Lamfers, M. L. M. | Search bioRxiv

bioRxiv2023

Identification of inter- and intra-tumoral molecular phenotypes steering temozolomide resistance in patient-derived glioblastoma cells.

BackgroundRadiation therapy and chemotherapy using Temozolomide are the standard adjuvant treatments for patients with glioblastoma. Despite maximal treatment prognosis is still poor largely due to the emergence of Temozolomide resistance. This resistance is closely linked to the widely recognized inter- and intra-tumoral heterogeneity in glioblastoma, although the underlying mechanisms are not yet fully understood. This study aims to investigate the diverse molecular mechanisms involved in temozolomide resistance. MethodsTo induce temozolomide resistance, we subjected 21 patient-derived glioblastoma cell cultures to Temozolomide treatment for a period of up to 90 days. Prior to treatment, the cells molecular characteristics were analyzed using bulk RNA sequencing. Additionally, we performed single-cell sequencing on four of the cell cultures to track the evolution of temozolomide resistance. ResultsThe induced temozolomide resistance was associated with two distinct phenotypic behaviors, classified as "adaptive" (ADA) or "non-adaptive" (N-ADA) to temozolomide. The ADA phenotype displayed neurodevelopmental and metabolic gene signatures, whereas the N-ADA phenotype expressed genes related to cell cycle regulation, DNA repair, and protein synthesis. Single-cell RNA sequencing revealed that in ADA cell cultures, one or more subpopulations emerged as dominant in the resistant samples, whereas N-ADA cell cultures remained relatively stable. ConclusionsThe adaptability and heterogeneity of glioblastoma cells play pivotal roles in temozolomide treatment and contribute to the tumors ability to survive. Depending on the tumors adaptability potential, subpopulations with acquired resistance mechanisms may arise. Further research is necessary to deepen our understanding of these mechanisms and develop strategies to overcome them.

cancer biology↗

bioRxiv2022

Novel kinome profiling technology reveals drug treatment is patient and 2D/3D model dependent in GBM

Glioblastoma is the deadliest brain cancer. One of the main reasons for poor outcome resides in therapy resistance, which adds additional challenges in finding an effective treatment. Small protein kinase inhibitors are molecules that have become widely studied for cancer treatments, including glioblastoma. However, none of these drugs have demonstrated a therapeutic activity or brought more benefit compared to the current standard procedure in clinical trials. Hence, understanding the reasons of the limited efficacy and drug resistance is valuable to develop more effective strategies toward the future. To gain novel insights into the method of action and drug resistance in glioblastoma, we established in parallel two patient-derived glioblastoma 2D and 3D organotypic multicellular spheroids models, and exposed them to a prolonged treatment of three weeks with temozolomide or either the two small protein kinase inhibitors enzastaurin and imatinib. We coupled the phenotypic evidence of cytotoxicity, proliferation, and migration to a novel kinase activity profiling platform (QuantaKinome) that measured the activities of the intracellular network of kinases affected by the drug treatments. The results revealed a heterogeneous inter-patient phenotypic and molecular response to the different drugs. In general, small differences in kinase activation were observed, suggesting an intrinsic low influence of the drugs to the fundamental cellular processes like proliferation and migration. The pathway analysis indicated that many of the endogenously detected kinases were associated with the ErbB signaling pathway. We showed the intertumoral variability in drug responses, both in terms of efficacy and resistance, indicating the importance of pursuing a more personalized approach. In addition, we observed the influence derived from the application of 2D or 3D models in in vitro studies of kinases involved in the ErbB signaling pathway. We identified in one 3D sample a new resistance mechanism derived from imatinib treatment that results in a more invasive behavior. The present study applied a new approach to detect unique and specific drug effects associated with pathways in in vitro screening of compounds, to foster future drug development strategies for clinical research in glioblastoma.

cancer biology↗