Introduction: While bevacizumab initially shows therapeutic response in glioblastoma (GBM), it quickly leads to a resistant and invasive phenotype. Here we describe stem cell enrichment in GBM in a multigenerational GBM xenograft model of anti-angiogenic resistance.
Methods: To study transcriptional changes, we performed a microarray gene expression analysis on multigenerational isogeneic xenograft models of bevacizumab-resistance(BevR) versus bevacizumab-sensitivity(BevS). Whole human genome microarray was performed on tumors from generations 1, 4 and 9, with gene expression bioinformatics analysis (Matlab2014a). Two-component normal mixture model identified genes exhibiting significant inter-generational variance between resistant versus responsive xenografts. To confirm increased stemness in bevacizumab-resistant tumors, total stem cell counts from patient tumor-derived neurospheres were performed. Neurosphere reformation assays were performed in multiple generations of xenograft-derived bevacizumab-resistant and responsive cells from which neurosphere diameter, neurosphere counts and absolute stem cell counts were analyzed.
Results: Gene set enrichment revealed significant overexpression across generations of putative tumor-stemness markers GLUT3, CD44, NT5E, SNAI2, ZEB1 and ZEB2 over nine generations of BevS versus BevR xenografts.
Transcriptional validation of BevS and BevR tumors and cells revealed GLUT3 and ZEB1 as the most significantly overexpressed across generations(p<0.001). Total stem cell counts from neurospheres revealed a higher count from bevacizumab-resistant GBMs (n=4) compared to bevacizumab-naïve recurrent GBMs (n=3; p<0.001), with a similar effect in cell counts from neurospheres derived from BevS and BevR cells (p<0.001). Neurosphere formation assay revealed larger diameters of BevR generation 9 neurospheres (p=0.002), while BevS cells yielded more neurospheres (p=0.03), suggesting higher proliferative capacity and hypoxic resistance of BevR glioma neurosphere.
Conclusions: These results suggest tumor progression under anti-angiogenic treatment is accompanied by a shift in genetic and phenotypic expression towards a more aggressive stem-cell enriched phenotype. Strategies perturbing the evolution of stemness characteristics should be developed and further validated to improve the durability of therapeutic response to anti-angiogenic therapies.
Patient Care: The work detailed in the abstract describes a genetic and phenotypic shift of GBM towards a stem cell enriched subtype with evolution of bevacizumab resistance. Glioblastoma is a disease with dismal outcomes and limited recent advancement in therapeutic approach. Our understanding of stem cell enrichment of bevacizumab resistant GBM opens a door for new therapeutics that could target evolution of GBM towards the stem cell enriched subtype and unleash the full potential of anti-angiogenic therapies.
Learning Objectives: By the end of this session, participants should be able to (1)Understand shift in gene expression of tumor stemness markers with evolution of bevacizumab resistance in GBM, (2) Describe cellular phenomenon validating stem cell enrichment in GBM resistance to bevacizumab (3) Discuss ZEB1 and GLUT3 as potential molecular targets to perturb GBM transition to the stem cell enriched subtype.