Sustained Growth of Primary Myeloma Cells in Coculture with Whole Donor Bone Marrow Is Associated with Induced Secretion of the Microenvironmental Mediator of Cytokinesis, Hemicentin-1

Primary human myeloma (MM) cells do not survive in culture while current systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animals. The aim of the study was to establish a coculture system for studying long-term growth of primary MM and their interaction with whole BM microenvironment, namely normal bone marrow (NBM) system . Whole BM cells from healthy donors (n=20) were cultured in medium supplemented with serum (10% v/v) pooled from MM patients for 7 days followed by coculture with CD138-selected primary MM cells (4:1 NBM:MM ratio) or MM cell lines (10:1 NBM:MM ratio) for ≥7 days. This NBM system is composed of adherent and non-adherent compartments. Adherent cells were mainly macrophages and mesenchymal stem cells (MSCs) whereas non-adherent cells contained typical hematopoietic cells including CD19+, CD3+, CD11b+ and CD33+ cells. Growth of MM cells was determined by CD45/CD38 flow cytometry and by bioluminescence of luciferase-expressing MM cells. MM cells or subset of MM cells from all patients (n=60) survived and grew in this system regardless of molecular risk or subtype, and MM growth was comparable to coculture with the supportive osteoclasts or MSCs. Adherent and non-adherent compartments supported MM cells which required patient’s serum for optimal growth. In 14 of 20 experiments, number of MM plasma cells, quantified by flow cytometry or bioluminescence analysis was increased by 58±12% (p<0.0005) in the NBM system and cell proliferation was evident by the loss of cell membrane PKH26 dye or by BudR uptake in dividing cocultured MM cells. Growth of OPM2, H929 and ARP1 lines was also stimulated in the NBM system which protected these cells from dexamethasone (1-2.5µM) but not bortezomib (0.01-5nM), while the effect of lenalidomide varied (0.1-5µM). For identifying secreted proteins that may mediate MM growth in the NBM system, supernatant were collected from serum-free culture of NBM, MM cells and NBM/MM coculture (18 hrs, n=3). Proteomics analysis performed on supernatant samples identified 1843 proteins. The clinical markers B2M and LDHA were present at high levels and were significantly higher by 2-2.4 folds in NBM/MM coculture compared to cultured NBM (p<0.04). Further filtration revealed 89 proteins that were significantly changed upon NBM/MM coculture but minimally detected in the MM cells culture: 14 were significantly lower and 75 were higher in NBM/MM cocultures compared to cultured NBM. These factors include mediators of extracellular matrix, immunity, and inflammation. A microenvironmental secreted factor that was not detected in the supernatant from MM cells or NBM but was secreted in cocultures was hemicentin-1 (HMCN1), a unique extracellular matrix protein directly involved in cytokinesis (Xu and Vogel, Curr Biol 2011) but has yet not been implicated in MM. Hemicentin-1 gene expression was detected in cultured NBM and MSCs but not in primary MM cells, MM lines or CD11b+ NBM cells. Induction of hemicentin-1 expression in MSCs after coculture with MM cells was validated by immunohistochemistry. Hemicentin-1 expression is higher in random bone biopsies from newly diagnosed MM patients (n=406) compared to donor biopsies (n=25, p<0.008) and highest in MM focal lesion biopsies (n=49, q<0.0005 vs. paired random bone biopsies). Higher baseline HMCN1 expression in biopsies was associated with inferior overall survival in TT3b clinical trial (p<0.027). The NBM system demonstrates the ability of primary MM plasma cells to interact with and to survive in coculture with healthy allogeneic adult BM through secretion of factors involved in immune evasion and extracellular matrix modification. Ongoing work is underway to unravel the role of hemicentin-1 in MM growth.