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Abstract
Background The plasticity of macrophage phenotype within the tumor microenvironment (TME) correlates with prognosis in non-small cell lung cancer (NSCLC).1 M2-like macrophages promote immunosuppression and facilitate tumor progression, while M1-like macrophages may drive an inflammatory antitumor immune response.2 Through a novel co-culture model comprised of cancer cells, cancer-associated fibroblasts (CAFs), and macrophages, we investigated whether NSCLC oncogenotype impacts macrophage phenotype and postulated that the immunosuppressive activity of macrophages is mediated through tumor-secreted soluble molecules. If identified and inhibited, these 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. Expression of salient macrophage genes (i.e. ARG1, NOS2, IL-1β, IL-6, CHIL-3, SOCS3) was investigated through species-specific qPCR. Whole-genome RNA sequencing (RNAseq) in select cases was conducted and cytokine arrays measuring expression of 40 inflammatory cytokines were performed. Positive controls included stimulation of macrophages with LPS and IL-4.
Results More than 70 NSCLC cell lines were characterized in the co-culture assay. Three highly reproducible clusters of macrophage phenotypes were identified: high Arginase (immunosuppressive), high IL-1β (inflammatory) and high SOCS3 (inflammatory, involved in JAK-STAT3 pathway) (figure 1).3 4 Major oncogenotypes (i.e. KRAS, TP53, STK11, EGFR, BRAF mutation) did not correlate with macrophage phenotype (figure 2). Analyses of differences between the 3 clusters is ongoing. 10 exemplar NSCLC lines representing each of these 3 clusters were selected 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, reflecting an M2-like phenotype.5 We also observed increased expression of human IL-6, IL-8, and MCP1, which are implicated in suppression of innate immune sensing of tumor cells (figure 3). RNAseq of CAF lines demonstrated mixed inflammatory and myofibroblastic phenotypes (figure 4), with increased expression of genes associated with macrophage recruitment and activation including: IL-6, CSF-1, CXCL6, CCL2, and CCL7.6
Three macrophage phenotypes induced in co-cultureHeatmap of mRNA expression from mouse macrophages co-cultured with human NSCLC cells and CAFs. mRNA expression of salient mouse macrophage genes depicted (x-axis) for each NSCLC cell line co-culture (y-axis).
Macrophage phenotype independent of oncogenotypePercentage of mutations of known human NSCLC oncogenes per mouse macrophage phenotype cluster.
Upregulation of macrophage-related cytokinesCytokine array assays demonstrating relative expression of cytokines and chemokines from individual cell types or multicellular co-cultures associated with macrophage recruitment and polarization
Mixed expression of iCAF and myCAF genes on RNAseqHeatmap of RNAseq transcriptome of human CAFs from co-culture model reflecting relative expression of known genes associated inflammatory (iCAF, top) and myofibroblastic (myCAF, bottom) phenotypes.
Novel co-culture model of NSCLC TMEDepiction of novel co-culture model with mouse bone-marrow derived macrophages, human NSCLC cells, and human CAFs with a representative immunohistochemical fluorescence image in vitro
Conclusions Through this novel co-culture model (figure 5), we demonstrate that patient-derived NSCLC cells reproducibly induce three major macrophage phenotypes in an oncotype-independent manner. Furthermore, cytokine release from NSCLC cells and CAFs is implicated in this process. This co-culture model provides a physiologically consistent experimental platform to identify tumor cell and CAF features that drive macrophage phenotype which may be suitable for targeted therapy.
Acknowledgements We thank the McDermott Center Next-Generation Sequencing Core at UT Southwestern. Figure 5 was created with Biorender.com
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