Ex vivo therapy-driven selection of glioblastoma stem-like cells models resistance evolution

Glioblastoma (GBM) arises from stem-like cells (GSCs) with intrinsic therapeutic resistance that can be positively selected by treatment, rendering recurrent GBM intractable. Mechanistic study of resistance evolution has been limited by the scarcity of matched primary/recurrent experimental models. We developed “resistant GSC families”, a within-patient matched platform in which temozolomide (TMZ)- and ionizing radiation (IR)-selected GSCs are derived ex vivo from the same primary GBM alongside treatment-naive controls (CTRL-GSCs). This design enables pressure-specific analysis of mechanisms associated with treatment selection, separating chemotherapy- and radiotherapy-associated changes often confounded in primary-versus-recurrent comparisons. In the absence of O6-methylguanine-DNA-methyltransferase (MGMT), TMZ selection was associated with either mismatch repair-dependent stable resistance or drug tolerance. Conversely, IR selection was linked to adaptive DNA damage response and increased radioresistance. Across treatments, emergent GSCs accumulate chromosomal alterations, share transcriptional programs involving DNA replication and genome maintenance, with IR-GSCs showing stronger enrichment of DNA double-strand-break repair pathways, and display increased receptor tyrosine kinase activity. Resistant GSC families enable mechanistic studies and hypothesis-driven testing of strategies relevant to GBM treatment.