A Thermo-responsive collapse system for controlling heterogeneous cell localization, ratio and interaction for three-dimensional solid tumor modeling.

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Cancer immunotherapy using engineered cytotoxic effector cells has demonstrated significant potential. The limited spatial complexity of existing in vitro models, however, poses a challenge to mechanistic studies attempting to approve existing approaches of effector cell-mediated cytotoxicity within a three-dimensional, solid tumor-like environment. To gain additional experimental control, we developed an approach for constructing three-dimensional (3D) culture models using smart polymers that form temperature responsive hydrogels. By embedding cells in these hydrogels, we constructed 3D models to organize multiple cell populations at specified ratios on-demand and gently position them by exploiting the hydrogel phase transition. These systems were amenable to imaging at low- and high-resolution to evaluate cell-to-cell interactions, as well as to dissociation to allow for single cell analyses. We have called this approach "thermal collapse of strata" (TheCOS) and demonstrated its use in creating complex cell assemblies on demand in both layers and spheroids. As an application, we utilized TheCOS to evaluate the impact of directionality of degranulation of natural killer (NK) cell lytic granules. Blocking lytic granule convergence and polarization by inhibiting dynein has been shown to induce bystander killing in single cell suspensions. Using TheCOS we showed that lytic granule dispersion induced by dynein inhibition can be sustained in 3D and results in a multi-directional killing including that of non-triggering bystander cells. By imaging TheCOS experiments, we were able to map a "kill zone" associated with multi-directional degranulation in simulated solid tumor environments. TheCOS should allow for the testing of approaches to alter the mechanics of cytotoxicity as well as to generate a wide-array of human tumor microenvironments to assist in the acceleration of tumor immunotherapy.