Adipocyte-Lineage Cells Support Growth and Dissemination of Multiple Myeloma in Bone

Abstract

Multiple myeloma (MM) cells reside in the bone marrow microenvironment and form complicated interactions with nonneoplastic, resident stromal cells. We previously found that aggressive MM cells shift osteoblast progenitors toward adipogenesis. In addition, adipocytes are among the most common cell types in the adult skeleton; both mature adipocytes and preadipocytes serve as endocrine cells that secrete a number of soluble molecules into the microenvironment. Therefore, we used a combination of in vivo and in vitro methods to test the hypothesis that an increase in adipocyte lineage cells feeds back to promote MM progression. The results of this study revealed that bone marrow from patients with MM indeed contains increased preadipocytes and significantly larger mature adipocytes than normal bone marrow. We also found that preadipocytes and mature adipocytes secrete many molecules important for supporting MM cells in the bone marrow and directly recruit MM cells through both monocyte chemotactic protein-1 and stromal cell-derived factor-1α. Co-culture experiments found that preadipocytes activate Wnt signaling and decrease cleaved caspase-3, whereas mature adipocytes activate ERK signaling in MM cells. Furthermore, mature adipocyte conditioned medium promotes MM growth, whereas co-culture with preadipocytes results in enhanced MM cell chemotaxis in vitro and increased tumor growth in bone in vivo. Combined, these data reveal the importance of preadipocytes and mature adipocytes on MM progression and represent a unique target in the bone marrow microenvironment.

Figure 1

Adipocyte lineage cells are altered in the bone marrow (BM) of patients with multiple myeloma (MM) compared with normal BM. A: BM biopsy specimens from healthy individuals and MM patients were stained for the preadipocyte marker preadipocyte factor (Pref-1). Cells positive for Pref-1 (arrows) were more abundant in MM patients compared with healthy BM specimens. Representative images are shown. B: Enumeration of Pref-1–positive cells from five random fields of view per section. C: Hematoxylin and eosin (H&E)–stained BM biopsies from healthy BM donors and MM patients. Very large adipocytes were more frequently observed in MM patients (arrows). D: Mature adipocyte area quantified from five random fields of view per section. Adipocytes in MM bone marrow were significantly larger than normal bone marrow. E: Distribution of mature adipocyte size as percentage of total adipocyte area. A higher percentage of larger adipocytes presented in MM bone marrow than in normal bone marrow. Data are expressed as means ± SEM (B and D). ^∗∗∗P < 0.001, ^∗∗∗∗P < 0.0001.

Figure 2

Adipocyte lineage cells secrete soluble molecules that attract multiple myeloma (MM) cells. A: Numbers of migrated mouse 5TGM1 MM cells. Preadipocyte (pAd) conditioned medium (CM), mature adipocyte (mAd) CM, or fresh medium (FM) were added in the bottom of each well as chemoattractants and 5TGM1 cells seeded in the inserts. The number of 5TGM1 cells migrated into the bottom of each well was counted after 24 hours. MM cells migrated significantly more toward both preadipocyte and mature adipocyte CM than to fresh medium, and preadipocyte CM consistently had the greatest chemoattraction. B: Similar results were observed using human CAG MM cells. Each sample was analyzed in triplicate and each assay was performed 3 times. Data are expressed as means ± SEM. ^∗P < 0.05, ^∗∗∗∗P < 0.0001.

Figure 3

Adipocytes secrete soluble molecules known to promote multiple myeloma (MM) progression and directly attract MM cells through monocyte chemotactic protein (MCP)-1 and stromal cell-derived factor (SDF)-1α. A: Cytokine array of conditioned medium (CM) collected from 3T3-L1 preadipocytes (pAds) or mature adipocytes (mAds). Molecules of particular importance are highlighted. B: Quantification of pAds versus mAds. C: Enzyme-linked immunosorbent assays for osteopontin (OPN), MCP-1, and SDF-1α in pAd and mAd CM confirmed cytokine array results. Each assay was analyzed in duplicate. D: Neutralizing antibodies against MCP-1 or appropriate isotype control were added to CM from preadipocytes, mature adipocytes, or fresh media (FM). Neutralizing MCP-1 resulted in significantly reduced migration of mouse 5TGM1 MM cells toward both pAd and mAd CM compared with isotype control. E: Similar results were observed when adding a SDF-1α neutralizing antibodies. F: Recombinant human preadipocyte factor (Pref-1) dose dependently acted as a chemoattractant for MM cells. Each sample was analyzed in triplicate, and each assay was performed at least 2 times. Data are expressed as means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001. GM-CSF, granulocyte-macrophage colony-stimulating factor; HGF, hepatocyte growth factor; IGF-1, insulin-like growth factor-1; M-CSF, macrophage colony-stimulating factor; MIP-2, macrophage inflammatory protein-2; MMP, matrix metalloproteinase; TGF-β, transforming growth factor-β; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor.

Figure 4

Adipocyte lineage cells differentially alter behavior and signaling of multiple myeloma (MM) cells. A: Western blotting after MM cell co-culture with preadipocytes (pAds) or mature adipocytes (mAds) for 3 days. The pAds activated β-catenin signaling and decreased cleaved caspase-3 levels in 5TGM1 MM cells, whereas the mAds activated ERK signaling in MM cells compared with those cultured alone. B: Quantification of Western blots from three independent experiments. C: MM cell migration assay. 5TGM1 MM cells cultured with pAds revealed enhanced migration compared with those cultured with mAds or in fresh medium (FM) alone. D: Cell proliferation rates. 5TGM1 MM cells cultured in mAd conditioned medium (CM) revealed increased growth compared with those cultured with pAd CM or FM. Each sample was analyzed in triplicate, and each assay was performed three times. Data are expressed as means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗∗P < 0.0001. p-ERK, phosphorylated ERK.

Figure 5

Preadipocyte (pAd) co-culture promotes myeloma progression in vivo. A: Representative bioluminescent images of mice at 4 weeks (left column) and 6 weeks (right column) after i.v. injection of mouse 5TGM1-luc multiple myeloma (MM) cells cultured alone or with pAds. Tumors were visible earlier and grew larger from pAd co-cultured MM cells compared with MM cells cultured alone. B: IgG2bκ serum levels, an indicator of total tumor burden, at time of sacrifice (6 weeks) of mice from panel A. C: IgG2bκ serum levels in mice injected with mouse 5TGM1-luc MM cells cultured alone or with mature adipocytes (mAds). n = 7 per group. Data are expressed as means ± SEM. ^∗∗P < 0.01.

Figure 6

Preadipocytes and mature adipocytes promote multiple myeloma (MM) progression. At the primary bone site, preadipocytes and mature adipocytes likely secrete pro-MM soluble molecules. Constant presence of mature adipocyte–derived molecules enhances MM cell growth, whereas preadipocytes educate MM cells for increased metastasis in new bone sites. In a secondary bone site, enhanced preadipocytes and mature adipocytes secrete chemoattractants, such as monocyte chemotactic protein (MCP)-1, stromal cell-derived factor (SDF)-1α, and preadipocyte factor (Pref)-1, to promote MM homing and subsequent growth. OPN, osteopontin; VEGF, vascular endothelial growth factor.