Multicellular Tumor Spheroids Combined with Mass Spectrometric Histone Analysis To Evaluate Epigenetic Drugs

Multicellular tumor
spheroids (MCTS) are valuable in vitro tumor models
frequently used to evaluate the penetration and efficacy
of therapeutics. In this study, we evaluated potential differences
in epigenetic markers, i.e., histone post-translational modifications
(PTMs), in the layers of the HCT116 colon carcinoma MCTS. Cells were
grown in agarose-coated 96 well plates, forming reproducible 1-mm-diameter
MCTS. The MCTS were fractionated into three radially concentric portions,
generating samples containing cells from the core, the mid and the
external layers. Using mass spectrometry (MS)-based proteomics and
EpiProfile, we quantified hundreds of histone peptides in different
modified forms; by combining the results of all experiments, we quantified
the abundance of 258 differently modified peptides, finding significant
differences in their relative abundance across layers. Among these
differences, we detected higher amounts of the repressive mark H3K27me3
in the external layers, compared to the core. We then evaluated the
epigenetic response of MCTS following UNC1999 treatment, a drug targeting
the enzymes that catalyze H3K27me3, namely, the polycomb repressive
complex 2 (PRC2) subunits enhancer of zeste 1 (EZH1) and enhancer
of zeste 2 (EZH2). UNC1999 treatment resulted in significant differences
in MCTS diameter under drug treatment of varying duration. Using matrix-assisted
laser desorption/ionization (MALDI) imaging, we determined that the
drug penetrates the entire MCTS. Proteomic analysis revealed a decrease
in abundance of H3K27me3, compared to the untreated sample, as expected.
Interestingly, we observed a comparable growth curve for MCTS under
constant drug treatment over 13 days with those treated for only 4
days at the beginning of their growth. We thus demonstrate that MS-based
proteomics can define significant differences in histone PTM patterns
in submillimetric layers of three-dimensional (3D) cultures. Moreover,
we show that our model is suitable for monitoring drug localization
and regulation of histone PTMs after drug treatment.