Nonadherent Spheres With Multiple Myeloma Surface Markers Contain Cells that Contribute to Sphere Formation and Are Capable of Internalizing Extracellular Double-Stranded DNA.

Abstract

The most prominent features of cancer stem cells are asymmetric cell division, tumorigenicity, and clonogenicity. Recently one more feature of poorly differentiated cell types of various origin, including cancer stem cells, has been described. Namely, these cells can internalize extracellular DNA natively, without additional transfection procedures. Using our approach to trace internalization of a TAMRA (carboxy tetramethyl-rhodamine [fluorescent dye])-DNA labeled probe by poorly differentiated cell types, we isolated and characterized the cells from free-floating spheres derived from the bone marrow clonogenic aspirate of a multiple myeloma patient. Nonadherent spheres display a B-cell phenotype (CD73/CD20+/CD45+/CD19dim). Further, free-floating spheres contain 1% to 3% cells with a clonogenic potential, and these cells display a marker of poorly differentiated cell types (TAMRA+). Upon association with a group of ∼ 10 free-floating TAMRA- cells, this peculiar cell type forms a sphere-forming cluster that initiates secondary aggregation of cells into a spheric structure. TAMRA+ and TAMRA- cells secrete distinct sets of cytokines indicative of the paracrine regulation. Grafting experiments of intact whole spheres versus cell suspensions prepared from dispersed spheres indicate that successful engraftment only occurs in the former case. Nonadherent 3-D cell colonies (spheres) encompass B cells with CD73/CD20+/CD45+/CD19dim phenotype, as well as double-stranded DNA-internalizing cells. The latter cell type appears to function as a sphere-forming center. Different cells in the spheres communicate with each other by secreting specific sets of cytokines. For successful engraftment and tumor growth in mice, intact spheres containing ∼ 106 cells must be used.