Filippi-Chiela, E. | bioRxiv Science

bioRxiv2026

Metformin modulates autophagy in heterozygous and CRISPR-edited TSC2 primary fibroblasts

BackgroundTuberous Sclerosis Complex (TSC) is a genetic disorder caused by variants in TSC1 or TSC2, leading to mTORC1 hyperactivation and autophagy suppression. Although TSC tumorigenesis typically follows a "two-hit" model, the role of TSC2 haploinsufficiency in autophagy regulation remains unclear. We evaluated autophagy markers in haploinsufficient and gene-edited TSC2 primary cells and investigated the role of metformin in modulating autophagy levels. MethodsPrimary fibroblast cultures were obtained from one healthy individual and three from patients carrying heterozygous germline TSC2 variants: the pathogenic variants c.1008T>G and c.4375C>T.A variant of uncertain significance (VUS) c.724A>T. CRISPR/Cas9-RNP editing was used to model loss of heterozygosity (LOH) in cell pools carrying each variant. Cultures were treated with rapamycin, HBSS, metformin, bafilomycin A1, or vehicle controls, and autophagy was assessed by autolysosomes formation by flow cytometry (acridine orange) and autophagosomes immunofluorescence (LC3 and p-S6K). ResultsIn wild-type cells, only HBSS increased autophagy-positive (acridine orange-positive) cells versus control (15.6% vs. 7.5%; p=0.003). In heterozygous pathogenic cells, rapamycin and metformin increased autophagic cells: c.1008T>G (16.2%, p=0.006; 17.6%, p=0.002) and c.4375C>T (12.5%, p=0.003; 13.3%, p=0.001), versus DMSO controls (9.2% and 7.1%, respectively). VUS c.724A>T cells, with rapamycin increasing autophagic cells (9.74% vs. 6.5%; p=0.0152). In CRISPR-edited cells, all treatments increased the number of autophagic cells compared to the heterozygous cells: c.1008T>G (rapamycin 27.1% vs. 16.7%, p<0.001; metformin 27.2% vs. 17.6%, p<0.001) and c.4375C>T (rapamycin 21.3% vs. 13.1%, p=0.0021; metformin 21.5% vs. 13.6%, p=0.0029). Editing also restored metformin responsiveness in VUS cells (12.5% vs. 8.4%; p=0.0055). Immunochemistry confirmed increased total LC3II and decreased p-S6K across treated cells compared to the control (DMSO). ConclusionThese findings demonstrate that TSC2 haploinsufficiency functionally impairs autophagy prior to second-hit loss. Metformin effectively restores autophagy with phenotypical changes of mTORC1 blockade, highlighting an accessible translational strategy to restore and induce autophagy in TSC cells.

molecular biology↗

bioRxiv2025

Functional autophagy gene set signature and state classification reveal a link between autophagy induction, lysosomal activity, and poor prognosis in glioblastoma

Autophagy is an essential mechanism for maintaining cell homeostasis and, when dysregulated, is related to various pathologies. In cancer, it functions as a double-edged sword, either as a tumor suppressor or as a tumor promoter. Elevated GFP-LC3 puncta and increased levels of autophagy-related proteins, commonly interpreted as markers of high autophagic activity, are often associated with poor prognosis in tumors. However, current assessments of autophagy largely rely on isolated cellular and protein-level observations. While transcriptomic approaches offer broader insights, they often utilize large gene signatures without adequately accounting for the dynamic nature of autophagic flux. In this study, we propose a functional gene set signature for autophagy based on the rational mapping of flux stages, enabling the inference of distinct autophagy states. Using a supervised machine learning approach, we developed two indices - AutoIndex and LysoIndex - that effectively distinguish between cells treated with autophagy inducers and inhibitors. Applying this framework, we analyzed autophagy states across four cancer types, comparing tumor samples to matched normal tissues and evaluating their association with patient survival. Among these, glioblastoma (GBM) exhibited the strongest correlation between inferred autophagy states and clinical outcomes. GBM samples characterized by high LysoIndex and low AutoIndex--corresponding to a transcriptomic induction state--were linked to significantly poorer survival. Compared to lower-grade gliomas, GBM showed elevated lysosomal activity, which may contribute to its more aggressive behavior. Additionally, GBM cell lines treated with temozolomide displayed signatures consistent with autophagy induction, supporting our models predictive relevance. These results reinforce the understanding and monitoring of autophagy, and suggest that, in the case of GBM, its negative influence may represent a potential target for novel therapeutic interventions.

cell biology↗