Fig 1: 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.
Fig 2: 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.
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