation and/or sialylation ([215]Figure 5D). ST3GAL1 overexpression was observed and may contribute to a premature stop in core 1 extension by O-3 sialylation, decreasing core 1 fucosylation. Interestingly, the combination of both hypoxia with low glucose impacted less on glycogenes remodeling than glucose suppression alone (normoxia-Glc). Notably, B3GNT6 and ST3GAL1 remained overexpressed ([216]Figure 5D). Downregulation of C1GALT1 and C1GALT1C1 was also observed ([217]Figure 5D), which may contribute to reduced core 1 synthesis and further elongation to core 2, impacting negatively on glycans extension. However, western blot analysis showed that, despite changes in glycogenes expressions, ST3Gal-I was the only main glycosyltransferase gatekeeper of O-glycans extension found elevated in stressed cells. ([218]Figure 5E). Also, while the focus was set on O-GalNAc glycosylation, we also characterized the N-glycome. Despite changes in N-glycogenes expression ([219]Table S7), no major alterations could be observed in relation to the main types of N-glycans (oligomannose, paucimannose, complex; [220]Table S10). Figure 5. [221]Figure 5 [222]Open in a new tab Hypoxia and low glucose impair O-glycans extension in BLCA, originating a simple cancer cell glycophenotype (A) BLCA cells exposed to hypoxia and low glucose exhibit less abundant, simpler, and shorter glycomes, lacking extensions beyond core 1 structures. nanoLC-MS/MS analysis shows that this glycophenotype is characterized by sialylated T antigens and core 3, likely due to decreased typical core 1 and 2 structures. DFX-treated cells, stabilizing HIF-1α, show no significant alterations in the glycome, suggesting that changes observed in stressed cells are not driven by HIF-1α. (B) Lectin affinity studies show significant upregulation of Tn and sialylated T antigens (recognized by PNA lectin after Neuraminidase [NeuAse] digestion) under stress, in accordance with MS-based glycomics. Notably, core 3 O-glycans (evaluated by GSL II lectin after PNGase F digestion) remain unchanged, highlighting that cellular stress primarily suppresses core 1/2 O-glycans, rather than increasing core 3 O-glycans. (C) Glucose suppression is the primary driver of glycome remodeling, which can be reversed by reoxygenation and restoration of glucose. (D) Glycogene remodeling is primarily driven by the combined effects of hypoxia and glucose deprivation and leads to a premature halt in glycans extension beyond core 1. C1GALT1C1, necessary for core 1 biosynthesis, is downregulated, whereas ST3GAL1, 3, and 4 are overexpressed, increasing sialylated T antigens and inhibiting core 2 formation. Downregulation of GCNT4 also contributes to core 2 inhibition. Interestingly, elevated GCNT1 and GCNT3 potentially counterbalance core 2 suppression. (E) Quantification of key enzymes involved in O-glycan elongation (C1GalT1; Cosmc; BGnT-6; C2GNT; ST3Gal-I) shows significant upregulation of ST3Gal-1 in stressed cells, consistent with transcriptomics. The others remain unchanged, indicating distinct regulation between glycogenes and glycosyltransferases under these conditions. Bold circles and triangles represent statistically significant changes in T24 and 5637 cell lines, respectively. Error bars represent mean ± SD for three independent experiments. Mann-Whitney Test was used for statistical analysis. Results were considered statistically significant when p < 0.05. Hypoxic bladder tumors share common molecular features with stressed cell lines We then assessed the translational character of our observations in patient samples. A cohort of BLCA surgical tissue sections without prior history of adjuvant treatments (n = 70), comprehending non-muscle (NMIBC; n = 30) and muscle invasive (MIBC; n = 40) tumors spanning all stages of the disease (Ta, T1, T2, T3, T4), was elected for this study. The tumors were screened for nuclear expression of HIF-1α and expression of the proliferation marker Ki-67 (HIF-1α^+ Ki-67^low; hypoxic phenotype). HIF-1α was detected in 10% of MIBC tumors, showing heterogeneous expression ranging from 20% to 60% of the tumor area ([223]Figure 6A). HIF-1α was not found in superficial lesions ([224]Figure 6A). Notably, 59% of studied MIBC were poorly proliferative, including the four hypoxic tumors, whereas only 15% of NMIBC presented a Ki-67 ^low phenotype. Also, HIF-1α-positive tumor areas did not express Ki-67, in agreement with the acquisition of quiescent phenotypes by hypoxic cells ([225]Figure 6B). However, no associations between the hypoxic phenotype and prognosis were found, as these were already highly aggressive lesions. Figure 6. [226]Figure 6 [227]Open in a new tab Hypoxic