Age-Associated Macrophage Polarization to an M2 Phenotype Drives Myeloma Proliferation in the Aged Bone Marrow By Loss of Tumor-Associated Phagocytosis

Multiple myeloma (MM) is a blood cancer characterised by the proliferation of malignant plasma cells within the bone marrow (BM). Cellular interactions in the BM microenvironment are hijacked by MM to support its survival and progression, demonstrating a functional role for immune cells in MM pathophysiology (García-Ortiz et al., 2021; Jibril et al., 2023). Macrophages differentiate into a range of polarisation states between the M1 and M2 extremes (Murray et al., 2014). M2-like macrophages are reported to resemble a pro-tumoral phenotype which supports remodelling of the immune BM microenvironment in MM (Guillerey et al., 2016; Sun et al., 2022). Further, with increasing age the senescence associated secretory phenotype (SASP) creates a pro-tumoral BM microenvironment (Campisi, 2013; Hellmich et al., 2020). To this end, age-related changes in the BM microenvironment have the potential to drive MM development. Here, we describe in vitro and in vivo how changes in macrophage phenotype during aging support MM development.

5TGM1^GFP+ myeloma cells were established in the aging KaLwRij mouse model (Radl et al., 1978). We and others have shown that 5TGM1^GFP+ myeloma cells do not engraft in young C57BL/6 mice but can engraft in obesity induced C57BL/6 mice (Lwin et al., 2015). To determine if the aging C57BL/6 BM is permissive to 5TGM1^GFP+ myeloma cells, we injected the cells intravenously into C57BL/6 mice aged 3-4 months (young) and 18-22 months (aged). After 30 days, BM, spleen and blood samples were harvested. Flow cytometry analysis of isolated BM and spleen showed successful 5TGM1^GFP+ engraftment in aged mice (7-34% BM GFP⁺; 3-26% splenic GFP⁺) but no engraftment was observed in young mice. To investigate the underlying mechanism permitting 5TGM1^GFP+ engraftment in the aged C57BL/6 BM microenvironment, BM was isolated from C57BL/6 mice aged 3-4 and 16-22 months, respectively. Flow cytometry analysis identified an elevated total CD45⁺ Gr1^- F4/80⁺ CD115^low macrophage count in aged mice compared to young (p<0.05), concomitant with a reduced M1 (CD45⁺ Gr1^- F4/80⁺ CD115^low CD86⁺) frequency (p<0.05) and a trend toward significance for an increased M2 (CD45⁺ Gr1^- F4/80⁺ CD115^low CD206⁺) frequency (p=0.057). Further, an overall elevated M2/M1 ratio was observed in aged mice compared to young (p<0.05). To explore the effect of M1 and M2 macrophages on 5TGM1^GFP+ viability, bone marrow-derived macrophages (BMDM) were cultured for 6 days as previously described (Toda et al., 2021), seeded at 5x10⁵ and stimulated with 100ng/mL LPS and 50ng/mL IFN-γ (M1 polarised) or 50ng/mL IL-4 and IL-13 (M2 polarised) for 24hr. After removing media, 7.5x10⁴ 5TGM1^GFP+ cells were co-cultured with M1 and M2 macrophages in DMEM, 10% FBS, 1% Pen/Strep. After 48hr, the percentage of 5TGM1 cells plated were reduced in M1 co-culture, when compared to 5TGM1-only culture or co-culture with M2 macrophages (p<0.01 and p<0.01, respectively). To investigate whether macrophages in the aged BM have impaired phagocytic capacity, pHrodo^TM Red E. coli bioparticles were incubated with BM from young and aged mice as above. Flow cytometry analysis found reduced pHrodo expression in F4/80⁺ macrophages in the aged BM relative to young (p<0.05), measured by mean fluorescence intensity (MFI).

Here, for the first time we show that aged C57BL/6 mice are permissive of 5TGM1^GFP+ cells. We show that this may in part be mediated by an elevated M2/M1 ratio in the aged BM microenvironment. We also demonstrate that macrophages in the aged BM have a reduced phagocytic capacity, suggesting that the BM microenvironment is immunosuppressed in the aged state. Taken together, these data suggest that a loss of M1-like phenotype or a shift toward the M2-like phenotype in the aged BM microenvironment may confer a permissive, anti-inflammatory state for MM development and proliferation.