Enhanced in vivo and ex vivo analysis enables deeper characterization of humanized mouse models for immuno-oncology research

Background We previously demonstrated that humanized mouse models can be generated using peripheral blood mononuclear cells (PBMCs), PBMC subpopulations such as T and NK cells, or CD34⁺ hematopoietic stem cells (HSCs), depending on the research objective. As next-generation immunotherapies advance-including checkpoint inhibitors (CPIs), engineered immune cells, and immune-cell engagers-establishing suitable in vivo and ex vivo analytical platforms becomes essential. Fully humanized mouse models containing both a human immune system and human tumors provide a more physiologically relevant setting for evaluating these therapies. Methods Humanized mice were established through intravenous transplantation of CD34⁺ cells, PBMCs, or purified NK or T cells into immunodeficient mice. Cell-derived xenograft (CDX) and patient-derived xenograft (PDX) tumors were engrafted either subcutaneously (s.c.) or orthotopically (intravenously or into the mammary fat pad). Tumor progression was assessed using caliper measurements (s.c.) or bioluminescence imaging (BLI) for orthotopic models. Quantitative immune cell composition in blood, bone marrow, spleen, and tumor tissue was analyzed by flow cytometry. 3D-Light-sheet fluorescence microscopy with the Ultramicroscope Blaze was established to enable spatial analysis and localization of immune-cell infiltration in tumors and spleen. Results Both CDX and PDX tumors from multiple cancer types successfully engrafted in humanized mice, with >70% engraftment success and no significant differences in tumor growth kinetics compared to non-humanized controls. Immunotherapy treatment revealed distinct responder and non-responder profiles, characterized by differential immune-cell infiltration patterns. Flow cytometry reliably monitored human immune reconstitution and quantified immune and tumor cell populations over time. Flow cytometry has also been used to follow therapeutic treatment effects. BLI provided a non-invasive method for longitudinal tumor assessment in orthotopic models. 3D-Light-sheet imaging with the Ultramicroscope Blaze enables the confirmation of FACS-based findings and visualized tumor-infiltrating lymphocytes (TILs), unlocking spatial mapping of immune-cell localization within tumors and lymphoid organs. Conclusions Continuous refinement of our humanized mouse models enables robust preclinical evaluation of emerging immunotherapies. Integration of spatial biology enhances mechanistic insight by providing high-resolution visualization of immune-cell behavior within the tumor microenvironment, strengthening the translational value of these platforms.