Live microscopy of multicellular spheroids with the multi-modal near-infrared nanoparticles reveals differences in oxygenation gradients

Abstract

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 emitting cell staining O2-sensitive nanoparticles, which enable measurements of spheroid oxygenation on a conventional fluorescence microscope. Nanosensor probes, termed MMIR (multi-modal infrared), incorporate a near-infrared O2-sensitive metalloporphyrin (PtTPTBPF) and a deep red aza-BODIPY reference dyes within a biocompatible polymer shell, allowing oxygen gradients quantification via fluorescence ratio and phosphorescence lifetime readouts. We optimized staining techniques and evaluated 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, spheroids size, and formation method influenced spheroid oxygenation. Unexpectedly, 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 towards 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 revealed the formation of 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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=38 SRC="FIGDIR/small/571110v2_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@49cef8org.highwire.dtl.DTLVardef@1b31072org.highwire.dtl.DTLVardef@fcaf4aorg.highwire.dtl.DTLVardef@1583b5f_HPS_FORMAT_FIGEXP M_FIG C_FIG