TLR4 inhibition potentiated anti-MM drug effect in MOLP-6-MSC coculture and decreased Vk*MYC mouse development. (A,B) Percentage of MOLP-6 cell inhibition after 7 days of coculture with HD or MM MSCs or without coculture. MSCs were untreated or treated with 1 µM C34, then MOLP-6 cells (5.10⁴) were added to each flask with 5 mL complete RPMI medium with or without 100 nM melphalan (A) or lenalidomide (B). For both cocultures, after 72 hours, 2 mL complete RPMI medium ± 1 µM C34 ± 100 nM melphalan or lenalidomide was added, respectively. Data are mean±SEM of percent inhibition calculated as 1-(number cocultured MOLP-6 ± melphalan or lenalidomide ± C34 at day 7/number untreated cocultured MOLP-6 at day 7) from 5 independent experiments. (C-I) C57Bl/6 WT mice were injected with Vk12653 myeloma cell line (2 × 10⁶ cells, iv). Ten days later, they were treated with 1 mg/kg C34 (C34 group, n = 14) or phosphate-buffered saline (PBS group, n = 14), twice a week for 2 weeks. Then, blood samples were collected and serum was harvested for protein electrophoresis. After treatment, the femur was harvested and flushed to extract BM. As a control, C34 effect on BM from WT mice is shown in supplemental Figure S2. Erythrocytes were lysed, and BM was stained with mouse antibodies as described in supplemental Table 1. (C) Schematic representation of the experimental design. (D) Representative electrophoresis and graph showing the presence of monoclonal Ig in the blood of PBS (Red) and C34 (Blue) groups after 2-week treatment. According to the manufacturer, serum proteins are separated into 6 major fractions (albumin, α-1, α-2, β-1, β-2, γ), and we detected and quantified monoclonal components for the diagnosis. Data are mean±SEM. Each point represents 1 sample. (E) Representative FACS plots showing the gating strategy to isolate MSCs. Viable cells were separated into 2 groups: plasma cells gated with double-positive CD155+/CD138+ cells and microenvironment CD155-cells. In this microenvironment, Lin-(B220, CD3e, Ter119, Gr1, CD11b), CD45.2-, CD31-, F4/80- allows for selecting MSCs with double-positive CD51+/Sca1+ cells. (F-H) Percentage of BM plasma cells (F) and BM MSCs (G) in C34-treated (Blue) or control (Red) groups among total BM cells. Data are mean±SEM from 3 independent experiments with n = 14 mice. (H) Correlation between BM plasma cell and BM MSC percentages. Each point represents 1 mouse. Pearson correlation coefficient, r = 0.7335. (I) Survival curve of Vk*MYC mice either untreated (PBS as control, Red) or treated by 10 mg/kg C34 (C34, Blue). Statistical differences between the 2 groups were determined with paired t test (A,B), Mann-Whitney test (C-G), and Gehan-Breslow-Wilcoxon test (I). *P < .05, **P < .01, ***P < .0001. ns, not significant.