HA promotes cluster formation of CD44⁺MM cells under shear stress. (A) The indicated MM cells were cultured at 1 to 5 × 10⁵ cells/mL on a feeder layer of UBE6T-7 BMSCs with or without exposure to shear stress for 1 hour. We continuously rotated 25 cm² culture flasks at 60 rpm on a horizontal rotator to generate 10 dyne/cm² shear force, which is virtually identical to the force from blood flow in the BM.^32,33 Representative phase-contrast images of MM cells are shown. Bar represents 200 μm. (B) The indicated MM cell lines were prestained with green or red MitoTracker, as indicated, and cultured at a 1:1 ratio in the presence of the supernatant from UBE6T-7 cells for 1 hour under shear stress. Representative phase-contrast images and fluorescence images are shown in the upper and lower panels, respectively. Nuclei were counterstained with DAPI (blue). Bar represents 100 μm. (C) Flow cytometric analysis of the expression of adhesion molecules on MM cells. The means ± SD (bars) of 3 independent experiments are shown. Isotype/isotype-matched controls. (D) KMS12-BM and MM.1S cells were treated with 100 μg/mL of isotype-matched immunoglobulin (IgG) or specific antibodies against CD44, CD49d, CD54, or CD319/SLAMF7 in the presence of UBE6T-7 supernatants for 2 hours, followed by 1-hour culture under shear stress. Representative phase-contrast images are shown. Bar represents 200 μm. (E) KMM.1 cells transduced with an empty vector (KMM.1-mock) or CD44v10-expression vector (KMM.1-CD44v10) were cultured in the absence or presence of UBE6T-7 supernatant for 1 hour under shear stress. KMS12-BM and RPMI8226 cells were cultured under the same conditions as positive and negative controls, respectively. Cytospin specimens were stained with anti-CD44 antibody and biotin-conjugated HABP followed by staining with Alexa Fluor 488-conjugated anti-mouse IgG (green) and Alexa Fluor 594-conjugated streptavidin (red). Nuclei were counterstained with DAPI (blue). Bar represents 25 μm. (F) Left panel: KMM.1-mock or KMM.1-CD44v10 cells were treated with HA at the indicated concentrations for 1 hour without BMSC supernatants under exposure to shear stress. Representative phase-contrast images are shown. Bar represents 200 μm. Right panel: The means ± SD (bars) of the cell cluster sizes are shown (n = 10-20). (G) Mononuclear cells isolated from EMD lesions of patients with MM were cultured with phosphate-buffered saline (PBS) or 500 ng/mL of HA for 1 hour under shear stress. Left panel: Representative phase-contrast images. Bar represents 100 μm. Right panel: Cytospin specimens were stained with FITC-conjugated anti-CD138 antibody (green) and PE-conjugated anti-CD44 antibody (red) or biotin-conjugated HABP, followed by staining with Alexa Fluor 594-conjugated streptavidin (red) and DAPI (blue). Bar represents 25 μm. (H) Serum HA concentrations in normal healthy volunteers (Normal) (n = 10), patients with MM without EMD [EMD (−)] (n = 5), and patients with MM with EMD [EMD (+)] (n = 3) were measured with an enzyme-linked immunosorbent assay. Bars indicate the means of each group. ∗P values were determined by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test.