Comparison of the biological characteristics of glioblastoma tumorspheres obtained from fresh and cryopreserved glioblastoma tissues

Abstract

Purpose: Glioblastoma (GBM) is the most common and aggressive primary brain tumor. Human GBM tumorspheres (TS) are essential for preclinical drug screening and establishing patient-derived xenograft (PDX) models, but their derivation is often limited to fresh tissue. Whether TS from cryopreserved tissues retain comparable molecular and biological properties to those from fresh tissues remains underexplored. We hypothesized that TS from cryopreserved tissues could provide a reliable alternative for TS derivation, thereby expanding accessibility for GBM research. Methods: TS isolation rates were compared across 39 primary GBM samples. Tumor tissues collected during surgical resection were divided into two groups: one processed immediately as fresh tissue, and the other cryopreserved for 1 month before processing. Gene expression profiling via RNA sequencing and biological comparisons, including cell proliferation, neuroglial differentiation, stemness, invasiveness, and responsiveness to radiation and temozolomide, were performed on three matched TS samples from each group. Tumorigenesis was also assessed using PDX models. Results: TS were successfully isolated from 64.1% of fresh and 58.9% of cryopreserved tissues. Gene expression profiling revealed similar expression patterns in TS derived from both tissue types, despite variations in cancer subtypes. Cell proliferation, neuroglial differentiation, stemness, or invasiveness rates did not differ significantly between TS derived from fresh and cryopreserved tissues. All three GBM TS exhibited comparable responsiveness to temozolomide and radiation, as well as similar tumorigenic potential in the PDX models. Conclusion: These findings suggest an alternative method for isolating TS when immediate processing is not feasible, offering a time-independent approach for GBM research.