Myeloma Cancer Stem Cells Research Articles

The Investigation of Mechanisms Underlying Hoechst 33342 Efflux in Myeloma ‘Side Population' Cells.

Abstract 2759Poster Board II-735A subpopulation of cells with characteristics of cancer stem cells can be identified by the side population (SP) phenotype that is based on the efflux of the fluorescent dye Hoechst 33342 and detected by flow cytometric analysis. We previously reported the identification of SP cells in 4 human myeloma cell lines (HMCL) and 18 of 21 bone marrow samples from patients with myeloma in which the percentage of SP cells ranged from 0 to 4.9% (median=0.3; mean=0.8; SD=1.2). We now report the mechanism underlying the Hoechst 33342 efflux in SP cells, which may be a contributor to the drug resistance in myeloma cancer stem cells (MCSC). SP and main population (MP) subsets of HMCL (KMS-11 and RPMI-8226), based on Hoechst-33342 staining, were analyzed or sorted by BD FACSAria™ II using a blue and red dual-wavelength analysis after UV excitation. In this study, we examined the activities of three main drug transporter classes associated to SP phenotype – ABCB1 (MDR1), ABCG2 (BCRP1) and ABCC (MRP). Inhibition of the SP phenotype using PSC833, Ko143, and sulfinpyrazone (ABCB1, ABCG2, and ABCC inhibitors respectively) were first tested. Only the Ko143 and sulfinpyrazone were able to inhibit the entire SP phenotype in HMCL. Next, the activity of each transporter in unfractionated, sorted-SP and sorted-MP cells was examined. Relative cellular accumulation of fluorescent substrate for each transporter, namely rhodamine123 1 μg/mL, mitoxantrone 20 μM, and fluo-3 am 1 μg/mL, was determined. Experiments were conducted in the presence or absence of specific inhibitors. There were significant increases of mitoxantrone (P=0.0033** and P=0.0262*) and fluo-3 am (P=0.0006*** and P=0.0152*) in unfractionated RPMI-8226 and KMS-11 in the presence of Ko143 and sulfinpyrazone respectively. In the presence of sulfinpyrazone, benzbromarone, or dipyridamole (ABCC inhibitors), there was a significant increase of fluo-3 am in both SP and MP cells (P<0.05) sorted from KMS-11, however, only the sorted-SP cells showed significant increase of mitoxantrone in the presence of Ko143 (P=0.0462*) (Figure 1A). This suggests that while both ABCC and ABCG2 activity were significant in HMCL, the ABCC activity was similar in both SP and MP cells, whereas the ABCG2 activity was higher in the SP cells. The addition of PSC833 did not increase the accumulation of rhodamine 123 in the unfractionated, sorted-SP, or sorted-MP cells. This confirmed that there is negligible ABCB1 activity in HMCL. Consistent with the functional assay, western blot results revealed no ABCB1 expression in both SP and MP cells from KMS-11. There was a moderate ABCC1 expression in both fractions, and more ABCG2 in SP than MP cells (Figure 1B). This report suggests that while ABCG2 and ABCC activity are present in myeloma cells, ABCG2 is the main contributor to defining the SP by Hoechst efflux and perhaps the molecular determinant of MCSC. However, whether the stem cell related characteristic differences between SP and MP cells are solely because of these transporters is far from conclusive. Understanding these principles will allow investigators to develop therapeutic approaches that capitalize on the uniqueness of MCSC, thereby targeting the malignant cell and sparing normal cells. In conclusion, this report elucidates a distinct drug transporter expression in myeloma SP cells that could be a crucial target in cancer drug development and future clinical trials in patients with myeloma. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Multiple Myeloma Cancer Stem Cell Animal Model.

