Disease-stage-dependent overexpression of TLR4 on MM MSCs modulated pro-MM factors implicated in crosstalk with MM cells. Fresh BM aspirates from HD MSCs and MM MSCs were analyzed at the stage of monoclonal gammopathy of undetermined significance (MGUS MSC), diagnosis (D MSC), complete remission (CR MSC), or early relapse (ER MSC) in the Institut Universitaire du Cancer de Toulouse-Oncopole (Toulouse). (A) TLR4 mRNA expression in BM MSCs from HD (n = 7), MGUS (n = 5), and MM (n = 7). Data are from U133+ 2.0 microarrays and previously described.⁸  Each point represents 1 sample; the horizontal bar is the mean. (B) Representative flow cytometry and graph showing the expression of isotype (dotted line) or TLR4 (solid line) in HD (Red) (n = 16) and MM (Blue) (n = 6) MSCs after primoculture (P1). Data are mean±SEM presented as rMFI = ratio of mean fluorescence intensity (MFI) of staining/MFI isotype control. Each point represents 1 sample. (C) Heatmap showing the fold change (pseudocolor scale with red for upregulation and blue for downregulation) of TLR4 expression in MSCs isolated from patients in CR, ER, D, or total MM MSCs (left) in comparison with MSCs isolated from D and HD MSCs, respectively (bottom). (D) Percentage of cells expressing TLR4 from ER (n = 6) and CR (n = 3) MM MSCs after primoculture (P1). Data are mean±SEM. Each point represents 1 sample. (E) CD49d, CD49e, CD54, and CD106 expression in HD (Red) and MM MSCs (Blue) after 1-hour stimulation with 1 µg/mL lipopolysaccharide (LPS) or not (untreated), a wash, and 2-day culture at 37°C 5% CO2. Data are mean±SEM of rMFI from 5 independent experiments. (F) IL-6 secretion by HD (Red, n = 3) and MM (Blue, n = 3) MSCs after 1-hour stimulation with 1 µg/mL LPS or not (untreated), a wash, and 2-day culture at 37°C 5% CO2. Data were analyzed by ELISA with a Varioskan scanning reader and represented as mean±SEM. (G) Heat shock protein 70 (Hsp-70) secretion by MM cell line (Green, MOLP-6), HD (Red), and MM MSCs (Blue) in normal culture condition. Data were analyzed by ELISA and represented as mean±SEM from 5 independent experiments. (H,I) CD54 expression (H) and IL-6 secretion (I) by HD and MM MSCs after 48-hour stimulation with human recombinant Hsp70 (1 µg/mL) or not (untreated) at 37°C 5% CO2. Data are mean±SEM of CD54 rMFI or IL-6 (pg/cell) secretion from 10 independent experiments. (J,K) Stroma-dependent (MOLP-6) (J) or stroma-independent MM cells (MM1S) (K) cell count after 7 days of coculture with HD or MM MSCs or without coculture. MSCs or MM cells (control) were untreated or treated with 1 µM TLR4 antagonist (C34). The number of MM cells was evaluated after staining with trypan blue and evaluated in a Malassez counting chamber on day 7. Data are mean±SEM from 3 independent experiments. (L) Stroma-dependent (MOLP-6) cell count after 7 days of coculture with C34 treated or not treated (untreated) HD or MM MSC, in presence or absence of human recombinant IL-6. Data are mean±SEM from 6 independent experiments. Statistical differences between 2 groups were determined by Mann-Whitney test (A,B,C,D), paired (E,F,H,I,J,K,L), or unpaired (F, G) t test. *P < .05, **P < .01, ***P < .001. ns, not significant. For the Figure 1F, paired t test is used to compare the HD or MM MSC untreated or treated with LPS, and unpaired t test is used for comparison between HD MSC and MM MSC.