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Background: Immunotherapy has revolutionized clinical outcomes for patients suffering from lung cancer, yet relatively few patients sustain long-term durable responses. Recent studies have demonstrated that the tumor immune microenvironment fosters tumorous heterogeneity and mediates both disease progression and response to immune checkpoint inhibitors (ICI). As such, there is an unmet need to elucidate the spatially defined single-cell landscape of the lung cancer microenvironment to understand the mechanisms of disease progression and identify biomarkers of response to ICI., Methods: Here, in this study, we applied imaging mass cytometry to characterize the tumor and immunological landscape of immunotherapy response in non-small cell lung cancer by describing activated cell states, cellular interactions and neighborhoods associated with improved efficacy. We functionally validated our findings using preclinical mouse models of cancer treated with anti-programmed cell death protein-1 (PD-1) immune checkpoint blockade., Results: We resolved 114,524 single cells in 27 patients treated with ICI, enabling spatial resolution of immune lineages and activation states with distinct clinical outcomes. We demonstrated that CXCL13 expression is associated with ICI efficacy in patients, and that recombinant CXCL13 potentiates anti-PD-1 response in vivo in association with increased antigen experienced T cell subsets and reduced CCR2+ monocytes., Discussion: Our results provide a high-resolution molecular resource and illustrate the importance of major immune lineages as well as their functional substates in understanding the role of the tumor immune microenvironment in response to ICIs., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Immune checkpoint inhibitors (ICIs) demonstrate unprecedented efficacy in multiple malignancies; however, the mechanisms of sensitivity and resistance are poorly understood and predictive biomarkers are scarce. INSPIRE is a phase 2 basket study to evaluate the genomic and immune landscapes of peripheral blood and tumors following pembrolizumab treatment., Methods: Patients with incurable, locally advanced or metastatic solid tumors that have progressed on standard therapy, or for whom no standard therapy exists or standard therapy was not deemed appropriate, received 200 mg pembrolizumab intravenously every three weeks. Blood and tissue samples were collected at baseline, during treatment, and at progression. One core biopsy was used for immunohistochemistry and the remaining cores were pooled and divided for genomic and immune analyses. Univariable analysis of clinical, genomic, and immunophenotyping parameters was conducted to evaluate associations with treatment response in this exploratory analysis., Results: Eighty patients were enrolled from March 21, 2016 to June 1, 2017, and 129 tumor and 382 blood samples were collected. Immune biomarkers were significantly different between the blood and tissue. T cell PD-1 was blocked (≥98%) in the blood of all patients by the third week of treatment. In the tumor, 5/11 (45%) and 11/14 (79%) patients had T cell surface PD-1 occupance at weeks six and nine, respectively. The proportion of genome copy number alterations and abundance of intratumoral 4-1BB+ PD-1+ CD8 T cells at baseline (P < 0.05), and fold-expansion of intratumoral CD8 T cells from baseline to cycle 2-3 (P < 0.05) were associated with treatment response., Conclusion: This study provides technical feasibility data for correlative studies. Tissue biopsies provide distinct data from the blood and may predict response to pembrolizumab.