Single cell and spatial profiling reveal molecular and immune evolution from precancers to invasive lung adenocarcinomas in genetic and carcinogen-induced mouse models

Abstract Introduction: An improved understanding of early lung carcinogenesis may facilitate development of novel diagnostic, screening, and prevention strategies to decrease lung cancer mortality. We have previously led a series of studies on the molecular and immune landscape of lung adenocarcinoma (LUAD) precursors. However, resected human specimens only provide molecular snapshots. Here, we sought to establish and characterize human-relevant murine lung precancer models to depict molecular evolution during early lung carcinogenesis and to provide novel insights for lung cancer interception. Methods: We have established 3 genetically engineered mouse models (GEMMs) (K: KrasLSL-G12D/+, KP: KrasLSL-G12D/+/Trp53R172H/+, KL: KrasLSL-G12D/+/Lkb1fl/fl) and 2 carcinogen-induced models (CITMs) (UWT: Urethane in wild type mice, URG: Urethane in Rag2−/− mice). Lung tissues were serially collected at multiple time points after induction and subjected to whole exome sequencing (WES), single cell RNA sequencing (scRNA-seq), spatial transcriptomics (Visium), and imaging mass cytometry (IMC). Results: Pathological review of specimens from 252 mice revealed normal lung, precancers and LUAD at different timepoints mirroring the evolution of human LUAD. Overall, the tumor burden was significantly higher in GEMMs than CITMs (p<0.05). Tumor mutation burden progressively increased with progression from precancers to LUADs across all models. scRNA-seq demonstrated a progressive decrease of CD4+ T cell, CD8+ T cell, NK cell, and B cell infiltration in all models except the B/T cell-deficient WRG model; and a progressive increase of neutrophils (in KL model) and macrophages (in K, KP, UWT and URG models) along with progression of precancers. These findings were confirmed by spatial Visium and IMC profiling. Infiltration of T cells, B cells and NK cells inside tumors was not different between GEMMs (K, KP, KL) and CITMs (UWT). However, UWT showed significantly higher density of immune cells at the peritumor regions (P<0.05). Compared to the immune-competent UWT model, the B/T-cell deficient URG model showed similar progression and tumor burden at early phases, but rapid progression and larger tumor burden in the later phases. URG had no mature B/T cells but significantly higher NK cell infiltration than UWT (p<0.05). Conclusion: We have established 5 murine carcinogen-dependent and -independent precancer models, morphologically resembling human LUAD and its precursors. Although progressive immunosuppression along with progression of lung precancers is universal across all models, the evolution patterns and the molecular/immune features underlying immunosuppression vary in different models, particularly between CITMs and GEMMs. These models may be valuable assets for studying early lung carcinogenesis and lung cancer interception. Citation Format: Bo Zhu, Jian-Rong Li, Hong Chen, Pingjun Chen, Junya Fujimoto, Yanhua Tian, Muhammad Aminu, Chenyang Li, Lingzhi Hong, Alexandre Reuben, Edwin Roger Parra Cuentas, Ou Shi, Monique Nilsson, Alissa Poteete, Shawna Hubert, Khaja Khan, Wei Lu, Daniel Kraushaar, Xingzhi Song, Jianhua Zhang, Don Gibbons, Luisa Solis Soto, Ignacio Wistuba, Jia Wu, John Heymach, Chao Cheng, Jianjun Zhang. Single cell and spatial profiling reveal molecular and immune evolution from precancers to invasive lung adenocarcinomas in genetic and carcinogen-induced mouse models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6513.