Cell Shape and Treatment Duration: How They Influence a Cancer Cell's Response to TNF

Abstract

The purpose of my research was to investigate the sources of heterogeneity in cellular decisions that are based on both external and internal cues. I used the signaling network induced by tumor necrosis factor (TNF) as a model system to examine how the duration of a stimulus and cell shape may affects signaling and cellular decisions. First, using a microfluidic device to achieve fine control of the ligand delivery to cells, my colleagues and I found that the duration of TNF stimulation is a factor that coordinates cell fate decisions in single cells. Specifically, we found that a few seconds of exposure to TNF is sufficient to activate the NF-κB pathway and induce apoptotic cell death and that, strikingly, a 1-min pulse of TNF can be more effective at killing cells than a 1-hour pulse. Second, to study the effects of cell shape, I used a two-pronged approach. Initially, I used live-cell imaging and single-molecule fluorescence in situ hybridization (smFISH) to examine whether descriptors of cell shape correlate with NF-κB nuclear translocation and NF-κB-dependent transcription in unperturbed populations of single cells. Next, I used surface micro-patterning to impose different geometry and degrees of spreading on cells and examine NF-κB-dependent transcription in these cells. I found that descriptors that quantify cell spreading, such as cell area and nuclear area, correlate with NF-κB nuclear translocation and NF-κB-dependent transcription. In addition, imposing bigger amount of spreading on cells increased the transcript abundance for two NF-κB-dependent genes, A20 and IκBα. In contrast, the relationships between geometry-related cell shape descriptors and NF- κB-dependent transcription are more subtle and complex. Importantly, despite observing the correlations between cell spreading and NF-κB activity, I found that the effects of cell shape on NF-κB dynamics and on NF-κB-dependent transcription were small. Together, my investigations of TNF-induced signaling have shown that while the duration of treatment encodes information used in the TNF-induced cell death decision, NF-κB dynamics and NF-κB-dependent transcription are quite robust to changes in cell shape.