Gli3 Is Required for M2 Macrophage Polarization and M2-Mediated Waldenström Macroglobulinemia Growth and Survival

Abstract

The macrophage polarization paradigm has become a forefront in cancer research in recent years, but the effects of this phenomenon have yet to be investigated in Waldenström Macroglobulinemia (WM). WM is an indolent, B-cell lymphoma, characterized by increased IgM production and infiltration of the bone marrow niche by malignant cells. While macrophage polarization is typically investigated as a singular point in time, it is important to understand that M2-type macrophages can switch to an M1 phenotype, or vice versa, based on environmental changes. M1 macrophages are typically pro-inflammatory and anti-tumor (although some cancers have reported an increased presence of M1 macrophages leading to worse clinical outcomes), and M2 macrophages are typically anti-inflammatory and pro-tumorigenic. Understanding which phenotype is prevalent in WM, as well as the mechanism behind increased tumorigenesis is important for guiding WM research and treatment. To assess the effects of macrophages on WM cell proliferation, we examined the effects of macrophage polarization using THP-1 cells, CD14+-derived macrophages from PBMCs and bone marrow-derived macrophages from C57BL/6 mice on WM cell growth and viability. WM cells grown in direct co-culture with macrophages exhibited increased proliferation compared to WM cells grown alone. WM cell viability was also enhanced when cells were directly co-cultured with macrophages. To investigate whether M1 or M2 macrophages were responsible for this increased proliferation and viability, we performed a qPCR analysis of macrophages in indirect and direct co-culture with WM cells. We found that WM cells induce a M2 phenotype upon direct co-culture, but this effect was not seen in indirect co-cultures. In previous work, we found that the transcription factor GLI3 plays a role in regulating cytokine expression and secretion in response to LPS stimulation. We performed RNA-seq on macrophages derived from mice lacking Gli3 in myeloid cells (M-Gli3-/-). Using a generalized linear model in edgeR, we identified 495 genes with significant interaction effects between genotype and LPS treatment. Ingenuity Pathway Analysis of the interaction genes revealed "Inflammatory Response” and "Immune Cell Trafficking” pathways as most significantly enriched. The 25 significant interaction genes on these pathways included 9 with a positive interaction and 16 with a negative interaction. Analysis also suggested Gli3 may play a role in M2 macrophage polarization. Bone marrow-derived macrophages were isolated from M-Gli3-/- and WT mice. We found that M-Gli3-/- macrophages were unable to polarize into the M2 phenotype and both proliferation and viability of WM cells cocultured with these macrophages did not increase. Our findings identify a novel role for Gli3 in regulating M2 polarization and subsequently a role for M2 macrophages in promoting WM growth and survival. This suggests therapeutic targeting of Gli3 in the tumor microenvironment may be beneficial in the treatment of WM.