Wissensbasierte Cytometry and imaging of tumour spheroids

Objective: In the past, the majority of antitumour compound screening approaches had been performed in two-dimensional cell cultures. Although easy to establish and standardize, this culture method provides results of limited significance, as cells are surrounded by an artificial microenvironment, are not exposed to oxygenation gradients, and lack cell-cell contacts. Such conditions directly affect the resistance to various anticancer drugs. Three-dimensional (3D) cell cultures maintain relevant biochemical and morphological properties of cancer cells and therefore more closely resemble the in vivo situation in avascularized tumour nodules. We aimed at the realization of an anticancer drug evaluation concept which employs imaging of reporter gene activity in transfected tumor cells growing in three dimensions.

Material and methods: We established protocols for the microwell plate based generation of homogeneously sized spheroids from different cancer cell lines. One of these was transfected with constructs encoding for Grx1-roGFP2, a green fluorescent protein- (GFP-) based glutathione-specific redox sensor which allows to detect alterations in the glutathione redox potential by ratiometric analysis. Subcellular localization of the sensor was verified by fluorescence microscopy, its functionality was analysed subsequent to oxidation/reduction by flow cytometry and microplate-based kinetic measurements. Ratio-imaging for the detection of redox sensor oxidation in 3D cell cultures was performed using a microscopic setup based on structured illumination and the application of non-phototoxic light doses.

Results: We established stable transfectants of U251MG glioblastoma cells homogeneously expressing Grx1-roGFP2 in the cytoplasm and mitochondria, respectively. The redox sensor was responsive to hydrogen peroxide and dithiothreitol when cells were grown in 2D cell culture. In spheroids, hydrogen peroxide treatment induced a rapid oxidation of the outermost 20–30µm, whereas inner regions remained unaffected.

Conclusion: This study provides a proof of concept for imaging of alterations in the glutathione redox potential in 3D cell cultures, which could be indicative for anticancer drug mediated effects.

Acknowledgement: This study was supported by grants from the Government of Baden-Württemberg and the European Union – Europäischer Fonds für die regionale Entwicklung. The authors thank Eva Winkler, Petra Kruse, Andrea Böhmler, and Claudia Hintze for expert technical assistance.