Abstract 1300: Single-cell and spatial transcriptomic profiling reveal the cascade of immune events during progression and metastasis of non-small cell lung cancer
Authors: Tian Yanhua , Li Jian-Rong , Zhu Bo , Fradette Jared , Chen Hong , Li Chenyang , Cui Sijia , Kraushaar Daniel , Fujimoto Junya , Rodriguez Leticia , Soto Luisa Maren Solis , Reuben Alexandre , Wistuba Ignacio , Gibbons Don , Chao Cheng , Zhang Jianjun 
Published: June 2023

Abstract
Introduction: Lung cancer is the leading cause of cancer-related deaths worldwide and metastasis accounts for 90% of cancer-related mortality. A better understanding of the cascade of molecular and immune events underlying evolution during tumor progression and metastasis may help develop new strategies to prevent and treat metastasis more effectively.
Methods: In this study, we utilized two mouse models of metastatic lung cancer: a genetically engineered mouse model (GEMM) with mutant Kras and Trp53 (KP, KrasLSL-G12D/+; Trp53fl/fl), which forms lung adenocarcinoma with high metastatic potential, and a syngeneic model using cell lines derived from the metastatic KP_GEMMs that faithfully develop lung metastases. Paired samples of the primary tumor, peripheral blood mononuclear cells, lung and subcutaneous tumors were serially collected at different time points (pre-metastatic, early-metastatic, and late-metastatic phases) and subjected to single-cell RNA sequencing (scRNA-seq), scTCR/BCR-seq, and spatial gene expression by using 10X Genomics (Visium).
Results: ScRNA analysis revealed large-scale comprehensive longitudinal changes in cell components throughout the invasion and metastasis cascade, which was validated by spatial gene expression. Furthermore, analysis of Visium data deduces the spatial information of these dynamic changes. We found that NK and B cells from both the lung tissues and PBMC mainly participated in the early-metastatic stage and vanished in the late-metastatic phase. We also identified a subset of tumor-associated Tregs (TA_Tregs) that existed in small amounts in the lung tissue in the pre-metastatic phase and greatly expanded in the late-metastatic phase. These cells were characterized by high expression of Treg-associated suppressive genes (Tnfrsf9, Tnfrsf4, Tnfrsf18, Traf1, Nfkbia), inhibitory molecules (Ikzf2, Il2ra, Tight, Pdcd1), cytokines and chemokines (Ccr5, Ccr8, Ccl5, Cxcr6) and other Treg effector molecules (Id2, Hif1a, Rora, Il1r2, Itgav, Stat1, Icos, Areg). The scTCR-seq results and trajectory analysis revealed the TA_Tregs might derive from minor clones of lung resident Tregs (LR_Tregs) and play an active role in remodeling an immunosuppressive microenvironment by closely interacting with other cell components in the TME.
Conclusion: By simultaneously characterizing cellular gene expression, immune repertoires, and their corresponding spatial information, we revealed the dynamic changes of the tumor immune microenvironment (TIME) during tumor progression and metastasis at single-cell resolution. The TIME of metastatic organs may be primed before the establishment of metastasis. Treatment targeting these molecular and immune events during pre- and early metastatic phases may provide a novel avenue to prevent metastasis of lung cancer.
Citation Format: Yanhua Tian, Jian-Rong Li, Bo Zhu, Jared Fradette, Hong Chen, Chenyang Li, Sijia Cui, Daniel Kraushaar, Junya Fujimoto, Leticia Rodriguez, Luisa Maren Solis Soto, Alexandre Reuben, Ignacio Wistuba, Don Gibbons, Cheng Chao, Jianjun Zhang. Single-cell and spatial transcriptomic profiling reveal the cascade of immune events during progression and metastasis of non-small cell lung cancer [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 1300.