Abstract BACKGROUND Galectins (GALs, LGALS) are a family of proteins recognized for their β-galactoside-binding activity, which underlies their diverse intra- and extracellular functions. Some members of the GAL family are associated with tumor development, including glioblastoma (GB). However, their precise role in tumorigenesis remains insufficiently explored. MATERIAL AND METHODS SUMO software was used to analyze publicly available TCGA and CCGA datasets. The scRNA-seq data analysis was conducted using the R toolkit Seurat and the SingleR package. As cellular models, we used patient-derived glioblastoma stem cells (GSCs) - BTSC73 and BTSC12. Genetic depletion was achieved using siRNA-mediated silencing and CRISPR-Cas9 genome editing. Pharmacological GAL-3 inhibition was achieved with the selective inhibitor GB1107. Cells were starved in DMEM containing penicillin-streptomycin. Cell viability was assessed by flow cytometry (FACS) using propidium iodide (PI) staining. Cell death was quantified using the Annexin/PI double staining assay. Cell cycle analysis was performed using PI staining followed by FACS. Proliferation was assessed by EDU staining. RESULTS We first conducted an in silico analysis for LGALS1, -3, -8, and -9 expression in GB. We found that a combination of high expression of these GALs is associated with the poorest prognosis in GB patients compared to other combinations. The scRNASeq analysis has revealed that GALs are distributed heterogeneously in GB, where GAL-3 is highly expressed in tumor cells, macrophages, and T-cells. We then conducted in vitro experiments to assess the role of GAL-3. Our results revealed that genetic silencing and pharmacological inhibition of Gal-3 lead to a decrease in BTSC12 and BTSC73 numbers, which is related to an effect on proliferation but not apoptosis. Cell cycle analysis demonstrated that CRISPR knockout cells may exhibit an accumulation in the G1 phase 22 hours after seeding. Notably, starvation of these cells resulted in the opposite phenotype, characterized by increased proliferation and a decreased G1-phase cell fraction. We suggest that these effects may be linked to a less effective G1 checkpoint, as indicated by elevated levels of γH2AX in knockout cells detected by Western blot analysis. Furthermore, TMZ treatment of BTSC73 following GAL-3 knockout resulted in a bigger decrease in cell viability compared to the control condition. CONCLUSION Our findings indicate that GAL-3 may contribute to GB development by regulating GSC proliferation, cell cycle progression, response to nutrient deprivation and TMZ treatment.
Sliusar et al. (Wed,) studied this question.