The power of polarization: tumor-associated macrophages and their cancerous counterparts

Abstract

Macrophages are ancient, phagocytic immune cells thought to have their origins 500 million years ago in metazoan phylogeny. Today, the understanding of macrophages has evolved to encompass their foundational roles in development, homeostasis, tissue repair, inflammation, and immunity. Notably, macrophages display high plasticity in response to environmental cues, capable of a strikingly wide variety of dynamic gene signatures and phenotypes. Macrophages are also involved in many pathological states, highlighting their critical role in multiple biological processes. In cancer, most tumor-associated immune cells are macrophages, coined tumor-associated macrophages (TAMs). While some TAMs can display anti-tumor properties such as phagocytizing tumor cells and orchestrating an immune response, most macrophages in the tumor microenvironment (TME) are immunosuppressive and pro-tumorigenic. Macrophages have been implicated in all stages of cancer, and many questions remain unanswered about how to best wield these powerful immune cells to treat cancer. In Chapter 2, we explore the poorly understood variability both between and within cancer types in their ability to maintain an immunosuppressive microenvironment and polarize TAMs. Identifying genetic biomarkers of cancers that strongly polarize TAMs will enable the discovery of novel therapeutic targets and support stratification of patients for appropriate immunotherapy. Our aim, therefore, was to correlate gene expression data from cancer cell lines with phenotypic changes in canine macrophages. We harvested standardized tumor-conditioned medium (TCM) from 25 canine cancer cell lines with bulk RNA-sequencing data available. Primary monocyte-derived macrophages were obtained from three canine donors and polarized with the twenty-five different TCM. After a washout period, supernatants were collected, and polarization changes of the macrophages were assessed via secretory products using ELISA and a multiplex cytokine/chemokine assay. For each analyte measured, a modified z-score was used to rank each cell line's ability to stimulate the secretion of that analyte from macrophages. Correlative analysis and pairwise comparison analysis (composed of "strong" and "weak" stimulators) were performed against the full transcriptome. Many significantly differentially expressed genes were identified in the cancer cell lines with the strongest ability to polarize macrophages. For example, cell lines with MVB12A upregulated, a gene involved in selecting cargo for exosomes, had the highest ability to stimulate vascular endothelial growth factor (VEGF) production from macrophages. This was subsequently confirmed by depleting the TCM of exosomes, resulting in a significant decrease in VEGF production. Multiple other genes involved in macrophage immunosuppression were upregulated, while those involved in cell adhesion and metastasis prevention were downregulated. In conclusion, we highlight that canine cancer cell lines have varying abilities to polarize primary macrophages, irrespective of histologic type, as measured by secretory products. The cell lines capable of potent polarization had relevant differentially expressed genes and pathways. These are currently being explored to identify novel biomarkers and therapeutic targets. In chapter 3, we highlight bringing a macrophage-targeting agent to patients via a clinical trial. Investigators at the National Institutes of Health (NIH) have recently demonstrated that the mannose receptor, CD206, which is commonly expressed on TAMs, is a druggable target. When stimulated, it can deplete TAMs and/or polarize them toward an antitumor phenotype in mouse models and human cells. However, the effects of CD206 targeting on canine macrophages are unknown. In collaboration with NIH intramural, a pilot clinical trial using the CD206 agonist NCGC00413972 (972) is underway in dogs with naturally occurring sarcomas. Given the propensity for CD206 activation to reprogram macrophages, we are using serial biopsies to analyze if CD206 will activate a shift in gene transcription that polarizes macrophages toward an M1-like phenotype resulting in a depletion of TAMs and ideally tumor regression. A starting dose is known from a small safety study in healthy beagle dogs, and so a rapid-dose escalation trial design was implemented. Enrolled dogs undergo tumor biopsy with CD206 immunohistochemistry to confirm that the target of 972 is present. Then, they are started on 972 with regular rechecks to monitor biochemical parameters, disease status, and constitutional status. This will give us rapid toxicity and efficacy data to potentially be applied towards human trials. Preliminarily, 972 appears well-tolerated with very few adverse effects. It has achieved stable disease in a majority of enrolled patients for at least 6 weeks. Patient enrollment is ongoing to continue to strengthen and mature this data. Finally, in chapter 4, we explore the unique situation when CD206 is expressed on malignant cells in histiocytic sarcoma (HS), a cancer of macrophage or dendritic cell origin. HS is an aggressive cancer with a grave prognosis in dogs and people. While CD206 agonism causes apoptosis and repolarization in primary macrophages, the effect of CD206 agonism on HS cells is unknown. The aim of this study was to evaluate the impact of CD206 agonism on HS cells to identify new therapeutic targets. Three HS cell lines were incubated with a small molecule agonist of CD206. Viability was assessed through the IncuCyte cell labeling system and resazurin blue fluorescence. Signaling pathways were interrogated via a luciferase reporter assay and western blots. CD206 agonism repeatably stimulated growth in all HS cell lines in contrast to its impact on primary macrophages. CD206 agonism also upregulated activation of nuclear factor kappa-light-chain-enhancer of activated b cells (NF-κB), extracellular signal-regulated kinase (ERK), and protein kinase b (AKT) signaling. These data indicate that HS growth is stimulated via agonism of CD206. NF-κB, ERK and AKT are all potential therapeutic targets that may be routinely upregulated during HS disease. Work is ongoing to explore the impact of inhibiting these targets and to demonstrate the effect of CD206 agonism on primary canine macrophages. In chapter 5, we discuss next steps for this research to further investigate these results discussed here. In conclusion, we present three varied but complementary looks at the power of polarization in both macrophages and their cancerous counterparts. Understanding and harnessing these pathways may lead to novel therapeutic strategies for cancers affecting both humans and dogs.