Clonogenic multiple myeloma progenitors, stem cell properties, and drug resistance

Abstract

Many agents are active in multiple myeloma, but the majority of patients relapse. This clinical pattern suggests most cancer cells are eliminated, but cells with the clonogenic potential to mediate tumor regrowth are relatively chemoresistant. Our previous data suggested that CD138(+) multiple myeloma plasma cells cannot undergo long-term proliferation but rather arise from clonogenic CD138(neg) B cells. We compared the relative sensitivity of these distinct cell types to clinical antimyeloma agents and found that dexamethasone, lenadilomide, bortezomib, and 4-hydroxycyclophosphamide inhibited CD138(+) multiple myeloma plasma cells but had little effect on CD138(neg) precursors in vitro. We further characterized clonogenic multiple myeloma cells and stained cell lines using the Hoechst side population and Aldefluor assays. Each assay identified CD138(neg) cells suggesting that they possess high drug efflux capacity and intracellular drug detoxification activity. We also found that multiple myeloma cells expressing the memory B-cell markers CD20 and CD27 could give rise to clonogenic multiple myeloma growth in vitro and engraft immunodeficient nonobese diabetes/severe combined immunodeficient mice during both primary and secondary transplantation. Furthermore, both the side population and Aldefluor assays were capable of identifying circulating clonotypic memory B-cell populations within the peripheral blood of multiple myeloma patients. Our results suggest that circulating clonotypic B-cell populations represent multiple myeloma stem cells, and the relative drug resistance of these cells is mediated by processes that protect normal stem cells from toxic injury.

Figure 1

Multiple myeloma cellular subsets display differential drug sensitivity. Clonogenic recovery of CD138⁺ (open bars) or CD138^neg (black bars) cells from the multiple myeloma cell lines RPMI 8226 (A) and NCI-H929 (B) or CD138^neg (C) multiple myeloma progenitors derived from four distinct clinical samples after treated with dexamethasone (Dex), lenalidomide, bortezomib, or 4HC. Values represent the mean of four experiments.

Figure 2

Multiple myeloma precursors display stem cell characteristics. A, expression of CD138 by RPMI 8226 and NCI-H929 side population (SP) or nonside population (G1) cells labeled with Hoechst 33342 and antihuman CD138. Shaded histogram, staining with an isotype control antibody. B, relative MFI of Aldefluor by RPMI 8226 and NCI-H929 CD138⁺ (open bars) and CD138^neg (black bars) cells. Values are mean of four experiments. C, cell cycle profile of RPMI 8226 and NCI-H929 CD138⁺ and CD138^neg cells after propidium iodide staining.

Figure 3

Multiple myeloma progenitors resemble normal memory B cells and display properties typical of normal stem cells. A, relative colony formation by CD138^neg CD34^neg bone marrow mononuclear cells (CD138^neg) isolated from four patients with multiple myeloma after depletion of additional cells expressing CD20, CD27, or CD3. Columns, mean; bars, SE. B, clonogenic recovery of CD138⁺ (open bars) or CD138^neg (black bars) cells from the multiple myeloma cell lines RPMI 8226 and NCI-H929 after treatment with rituximab (Ritux), alemtuzumab (Alemtuz), and/or human complement (C’). Values represent means of 4 experiments. C, clonogenic recovery of CD138^neg multiple myeloma progenitors derived from primary clinical specimens after antibody treatment with (open bars) or without (black bars) human complement. D, engraftment of NOD/SCID mice with peripheral blood memory B cells derived from patients with multiple myeloma. Flow cytometric analysis of NOD/SCID mouse bone marrow cells for expression of human CD138 and intracellular Ig λ light chain (left) or CD19 and surface Ig λ light chain (right) after injection of peripheral blood memory B cells. E, comparison of capillary electorphoretic profiles of Ig heavy chain CDR3 amplification products (black arrow) obtained by PCR of CD138⁺ multiple myeloma plasma cells isolated from the primary clinical bone marrow specimen or from bone marrow cells collected from a mouse injected with memory B cells from the same patient. Open arrow, a control PCR reaction product.

Figure 4

Circulating multiple myeloma stem cells display properties typical of normal stem cells. A, expression of CD27 and surface Ig light chain expression by peripheral blood B cells with the side population or nonside population phenotype derived from a representative multiple myeloma patient. B, expression of CD27 and surface Ig light chain expression by peripheral blood B cells with high or low Aldefluor fluorescence derived from a representative multiple myeloma patient.