Figure 7. Adipocytes inhibit MM cell apoptosis and induce MM drug resistance in vivo.

SCID mouse femurs were injected with MM cells ARP-1 or MM.1S or co-injected with MM cells and GFP-labeled mature human adipocytes, and the mice were treated with or without melphalan (Mel). After treatment, BM cells from SCID mouse femurs were flushed out and labeled with anti-human CD138 antibody. The percentages of human CD138⁺ cells are shown in (A). The cells were further sorted by anti-human CD138-coated magnetic beads. An aliquot of CD138⁺ cells was examined with the annexin V-binding assay for apoptosis, and the percentages of apoptotic cells are shown in (B). An aliquot of CD138⁺ cells was stained with the MDC assay for autophagy, and the percentages of MDC⁺ cells are shown in (C). An aliquot of CD138⁺ cells isolated from the BM aspirates of ARP-1 tumor-bearing mice was examined with the Western blot analysis and the levels of phosphorylated (p) Stat3 are shown in (D). In addition, the aliquot of CD138⁻ cells was labeled with anti-GFP antibody to detect the injected GFP-carried adipocytes (E) or with anti-human aP2 antibodies to detect injected adipocytes (F), and counted by flow cytometry. (G) Concentration of human leptin or human adipsin in the serum of mouse injected with or without human adipocytes as measured by ELISA. (H) Representative images of immunohistochemical staining show the expression of perilipin (an adipocyte marker) and CD138 (a MM marker) in the BM of SCID mice bearing ARP-1 cells with mature adipocytes at week 2 or week 4 post cell injection. (I) Representative images of radiography show the osteolytic bone lesions in the femurs of the ARP-1 tumor-bearing mice four weeks post cell injection. The image from the mice without MM cells served as control. Red arrows indicate osteolytic lesions. In addition, MM ARP-1 cells with or without mature adipocytes (ADs) were subcutaneously injected into SCID mice. 4 weeks after the cell injection, mice were intraperitoneally injected with 50 μg/mouse of melphalan (Mel) or PBS (control), twice a week for 3 weeks. (J) Shown are tumor volume changes in the mice after the treatment. The results represent average values from three independent experiments of five mice per group. **P < 0.01.