Unique gene patterns lead to distinct functional phenotypes and chemosensitivity profiles among subclones obtained from a single glioblastoma cell line

• • Single-gene knockdown of ITGA11 or ITGA6 in mixed U87 cells failed to alter phenotype or chemosensitivity, whereas combined knockdown significantly inhibited tumor growth and increased chemotherapy sensitivity, demonstrating that functional redundancy and subclonal masking necessitate multi-target intervention. • • CF5 and G11 subclones exhibited opposite phenotypes: CF5 showed stronger proliferation and chemoresistance, while G11 displayed greater motility and invasion, indicating that a single GBM cell line harbors subpopulations with divergent drug responses and metastatic potential. • • Transcriptomic profiling revealed that downregulated genes in individual clones were significantly enriched in extracellular matrix (ECM)-related gene sets, implicating ECM remodeling as a key axis of subclonal functional divergence. • • The study employed a subcutaneous tumor model for therapeutic evaluation, which, despite not fully mirroring the brain microenvironment, enabled straightforward monitoring of tumor dimensions and growth dynamics, facilitating immediate assessment of treatment efficacy.