| Autor: |
Debruyne AC; Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, 9000 Ghent, Belgium., Okkelman IA; Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, 9000 Ghent, Belgium.; Ghent Light Microscopy Core, Ghent University, 9000 Ghent, Belgium., Heymans N; Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, 9000 Ghent, Belgium., Pinheiro C; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, 9000 Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium., Hendrix A; Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, 9000 Ghent, Belgium.; Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium., Nobis M; Intravital Imaging Expertise Center, VIB Center for Cancer Biology, KU Leuven, 3000 Leuven, Belgium., Borisov SM; Institute of Analytical Chemistry and Food Chemistry, Graz University of Technology, Stremayrgasse 9, Graz 8010, Austria., Dmitriev RI; Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, 9000 Ghent, Belgium.; Ghent Light Microscopy Core, Ghent University, 9000 Ghent, Belgium. |
| Abstrakt: |
Assessment of hypoxia, nutrients, metabolite gradients, and other hallmarks of the tumor microenvironment within 3D multicellular spheroid and organoid models represents a challenging analytical task. Here, we report red/near-infrared (NIR) emitting cell staining with O 2 -sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed "MMIR" (multimodal infrared), incorporate an NIR O 2 -sensitive metalloporphyrin (PtTPTBPF) and deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing for oxygen gradient quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated the nanosensor probe characteristics and cytotoxicity. Subsequently, we applied nanosensors to the live spheroid models based on HCT116, DPSCs, and SKOV3 cells, at rest, and treated with drugs affecting cell respiration. We found that the growth medium viscosity, spheroid size, and formation method influenced spheroid oxygenation. Some spheroids produced from HCT116 and dental pulp stem cells exhibited "inverted" oxygenation gradients, with higher core oxygen levels than the periphery. This contrasted with the frequently encountered "normal" gradient of hypoxia toward the core caused by diffusion. Further microscopy analysis of spheroids with an "inverted" gradient demonstrated metabolic stratification of cells within spheroids: thus, autofluorescence FLIM of NAD(P)H indicated the formation of a glycolytic core and localization of OxPhos-active cells at the periphery. Collectively, we demonstrate a strong potential of NIR-emitting ratiometric nanosensors for advanced microscopy studies targeting live and quantitative real-time monitoring of cell metabolism and hypoxia in complex 3D tissue models. |
