Establishment and characterization of an orthotopic implanted lung cancer model to mimic human tumor structure, microenvironment, and metastatic spread.

[BACKGROUND] Subcutaneous (SC) lung tumor models are widely used in preclinical studies due to their technical simplicity but fail to recapitulate the complex microenvironment, immune landscape, and metastatic behavior of human lung cancers. These limitations hinder the translational value of such models, particularly in evaluating immunotherapies and metastasis-related mechanisms. There is a critical need for more physiologically relevant models that better reflect clinical tumor characteristics and disease progression. To address these limitations, we sought to develop a reproducible orthotopic lung cancer (LuO) model that enables detailed study of tumor progression, immune infiltration, and metastatic dynamics.

[METHODS] We established and characterized a thoracotomy-based LuO model using a panel of human and murine lung cancer cell lines implanted into the pulmonary parenchyma of immunodeficient and syngeneic mice. Tumor progression was monitored longitudinally using bioluminescence imaging (BLI) and micro-computed tomography (CT). Comparative analyses with SC tumors were performed using immunohistochemistry, multiplexed immunofluorescence, transcriptomic and proteomic analyses. Circulating tumor cells (CTCs) and spontaneous metastases were isolated and functionally characterized.

[RESULTS] The orthotopic model reliably generated solitary intrapulmonary tumors that closely mimic human lung cancer in growth pattern, vascularization, and progression. Compared to SC tumors, orthotopic tumors exhibited significantly enhanced vascular density, reduced hypoxia and DNA damage, and increased proliferation. Immune profiling revealed enriched and spatially organized infiltrates of CD4, CD8 T cells, dendritic cells (DCs), and myeloid populations in orthotopic tumors, forming structures analogous to those found in patient tumors. Moreover, orthotopic tumors released CTCs capable of forming spontaneous and site-specific metastases to clinically relevant organs. Transcriptomic and proteomic profiling of metastasis-derived cell lines uncovered conserved pro-metastatic signatures and niche-specific adaptations.

[CONCLUSIONS] This LuO model offers a reproducible, clinically relevant platform that captures important aspects of human lung cancer biology, including immune landscape, tumor microenvironment (TME), and metastatic progression. Its superior anatomical and immunological fidelity makes it a valuable preclinical tool for evaluating therapeutic strategies and dissecting molecular mechanisms of metastasis.