Characterization of myeloma (MM) stromal cells. (A) Stromal cells from patients with MM or healthy subjects were cocultured with the MM1.S or ANBL-6 MM cell line for 3 days. MM cell growth was determined by counting MM cells after removing the MM cells by vigorously pipeting. *P < .05 compared with normal. (B) The levels of VCAM-1 expression in primary normal and myeloma-derived stromal cells from 13 MM patients and 11 healthy controls were determined by Western blot analysis as described in “Methods.” The ratio of VCAM-1 to β-actin was compared by densitometry. Results are presented as mean (± SEM). *P < .05 compared with normals. (C) IL-6 production in the same samples was determined by ELISA. (D) FACS analysis of MM and normal stromal cells. Bone marrow mononuclear cells from patients and healthy subjects were cultured at 10⁶/mL in IMDM plus 10% FCS. After 3 days in culture, nonadherent cells were removed, and the media was replaced. The cultures were fed 2 times a week until confluence or 21 days. Stromal cells were detached using trypsin and counted using trypan blue exclusion, and the presence of residual MM cells determined by CD138 staining. The stromal cells were then cultured at 10³ cells/cm² under the same conditions for 3 days and used for each experiment. Cells (10⁴) were incubated with the specific antibody for 15 minutes, and the percentage of positive cells were determined by FACS. Similar FACS results were seen in experiments using stromal cells from 3 different patients and healthy subjects.