Establishment of An HS23 Stromal Cell-Dependent Myeloma Cell Line: Fibronectin and IL-6 Are Critical for Survival

Abstract 2949

Adhesion of multiple myeloma (MM) cells to bone marrow stromal cells triggers cytokine-mediated tumor cell growth, survival, and drug resistance. In particular, integrin a₄b₁, very late antigen 4 (VLA-4)-mediated fibronectin adhesion confers a survival advantage to myeloma cells. One of the problems in treating patients with MM is that it is very hard to eliminate residual myeloma cells, even following high-dose chemotherapy followed by auto-stem cell transplantation. Importantly, cell adhesion-mediated drug resistance (CAM-DR) must be overcome in order to eliminate the minimal residual disease of MM. Here we characterized a multiple myeloma cell line, MSG1, which depends on HS23 stromal cells for its survival, was established from the pleural effusion of a patient with MM who expressed the M-protein of IgA-λ in his serum. During the first two months of culture, the myeloma cells survived on adhesive cells from the pleural effusion and subsequently, they continued to proliferate on HS23 stromal cells but not on HS27A stromal cells (both stromal cells were established by Torok-Storb B, Blood 1995). The phenotype of the established MSG1 cell line was: CD138⁺, CD38⁺⁺, CD19⁻, CD56⁻, VLA-4⁺, VEGFR1⁺, and VEGFR2⁺. Furthermore, immunohistochemical staining demonstrated expression of IgA and λ chain in the cytoplasm. Karyotype analysis indicated complex chromosomal abnormalities, basically hypertriploidy including the deletion of chromosome 13 and 17, and c-myc translocation. MSG1 cells continued to proliferate, not only when co-cultured with HS23 cells, but also when cultured on fibronectin-coated plates with the supernatant of HS23 cells or RPMI1640 medium supplemented with 10% FBS (control medium) containing IL-6 (10 ng/ml). Notably, MSG1 could not survive in control medium containing IL-6 or in HS23 supernatant unless bound to fibronectin, which was also expressed on HS23 and HS27A cells. IL-6 and VEGF production were detected in the supernatants of both HS23 and HS27A stromal cells (36.8±4.5 pg/ml and 131±5.8 pg/ml; 13.2±1.9 pg/ml and 16664±418 pg/ml, respectively). Next, we analyzed the effect of tocilizumab, an anti-IL-6R antibody, and bevacizumab, an anti-VEGF antibody on MSG1 survival. Tocilizumab (50 μ g/ml) inhibited MSG1 survival when cultured on fibronectin-coated plates in control medium containing IL-6 (10 ng/ml), and tocilizumab (10 μ g/ml) inhibited MSG1 survival when cultured on HS23 stromal cells. However, bevacizumab (500 μ g/ml) did not show such inhibition. Therefore, MSG1 survival depends on HS23 stromal cells: in other words, it depends on binding to fibronectin and IL-6. If these factors induced CAM-DR in myeloma cells, MSG1 may be a unique myeloma cell line that will be useful for analysis of CAM-DR, and tocilizumab might be a useful drug for treatment of MM. Furthermore, since MSG1 could survive on irradiated HS27A cells, and since HS23 and HS27A express similar adhesion molecules (Torok-Storb B et al., Blood 1995), these data suggest that HS27A might secrete factors that are detrimental to MSG1 survival. The identification of such an inhibitory factors could be of interest in terms of the regulation of myeloma proliferation.