Generation of Heterotypic Primary Human Spheroids from Glioblastoma Resections and (Pre-)Clinical Applications

The rapid expansion of individualized treatment strategies necessitates advanced patient-specific screening platforms recapitulating tumor complexity. Here, we present such a platform preserving heterotypic cellular interactions and three-dimensional architecture which are both critical for predicting therapeutic responses. This cost-effective and versatile platform allows the rapid generation and functional testing of spheroids derived from primary human glioblastoma specimens. To maximize accessibility and ease of integration, we took advantage of commercially available kits to optimize the protocol of spheroid generation we had previously established. This enabled the robust, adaptable, and reproducible formation of homogeneous, multicellular spheroids within a few days. Spheroids generated from one patient specimen were structurally stable and showed a high degree of homogeneity over time. Immunohistochemical and flow cytometric analyses further revealed patient-specific cellular heterogeneity, underscoring the platform's ability to preserve clinically relevant tumor features. Functionally, we demonstrate the applicability of this system for drug response profiling by assessing invasion dynamics following treatment with clinically relevant compounds. Collectively, our results establish a scalable and adaptable 3D screening platform that enables rapid, patient-specific phenotypic and functional analyses. This approach provides a powerful tool to complement existing clinical workflows and holds promise for improving the prediction of therapeutic responses in glioblastoma.