Introduction: Macrophages are key architects of the immune landscape in the tumor microenvironment (TME), and the plasticity of macrophage phenotype correlates with prognosis in non-small cell lung cancer (NSCLC). M2-like macrophages promote immunosuppression and facilitate tumor progression, while M1-like macrophages may drive an inflammatory antitumor immune response. We investigated if NSCLC oncogenotype impacts macrophage phenotype in a novel tumor cell, cancer-associated fibroblast (CAF) and macrophage co-culture model. We hypothesize that macrophage-mediated immunosuppression is driven in part through tumor-secreted signaling molecules that, if identified and blocked, may re-sensitize cancer cells to immune surveillance and enhance antitumor immunity. Methods: We developed an in vitro co-culture system (patient-derived NSCLC cells, human CAFs, and mouse macrophages) to interrogate impact of NSCLC cells and CAFs on macrophage phenotype. Through species specific qPCR we measured the expression salient macrophage genes (i.e. Arginase, NOS2, IL-1β, IL-6, CHIL-3, SOCS3) in the co-cultures. We also performed whole genome RNA sequencing (RNAseq) in select cases and evaluated protein expression using cytokine arrays measuring expression of 40 inflammatory cytokines. Positive controls included stimulation of macrophages with LPS and IL-4. Results: We characterized the effect of over 70 NSCLC cell lines in the co-culture assay. Three highly reproducible clusters of macrophage phenotypes were identified: high Arginase (immunosuppressive), high IL-1β (inflammatory) or high SOCS3 (inflammatory, involved in JAK-STAT3 pathway). Major NSCLC oncogenotypes (i.e. KRAS, TP53, STK11, EGFR, BRAF mutation) did not correlate with macrophage phenotype. Analyses of differences between the 3 clusters is ongoing. We selected 10 exemplar NSCLC lines representing each of these 3 clusters for RNA sequencing (mouse genes) and cytokine array protein (human) profiling. Across all clusters, we found suppression of macrophage endocytosis pathways and activation of scavenger receptor A (SRA) signaling (M2-like phenotype) and increased expression of human IL-6, IL-8, and MCP1, which are implicated in suppression of innate immune sensing of tumor cells. RNAseq of CAF lines demonstrated mixed inflammatory and fibroblastic phenotype, with increased expression of genes associated with macrophage recruitment and activation including: IL-6, CSF-1, CXCL6, CCL2, and CCL7. Conclusions: We demonstrate in a novel co-culture model that patient-derived NSCLC cells reproducibly induce three major macrophage phenotypes. Cytokine release from NSCLC cells and CAFs are implicated in this process. This co-culture model provides a robust, physiologically consistent experimental platform to identify tumor cell and CAF features that drive macrophage phenotype. Citation Format: Josiah Flaming, Raghav Chandra, Luc Girard, Debolina Ganguly, Jason Toombs, John D. Minna, Rolf A. Brekken. Lung cancer cells and cancer-associated fibroblasts drive macrophage polarization in a co-culture model [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO021.