Assistant Professor New Jersey Institute of Technology Newark, New Jersey, United States
Introduction: : Epithelial-to-mesenchymal transition (EMT) is a hallmark of cancer progression. Pathological tissue stiffening in tumors alters cellular behavior via mechanotransduction, initiating EMT, and driving cancer progression through complex phenotypic transitions. The cellular glycocalyx, a glycan-rich matrix on the cell surface, buffers and mediates these changes in addition to other cellular interactions with the extracellular matrix (ECM) and other cells. The glycocalyx sugar composition and architecture critically influence tumor phenotype and mechanics. A key regulation of the glycocalyx is sialylation, an enzymatic addition of sialic acid to the terminal ends of glycoconjugates by sialyltransferases. These modifications influence cell surface charge, receptor activation, and cellular plasticity. Several studies have highlighted the role of sialylation in cancer progression; however, standard in vitro models for most cancer research do not mimic the microenvironmental characteristics of human tissue. Most cancer research relies on tissue culture polystyrene surfaces, which are significantly stiffer than physiological tissues. This can artificially enhance stress fiber formation, alter cytoskeletal organization, and influence glycosylation patterns in ways not observed in vivo. Additionally, many studies use culture media with higher-than-physiological glucose concentrations that overstimulate the physiologically relevant metabolic pathways. This is particularly concerning in glycosylation studies, as the availability of nucleotide-sugar donors, derived from glucose metabolism, directly regulates glycan biosynthesis. In this study, we hypothesize that glucose concentration and availability within the cellular microenvironment critically modulate glycosylation in mammary epithelial cells in response to mechanical cues from the ECM.
Materials and
Methods: : To test the hypothesis that physiological glucose concentrations constrain ECM stiffness-driven glycocalyx remodeling, MCF-10A cells were cultured on polyacrylamide (PA) hydrogels of varying stiffnesses (400 Pa, 1 kPa, 6 kPa, and 60 kPa) to mimic healthy and tumor-like ECM. Cells were maintained under low (5 mM) and high (25 mM) glucose conditions. Transcriptomic profiling data for the MCF-10A cells cultured under these defined conditions were obtained through collaborative efforts and analyzed using BioJupies, an automated cloud-based RNA-seq processing platform. Differential gene expression analysis and principal component analysis were performed to identify genes responsive to mechanical and metabolic cues. Among the top 2,500 differentially expressed genes under physiological glucose conditions, ST6GalNAc4 and ST6GalNAc6 were selectively upregulated in response to increased ECM stiffness. We also discovered that cells cultured under high stiffness (60 kPa) with 5mM glucose concentration and those cultured under low stiffness (400 Pa) with higher glucose concentration (25 mM) exhibit similar global gene expression profiles, as indicated by their proximity in the principal component analysis (PCA) plot (Fig. 1). These findings support our hypothesis that glucose concentration modulates the transcriptional responsiveness of glycosylation enzymes to mechanical stress. High glucose conditions exaggerated these responses, highlighting the risk of hyperglycosylation artifacts in standard in vitro models. To validate these observations, CRISPR based gene editing and lentiviral overexpression systems will generate gain- and loss-of-function cell lines for ST6GalNAc4 and ST6GalNAc6 in MCF-10A and MDA-MB231 cells. Knockouts will be verified by genomic sequencing, while overexpression will be confirmed via immunoblotting and lectin-binding assays.
Results, Conclusions, and Discussions:: Analysis from our RNA-seq data proves that glucose concentration is an important regulator of cellular behavior, exerting influence that is comparable to ECM stiffness – an established driver of mechanotransduction that has dominated recent studies. Furthermore, while most glycosylated-related genes, including members of the sialyltransferase family, exhibit minimal response to increasing ECM stiffness under low glucose conditions, ST6GALNAC4 and ST6GALNAC6 were differentially expressed, indicating that glucose availability modulates the mechano-responsiveness of these genes. These findings challenge existing reports, which may be confounded, potentially, by hyperglycosylation artifacts arising from high non-physiological glucose conditions which may obscure true mechanotransducive responses. This highlights the importance of mimicking physiological conditions in in vitro models to improve the translational relevance of cancer mechanobiology studies. By identifying ST6GALNAC4 and ST6GALNAC6 as co-regulated by mechanical and metabolic inputs, we provide a foundation for more predictive models of tumor progression. We aim to use these sialyltransferases to elucidate the intricate interplay between glucose metabolism and ECM stiffness in regulating glycosylation dynamics in mammary epithelial cells. We aim to uncover how ST6GalNAc4 and ST6GalNAc6 influence glycocalyx remodeling, cellular phenotype, and ultimately, EMT. This work will advance our understanding of how glucose metabolism and ECM stiffness collectively shape mammary epithelial cell behavior and may inform the development of more effective therapeutic strategies.
Acknowledgements and/or References (Optional):: This work was supported by New Jersey Health Foundation Grant PC 26-24 to ABJ
