Increased M2 Macrophages in Multiple Myeloma Patients with Progressive Disease and Down-Regulated Polarization with the JAK2 Inhibitor Ruxolitinib

Introduction: Macrophages polarize into pro-inflammatory M1 or alternative M2 states with distinct phenotypes and physiological functions. M2 cells promote tumor growth and metastasis through secretion of growth factors. However, crosstalk between tumor cells and macrophages in the development of M2 polarization has not been well demonstrated. We evaluated the proportion of M2 macrophages in bone marrow (BM) from patients (pts) with multiple myeloma (MM), the effects of MM cells on M1 and M2 differentiation, and the role of Trib1 in M2 differentiation in MM BM. Since the JAK-STAT signaling pathway plays key roles in the cell growth and differentiation of macrophages, we evaluated the effects of the JAK2 inhibitor ruxolitinib (RUX) on M2 polarization in MM.

Methods: Using immunofluorescence (IFC) analysis, we determined the proportion of M1 and M2 macrophages in BM biopsies from MM pts with progressive disease and in remission. The BM biopsy samples were stained with antibodies directed against human iNOS and CD86 for M1 and arginase 1(ARG1) and CD36 for M2 cells, following a standard IFC protocol. Monocyte/macrophage phenotypes for M1 and M2 subtypes in mononuclear cells isolated from MM BM aspirates were also examined using flow cytometric analysis with these same antibodies. Human monocytes isolated from normal subjects or the THP1 monocyte cell line were co-cultured with MM cell lines or primary MM tumor cells with or without exposure to low concentrations (IC20) of ruxolitinib (RUX) using Transwell plates. The percentage of M1 and M2 macrophages was determined using flow cytometric analysis. Total RNA was extracted from monocytes followed the manufacturer’s directions. Quantitation PCR were measured with TaqMan technology performed in an OneStepPlus instrument.

Results: IFC demonstrated that the percentage of M2 macrophages (CD36⁺/ARG1⁺) was markedly increased in BM sections from MM pts with progressive disease compared with those pts in remission. The flow cytometric data also showed the percentage of M2 (CD36⁺/ ARG1⁺) macrophages in BM was significantly increased in MM patients with progressive disease (n=20) compared to those in remission (n=9; P=0.005) whereas there was no significant difference in the percentage of M1 (CD86⁺/iNOS⁺) macrophages in BM derived from MM patients with progressive disease compared to those in remission. The results of both RT-PCR and Quantitation PCR showed Trib1 gene expression levels were higher among patients with progressive disease compared to those in remission. In contrast, the gene expression of Trib2 and Trib3 was not related to the MM patient’s clinical status. To determine whether MM tumor cells affected monocyte/macrophage differentiation and Trib gene expression, we co-cultured MM cell lines or fresh MM tumor cells with purified healthy human monocytes using Transwell plates. The percentage of M2 cells markedly increased whereas the proportion of M1 cells decreased. The expression of Trib1 increased during the 7 days of co-culture whereas there was no change in the expression of Trib2 and Trib3. Moreover, when direct cell-to-cell contact occurred between the MM cells and the monocytes, the percentage of M2 macrophages markedly increased after 7 days of incubation. It has been reported that Trib-1 mediated regulation of the MAPK/ERK pathway in a murine model and the Ras/Raf-1/MEK1/ERK cascade culminates in up-regulated expression of the gene encoding STAT3 whereas recruitment and activation of tyrosine kinase JAK-2 phosphorylates it. Thus, we investigated the effects of the JAK2 inhibitor RUX on M2 differentiation induced with MM tumor cells. The percentage of M2 cells was decreased when the monocytes that were co-cultured with MM tumor cells were treated with a low concentration (IC20) of RUX. Trib1 gene expression of the monocytes treated with RUX was also reduced comparing to the cells untreated with JAK2 inhibitor.

Conclusion: Our results show that MM cells induce monocytes to become M2 macrophages and increase Trib1 gene expression providing a positive feedback loop on Trib1 expression, monocyte differentiation and tumor cell growth. M2 cells are present at high levels in BM derived from MM pts with progressive disease compared to those in remission. Notably, the JAK2 inhibitor RUX shows inhibition of both M2 macrophage polarization and Trib1 gene expression in MM, and these results suggest this drug may be effective for treating MM.