BLCA, characterized by high nuclear HIF-1α expression and low proliferation, shares malignant molecular features with hypoxic and glucose-deprived cells in vitro, including simple glycophenotypes (A and B) Roughly 10% of MIBC tumors display a hypoxic fingerprint (HIF-1α^positive/Ki-67^low) that was not observed in NMIBC and most MIBC tumors (HIF-1α^negative/Ki-67^high), indicating a potential link to aggressiveness. (C) Hypoxic tumors display significantly higher AMPK phosphorylation compared to proliferative cases, denoting a catabolic state. (D) Hypoxic tumors show distinct cellular signaling pathway activation compared to proliferative tumors. PCA for phosphoproteomics data indicates that PC1 (58% variance) primarily separates hypoxic from proliferative tumors, whereas PC2 (15% variance) highlights marked differences among hypoxic tumors. (E) Kinase-Substrate enrichment analysis supports major cell rewiring in hypoxic tumors. Kinases color-coded in red are significantly activated, whereas blue is significantly inactivated. (F) Hypoxic tumors share common kinase activation patterns with stressed BLCA cells in vitro. (G) KEGG pathway enrichment analysis indicates significant alterations in cell signaling pathways, promoting cell motility, cellular senescence, and autophagy in hypoxic tumors as found in stressed cells in vitro. (H) Hypoxic tumors present simple O-glycophenotypes compared to proliferative tumors. nanoLC-MS/MS reveals more homogeneous O-glycome in hypoxic tumors with scarce core 2 glycans. We represent the most abundant structures also found in cell lines, keeping reference to their original relative abundance in relation to all identified glycan species. (I) Hypoxic tumors N-glycome is enriched for oligomannose N-glycans, whereas proliferative tumors are enriched for complex N-glycans. (J and K) Hypoxic tumors show higher levels of Tn and sialylated T antigens and lower levels of sialylated Lewis antigens in O-glycans compared to proliferative tumors, reinforcing the primary suppression of O-glycan extension. NeuAse means sialidase neuraminidase. (L) In hypoxic tumors, Tn and sialylated T antigens co-localize with high HIF-1α. Normoxic, proliferative tumors lack HIF-1α and show low levels of sialylated T antigens and no Tn antigens. Healthy urothelium from non-cancerous individuals served as a negative control for HIF-1α, low Tn, and sialylated T antigens expression. Unpaired t test and Mann-Whitney test were used for statistical analysis. Results were considered statistically significant when p < 0.05. We then elected for downstream molecular studies of tumors showing diffuse and intense nuclear HIF-1α expressions (>50% of the tumor area). These tumors showed increased AMPK phosphorylation and pAMPK/AMPK ratios in comparison to highly proliferative lesions ([228]Figure 6C), consistent with a catabolic state. Subsequent phosphoproteomics highlighted marked differences between hypoxic and non-hypoxic tumors and striking similarities between hypoxic tumors and cell lines in terms of kinase activation ([229]Figures 6D and 6F; [230]Tables S5 and [231]S6). Namely, we found increased PRKCZ and BRAF phosphorylation, contrasting with significantly lower SRC, CDK1, MTOR, CDK2, RPS6KA1, and MAPK3 activation ([232]Figures 6E and 6F). Likewise, altered pathways were also common, with emphasis on endocytosis, cell-cycle arrest, senescence, autophagy, and invasion/cell motility ([233]Figure 6G). Furthermore, hypoxic tumors exhibited elevated expression of hypoxia-associated poor-prognosis markers TMEM and SLC2A3 ([234]Figures 2F and [235]S9). Moreover, 75% of hypoxic tumors co-overexpress at least two out of four hypoxia-linked genes with prognostic significance ([236]Figure 2F), in contrast to 33% under normoxia, underscoring their molecular resemblance to stressed cells in vitro. The glycome of hypoxic tumors was subsequently characterized by mass spectrometry, revealing the sialyl-T antigen as the predominant glycan and lower levels of core 2 O-glycans compared to other shorter glycans ([237]Figure 6H; [238]Table S11), resembling stressed cell lines. The major difference was the absence of core 3 as well as fucosylated T antigens, invariably present in cell models. Trace amounts of STn were also detected ([239]Table S11). Contrastingly, HIF-1α^negative/Ki-67^high tumors were more heterogeneous, with some tumors showing hypoxic-like glycopatterns, whereas others exhibited a high percentage of core 2 elongations and lower sialyl-T expressions ([240]Figure 6H), suggesting that other factors may also drive altered glycosylation. Semi-quantitative assessment of major glycans linked to hypoxia by lectin immunohistochemistry further