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Background Chimeric Antigen Receptor (CAR) T cell therapies have demonstrated remarkable clinical success in hematologic malignacies, yet their efficacy in solid tumors is influenced by cellular heterogeneity of the immune and stromal cells comprising the tumor microenvironment (TME). Advanced spatial profiling technologies of the TME are essential to dissect these complexities. The MACSima™ Imaging Platform enables high-content, multiplexed analysis of tissue sections, offering a powerful approach to characterize CAR T cell products and their interactions within the TME.Methods In this study we used an automated, closed, and good manufacturing practice (GMP)-compliant process employing the CliniMACS Prodigy® Platform to generate CAR T cells. We employed the MACSima™ Spatial Multiplexing platform for in depth characterization of CAR T cell products both in vitro and in vivo. In vitro, CAR T cells were profiled using iterative immunofluorescence staining credentialing the ability to assess activation, exhaustion, and T cell phenotype markers. In vivo, CAR T cells were administered in a xenograft mouse model. Tissue sections from tumor-bearing mice were analyzed using RNA probes specific to the CAR transgene to detect and localize CAR T cells. Concurrently, an extensive panel of antibodies was used to spatially characterize the TME, identifying immune infiltrates, stromal components, and checkpoint molecule expression.Results In vitro analysis revealed heterogeneous CAR T cell populations, with distinct subsets expressing activation markers, exhaustion markers, and memory-associated markers. In xenograft tissues, CAR T cells were successfully detected using RNA probes in situ, demonstrating their infiltration into tumor sites. Tumor cells characterization and TME profiling highlighted diverse immune cell populations and stromal architecture. In addition, immune cells were analyzed for checkpoint molecule as well as cytokine expression providing insights into potential mechanisms of resistance and therapeutic response.Conclusions The MACSima™ platform enables comprehensive spatial and phenotypic characterization of CAR T cell products and their in vivo behavior. Integration of RNA-based CAR detection with multiplexed antibody staining offers a powerful approach to study CAR T cell localization and function within the TME. These insights are critical for optimizing CAR T cell design and improving therapeutic outcomes.Ethics Approval Has been approved by the ethics review committee at Miltenyi Biotec, Bergisch Gladbach, Germany for studies involving non-human primates.Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal