Healthy Donor Whole Bone Marrow Cells Preconditioned With Myeloma Patient Serum Support Long-Term Survival Of Primary Myeloma and Reveal Altered Microenvironmental Pathways

Primary human myeloma (MM) cells do not survive in culture while current in vitro and in vivo systems for growing these cells are limited to coculture with specific bone marrow (BM) cell type or growth in immunodeficient animal model. The aim of the study was to determine long-term survival and interaction of primary MM plasma cells with a healthy adult human BM that include immune cells capable of functional activation. This system is different from the autologous BM culture that is already affected by the disease. Whole BM cells from healthy donors were cultured in αMEM medium supplemented with 10% FBS and 10% serum pooled from MM patients. Following 7-9 days the cultures were composed of adherent and nonadherent cellular compartments. The nonadherent compartment contained typical BM hematopoietic cells such as monocytes, B and T lymphocytes and NK and normal plasma cells as assessed by flow cytometry, while the adherent compartment contained cells that morphologically resemble macrophages, osteoclasts, megakaryocytes and fibroblast-like cells. At this culture stage, CD138-selected MM cells from 20 patients were added to the BM cultures (4:1 BM:MM cell ratio) and survival and growth of MM cells were determined after 7 days by assessing proportion of CD45^{low/intermediate}/CD38^high MM plasma cells among total number of cells. MM and BM cell viability was constantly high (>90%) in cocultures. Subsets of primary MM plasma cells, regardless of molecular risk or subtype, were survived and detected in all cases while in 14 of 20 experiments, number of MM plasma cells was increased by 58±12% (p<0.0005, n=14). MM cell proliferation following long-term coculture was evident by the loss of cell membrane PKH26 dye or by BudR uptake in dividing cocultured MM cells. Growth of primary MM was superior in cocultures supplemented with patient serum compared to healthy donor serum. In additional study, we stably infected IL6- or stroma-dependent MM lines, or two primary MM cell cases capable of passaging in SCID-hu mice with EGFP/luciferase construct and demonstrated increased MM cell growth in all experiments in coculture using bioluminescence analysis (statistical significance range: p<0.04 to p<0.0003). Growth of OPM2 MM line was also enhanced in coculture compared to culture alone. The coculture conditions protected OPM2 cells from dexamethasone but not bortezomib while proportion of MM cell killing by lenalidomide was enhanced compared to culture of OPM2 cells alone. To assess the effect of MM cells on BM cells in coculture, global gene expression profile was performed on BM cells cultured alone or plasma cell-depleted BM after coculture with MM cells from 4 patients. Among the top underexpressed genes we identified immunoglobulin genes related to polyclonal plasma cells, extracellular factors associated with osteoblastogenesis (e.g. MGP, IGFBP2), WNT signaling (e.g. SOX4, LRP1, LRP6) and TGFb bioavailability (e.g. FBN1, LTBP1). Top upregulated genes include immuneregulatory factors such as PROK2, LRG1, OLFM4 and IL16, and cellular markers (e.g. ARG1 expressed by MDSCs). This culture system demonstrates the ability of primary MM cells to interact with and to survive in coculture with healthy adult BM that was first cultivated by patients' serum and is appropriate for studying MM-microenvironment interaction, characterization of MM cell subpopulations capable of long term survival and targeted therapy.