reinforced the adoption of a shorter glycophenotype by hypoxic tumors ([241]Figures 6J and 6K). Specifically, hypoxic tumors exhibited significantly elevated expressions of Tn and sialyl-T antigens compared to HIF-1α^negative/Ki-67^high tumors ([242]Figures 6J and 6K). Concurrently, these tumors exhibited decreased levels of sialylated Lewis antigens following PNGase F digestion, indicating a loss of O-glycans complexity as observed in hypoxic cell lines in vitro. Double immunofluorescence confirmed higher levels of Tn and sialylated T antigens in hypoxic tumor areas ([243]Figure 6L), In addition, we performed a broad characterization of the N-glycome. Despite significant structural diversity, hypoxic tumors were enriched for oligomannose N-glycans ([244]Figure 6I; [245]Table S12), which were not evident in vitro. Collectively, these observations highlight fundamental microenvironment-driven molecular grounds shared by cell models and tumors, including the adoption of an immature O-glycocalyx and potentially N-glycosylation, which warrants deeper investigation. Stress-induced glycome alterations contribute to cancer aggressiveness T24 cells were glycoengineered ([246]Figures S10 and [247]S11) to display distinct simple O-glycophenotypes and gain more knowledge on the biological role played by altered glycosylation in BLCA. Notably, we also tried to edit 5637 cells without success. We started by overexpressing ST3GAL1 ([248]Figure S12), which decreased the T antigen and core 1 fucosylation but not core 2-derived glycans, thus not reflecting the hypoxic phenotype ([249]Table S13). Therefore, we glycoengineered cells to hamper O-glycan extension beyond core 1 and core 2 by suppressing C1GALT1 ([250]Figure S10) and GCNT1 ([251]Figure S11), respectively, using validated gRNAs[252]^56 through CRISPR-Cas9 technology. Considering immunoassays using lectins ([253]Figures 7A and [254]S13) and MS analysis ([255]Figure 7B), we observed a significant increase in Tn antigen levels in C1GALT1 knockouts. Notably, the reduced diversity of O-glycans, due to the inability to detect the Tn antigen by MS, led to spectra dominated by core 3 structures, despite complementary flow cytometry indicating their mostly residual presence ([256]Figure S13). Interestingly, we also observed marginal expression of STn in these cell lines ([257]Figure S13). Collectively, this demonstrated reduced levels of O-6 GalNAc sialylation and core 3 biosynthesis, despite losing capacity to produce core 1 structures ([258]Figures 7A, 7B, and [259]S13). As a result, we have established a suitable and glyco-homogeneous model for investigating the role of the Tn antigen, which is enriched in stressed cells ([260]Figure 5) as well as hypoxic tumors ([261]Figure 7). Figure 7. [262]Figure 7 [263]Open in a new tab Hypoxia and low-glucose-induced simple glycophenotypes drive relevant cancer-associated hallmarks (A and B) T24 C1GALT1 KOs show complete abrogation of O-glycans extension beyond the Tn antigen, mirroring a major alteration observed in hypoxic tumors. Glycoengineered cells homogeneously express the Tn antigen and show low levels of core 3. Mock controls' glycosylation closely resembles T24 wild-type cells. (C) C1GALT1 KOs display reduced proliferation compared to controls under normoxia. Collectively, altered glycosylation impacts proliferation more when compared to hypoxia and glucose deprivation. (D) Under normoxia and hypoxia with glucose deprivation, C1GALT1 KOs glycoengineered cells display significantly enhanced invasion, suggesting a critical role of C1GALT1 in modulating invasive behavior under stress. (E and F) C1GALT1 KOs demonstrate higher resistance to cisplatin (E) and anoikis compared to mock controls (F). (G and H) In CAMs, C1GALT1 KOs give rise to smaller tumors (G), in agreement with proliferation studies in vitro (C), showing less cohesive features and invasive patterns compared to control (H). (I and J) T24 GCNT1 KOs exhibit complete abrogation of O-glycans extension beyond core 1, mirroring another major alteration observed in hypoxic tumors. As a result, glycoengineered cells express high levels of sialylated T antigens, namely sialyl-T, but do not present core 2-derived glycans. Mock controls' glycosylation closely resembles T24 wild-type cells. (K and L) GCNT1 KOs display reduced proliferation compared to controls (K) and increased invasion (L) under normoxia, resembling C1GALT1 KOs. A higher invasion is also observed under hypoxia-Glc. (M and N) GCNT1 KOs demonstrate similar resistance to cisplatin (M) but higher resistance to anoikis compared to mock controls (N). (O and P) In CAMs, GCNT1 KOs give rise to smaller (O) and more invasive