Dissecting inherent intratumor heterogeneity in patient-derived glioblastoma culture models

Abstract

Background. Molecular profle of glioblastoma multiforme (GBM) revealed 4 subtypes, 2 of which, proneural and mesenchymal, have been predominantly observed, with the latter displaying a more aggressive phenotype and increased therapeutic resistance. Single-cell RNA sequencing revealed that multiple subtypes actually reside within the same tumor, suggesting cellular heterogeneity in GBM. Further, plasticity between these 2 subtypes is observed during tumor recurrence and in response to radiation therapy. Methods. Patient-derived GBM stemlike cells were cultured as neurospheres. These cells were differentiated in serum by attaching to the culture dishes. The "?oating" cells that were not attached/differentiated were harvested from the conditioned medium. The characteristics of these cells were studied with limiting dilution assays and immuno?uorescence staining. Cell growth and nuclear factor-kappaB (NFkB) activation were monitored using bioluminescent assays as well as quantitative polymerase chain reaction and western blotting. In vivo tumorigenesis was evaluated in orthotopic xenograft models using bioluminescence imaging. Results. Patient-derived GBM stemlike cells undergo differentiation by attaching to the culture dish in serumcontaining medium. We observed that a small subset of these cells escape this adhesion/differentiation and grow as?oating cells. These cells displayed enhanced cancer stem cell properties with a molecular and phenotypic mesenchymal signature, including resistance to radiation and targeted therapies, a more aggressive tumor formation, and NFkB activation. Conclusion. Our results endorse inherent intratumor molecular subtype heterogeneity in glioblastoma and provide a valuable approach to study phenotypic plasticity, which could be applied to fnd novel therapeutic strategies to eradicate this aggressive tumor and can be extended to other cancer types.