Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer and HIV sanctuary sites. Here, we describe a CAR RNA FISH histocytometry platform combined with a dnnRRS image analysis algorithm to quantitate spatial distribution and in vivo functional ability of a CAR T cell population at a single cell resolution. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions engineered cells in situ with their target cells in tissues with high statistical power and thus, as an important tool for preclinical and potentially clinical assessment of CAR T cell effectiveness.
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