tumors compared to controls (P). Error bars represent mean ± SD for three independent experiments. Unpaired t test, two-way ANOVA followed by Tukey’s multiple comparison test, and Wilcoxon test were used for statistical analysis. Results were considered statistically significant when p < 0.05. GCNT1 KOs exhibited a glycome enriched for monosialylated T antigens and a complete absence of core-2-derived glycans ([264]Figures 7I, 7J, and [265]S13; [266]Table S13). Additionally, low levels of core 3 were also observed, whereas Tn and STn levels were low. While presenting a more heterogeneous nature in comparison to C1GALT1 KOs, this model provides an opportunity to address the functional role of the ST antigen in a context deprived of more elongated glycans, which constitutes the main structural feature of stressed cells and hypoxic tumors. Interestingly, ST3GAL1 overexpression on T24 wild-type cells decreased core 1 and fucosyl-core 1, increasing the percentage of sialylated core 2 glycans, whereas for GCNT1 KO cells it increased T antigen di-sialylation ([267]Table S13). These observations strongly suggest that ST3GAL1 elevation alone cannot account for the stress-associated glycome. C1GALT1 and GCNT1 KO cells, resembling relevant glycophenotypic signatures of hypoxic cells, were further used to interrogate the role played by glycosylation in decisive aspects of the disease, namely capacity to proliferate, invade, grow without anchorage, potentially metastasize, and tolerate chemotherapy agents ([268]Figure 7). Both were less proliferative in vitro under normoxia as well as hypoxia and low glucose ([269]Figures 7C and 7K). A higher capacity to grow in an anchorage-independent manner and resist anoikis was also observed, supporting increased metastatic potential ([270]Figures 7F and 7N). C1GALT1 KO cells were more resistant to cisplatin over a wide range of drug concentrations and presented higher IC50 than its control ([271]Figure 7E). Increased invasion was also observed under normoxia and microenvironmental stress ([272]Figure 7D). These findings substantiate a role for the Tn antigen in urothelial cancer cells analogous to the one found for other simple cell models from various solid tumors, such as C1GALT1 (T-synthase) knockouts[273]^57^,[274]^58 and C1GALT1C1 (COSMC) knockouts.[275]^59^,[276]^60^,[277]^61 This reinforces the notion of a pan-carcinomic function of Tn antigen driving disease progression. Complementary phosphoproteome characterization confirmed enrichment for pathways consistent with these functional alterations ([278]Figures S14A and S14B), associated to activation of relevant tyrosine kinases also found in hypoxic cells and tumors ([279]Figure S14C), suggesting common molecular grounds supported by immature glycosylation. Interestingly, much less understood GCNT1 KOs also presented low proliferation and higher invasion capacity in normoxia and hypoxia plus low glucose ([280]Figure 7L) but not increased resistance to cisplatin ([281]Figure 7M). However, the molecular pathways governing glycan-linked functional alterations differ significantly from those in Tn-overexpressing cells ([282]Figures S14D–S14F), thus emphasizing the crucial role of glycosylation in fine-tuning molecular functions. Finally, we explored the in ovo chick chorioallantoic membrane (CAM) assay to assess the biological role of glycans in vivo. Notably, T24 CAM models have been previously developed and proven to reflect many histological and molecular features of human bladder tumors,[283]^62 providing relevant platforms for functional studies. C1GALT1 and GCNT1 KO CAM models exhibited significantly smaller tumors when compared to controls, which was consistent with observations in vitro ([284]Figures 7G and 7O). Interestingly, despite displaying much smaller lesions, C1GALT1 KO tumors invaded similarly to controls and exhibited a less cohesive phenotype characterized by a higher number of isolated tumor niches, suggesting higher motility ([285]Figure 7H). GCNT1 KO models invaded more, reaching the lower allantoid epithelium (endoderm) and, in some cases, even expanding beyond that ([286]Figure 7P). In summary, we have highlighted the different contributions of distinct glycosylation patterns associated with hypoxia and glucose suppression to cancer progression and potentially dissemination. Our findings suggest that the loss of glycan heterogeneity and glycan shortening, regardless of the specific nature of involved glycans, leads to decreased cell proliferation while enhancing invasion, metastasis potential, and resistance to chemotherapy. This suggests a role in promoting hypoxia-related aggressiveness. Discussion and concluding