ESTABLISHMENT AND CHARACTERIZATION OF A HUMAN STROMA-DEPENDENT MYELOMA CELL-LINE (MM5.1) AND ITS STROMA-INDEPENDENT VARIANT (MM5.2)

Although IL-6 has been identified as a major growth factor in multiple myeloma (MM), it is believed that maintenance of tumor growth in vivo depends on one or more additional stroma-derived factors, We describe a new human myeloma cell line (MM5.1) that can be maintained in the p resence of bone marrow-derived stromal cell layers, and not only when cultured with exogeneous IL-6. This cell line expresses the same immun oglobulin kappa light chain RMA sequence as the patient's original tum or cells, has a plasma cell morphology and expresses plasma cell antig ens (cytoplasmic kappa light chain, CD38, BB4), Without the presence o f stromal factors, MM5.1 cells become apoptotic. A low proliferative e ffect was observed in the presence of oncostatin M (OSM) but other cyt okines (IL-10, IL-ll, stem cell factor (SCF) and leukemia inhibitory f actor (LIF)) had no effect at all, We observed that MM5.1 cells also g row when physically separated from stromal cell layers by a 0.45 mu m microporous membrane or when cultured in conditioned medium from strom al marrow cells, Unexpectedly, the growth in stromal supernatants was markedly inhibited by an anti-IL-6 antiserum and an anti-IL-6 receptor transducer chain (gp130) mAb in a dose-dependent manner. This implies that MM5.1 cells are IL-6 responsive only when exposed to one or more additional soluble factor(s) derived from bone marrow stroma. Cocultu ring MM5.1 cells with IL-S and cytokines that were described to increa se the IL-6 responsiveness of myeloma cells (G-CSF, GM-CSF and IL-3) h ad no effect on the growth or survival, A strong proliferative effect was observed when MM5.1 cells were cultured with IL-6 and soluble IL-6 receptor (sgp80). However no sgp80 could be detected in stromal super natants using a sensitive immunoassay. This indicates that sustained p roliferation of the MM5.1 cell line depends on a combination of IL6 an d at least one, thus far unidentified, stroma-derived factor, After mo re than 1 year in continuous culture, we could obtain a variant of the line (MM5.2) that shows an improved growth rate and grows stroma inde pendently, Molecular analysis revealed clonal identity with the early passage form and Epstein-Barr virus antigen expression was negative. T he two variants of this cell line offer a useful model to identify mol ecular mechanisms involved in clonal evolution towards stroma-independ ent growth of myeloma cells.