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Tumor and microenvironment reprogramming during radiochemotherapy in glioma using a combined diagnostic imaging and single-cell RNA sequencing approach

ABSTRACT: Glioblastoma (GBM) is the most common primary brain tumor in adults with a median survival of 11-12 months. Standard therapy consists of radiotherapy (RT) plus chemotherapy with Temozolomide (TMZ), and no alternative treatment is available. We assessed changes in tumor microenvironment (TME) after RT associated with TMZ and metformin (MET) in a syngenic murine GBM model using single-cell RNA-sequencing (scRNA-seq). RT induced an enrichment of GO annotation related with cell cycle, translation and ribosome biogenesis in all tumor clusters, except the radio resistant one where an increase in the inflammatory phenotype was observed. Resident GAM were reduced after RT alone and differentially modulated by RT-drug combinations. After RT alone, we detected an increase in GAM markers and a downregulation of glycolytic metabolism in both microglial and peripheral macrophage subclusters. Association of TMZ increased the pro-inflammatory phenotype of some GAM clusters in comparison to RT. A different signature was observed for the TMZ-MET association but limited to peripheral infiltrating GAM.

ORGANISM(S): Mus musculus

PROVIDER: GSE256358 | GEO | 2025/06/18

REPOSITORIES: GEO

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Project description:Background. Glioblastoma multiforme (GBM) exhibits a cellular hierarchy with a subpopulation of stem-like cells known as glioblastoma stem cells (GSCs), that drive tumor growth and contribute to treatment resistance. NAD(H) emerged as a crucial factor influencing GSCs. Methods. A multistep process of machine learning algorithms was implemented to construct the glioma stemness-related score (GScore). Further in silico and patient tissue analyses validated the predictive ability of the GScore and identified a potential target, CYP3A5. Loss-of-function or gain-of-function genetic experiments were performed to assess the impact of CYP3A5 on the self-renewal and chemoresistance of GSCs both in vitro and in vivo. Mechanistic studies were conducted using nontargeted metabolomics, RNA-seq, seahorse, transmission electron microscopy, immunofluorescence, flow cytometry, ChIP‒qPCR, RT‒qPCR, western blotting, etc. The efficacy of pharmacological inhibitors of CYP3A5 was assessed in vivo. Results. Based on the proposed GScore, we identify a GSC target CYP3A5, which is highly expressed in GSCs and temozolomide (TMZ)-resistant GBM patients. This elevated expression of CYP3A5 is attributed to transcription factor STAT3 activated by EGFR signaling or TMZ treatment. Depletion of CYP3A5 impairs self-renewal and TMZ resistance in GSCs. Mechanistically, CYP3A5 maintains mitochondrial fitness to promote GSC metabolic adaption through the NAD⁺/NADH-SIRT1-PGC1α axis. Additionally, CYP3A5 enhances the activity of NAD-dependent enzyme PARP to augment DNA damage repair. Treatment with CYP3A5 inhibitor alone or together with TMZ effectively suppresses tumor growth in vivo. Conclusion. Together, this study suggests that GSCs activate STAT3 to upregulate CYP3A5 to fine-tune NAD⁺/NADH for the enhancement of mitochondrial functions and DNA damage repair, thereby fueling tumor proliferation and conferring TMZ resistance, respectively. Thus, CYP3A5 represents a promising target for GBM treatment.

Project description:Glioblastoma (GBM) carries a dismal prognosis largely due to acquired resistance to the standard treatment, which incorporates the chemotherapy temozolomide (TMZ). Inhibiting the proteasomal pathway is an emerging strategy, where combination treatments are under clinical investigation. We hypothesized that pre-treatment of GBM with bortezomib (BTZ) might sensitize glioblastoma to TMZ by abolishing autophagy survival signals to augment DNA damage and apoptosis. P3 patient-derived GBM cells as well as the tumor cell lines U87, HF66, A172 and T98G were investigated for clonogenic survival after single or combined treatment with TMZ and BTZ in vitro. Change in autophagic flux was examined after experimental treatments in conjunction with inhibitors of autophagy or downregulation of autophagy-related genes -5 and -7 (ATG5 and ATG7, respectively). Autophagic flux was increased in TMZ-resistant P3 and T98G cells as indicated by diminished levels of the autophagy markers LC3A/B-II and increased STX17, higher protein degradation and no formation of p62 bodies nor induction of apoptosis. In contrast, BTZ treatment attenuated ULK1 mRNA, total and phosphorylated protein, and accumulated LC3A/B-II, p62 and autophagosomes analogously to Baf1 and chloroquine autophagy inhibitors. These autophagosomes did not fuse with lysosomes, indicated by attenuated STX17 expression and reduced degradation of long-lived proteins, which culminated in enhanced caspase-3/8 dependent apoptosis. BTZ synergistically enhanced TMZ efficacy, attenuated tumor cell proliferation, triggered ATM/Chk2 DNA damage signalling to further augment caspase-3/8 mediated apoptosis in the TMZ resistant P3 and T98G GBM cells. Genetic or chemical inhibition of autophagy (with CRISPR-CAs9 ATG5, ATG7 shRNA, MRT68921 or VPS34-IN1) abrogated BTZ efficacy and rescued BTZ+ TMZ treated GBM cells from death. We conclude that Bortezomib ameliorates temozolomide resistance through ATG5/7-dependent abrogated autophagic flux and may be amenable in combination treatment regimens for TMZ refractory GBM patients.

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Tumor and microenvironment reprogramming during radiochemotherapy in glioma using a combined diagnostic imaging and single-cell RNA sequencing approach

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