B31 Development of Multicell-Type Organoid Cultures for Preclinical Studies of Immunotherapeutics for Lung Cancer

Abstract

Macrophages are key regulators of the immune landscape within the tumor microenvironment (TME). The plasticity of macrophage phenotypes in the TME has previously been correlated with prognosis within non-small cell lung cancer (NSCLC). Depending on their phenotype, macrophages in the TME can secrete protumor cytokine and chemokines, ultimately suppressing the function of other immune cells in the TME. The purpose of our study was to explore the ability of individual NSCLC preclinical models to alter macrophage phenotype in organoid cultures and to relate effects on macrophages to the molecular characteristics of different NSCLCs. We hypothesize that immune suppression occurs through tumor-secreted signaling molecules, and if blocked, macrophage suppression can be alleviated, resulting in a better antitumor immune response. We developed an in vitro organoid coculture system (NSCLC tumor cells, human cancer-associated fibroblasts, CAFs, and mouse macrophages) to interrogate cancer cell features causing heterogeneity of macrophage phenotypes across a panel of NSCLCs. We measured (with 4-7 replicates for each NSCLC): mRNA expression in mouse macrophages with a panel of qPCR probes for important macrophage-related genes (Arg, NOS2, IL1beta, IL-6, CHIL-3, SOCS3), and in selected cases whole-genome RNAseq; and protein expression using cytokine arrays measuring expression of 40 inflammatory cytokines. Positive controls were stimulation with LPS and IL-4. Using our platform, we characterized 70 NSCLC patient-derived lines by their ability to alter mouse macrophage phenotype. We found: 1. the macrophage phenotypes induced by any one NSCLC were highly reproducible; 2. three major clusters of cancer polarized macrophage phenotypes: high Arg (immune suppressive), high IL-1beta (inflammatory) or high SOCS3 (cGAS-STING pathway) expression; and 3. the major oncogenotypes (KRAS, TP53, STK11, EGFR, BRAF) have no correlation to the induced macrophage phenotype. We selected 7 NSCLC “exemplar” lines representing each of these 3 clusters for RNA sequencing (mouse genes) and cytokine array protein (human) profiling. Across all clusters we found: 1. suppression of macrophage endocytosis pathways and activation of scavenger receptor A (SRA) signaling (M2 immune suppressive phenotype); and 2. increased expression of human IL6, IL8, and MCP1 proteins, which have been implicated in suppressing innate immune tumor sensing. Analyses of differences between the 3 clusters is ongoing. Patient-derived NSCLC preclinical models have reproducible effects on altering macrophage phenotypes in organoid cultures. Three major classes of NSCLC initiated macrophage alteration, which are not linked to oncogenotype. Cytokines secreted by the NSCLCs appear responsible for these macrophage changes, and this system provides an experimental mechanism to systematically test each as potential therapeutic targets.