Source: Dissertation Abstracts International, Volume: 79-11(E), Section: B.

Adviser: Kathryn Miller-Jensen.

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Macrophages are cells of the innate immune system that have diverse functional roles in development, tissue repair, homeostasis, and immunity, and their dysregulation has been implicated in a wide range of diseases. To perform their diverse set of functions, macrophages must be able to respond to multiple conflicting stimuli in their microenvironment. However, it is unclear how individual macrophages exposed to conflicting cues coordinate a cohesive immune response. Furthermore, macrophages have been identified as important regulators of tumor microenvironments, a complex tissue containing many conflicting stimuli. Understanding the macrophage response in complex tissues is a key step to design therapeutic strategies to fight diseases like cancer. The work presented here investigates the polarization response of macrophages in complex microenvironments. Through a combination of systems-level approaches in both in vitro and ex vivo experiments, we investigate the role of macrophages in immunity and their potential ability to induce an anti-tumor response in melanoma.

We first examined how macrophages respond to conflicting cues in a controlled, in vitro model of macrophage polarization using opposing inflammatory (LPS+IFNgamma) and anti-inflammatory (IL-4) cues. We found that macrophages stimulated with opposing cues display extensive crosstalk between inflammatory (M1-like) and anti-inflammatory (M2-like) programs, but cell isolation decreased the cross-inhibition between these two programs. Furthermore, although isolated macrophages stimulated with LPS+IFNgamma plus IL-4 simultaneously activated intracellular signaling pathways downstream of both IFNgamma and IL-4, most of these macrophages become single-responder cells exclusively secreting either pro- or anti-inflammatory cytokines. High-dimensional clustering and progression analysis revealed that macrophages choose either an M1-like or M2-like polarization trajectory, and few cells induce a mixed polarization state. Together, these results suggest that, when simultaneously presented with opposing cues, the response of individual macrophages display orthogonal responses while the ensemble of macrophages produce a mixed population response. This data provides insight into how individual macrophages integrate signals in complex microenvironments.

Next, we examined the functional role of macrophages in a tumor microenvironment. The recent success of checkpoint inhibitor therapy (CIT) has revolutionized cancer immunotherapy, however, a significant fraction of patients do not respond to CIT. In these patients, tumor-associated macrophages (TAMs) are promising targets to induce an anti-tumor immune response. To this end, we used an autochthonous, poorly immunogenic mouse model of melanoma to study the functional secretion of macrophages in a tumor microenvironment. Using a single-cell secretion assay, we show that TAMs display an M2-like polarization state secreting mostly CCL17, CCL22, MMP9 and Chi313 in untreated tumors. Furthermore, using a myeloid-targeted combination therapy (agonistic CD40 and CSF1R inhibition), we demonstrate a potent suppression of tumor growth. TAM functional analysis revealed a loss of macrophages secreting MMP9, as well as an induction of a novel subpopulation of macrophages co-secreting TNFalpha, IL-6, IL-12 and Chi313. Tumor suppression was dependent on TNFalpha, mostly secreted by TAMs, and on IFNgamma, mostly produced by T-cells. This study presents the first single-cell functional secretion study of macrophages isolated directly from an endogenous melanoma model, and demonstrates the potential for targeting macrophages to modulate an effective immune response in previously "cold" tumor microenvironments.

Together, the work presented in this thesis uses novel approaches to characterize macrophage responses in complex tissue microenvironments. Macrophages responding to simultaneous opposing cues display orthogonal functional responses, while targeting TAMs in vivo induces a mixed functional response and potent antitumor immunity in tumor microenvironments. These insights will help design novel therapies to modulate macrophage function in disease.