A general method for studying autocrine signaling and its impact on cancer cell growth

Autocrine signaling plays essential roles in providing self-sustaining growth signals to cancer cells. Since the introduction of the autocrine hypothesis in 1980s, the contribution of autocrine signaling in cancer medicine has been limited to cancer tissues with adequately characterized mitogenic pathways. Its closed-loop nature and complex interplay with other environmental cues prevents the experimental study of unknown autocrine loops, requiring specific perturbing agents to inhibit the underlying ligand/receptor interactions. Recent studies reported the ability of drug-resistant cancer cells to acquire mitogenic signals from previously neglected autocrine loops, causing tumor recurrence. Methods that can evaluate autocrine-loop dependency in more diverse cancer tissues will help identify other means that autocrine signaling employs to maintain cancer growth. This thesis presents the use of cell-patterning methods as a tool for modulating intrinsically generated diffusive signaling cues. Such technology enables the investigation of autocrine loops without the need for specific therapeutics or prior knowledge of underlying ligand/receptor pairs. To achieve this goal, the first aim of this thesis is to determine characteristics of autocrine signaling that pertain to modulation of intercellular spacing, using existing investigation methods. In addition to demonstrating the limitation of conventional methods in examining unknown autocrine loops, we showed that changes of intercellular spacing in randomly plated culture cannot specifically modulate autocrine activity, due to the concurrent changes of other environmental cues. The second aim of this thesis is to establish engineering tools for 1) ensuring modification of only autocrine loops with the modulated cell arrangement and 2) providing prediction of autocrine activity changes with varying intercellular spacing. We illustrated cell-patteming approaches for introducing spatial regularity to standard cell culture. We then developed a stochastic model to predict changes of ligand/receptor binding with varying cell arrangement designs. We determined the spatial requirement for autocrine activity to transition from the isolated to the communicative mode. The model also helps determine cell-patterning designs that can potentially maintain uniform impacts of non-diffusive signaling cues while enabling specific modulation of autocrine signaling. In the last aim of this thesis, we evaluated the ability of regularly-shaped cell arrays to demonstrate the impact of autocrine signaling in supporting cancer growth. In comparison to randomly-plated culture, the cellpatterning platform exhibited growth change with altering intercellular spacing that better corresponds with the predicted and measured changes of autocrine ligand capture. With increasing global cell density, we also showed that regularly-shaped cell arrays acquire more uniform distribution of local cell density, while the randomly-plated cells exhibit distinct changes of local cell density. We present in this thesis the first method for the modulation of combined autocrine activity while ensuring minimal concurrent alteration of non-diffusive cues without the need of specific perturbing agents. Thesis supervisor: Joel Voldman Title: Associate Professor of Electrical Engineering and Computer Science, MIT Thesis chair: William M. Deen Title: Professor of Chemical Engineering, MIT Thesis committee member: Peter K. Sorger Title: Professor of Systems Biology, HMS and Professor of Biological Engineering, MIT Thesis committee member: Douglas A. Lauffenburger Title: Professor of Biological Engineering, MIT