Temporal multi-omic profiling reveals chemoradiotherapy-specific dualistic metabolic rewiring that supports glioblastoma tumor recurrence (scRNA-Seq)
Tumor recurrence afflicts over 95% of glioblastoma (GBM) patients, contributing to the disease's high fatality rates. To unravel the molecular mechanisms behind post-therapy recurrence, we employed a clinically-relevant chemoradiotherapy model, studying the temporal molecular metabolic evolution of patient-derived GBM samples in vitro and in vivo. Leveraging unbiased multi-omics methods, including single-cell and bulk transcriptomics, untargeted metabolomics, and stable isotope tracing, we revealed a dynamic metabolic rewiring favoring one-way anaplerotic pyruvate metabolism via carboxylation across GBM samples. This conserved adaptation, confirmed in independent datasets, resulted in reduced glucose-derived acetyl-coA production, hindering histone acetylation and silencing neural-differentiation genes NEUROD1 and DCX. Pharmacological intervention targeting this metabolic shift reduced recurrent GBM cell aggressiveness, prolonging survival in preclinical GBM xenograft tumors treated with chemoradiotherapy. These findings illuminate a potential metabolic therapeutic avenue to enhance current strategies, addressing disease recurrence and offering much-needed improvements in survival outcomes for GBM patients suffering from this dismal disease. Overall design: GBM cells received 25 µM of temozolomide and controls received equal volumes of DMSO vehicle. Immediately following TMZ treatment, cells were exposed to 1 Gy of X-ray irradiation using the Rad Source 2000 and incubated at 37°C in a 5% CO2 incubator for 1 hour. To mimic the exposure time of chemotherapy received by patients, where the half-life of TMZ is approximately 1-2 hours in circulation, the drug treatment was washed out of the patient-derived GBM cells after 1 hour and replaced with fresh media. The concurrent chemoradiotherapy treatment regimen was repeated daily for 5 consecutive days. After the 5th treatment day, patient-derived GBM cells received a 2-day treatment holiday prior to collection and subsequent analysis. At days 0 (Naïve), 1, 3, 5, and 7 (Resistant), during this treatment protocol, samples were collected and living persisting cell populations were isolated by eliminating dead cells through magnetic bead sorting. Cellular populations consisting of living single cells were submitted for Single cell 3' RNA-Seq using 10X Genomics Chromium (10X Genomics) and Illumina next generation sequencing technologies (Illumina Nextseq 500 Sequencer).