Figure 1.
Figure 1. Direct cell-cell interaction between multiple myeloma cells and BMSCs causes strong de novo multidrug resistance. (A) Adherence to fibronectin (FN) induces modest CAM-DR. NCI-H929 (NCI) and OPM-2 myeloma cells were preincubated on FN-coated well plates for 24 hours and subsequently incubated for 24 hours either with 50 μM melphalan (Mel) or control medium (Co). Apoptosis was determined by annexin V (aV)/propidium iodide (PI) staining. (B) Strong reduction of cell death of all tested myeloma cell lines on adherence to HS-5 stromal cells. The 4 myeloma cell lines NCI-H929 (NCI), U266, RPMI-8226 (RPMI), and OPM-2 were treated with 30 μM melphalan for 48 hours in the absence or presence of HS-5 BMSCs. Myeloma cells were gated with CD38 or CD138 and cell death/apoptosis was determined by PI uptake. (C) Melphalan-induced apoptosis is strongly reduced in myeloma cells on adherence to HS-5 stromal cells. NCI-H929 myeloma cells (NCI) were treated with 5 μM melphalan for 2 days in the presence or absence of a confluent HS-5 stromal cell layer (suspension versus HS-5). Myeloma cells were gated with CD38 and cell death by PI uptake and apoptosis by aV-FITC binding were determined. (D) Cell adhesion induces multidrug resistance in multiple myeloma cells. NCI-H929 myeloma cells (NCI) were treated for 48 hours with 30 μM melphalan (Mel), 100 μM treosulfan (Treo), 10 μM doxorubicin (Doxo), 4 nM bortezomib (PS341), 3 μM dexamathasone (Dexa), or were incubated with medium alone (Co). Experiments were performed in the absence or presence of a confluent HS-5 layer (suspension versus HS-5). CD38+ cells were gated and PI uptake was determined by flow cytometry. (E) Primary BMSCs from patients induce strong CAM-DR. NCI-H929 cells (NCI) were treated with 30 μM melphalan (Mel) for 48 hours in the absence or presence of primary hBMSC cultures (suspension versus hBMSCs). CD38+ cells were gated and PI uptake was determined. The mean values and SDs of 3 experiments with hBMSCs from 3 consecutive patients are shown. (F) Continuous presence of BMSCs is required for CAM-DR. NCI-H929 (NCI) myeloma cells were treated for 48 hours with 30 μM melphalan. Myeloma cells were coincubated with a confluent HS-5 monolayer over the last 8 (8 h), 24 (24 h), or 36 (36 h) hours of the 48-hour incubation period. Myeloma cells were gated with CD38 and cell death was determined by flow cytometry (PI). (G) CAM-DR is dependent on direct cell-cell contact. NCI-H929 (NCI) myeloma cells were incubated for 4 days with 5 μM melphalan (Mel) either in the absence of stromal cells (suspension), or on a confluent HS-5 monolayer (HS-5), or in the same well with a confluent HS-5 monolayer but separated by cell culture inserts (HS-5 + inserts). Myeloma cells were gated with CD38 and the number of PI-positive cells was determined. (H) Conditioned medium does not overcome CAM-DR. NCI-H929 myeloma cells (NCI) were treated for 2 days with 30 μM melphalan (Mel). Co indicates untreated control. The standard RPMI/FCS medium was mixed with conditioned medium from confluent growing HS-5 cells (HS-5 medium) or from HS-5/NCI-929 coculture (HS-5/NCI medium) in the indicated ratios (50%, 75%, 100%). PI positivity and aV-FITC binding of myeloma cells was determined by flow cytometry. (I) The adhesion molecules ICAM-1 and VCAM are up-regulated on HS-5 BMSCs on melphalan and treosulfan treatment. HS-5 BMSCs were treated with increasing concentrations of melphalan (Mel) and treosulfan (Treo) for 48 hours. Surface expression of ICAM-1 and VCAM was determined by flow cytometry. Dose-dependent up-regulation was observed. The flow cytometry analysis for 10 μM (gray area) in comparison to untreated control (black line) is shown. Mean values with standard deviations and P values are shown in the figure. n.s. indicates not significant.

