Article
Correlative biomechanical and biochemical analysis of glioblastoma cell cultures using brillouin and raman spectroscopy
Korrelative biomechanische und biochemische Analyse von Glioblastomzellkulturen mittels Brillouin- und Ramanspektroskopie
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Published: | May 25, 2022 |
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Objective: Biomechanics of tumor cells influence proliferation, migration and metastasis of brain tumor cells. So far, the measurement of cell stiffness in tumor models has been limited to cell surface measurements due to the use of contact-based techniques such as AFM. Here, we overcome this limit by using optical Brillouin spectroscopy to perform subcellular investigation of cell compartments in adherent cells and spheroid cultures of glioblastoma. We correlated biomechanical with biochemical information obtained from simultaneous and co-localized Raman spectroscopy for interpreting stiffness changes.
Methods: U87-MG cells were cultured as adherent cells (n=11) and spheroids (n=9). Additionally, five primary glioblastoma cell lines were analyzed (each cell line n=10-12). Brillouin and Raman spectroscopy was performed in a combined setup using 780 nm excitation wavelength. The Brillouin shift, which provides information about the stiffness, was computed by fitting each of the 2500 single measurements per sample with Lorentzian functions. Raman spectra in the range 700-1800 cm-1 were baseline corrected, normalized and analyzed by k-means clustering.
Results: U87-MG adherent cells showed smaller Brillouin shifts and values displayed a higher variability compared to spheroids (5.319 ± 0.033 GHz vs. 5.408 ± 0.015 GHz, mean ± SD, n=11 and 9, unpaired t-test p<0.001). Cluster analysis of Raman spectra of adherent cells was used to identify cell compartments. Nucleus, perinuclear region and cytoplasm exhibited different mean Brillouin shifts (5.33 GHz, 5.29 GHz and 5.23 GHz, respectively), thus indicating that the stiffness of nucleus is highest. For spheroids, larger Brillouin shifts were retrieved in regions with high content of proteins (identified by Raman bands at 1006 and 1607 cm-1) as well as of lipids (Raman bands at 1440 and 1750 cm-1). Also in primary cell lines the Brillouin shift correlated with the amount of lipid droplets.
Conclusion: Adherent cells and cell spheroids have different biomechanical properties, which are driven by culturing methods and can be explained at subcellular level by different biochemical composition. Brillouin spectroscopy of cell spheroids delivers more reproducible results, but the Brillouin shift is highly sensitive to lipid content in the cells. Further analysis shall clarify by which mechanisms the lipids exactly induce the apparent stiffness increase and how this relates to tumor properties.