remarks Hypoxia and glucose deprivation are relevant features of solid tumors, arising from sustained proliferative signaling and defective neo-angiogenesis, which decisively shape the molecular and functional phenotypes of tumor cells. The contribution of hypoxia to disease severity, even though extensively studied and rather well known, has been addressed without considering the concomitant influence of low glucose. Acknowledging the complex microenvironmental challenges experienced by cells striving to survive in poorly vascularized tumor areas, the present study is devoted to understanding cells’ plasticity and adaptability to these selective pressures, providing the means for more educated and precise interventions. We have comprehensively addressed the impact of microenvironmental stressors (hypoxia and low glucose) at the transcriptome, metabolome, and phosphoproteome levels, which ultimately led us to interrogate the glycome and its biological implications in disease. Strikingly, BLCA models of distinct genetic and molecular backgrounds responded similarly to microenvironmental challenges, denoting common biological grounds facing stress. Cells tolerated low levels of oxygen and glucose, maintaining viability, and avoiding apoptosis, while dramatically decreasing proliferation. Concomitantly, cells became more invasive, denoting an active strategy to escape suboptimal growth conditions. Stress also increased cells' capacity to tolerate the chemotherapy drug cisplatin, possibly explaining the resilient nature of poorly vascularized bladder tumors.[287]^63^,[288]^64 Interestingly, this aggressive behavior was completely reverted by re-oxygenation and glucose restoration without compromising cell viability. This further highlighted relevant molecular plasticity accommodating microenvironmental challenges. Under stress, despite the low levels of oxygen, cancer cells adopted mitochondrial fatty acid β-oxidation and oxidative phosphorylation rather than anaerobic glycolysis as the main bioenergetic pathway, explaining the tremendous decrease in lactate production by these cells. Similar observations were made for prostate cancer, where fatty acid oxidation is a dominant bioenergetic pathway rather than glycolysis, showing potential to be the basis of imaging diagnosis and targeted treatment.[289]^65 To some extent, this also challenges the pivotal role attributed to lactate in the aggressiveness of hypoxic cancer cells in many studies.[290]^66 We have also found strong indications of mitochondrial autophagy, which has been previously described as a survival mechanism of cancer cells under nutrient deprivation, driving invasion, immune escape, and metastasis.[291]^11 Notably, most of the stress-induced molecular alterations found in vitro were observed in highly hypoxic and aggressive bladder tumors, providing a basis for patient stratification and targeted interventions. Finally, we have shown that both stressed BLCA and hypoxic tumors exhibit immature glycophenotypes, characterized by the incapacity to display extended and heterogeneous O-glycans. In previous studies, we have linked short glycoforms to advanced stages and poor prognosis,[292]^16^,[293]^67^,[294]^68^,[295]^69 without providing a clear rationale for their overexpression in BLCA. Here, we demonstrate that glycocalyx remodeling is an integral component of the adaptive responses of cancer cells to hypoxia and glucose deprivation driving disease aggressiveness. We show that microenvironmental stress induces major metabolic changes that have direct impact on key sugar nucleotides required to support O-glycosylation. Namely, we found lower UDP-GalNAc, Neu5Ac, and UDP-Glc, which may ultimately compromise O-GalNAc glycosylation initiation, elongation, and capping. Decreased gluconic acid and D-ribulose 5-phosphate as well as AMP, UMP, GMP, and GDP also evidenced alterations in the pentose phosphate pathway required to generate sugar nucleotide precursors of glycosylation. In addition, we found profound disorganization of secretory organelles, which have been previously linked to major alterations in O-glycosylation. We have further observed major downregulation of multiple polypeptide GalNAc-Ts responsible for O-glycosylation initiation, dictating glycosites distribution and density. These events likely contribute to the decreased O-GalNAc glycosylation observed in stressed cells, as well as the reduced structural diversity. Finally, we observed increased expression of ST3Gal-I, which perfectly aligns with the over-sialylation of T antigen observed in stressed cells, ultimately resulting in a premature termination of O-glycans extension. We believe that these events may act synergistically to drive glycocalyx remodeling. Changes in protein O-glycosylation may be part of a wide array of responses that make BLCA cells more capable of resisting microenvironmental stress. Specifically, these enabled cells to halt proliferation, resist anoikis, and tolerate chemotherapy. Shorter and more homogeneous O-glycosylation also provides a stress escape mechanism by promoting less cohesive and invasive traits. These observations are in perfect agreement with other reports from simple cell cancer models of different origins, highlighting their pan-carcinomic nature.