Abstract
Background: Tumor organoids recapitulate the three-dimensional architecture, cellular composition, and immune microenvironment of the originating tumor, offering a reliable in vitro platform for immuno-oncology research. However, understanding the spatial organization of tumor–immune interactions within these models requires technologies that preserve tissue context. Objectives: This narrative review critically assesses how spatially resolved transcriptomics (SRT) is integrated into co-cultures of immunocompetent tumor organoids to reveal the spatial organization of the tumor immune microenvironment. It further evaluates the principal SRT platforms currently applied to organoid research and their suitability for 3D models. Methods: PubMed and Scopus were searched for English-language articles published primarily between 2020 and 2026, using combinations of keywords related to spatial transcriptomics, tumor organoids, immune microenvironment, and immunotherapy. Reference lists of identified reviews were also screened. Results: The review examines array-based methods (Visium, Slide-seqV2, Stereo-seq) and imaging-based approaches (MERFISH, Xenium, CosMx), comparing their resolution, gene coverage, and compatibility with 3D organoid culture. Spatial profiling has revealed recurring organizational features, including distinct immune niches, chemokine barriers that exclude lymphocytes, hypoxia-polarized myeloid populations, and tertiary lymphoid structure (TLS)-like aggregates within organoid structures. Translational implications include the recreation of clinical tumor immune phenotypes, discovery of predictive biomarkers of immunotherapy response, and characterization of resistance determinants in patient-derived organoid models. Conclusion: SRT has matured from a proof-of-concept tool into an integrated discovery framework for tumor–immune interactions. Closing the translational gap will require live-cell spatial imaging, multi-omic integration, and prospective clinical cohort studies that link organoid profiles to patient outcomes.