A Single-Cell Spatially Resolved Atlas of Immune-Mediated Control of Lung Adenocarcinoma Using 1,000-Plex Single-Cell Spatial Molecular Imaging in a Transgenic Mouse Model

Abstract Background: Lung adenocarcinoma is one of the leading causes of cancer-related mortality. One of the most frequently occurring mutations in lung cancer is KRAS mutations. Recent development of chemical inhibitors that specifically target oncogenic variants of Ras, particularly the commonly mutated KRAS-G12D isoform, represents a significant breakthrough in targeted therapeutics. The tissue-level mechanisms underlying the cell-autonomous and non-cell-autonomous effects of KRas-G12D inhibitors are poorly understood. Additionally, the effectiveness of KRas-G12D inhibitors in lung cancer models remains unknown. To address these gaps in knowledge, we analyzed the spatial interactions between cancer cells and the surrounding tissue microenvironment during the process of tumor eradication mediated through KRas-G12D inhibitors. Methods: We utilized a genetic mouse model of non-small cell lung cancer (NSCLC), driven by the activation of KRas-G12D in combination with the loss of p53. We investigated the immune-mediated tumor recognition following the targeting of KRas-G12D in an immunocompetent setting. Lung samples were collected from control mice and treated. Spatial transcriptomic analysis (CosMxTM SMI 1,000-plex Mouse Universal Cell Characterization Panel) were employed to obtain a high-plex, single-cell, temporal and spatially resolved molecular atlas of lung tumor regression. Results: Prior to Kras inhibitor treatment, mice displayed both LuAD and lung adenomas. Lesion size and histological grade progressively decrease during pharmacological treatment with Kras inhibitor. 906 unique genes were detected above background on intact FFPE tissue slides, along with 4 protein markers for optimal single-cell segmentation results. Concordance between CosMx SMI and scRNAseq was assessed and demonstrated high assay sensitivity and specificity of the CosMx assay. LUAD cell subtypes were identified and showed distinct spatial features. Molecular states, and receptor-ligand interactions, within niche-specific signaling networks were investigated to underline molecular mechanism of immune attack and eventual eradication of tumors. Conclusion: This study provides novel insights into the temporal and spatial dynamics of KRas-G12D inhibitor-mediated tumor regression in lung cancer, shedding light on the previously unknown cell-cell interactions occurring during this process. By investigating these spatiotemporal aspects, we aim to enhance our understanding of lung cancer biology and potentially identify new immunotherapeutic biomarkers. Moreover, the research tools used in this study have implications for the design of future preclinical studies exploring the potential of immuno-oncology combination therapies. FOR RESEARCH USE ONLY. Not for use in diagnostic procedures. Citation Format: Tito Panciera, Francesca Zanconato, Michelangelo Cordenonsi, Mattia Forcato, Giada Vanni, Silvio Bicciato, Julian Preciado, Saskia Ilcisin, Katrina V. Raay, Margaret Hoang, Michael Patrick, Shanshan He, Joseph Beechem, Stefano Piccolo. A single-cell spatially resolved atlas of immune-mediated control of lung adenocarcinoma using 1,000-plex single-cell spatial molecular imaging in a transgenic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1138.