Direct cell-cell interaction between multiple myeloma cells and BMSCs causes strong de novo multidrug resistance. (A) Adherence to fibronectin (FN) induces modest CAM-DR. NCI-H929 (NCI) and OPM-2 myeloma cells were preincubated on FN-coated well plates for 24 hours and subsequently incubated for 24 hours either with 50 μM melphalan (Mel) or control medium (Co). Apoptosis was determined by annexin V (aV)/propidium iodide (PI) staining. (B) Strong reduction of cell death of all tested myeloma cell lines on adherence to HS-5 stromal cells. The 4 myeloma cell lines NCI-H929 (NCI), U266, RPMI-8226 (RPMI), and OPM-2 were treated with 30 μM melphalan for 48 hours in the absence or presence of HS-5 BMSCs. Myeloma cells were gated with CD38 or CD138 and cell death/apoptosis was determined by PI uptake. (C) Melphalan-induced apoptosis is strongly reduced in myeloma cells on adherence to HS-5 stromal cells. NCI-H929 myeloma cells (NCI) were treated with 5 μM melphalan for 2 days in the presence or absence of a confluent HS-5 stromal cell layer (suspension versus HS-5). Myeloma cells were gated with CD38 and cell death by PI uptake and apoptosis by aV-FITC binding were determined. (D) Cell adhesion induces multidrug resistance in multiple myeloma cells. NCI-H929 myeloma cells (NCI) were treated for 48 hours with 30 μM melphalan (Mel), 100 μM treosulfan (Treo), 10 μM doxorubicin (Doxo), 4 nM bortezomib (PS341), 3 μM dexamathasone (Dexa), or were incubated with medium alone (Co). Experiments were performed in the absence or presence of a confluent HS-5 layer (suspension versus HS-5). CD38+ cells were gated and PI uptake was determined by flow cytometry. (E) Primary BMSCs from patients induce strong CAM-DR. NCI-H929 cells (NCI) were treated with 30 μM melphalan (Mel) for 48 hours in the absence or presence of primary hBMSC cultures (suspension versus hBMSCs). CD38+ cells were gated and PI uptake was determined. The mean values and SDs of 3 experiments with hBMSCs from 3 consecutive patients are shown. (F) Continuous presence of BMSCs is required for CAM-DR. NCI-H929 (NCI) myeloma cells were treated for 48 hours with 30 μM melphalan. Myeloma cells were coincubated with a confluent HS-5 monolayer over the last 8 (8 h), 24 (24 h), or 36 (36 h) hours of the 48-hour incubation period. Myeloma cells were gated with CD38 and cell death was determined by flow cytometry (PI). (G) CAM-DR is dependent on direct cell-cell contact. NCI-H929 (NCI) myeloma cells were incubated for 4 days with 5 μM melphalan (Mel) either in the absence of stromal cells (suspension), or on a confluent HS-5 monolayer (HS-5), or in the same well with a confluent HS-5 monolayer but separated by cell culture inserts (HS-5 + inserts). Myeloma cells were gated with CD38 and the number of PI-positive cells was determined. (H) Conditioned medium does not overcome CAM-DR. NCI-H929 myeloma cells (NCI) were treated for 2 days with 30 μM melphalan (Mel). Co indicates untreated control. The standard RPMI/FCS medium was mixed with conditioned medium from confluent growing HS-5 cells (HS-5 medium) or from HS-5/NCI-929 coculture (HS-5/NCI medium) in the indicated ratios (50%, 75%, 100%). PI positivity and aV-FITC binding of myeloma cells was determined by flow cytometry. (I) The adhesion molecules ICAM-1 and VCAM are up-regulated on HS-5 BMSCs on melphalan and treosulfan treatment. HS-5 BMSCs were treated with increasing concentrations of melphalan (Mel) and treosulfan (Treo) for 48 hours. Surface expression of ICAM-1 and VCAM was determined by flow cytometry. Dose-dependent up-regulation was observed. The flow cytometry analysis for 10 μM (gray area) in comparison to untreated control (black line) is shown. Mean values with standard deviations and P values are shown in the figure. n.s. indicates not significant.

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