Critical to advancing immunotherapy and cell therapy in cancer is developing a deeper understanding pf the dynamics of immune cell-mediated cytotoxicity. The results from the multidisciplinary effort reported here include numerous measurements and movies of immune cell-mediated cytotoxicity with striking examples of serial killing, foraging, path-tracking, cytokine gradients at tumor margins, and killing dynamics, in some cases revealing peak apoptotic signatures just minutes after T Cell engagement.

In vitro studies of immune cell killing are traditionally performed using time-lapse imaging and biochemical assays, but these methods are often limited by spatial and temporal resolution, throughput, and the ability to extract the dynamics of cellular interactions. This study integrates high-resolution and high-speed laser scanning confocal microscopy with artificial intelligence (AI), and machine learning (ML) approaches to provide a high-resolution data-driven analysis of immune cell killing dynamics in vitro.

We have engineered a perfusion-enabled 3D culture system integrated microscopy to assess cellular dynamics for extended periods of time. Perfusion culture maintains the interstitial flow of liquid culture media, clearing the microenvironment of toxic metabolites and reactive oxygen species. This platform uses a Liquid-Like Solids (LLS) to mimic the transport dynamics of a capillary bed. Integrated microscopy allows in situ quantification of spatiotemporal cytokine concentrations, immune cell tracking, immune cell killing dynamics, and invasion dynamics.