Comprehensive analysis of resistance signaling in trail-induced apoptosis with single-cell mass cytometry [electronic resource]
Keywords: Microbiology and Immunology.

Resistance to cancer therapies has been understood as a genetic process driven by mutations in therapeutic targets and associated regulators. Mutation-independent, "non-genetic" resistance has recently been implicated as an additional and significant hindrance to therapeutic efficacy. In order to develop therapies able to combat non-genetic resistance, it is necessary to understand what signaling states enable resistance. Non-genetic resistance is challenging to study since it occurs non-uniformly, even in cell lines, and can involve the interplay of multiple survival pathways on the functional level. Here, mass cytometry is used to dissect the signaling state of cells resistant to TRAIL-induced apoptosis with single-cell resolution. The first robust viability stain for mass cytometry was developed to separate mutation-independent resistant cells, "persisters, " from apoptotic cells. The subset of persisters enabling resistance was identified using mass cytometry. Interrogation of the signaling state of these persisters elucidated how resistance is maintained on the functional level and permitted rational design of combinatorial therapies able to halt non-genetic resistance. Chapter 1 details the current theoretical and empirical studies on non-genetic resistance in cancer. Theories on how non-genetic resistance is maintained are discussed, including the Waddington Landscape and Regulated Stochasticity. The ongoing debate on whether non-genetic resistance is selected or induced and if Darwinian or Lamarkian mechanisms are involved is described. The potential for mass cytometry to address questions in the field is presented. Chapter 2 describes the optimization of a simple and rapid viability staining protocol for mass cytometry, using cisplatin. Cisplatin staining offers a robust isotopic indicator of cellular viability, allowing identification of cells that were viable at the beginning of an experiment, even after formaldehyde fixation, methanol permeabilization, and intracellular antibody staining. This approach enabled the use of mass cytometry for measuring intracellular signaling network state in tandem with viability, and was crucial for discriminating persisters from apoptotic cells. In Chapter 3, the first study of non-genetic resistance to TRAIL-induced apoptosis using single-cell mass cytometry is presented. The progression of cells through apoptosis is described using 20 intracellular signaling markers covering apoptosis, survival signaling and associated regulatory changes to create a map of apoptotic progression that can identify cells primed for apoptosis within 30 min after TRAIL treatment. There is a group of cells, persisters, that does not enter the apoptotic cascade and can survive TRAIL treatment. The persisters have no genetic differences from cells that become apoptotic. The signaling state of the persisters is found to occupy a broad, but not entirely uniform, distribution of the basal signaling state. Targeting survival pathways constricts the distribution of persister signaling states; increased constriction correlates with decreased resistance. The effect of single inhibitors on signaling space is predictive of combinatorial effects, even for inhibitors that have complex effects on survival. The methods developed here will facilitate other high-dimensional single-cell studies of non-genetic resistance and these findings may have broad applicability for targeting persisters enabling resistance.