Macrophages in Waldenstrom Macroglobulinemia: Implications for the Tumor Microenvironment

Waldenstrom Macroglobulinemia (WM) is a non-Hodgkin B-cell lymphoma, characterized by infiltration of the bone marrow with plasma cells and lymphocytes. The microenvironment surrounding malignant cells (tumor microenvironment; TME) has been shown to play an important role in the progression of WM, however, the effects of macrophages on WM biology have yet to be elucidated. Here, we show that macrophages are present in the WM TME, and that the direct co-culture of M0 and M2, but not M1 macrophages, derived from THP-1 human cell line, healthy human donor peripheral blood, or murine bone-marrow derived macrophages, with WM cell lines, promotes WM cell growth and survival, in vitro. First, to assess whether macrophages could be involved in WM progression, confirmed cases of lymphoplasmacytic lymphoma (LPL)/WM were identified by pathology and clinical record (Kingston General Hospital, Kingston, ON, Canada). Several bone marrow and lymph node (when available) sections were retrieved and stained for human macrophage antigens CD14 and CD68. We show that macrophages are present in the WM microenvironment, and are found in close proximity to malignant cells. We also show that some macrophages were present and associated with WM cells in tumor samples from SCID mice that were injected with WM cell lines (BCWM.1 and RPCI-WM1) and bone marrow stromal cells (HS-5) stained with H&E staining. To assess the effect of these WM-associated macrophages, macrophages were first derived from THP-1 human cell line monocytes, healthy human peripheral blood donor, or C57BL6J bone marrow aspirate. Macrophages were treated with the appropriate cytokine cocktail to induce M1 or M2 polarization states. M0 and M2 macrophages derived from all three sources, directly co-cultured with WM cells (1:1) for 72 hours, increased the proliferation and survival of WM cells, compared to WM cells grown alone. Additionally, M1 macrophages did not induce a proliferative effect, and in some cases, decreased the proliferation and viability of WM cells. M2 macrophages have been shown to have a pro-proliferative effect in several other cancers, but M0 macrophages were not expected to have this effect. To address why M0 macrophages were also inducing this pro-proliferative and pro-survival effect, WM cells were co-cultured for 72 hours with M0 macrophages generated from cell line, human donor, or murine bone marrow. Co-culture of WM cells with M0 macrophages induced the expression of M2-associated genes in these macrophages, indicating that WM cells are preferentially inducing macrophages towards an M2 phenotype to further aid themselves in progression. Our group has previously investigated the role of GLI transcription factors in myeloid cell biology and found that activation of macrophages with LPS induces GLI3 expression. To investigate mechanisms underlying the pro-tumorigenic macrophage-malignant cell relationship, we performed an analysis of RNA-sequencing data using macrophages from mice lacking Gli3 in myeloid cells (M-Gli3-/- mice) stimulated with LPS. Through this, a subset of genes that are differentially expressed in the absence of GLI3 were identified and it suggested that GLI3 may play role in promoting M2 macrophage polarization. Confirming our results, we found that GLI3 expression is induced in M2 but not M1 macrophages and this is supported by public RNA-sequencing data showing an induction in Gli3 expression in murine M2 macrophages. We found that macrophages from M-Gli3-/- mice did not induce WM cell proliferation and there was a significant reduction in the survival of WM cells cocultured with M2 macrophages from M-Gli3-/- mice compared to WT mice. In addition, M-Gli3-/- M0 macrophages cultured in M2 polarizing media did not express Cd163, an M2 marker, despite the induction of iNos expression (M1 marker) when M0 cells were cultured in M1 polarizing conditions. Finally, macrophages lacking Gli3 were unable to increase the proliferation and survival of WM cells, implicating a role for GLI3 in macrophage polarization and subsequent effects on WM cell growth and proliferation. Taken together, these results suggest a role for M2 macrophages in promoting WM cell growth and survival and identify GLI3 as a modulator of macrophage polarization in the WM tumor microenvironment.