Multiple myeloma (MM) originates in the bone marrow where adipocytes occupy 65% of the cellular volume in a typical myeloma patient. Cancer associated adipocytes support the initiation, progression, and survival of solid tumors via mechanisms including adipokine secretion, modulation of the tumor microenvironment, and metabolic reprogramming of cancer cells. Although MM cells are surrounded by abundant bone marrow adipocytes (BMAd), the nature of their interaction remains unclear. Recent studies have elucidated the role of BMAds in supporting the survival of MM cells, in part, through secreted adiponectin. Increased fatty acid (FA) metabolism may result in metabolic reprogramming of cancer cells impacting their growth and survival. Here, we hypothesize that MM cells extract FA from adipocytes for their growth.
We first characterized mesenchymal stem cells (MSCs) from MGUS, smoldering MM (SMM), and newly diagnosed MM (NDMM) patients by flow cytometry analysis. MSCs showed significant increase in Pref1, leptin receptor and perilipin A, suggesting increased adipogenic commitment. MSCs from healthy donors (HD), MGUS, SMM, and NDMM patients were induced to differentiate into adipocytes and then co-cultured with human MM MM.1S cells. After 72 hr of co-culture, CyQUANT assay demonstrated significant increase in proliferation of MM.1S cells in the presence of BMAd from HD; this was further increased in the presence of BMAd from MGUS/SMM and NDMM. These data suggest that the BMAd support the growth of MM cells and this effect is more pronounced in patient derived BMAd. A PCR-array targeting lipid metabolism on BM fat aspirates showed significant deregulation of genes involved in FA synthesis and lipolysis. Taken together, our data suggest that BMAd in MM patients are altered to further support the aggressive expansion of MM cells.
The proliferative-supportive role of adipocytes was further validated in co-culture of OP9 murine BM stromal preadipocytes with 5TGM1 murine MM cells. To study the bidirectional interaction of MM/ BMAd, mature OP9 adipocytes were co-cultured with 5TGM1 or human OPM2 MM cells for 24 hr. Intracellular lipid droplets were labelled with Deep Red LipidTox stain. The lipid droplet sizes were significantly decreased in the presence of both 5TGM1 and OPM2 cells compared to OP9 alone. The decrease in lipid size suggested that MM cells may induce lipolysis in adipocytes. Indeed, 24hr co-culture of 5TGM1 cells with OP9 mature adipocytes significantly increased lipolysis 3-fold as measured by glycerol secretion in conditioned media. Co-culture of OP9 adipocytes with other MM cell lines of human origin, MM.1S, INA6, KMS-12 PE, and OPM2 also significantly increased the glycerol production as much as 4-fold. Taken together these data indicate that MM cells induce lipolysis in adipocytes. In contrast, treatment of 5TGM1 cells with synthetic catecholamine isoproterenol did not induce lipolysis, or glycerol production, indicating lack of triglyceride storage. Next, we hypothesized that the free FAs released from adipocytes are taken up by MM cells for various biological processes. To test this, 5TGM1, MM.1S and OPM2 cells were incubated with BODIPY-C12 and BODIPY-C16, the BODIPY-fluorophore labelled 12-carbon and 16-carbon long chain FA. All MM cells showed saturated uptake of the FA within 10 minutes suggesting that MM cells have efficient FA transporters. To confirm this uptake, unstained 5TGM1, OPM2 and KMS12 PE cells were co-cultured with the LipidTox-labelled OP9 mature adipocytes. After 24 hours, flow cytometric analysis showed LipidTox signal in MM cells. These data demonstrate that FAs released by MM induced adipocyte lipolysis are taken up by MM cells. Long-chain FAs such as BODIPY-C12 and BODIPY-C16 are transported into cells through FA transporter protein (FATP) family of lipid transporters. We therefore analyzed patient samples which showed that CD138+ plasmacells and myeloma cells expressed high levels of FATP1 and FATP4 whereas, their expression was absent in lineage-sibling T-cells. Moreover, pretreatment with Lipofermata, a FATP inhibitor, was able to decrease the uptake of BODIPY-C12 and -C16 in 5TGM1 cells.
Taken together, our data show that myeloma cells induce lipolysis in adipocytes and the released free FAs are then uptaken by myeloma cells through FATPs. Inhibiting myeloma cell induced lipolysis or uptake of FA through FATPs may be a potential anti-tumor strategy.
Disclosures
Fulzele: FORMA Therapeutics, Inc: Current Employment, Other: Shareholder of Forma Therapeutics. Raje:Amgen: Consultancy; bluebird bio: Consultancy, Research Funding; Caribou: Consultancy, Membership on an entity's Board of Directors or advisory committees; Immuneel: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Consultancy; Celgene: Consultancy; Immuneel: Consultancy; Janssen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy.
Role of B-Cell–Activating Factor in Adhesion and Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment
