Exploiting Multi-Cell Type Cultures to Elucidate Tumor Cell Features That Impact Macrophage Phenotype

Lung cancer is expected to kill ~150,000 people this year, encompassing 25% of all cancer related deaths making lung cancer the leading cause of cancer-related mortality in men and women. Lung cancer is divided into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) which represent 80-85% and 15-20% of cases, respectively. My dissertation project focused on understanding how to model the interactions between lung cancer cells, fibroblasts and immune cells. Immune cells are critical components of the tumor microenvironment (TME) that contribute to tumorigenesis, angiogenesis and metastasis. Macrophages are key regulators of the immune landscape within the TME. The plasticity of macrophage phenotypes in the TME correlates with prognosis of NSCLC. Depending on their phenotype, macrophages in the TME can secrete pro-tumor cytokines and chemokines, ultimately suppressing the function of anti-tumor immune cells in the TME. The purpose of my project was to investigate if and how NSCLC cells alter macrophage phenotype in multi-cellular co-cultures and to relate effects on macrophages to the molecular characteristics of different NSCLCs. The central hypothesis of the project is, tumor cell characteristics drive macrophage polarization in the TME, and this can be captured using a multicellular co-culture model. Given the central importance of macrophages to the TME and the immune landscape of NSCLC, an understanding of the tumor cell characteristics associated with immune suppressive or immune stimulatory macrophage phenotype could be exploited from a therapy perspective in the future. To address this hypothesis, an in vitro co-culture system (NSCLC tumor cells, human cancer associated fibroblasts (CAFs), and mouse macrophages) was developed to interrogate cancer cell features driving heterogeneity of macrophage phenotypes across a panel of NSCLCs. We measured: mRNA expression in mouse macrophages with a panel of qPCR probes for genes associated with distinct macrophage phenotypes (Arg1, iNOS, Il-1β, Il-6, Ym-1, Socs3). This system was validated by comparison of macrophage phenotypes represented in the TME of lung cancer xenografts grown in athymic nude mice. Using our platform, we evaluated ~80 NSCLC patient derived lines for their effect on mouse macrophage phenotype. We identified three main macrophage phenotypes across this panel of NSCLCs. To identify cancer cell biomarkers for macrophage polarization, we interrogated molecular characteristics of the cancer lines. Additionally, we expanded the functionality of the platform to assess the effects of pharmacologic agents on macrophage phenotype. As a proof of principle, a small panel of known immune stimulating compounds was tested in the in vitro co-culture platform and validated in human tumor xenografts. Finally, we identified a few novel compounds that show selective cancer cell toxicity and reprogram macrophage phenotype. In conclusion, we built a reproducible in vitro platform to interrogate macrophage polarization in the TME. We leveraged this platform to identify three dominant macrophage phenotypes induced by NSCLC cells and CAFs. We found that no cancer cell molecular characteristic alone drives macrophage polarization. Finally, we illustrate the significance of this platform for immune stimulating drug identification; we identified two novel chemicals that repolarize macrophages and kill cancer cells simultaneously.