The administration of HA facilitates EMD development in a syngeneic murine MM model. (A) MOPC315.BM-Luc cells were cultured at 1 × 105 cells/mL with various concentrations of HA under shear stress for 1 hour. Left panel: Representative phase-contrast images at the indicated concentrations of HA. Bar represents 200 μm. Right panel: The means ± SD (bars) of cell cluster sizes (n = 8-20). (B) We intravenously injected 5 × 105 luciferase-expressing MOPC315.BM.Luc cells or vehicle alone (PBS) into BALB/c mice, started the administration of the vehicle (PBS) or 50 μg/kg of HA 4 weeks after transplantation, and continued the treatments twice a week for an additional 4 weeks. Kaplan-Meier survival curves of the PBS group (PBS-injected and PBS-administered mice) (n = 5), the MOPC + PBS group (MOPC315.BM.Luc-transplanted and PBS-administered mice) (n = 9), the HA group (PBS-injected and HA-administered mice) (n = 5), and the MOPC + HA group (MOPC315.BM.Luc-transplanted and HA-administered mice) (n = 10). P values and the hazard ratio (HR) were determined between the MOPC + PBS group and the MOPC + HA group by the log-rank test. (C) The numbers of EMD lesions in each mouse were counted. The P value was determined by Student t test. (D) Representative photographs of whole-body ventral images obtained by the IVIS Imaging System on Day 63 (indicated by an arrow in B) before and after euthanasia in the left and right panels, respectively (original magnification, ×2). Luciferase-expressing soft tissue tumors are circled and numbered. (E) Representative photographs and ex vivo images of soft tissue tumors, designated #1 to approximately #4 in D, spleen, and liver resected from the PBS-1 and HA-4 mice (original magnification, ×2). See supplemental Figure 3D for all mice and supplemental Figure 3F for quantification. (F) Spleen, liver, soft tissue tumors, and (G) BM sections were obtained from mice in D and stained with hematoxylin-eosin (HE) or with FITC-conjugated anti-human CD138 antibody (green) and biotin-conjugated HABP, followed by the staining with Alexa Fluor 584-conjugated streptavidin (red) and DAPI (blue).