Abstract 1848Poster Board I-874Multiple myeloma (MM) is the second most common hematologic malignancy and characterized by clonal proliferation of CD138+ bone marrow plasma cells. Despite various treatment options few patients with MM have been cured. Furthermore, high relapse rates and recent evidence from xenogeneic transplantation models and primary MM marrow samples indicate that a rare population of cells or MM cancer stem cells (MM CSCs) within the marrow regenerates itself and may be responsible for drug resistance. These MM CSCs are phenotypically similar to memory B cells (CD138- CD34-CD19+) but differ in that they have the capacity to regenerate themselves or self-renewal. However, most of the reports on MM CSC animal models are established in NOD/SCID mice that require a larger number (1 – 10 × 106) of bead sorted cells for each animal. In addition, the latency of MM induction (4 – 6 months) in NOD/SCID mouse models and lack of in vivo tracking of the malignant clone preclude robust pre-clinical testing of novel therapeutic strategies that target MM CSC.Mononuclear cells were isolated from autologous mobilized peripheral blood of at least four primary MM patients after Ficoll gradient centrifugation followed by immunomagnetic bead depletion of CD34+ and CD138+ cells and/or further sorted using a FACSAria. The CD138-CD34- population was transduced with lentiviral luciferase GFP (GLF) and transplanted (10,000 to 106 cells per mouse) intrahepatically into neonatal RAG2-/- gamma chain-/- (RAG2-/-gc-/-) mice. Engraftment was compared to mice transplanted with either CD34+ or CD138+ cells. Mice were imaged with an in vivo imaging system (IVIS) to detect bioluminescent engraftment. Results showed that a relatively rare CD138- CD27+ population, resembling memory B cells (∼1.2%), persists in MM autografts and can engraft immunocompromised mice more rapidly and effectively than the CD138+ (Lin+) population of mature plasma cells. This data supports the persistence of CSCs despite high dose chemotherapy further underscoring the need for CSC targeted therapy. Bioluminescence was detected in live mice transplanted with as little as 60,000 cells of CD138- CD34- population and as soon as 4 weeks after transplantation. FACS analysis of these mice demonstrated successful engraftment with the presence of CD45+ and CD138+ population in bone marrow, spleen and liver and bioluminescence was also detected in the secondary transplantation of cells from MMCSC primary engraftment demonstrating the self-renewal capacity of this rare CD138- CD27+ population. Our results suggest that by utilizing a lentiviral GFP-luciferase system in a highly immunocompromised mouse strain fewer cells will be required to monitor MM engraftment and perhaps hasten disease development. Further studies to confirm the expression of selected IgG genes from myeloma cells and to characterize the self-renewal capacity with genes involved in developmental signaling such as sonic hedgehog and wnt pathways are underway. Disclosures:Goff:Coronado Biosciences: Research Funding.

CD200 Is Preferentially Expressed in CD138 dim Multiple Myeloma Cells: Implications for a Myeloma Stem Cell Subpopulation with Potential Immune-Resistance.

Abstract 4877 IntroductionThe CD138negCD20+ cell population has been suggested as the putative multiple myeloma (MM) cancer stem cells, both in MM cell lines and in patient specimens. CD200 is an immunosuppressive membrane glycoprotein reported to be co-expressed with other stem-cell markers in prostate, breast, brain, and colon cancers. Also, CD200 is a negative prognostic factor for MM. It remains unknown whether CD200 is expressed in the CD138negCD20+ MM cell population, the putative MM cancer stem cells. Here we addressed this question by assessing CD200 expression in different subpopulations of MM cells according to their CD138 expression levels. MethodsTwo MM cell lines (RPMI8226 and NCI-H929) were co-stained with FITC-labeled anti-CD200, PE-labeled anti-CD20, and APC-labeled anti-CD138 antibodies for multicolor flow cytometry analysis. The cells were gated into 4 subpopulations (h1>h2>h3>h4 corresponding to CD138 expression levels: high>dim>low>negative). In each gated cell population, CD200 expression was assessed, and the CD200+ and CD200neg cell subpopulations were further gated for analysis of their CD20 expression levels. ResultsIn the RPMI 8226 cell line, the distribution of gated h1, h2, h3, and h4 populations was 87.51%, 5.86%, 5.26%, and 1.29%, respectively. In the h2 (CD138dim) population, 22.88% of the cells were CD200+. In contrast, CD200 was expressed at much lower levels in the other three populations, ranging from 7.28% (h1), 6.65% (h3), to 0.48% (h4). In CD200+ cells from the gated h1, h2, h3, and h4 population, CD20+ cells were 19.50%, 23.03%, 27.67%, and 18.75%, respectively, while in the CD200neg cells, CD20+ cells were 20.79%, 22.50%, 25.08%, and 28.02%, respectively. In the NCI-H929 cell line, 18.59% of the cells in the h2 population were CD200+, whereas only 1.61%, 1.69%, and 0.47% of the cells in the h1, h3, and h4 populations were CD200+. In each population, there were more CD20+ cells in CD200+ cells than in CD200neg cells, which were 24.52% vs 0.04% (h1), 41.94% vs 11.34% (h2), 11.11% vs 3.06% (h3), and 50.00% vs 2.25% (h4), respectively. ConclusionsThese results demonstrate that the immunosuppressive molecule CD200 is preferentially expressed in a CD138dim subpopulation of multiple myeloma cells. Depending on cell line, the putative myeloma stem cell marker CD20 is either preferentially, or not preferentially, expressed in the CD200+ cells, suggesting that an immune-resistant subpopulation of MM stem cells might exist, which may have important implications for designing immunotherapeutic approaches to treat this disease. DisclosuresGoldenberg: Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.

Multiple Myeloma Cancer Stem Cells

Multiple myeloma is characterized by the clonal expansion of neoplastic plasma cells within the bone marrow, elevated serum immunoglobulin, and osteolytic bone disease. The disease is highly responsive to a wide variety of anticancer treatments including conventional cytotoxic chemotherapy, corticosteroids, radiation therapy, and a growing number of agents with novel mechanisms of action. However, few if any patients are cured with these modalities and relapse remains a critical issue. A better understanding of clonogenic multiple myeloma cells is essential to ultimately improving long-term outcomes, but the nature of the cells responsible for myeloma regrowth and disease relapse is unclear. We review evidence that functional heterogeneity exists in multiple myeloma and discuss potential strategies and clinical implications of the stem-cell model of cancer in this disease.