Abstract PR017: Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics

Abstract

Abstract Introduction: Tumors systemically remodel the immune system during metastasis. Developing anti-metastatic immunotherapies that target these tumor-immune interactions to make the metastatic niche hostile colonization would represent a paradigm shift in cancer treatment. However, traditional high-throughput screening (HTS) platforms that use simple read-outs and contrived in vitro systems are ill-suited to identify such therapies. In contrast, HTS platforms that accurately model the metastatic niche and use high-dimensional read-outs such as multiplexed single-cell RNA-sequencing (scRNA-seq) can accurately profile nuanced perturbation responses in individual immune cell types and therefore have the potential to discover anti-metastatic immunotherapies. In this study, we describe the development of an ex vivo lung tissue culture HTS platform that preserves immune gene expression signatures observed in the in vivo metastatic niche and is amenable to single-cell chemical transcriptomic analysis using MULTI- seq (McGinnis et al., Nature Methods, 2019). Inspired by our recent description of myeloid cell TLR-NFκB inflammation during breast cancer lung metastasis (McGinnis et al., Cancer Cell, 2024), we then performed the first anti-metastatic immunotherapy drug screen and identified TLR-NFκB inhibitors that effectively operate on all desired myeloid cell types in the lung metastatic niche. Methods and Results: Building upon existing techniques for culturing precision-cut lung slices (Wu et al., Journal of Experimental Medicine, 2024) and patient- derived tumor fragments (Voabil et al., Nature Medicine, 2021), we established and optimized an ex vivo lung tissue culture system that enables 48-hour culturing of lung slices isolated from 4T1 tumor-bearing mice. scRNA-seq profiling revealed successful capture of all relevant immune cell types and retention of metastasis-associated gene expression programs (e.g., myeloid TLR-NFκB inflammation and neutrophil degranulation) following culture. We then scaled our platform to address how myeloid cells in the lung metastatic niche respond to perturbations targeting every component of the canonical TLR-NFκB signaling cascade. Cell-type-specific perturbation modeling and quantification of TLR-NFκB signaling inhibition identified compounds that optimally perturb all intended myeloid cell types (e.g., tissue-resident macrophages, neutrophils, and monocytes) and represent prioritized candidates for future in vivo validation studies to assess impact on metastatic disease progression. Conclusions: In this study, we describe the development and application of a novel HTS platform that couples ex vivo lung tissue cultures with single-cell chemical transcriptomics to identify anti-metastatic immunotherapy candidates. We demonstrate how our platform can identify TLR-NFκB signaling inhibitors that optimally operate in the lung metastatic niche, paving the way for future interrogation of additional metastasis-associated immune cell signaling pathways in different disease and tissue backgrounds. Citation Format: Chris McGinnis, Winnie Yao, Ansuman Satpathy. Anti-metastatic immunotherapy discovery using ex vivo lung tissue cultures and high-throughput single-cell chemical transcriptomics [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr PR017.