Figure 3.
CD44 is required for HA-induced cluster formation in vitro and EMD development in a mouse MM model. (A) We established MOPC315.BM-Luc sublines, in which CD44 was deleted using the CRISPR/Cas9 system (CD44-KO) or an empty vector was stably transfected (Mock). Mock and CD44-KO 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 of MM cells. Bar represents 100 μm. Right panel: The means ± SD (bars) of the size of cell clusters (n = 8-20). (B) We intravenously injected 5 × 105 CD44-KO cells 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 (n = 8) and the HA group (n = 8). P values were determined 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) MOPC315.BM-Luc cells were cultured with the indicated concentrations of the anti-CD44 antibody Hermes-1 in the presence of HA under shear stress for 1 hour. The means ± SD (bars) of the size of cell clusters are shown (n = 8-20). ∗P < .05 by one-way ANOVA with Tukey’s multiple comparison test. (E) MOPC315.BM-Luc cells were cultured with the indicated concentrations of control IgG or Hermes-1 (anti-CD44). Cell proliferation was assessed after 72 hours and is expressed as a percentage of the values for corresponding untreated cells. The means ± SD (bars) of 3 independent experiments are shown. (F) We intravenously transplanted 5 × 105 MOPC315.BM.Luc cells into BALB/c mice. Beginning 4 weeks after transplantation, mice were administered 10 mg/kg of isotype-matched immunoglobulin (IgG) or the anti-CD44 antibody Hermes-1 (Anti-CD44) along with 50 μg/kg HA twice a week for an additional 4 weeks.38 Representative overlay photographs taken by the IVIS-CT Imaging System on Day 70 are shown with the location of the intramedullary tumors, splenic tumors, and EMD lesions. (G) The numbers of EMD lesions in each mouse were counted. The P value was determined by Student t test.

CD44 is required for HA-induced cluster formation in vitro and EMD development in a mouse MM model. (A) We established MOPC315.BM-Luc sublines, in which CD44 was deleted using the CRISPR/Cas9 system (CD44-KO) or an empty vector was stably transfected (Mock). Mock and CD44-KO 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 of MM cells. Bar represents 100 μm. Right panel: The means ± SD (bars) of the size of cell clusters (n = 8-20). (B) We intravenously injected 5 × 105 CD44-KO cells 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 (n = 8) and the HA group (n = 8). P values were determined 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) MOPC315.BM-Luc cells were cultured with the indicated concentrations of the anti-CD44 antibody Hermes-1 in the presence of HA under shear stress for 1 hour. The means ± SD (bars) of the size of cell clusters are shown (n = 8-20). ∗P < .05 by one-way ANOVA with Tukey’s multiple comparison test. (E) MOPC315.BM-Luc cells were cultured with the indicated concentrations of control IgG or Hermes-1 (anti-CD44). Cell proliferation was assessed after 72 hours and is expressed as a percentage of the values for corresponding untreated cells. The means ± SD (bars) of 3 independent experiments are shown. (F) We intravenously transplanted 5 × 105 MOPC315.BM.Luc cells into BALB/c mice. Beginning 4 weeks after transplantation, mice were administered 10 mg/kg of isotype-matched immunoglobulin (IgG) or the anti-CD44 antibody Hermes-1 (Anti-CD44) along with 50 μg/kg HA twice a week for an additional 4 weeks.38 Representative overlay photographs taken by the IVIS-CT Imaging System on Day 70 are shown with the location of the intramedullary tumors, splenic tumors, and EMD lesions. (G) The numbers of EMD lesions in each mouse were counted. The P value was determined by Student t test.

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