The Role of Tumor Associated Macrophages in Multiple Myeloma and Its Pathophysiological Effect on Myeloma Cells Survival, Apopotosis and Angiogenesis

Objective

The aim of this study is to explore the role of tumor associated macrophages (TAMs) in the prognosis, early treatment response of multiple myeloma and to investigate the role of TAMs on the proliferation, apoptosis£¬oncogene expression and chemotaxis of myeloma cells.

Methods

1 In vivo we retrospectively collected and analyzed 240 patients initially diagnosed wih multiple myeloma and their bone marrow biopsy tissue from Jan, 2009 to June, 2014 in West China Hospital, Sichuan University, China. All the patients enrolled in this study were followed up till April, 2015. We observed and quantified the involvement of macrophage (M¦µ), classic activated macrophage (M1 M¦µ) and alternatively activated macrophage (M2 M¦µ) in bone marrow by immunohistochemical staining of anti-CD68 monoclonal antibody, anti-iNOS monoclonal antibody and anti-CD163 monoclonal antibody, respectively. We analyzed the relation between macrophage involvement with International Staging System (ISS) and the clinical response as well. The effect of different type macrophage involvement on prognosis, progression-free survival and overall survival were estimated. Time-to-event data were analyzed with the Kaplan–Meier method, and the differences were calculated using the Log-rank and Breslow tests. Cox proportional-hazards models were used to estimate hazard ratios and 95% confidence intervals for the main comparisons.

2   In vitro we induced human peripheral blood mononuclear cell£¨PBMC£© and human monocytic THP-1 cells to M2 macrophages with M-CSF or PMA in the presence of IL-4/13 in vitro. Macrophages were identified by morphology and flow cytometry. Two myeloma cell lines (RPMI 8226 and U266) were cocultured with M2 macrophages by using a transwell system. We measured myeloma cells proliferation through CCK-8 method and the pro-inflammatory cytokines expression (TNF-¦Á and IL-6) by ELISA. Real time PCR was applied to measure chemokines (CCL2 and CCL3), chemokine receptors (CCR2, CCR1, CCR5), vascular endothelial growth factor (VEGFA, VEGFB and VEGFC), VEGF receptors (VEGFR1-3), proto-oncogene serine/threonine-protein kinase Pim (PIM1-3). In addition, flow cytometry was used to analyze the apoptosis of myeloma cells induced by dexamethasone.

Results

1 patients with high M2 macrophage involvement (>40/hp) in bone marrow showed poorer response (including complete response and partial response after 3 cycles of chemotherapy) to Dexamethasone-containing regimen (23.9% versus 73%, P=5x10^-13). On the contrary, the patients with high M1 macrophage involvement demonstrated much better response to regimen than low M1 macrophage (69.6 versus 40.6%, P=5x10^-5).

2 Both progression-free survival and overall survival were significantly shorter with high M2 macrophage involvement than low involvement (median progression-free survival, 12.9 months vs. 39 months; hazard ratio for progression, 1.77, 95% confidence interval [CI], 1.14 to 2.74; P=0.01; and overall survival, 4.9 months vs. 59.2 months; hazard ratio for death, 2.63; 95% CI, 1.75 to 3.95; P<0.001).

3 In vitro M2 macrophage stimulate myeloma cell proliferation.

4 In vitro M2 macrophage protect myeloma cells from dexamethasone induced apoptosis.

5 In vitro M2 macrophage promote myeloma cells secreting higher level of IL-6, TNF-¦Á and higher expression of CCL2, CCL3, CCR2, CCR5, VEGFA, VEGFR-1,-2, PIM-1, PIM-2 compared with the non-macrophage coculture system.

Conclusion

TAMs are associated with early clinical response and prognosis. Notably, M2 macrophages involvement has been shown strongly negatively associated with progression-free survival and overall survival. M2 macrophages promote myeloma cells proliferation and protect from apoptosis through a very complex mechanism involving pro-inflammatory cytokines IL-6 and TNF-¦Á, chemokines and related receptors such as CCL2, CCL3, CCR2 and CCR3, VEGF, VEGFR and PIM1, PIM2.

Fig 1  Kaplan-Meier Analysis of PFS and OS in multiple myeloma patients in total Macrophage subgroups (A), M1 subgroups (B) and M2 subgroups(C).

  

Fig 2 Macrophages promote myeloma cells proliferation.