Investigating Macrophage Molecular and Functional Diversity in Tumor Immunity

PROJECT SUMMARY Macrophages (MF) are one of the largest immune cell components of tumor lesions, where their numbers can even exceed cancer cells. MF play a key role in shaping the composition of the tumor microenvironment (TME), the modulation of tumor innate and adaptive immunity, and the response to cancer immunotherapy. Because of their critical roles, MF are an important target for cancer treatment. However, modulating tumor- associated MF has proved extremely difficult. This is in large part because we still do not have a complete understanding of the tumor MF compartment. In order to develop ways to modulate tumor MF and promote cancer immunity, it is vital we gain a deeper understanding of the molecular and functional diversity of MF in their tissue context. Using mass cytometry (CyTOF) and single cell RNA-seq (scRNA-seq), we initiated deep characterization of the immune composition of early human non-small cell lung carcinomas (NSCLC). We uncovered evidence of multiple distinct MF populations enriched in human tumors. Notably, related MF clusters were also identified in mice lung cancer lesions. Using fate mapping and scRNA-seq, we discovered these discrete MF populations differ in developmental origin and have a distinct distribution in the TME. When the different subsets of MF were depleted, tumor growth was impaired, but the alterations in TME differed, suggesting distinct mechanisms of activity. Based on our findings, we hypothesize that the unique subsets of MF have differential molecular states and mediate differential contributions to tumor growth, organization, immunity, and response to PD1 blockade. To address our hypothesis, we will: (1) comprehensively map the MF compartment of human lung tumors, at the single cell level and with spatial resolution, at baseline and during treatment with PD1 blockade, in NSCLC patients enrolled in a neoadjuvant immunotherapy clinical trial, (2) assess the functional contribution of distinct MF subsets to tumor tissue remodeling and immune cell dynamics in the TME, and (3) determine the contribution of distinct MF subsets to lung tumor immunity. We will also (4) investigate the function and activity of a specific cell surface receptor, Trem2, which we found to be exclusively expressed on monocyte-derived MF in both human and mouse lung tumors, and whose knockout impaired lung tumor growth similar to MF depletion; suggesting an important and potentially targetable molecule in MF control of tumor growth. The outcome of these studies will (i) uncover the molecular and functional diversity of the MF compartment of human lung tumors, (ii) determine how distinct MF subsets, and MF-specific genes, influence tumor growth, the TME state, and tumor immunity, and (iii) provide insight into to how MF subsets influence, and are influenced by, PD1 blockade in human NSCLC. These studies have the potential to help us understand some of the factors that contribute to tumor response and resistance to immune editing, and aid in the further development and clinical use of cancer immunotherapy strategies.