Exploring the biomechanical complexity of glioblastoma spheroids and organoids with co-localized Brillouin and Raman microspectroscopy.

Brillouin microscopy allows mechanical investigations of biological materials at the subcellular level and can be integrated with Raman spectroscopy for simultaneous chemical mapping, thus enabling a more comprehensive interpretation of biomechanics. The present study investigates different in vitro glioblastoma models using a combination of Brillouin and Raman microspectroscopy. Spheroids of the U87-MG cell line and two patient-derived cell lines as well as patient-derived organoids were used. Brillouin microscopy provided maps of viscoelastic parameters, while Raman spectroscopy identified key biochemical components such as proteins, lipids, glycogen and cholesterol. Cluster analysis of the Raman spectra allowed the categorization of biochemical groups and the correlation of their Brillouin shift and bandwidth across the different glioblastoma models. The results showed that spheroids from the same cell line exhibited relatively homogeneous biomechanical properties, while differences existed between different cell lines. In contrast, organoids from the same patient exhibited greater mechanical and biochemical heterogeneity. Brillouin shift and bandwidth showed significant variation among Raman clusters, highlighting the need to consider biochemical composition in biomechanical assessments. The cytoplasmic protein cluster was biochemically and biomechanically consistent across models, while lipid- and glycogen-related clusters varied. The approach used in this study facilitates the interpretation of Brillouin data in heterogeneous biological systems and allows comparisons between different models. The results emphasize the need for multimodal analysis for correct interpretation of biomechanical measurements in complex tissues and for comparison between heterogeneous samples.