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Background Humanized immune system mice are increasingly used to evaluate cancer immunotherapies and avoid the need for mouse-specific drug surrogates. However, most preclinical studies using these models rely on subcutaneous tumor engraftment, a site that fails to replicate organ-specific interactions with stroma, infiltrating immune cells, and vasculature, and rarely supports metastasis. Orthotopic engraftment addresses these limitations by reproducing a more tissue-specific tumor microenvironment and enabling spontaneous distant metastasis, thereby offering a more physiologically relevant context for assessing therapeutic responses.Methods Here, we established a human non-small cell lung cancer model in CD34+ humanized NCG mice by intercostal injection of A549-GFP-Luc cells into the left lung lobe following a local skin incision.Results Within 6 weeks, 100% of engrafted mice exhibited macroscopic primary lung tumors (detectable by bioluminescence and histology) and 85% developed spontaneous liver metastases. Using histopathological analysis, we showed that primary and metastatic sites contained clear peritumoral collagen fibrogenesis and were infiltrated by immune cells, including human T (CD3+) and myeloid (CD68+) cells. Although total immune cell counts in the lungs and livers were not affected by tumor engraftment, T and NK subsets underwent phenotypic changes, including activation of CD4+ helper T cells (marked by increased CD25 expression) and NK cells (marked by increased CD16+ expression) in tumor-bearing versus non-engrafted lungs.Conclusions Our results suggest that lung orthotopic engraftment of human tumors in humanized mice leads to a responsive and microenvironment-specific immune response, thereby providing a reliable preclinical platform to evaluate immunotherapies and biologics.Ethics Approval This work does not involve human subject research. Animal experimentation procedures have been approved by the French Ministry of Higher Education and Research (DAP authorization 38383 – 2022082413416895) and the Departmental Directorate of Population Protection in Haute Savoie, France (DDPP authorization A 7418 324). TransCure bioServices is an an AAALAC-accredited institution.