biostudies-literatureEichholz KNational institute of healthNIAID NIH HHSNCI NIH HHSGilead Sciences12921https://www.ebi.ac.uk/biostudies/studies/S-EPMC8217486biostudies-literatureUnknown11(1)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. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. 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 of genetically engineered cells in situ wiScientific reportsA CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue.PMC8217486R01 AI143773P30 CA015704HIV Cure Grant ProgramUM1 AI126623UM1 A126623Li AZZhu JJensen MCCorey LDiem KEichholz KfalseA CAR RNA FISH assay to study functional and spatial heterogeneity of chimeric antigen receptor T cells in tissue.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. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. 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 of genetically engineered cells in situ wi2021-01-01T00:00:00Z2021 Jun2026-05-08T18:57:48.133Z2022-02-11T11:00:20.324ZS-EPMC82174863415523510.1038/s41598-021-92196-x