Reversible Intracellular Gelation Reduces MCF10A Spheroid Growth

In nature, some organisms can survive in extreme environments by inducing a biostatic state within the organism’s molecular structure. Synthetic biostatic states in adherent mammalian cells have been previously achieved via intracellular network formation. This is attained by using bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) reactions between functionalized poly(ethylene glycol) (PEG) macromers introduced to the cell by a lipofectamine aided transfection. These macromers are able to spontaneously crosslink within the cytosol. In this work, the effects of intracellular network formation in a complex 3D epithelial MCF10A spheroid model are explored. Transfected cells are encapsulated in a proteinous 3D matrix, Matrigel, and overall spheroid area is reduced by ~50% compared to controls. Interestingly, intracellular network formation also induced a change in cell cycle state. Network formation results in a higher quiescent cell population indicated by the loss of phospho-Rb and a gain in p21 expression within single cell analysis of spheroids. After lipofectamine-aided transfection with SPAAC macromers, the formed network reduces overall bioenergetic (ATP/ADP) levels and functional metabolic rates, while also inducing quiescence -like effect in the mitochondrial electron transport chain. These effects are confirmed by Fluorescence Lifetime Imaging Microscopy (FLIM) and Seahorse Cell Metabolic Analysis. To enable reversibility of the observed biostasis effect within the model, a photodegradable nitrobenzyl moiety is incorporated into an azide containing macromer. This allows the PEG network to experience photoinduced degradation. The degraded network allows for continued proliferation and a return to normal spheroid growth. Following light exposure at day 12, growth and metabolic rates return to control levels, while SPAAC treated spheroids that did not receive light exposure (i.e., spheroids containing intact intracellular networks) remain smaller and less metabolically active through this same period. These results demonstrate that photodegradable intracellular polymer networks in 3D spheroid culture is a novel method that can control metabolic states and induce a reversible quiescent state.