[296]^59^,[297]^60^,[298]^70 These are seminal findings that offer new insights into understanding the drivers of cancer-associated glycosylation, which have so far been linked to functional mutations in COSMC[299]^71 and, more recently, pro-inflammatory immune responses.[300]^72 Future studies must now focus on conducting an in-depth characterization of the hypoxia-related glycoproteome. This will serve as a starting point for better understanding the molecular mechanisms underlying glycan-triggered aggressiveness, which will be crucial for precise cancer targeting. We also argue that the functional implications arising from stress-induced glycan-remodeling may extend beyond those reported in this study, also serving as a mechanism for evading the immune system. Namely, the Tn antigen found in hypoxic cells is considered a key mechanism of cancer immunosuppression, impairing the maturation of macrophages and dendritic cells by interacting with immune system lectins.[301]^73 Investigations on the hypoxia-glycome-immune response axis will likely offer novel insights into the capacity of cells to thrive under stress conditions. In summary, this study offers a comprehensive exploration of the molecular and functional adaptations undergone by bladder cancer cells when confronted with hypoxia and glucose deprivation, two microenvironmental features that are rarely analyzed together despite their physiological coexistence in tumors. Furthermore, we have addressed a relatively unexplored dimension of molecular adaptation to microenvironmental stress, focusing on glycocalyx remodeling and its underlying functional consequences for cancer cells. Our study offers insights into the context of immature glycosylation in BLCA, a factor consistently associated with unfavorable prognosis. It contributes to our understanding of the mechanisms driving BLCA invasion and dissemination, with potential implications for precision therapeutics. Furthermore, it offers seminal observations to better comprehend glycocalyx remodeling beyond genetic drivers, underscoring the necessity for a more structured understanding of the physiological dimension of glycosylation. Limitations of the study The present study provides an in-depth multi-omics characterization of major cellular molecular rewiring in response to hypoxia and glucose deprivation stressors, emphasizing the impact on the glycocalyx and associating these changes with cancer aggressiveness. However, it does not provide causal connections or molecular clarification of the underlying mechanisms behind how hypoxia and metabolic changes drive glycan alterations toward more aggressive cancer phenotypes. Another key limitation is its sole reliance on in vitro cell models to simulate the complex tumor microenvironment, which may not fully capture the heterogeneity and complexity of human tumors, limiting the direct translational potential of the findings. Resource availability Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, José Alexandre Ferreira (jose.a.ferreira@ipoporto.min-saude.pt). Materials availability All unique models generated in this study are available from the [302]lead contact with a completed materials transfer agreement. Data and code availability * • Gene expression data are deposited at Gene Expression Omnibus (GEO) repository ([303]https://www.ncbi.nlm.nih.gov/geo/), metabolomics data are deposited at Metabolomics Workbench ([304]https://www.metabolomicsworkbench.org), glycomics data are deposited at GlycoPost ([305]https://glycopost.glycosmos.org/), and proteomics/phosphoproteomics data are deposited at PRIDE ([306]https://www.ebi.ac.uk/pride/). All data are publicly available as of the date of publication. Accession numbers and codes are listed in the [307]key resources table. * • The remaining data and information are available within the main text or [308]supplemental information. Any additional information required to reanalyze the data reported in this paper is available from the [309]lead contact upon request. * • This paper does not report original code. Acknowledgments