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Purpose: Current lung cancer organoid models often fail to replicate the complex tumor immune microenvironment, reducing their predictive value for immunotherapy and radiotherapy. Therefore, it is crucial to establish an optimized lung cancer organoid model which could recapitulate the tumor immune microenvironment, enabling more accurate evaluation of therapeutic responses. Methods: We developed an optimized air-liquid interface (ALI) culture method to generate patient-derived lung cancer organoids (ALI-LUOs) from 19 lung cancer samples. The tumor microenvironment, including immune and stromal components, was characterized using immunofluorescence, flow cytometry, and single-cell RNA sequencing. The organoids were further used to assess responses to ¦ÁPD-1 therapy and radiotherapy. Results: The optimized method significantly improved organoid formation efficiency while preserving immune cell viability for up to 30?days. Immune and fibroblast populations were confirmed by immunofluorescence and flow cytometry. Single-cell RNA sequencing demonstrated that ALI-LUOs accurately replicate the tumor immune landscape. Key tumor immunity pathways such as cGAS-STING could be captured by ALI-LUOs. Importantly, ALI-LUOs modeled clinical responses to immune checkpoint inhibitors and radiotherapy with high fidelity. Conclusions: The ALI-LUOs, developed through an optimized culture method, faithfully capture the key characteristics of lung cancer, including its immunosuppressive tumor microenvironment. Our findings highlight this modified ALI-LUOs as a valuable preclinical platform for evaluating antitumor immunity and refining lung cancer treatments.

AbstractINTRODUCTIONRecent studies have identified important species©\dependent differences in the response of microglia to ¦Â©\amyloid (A¦Â) pathology. Yet, whether human microglia also interact differently with the pathognomonic combination of amyloid and tau pathologies that occur in Alzheimer's disease (AD) remains unclear.METHODSWe generated a xenotolerant mouse model of AD that develops both plaque and tangle pathologies, transplanted stem cell©\derived microglial progenitors and examined the interactions between human microglia and AD pathologies with scRNA sequencing, immunohistochemistry, and in vitro modeling.RESULTSThe combined amyloid and tau pathologies induced robust type©\I interferon and proinflammatory cytokine responses, as well as an increased adoption of a distinct ¡°rod¡± morphology in human microglia. The rod morphology could be induced with type©\I interferon treatment in vitro.DISCUSSIONWe provide new insights into human microglial responses to combined AD pathologies and a novel platform to investigate and manipulate human microglia in vivo.Highlights Amyloid pathology promotes the rapid development of neurofibrillary tangles and neuronal loss in a novel chimeric model of AD.Combined Alzheimer's disease pathologies lead to an expansion of disease©\associated microglia (DAM) and exacerbate Interferon©\responsive and cytokine/chemokine©\enriched states in xenotransplanted human microglia.The combination of amyloid and tau promotes the development of a distinctive rod microglial phenotype that closely correlates with tau pathology and neurodegeneration.Rod morphology and transcriptional changes can be modeled in vitro by treatment of induced pluripotent stem cells (iPSC) ©\microglia with type©\I interferons.

Various inhibitors targeting T-cell immunoglobulin and mucin-containing molecule 3 (TIM3) aimed at reversing T-cell exhaustion for better immunotherapy outcomes have demonstrated limited clinical efficacy as monotherapy, with the underlying mechanisms remaining ambiguous. TIM3 is markedly expressed in dendritic cells (DCs), and the inconsistent research findings on its role in myeloid cells underscore its vital function within DCs. Through the establishment of an in vitro differentiation model generating mature dendritic cells (mDCs) under TIM3-targeted interventions, combined with an RNA sequencing analysis, this investigation systematically examined TIM3-mediated regulation and ligand interactions in human primary DCs. The findings indicate that TIM3 inhibition hinders DC maturation, which subsequently diminishes the antigen-presenting capacity of DCs, ultimately leading to immune suppression in T cells. These findings collectively establish TIM3 as a regulator of DC differentiation that promotes DC maturation while optimizing the antigen-processing and presentation capacity. This study elucidates the rationale behind the suboptimal efficacy of TIM3 inhibitors and advocates for retaining TIM3 signaling pathways in DCs.