Spatial Stable Isotopic Labeling by Amino Acids in Cell Culture: Pulse-Chase Labeling of Three-Dimensional Multicellular Spheroids for Global Proteome Analysis

Three-dimensional cell cultures, or spheroids, are important model systems for cancer research because they recapitulate chemical and phenotypic aspects of in vivo tumors. Spheroids develop radially symmetric chemical gradients, resulting in distinct cellular populations. Stable isotopic labeling by amino acids in cell culture (SILAC) is a well-established approach to quantify protein expression and has previously been used in a pulse-chase format to evaluate temporal changes. In this article, we demonstrate that distinct isotopic signatures can be introduced into discrete spatial cellular populations, effectively tracking proteins to original locations in the spheroid, using a platform that we refer to as spatial SILAC. Spheroid populations were grown with light, medium, and heavy isotopic media, and the concentric shells of cells were harvested by serial trypsinization. Proteins were quantitatively analyzed by ultraperformance liquid chromatography-tandem mass spectrometry. The isotopic signatures correlated with the spatial location and the isotope position do not significantly impact the proteome of each individual layer. Spatial SILAC can be used to examine the proteomic changes in the different layers of the spheroid and to identify protein biomarkers throughout the structure. We show that SILAC labels can be discretely pulsed to discrete positions, without altering the spheroid's proteome, promising future combined pharmacodynamic and pharmacokinetic studies.