Figure 7.
Targeting CD44 signaling overcomes cluster-mediated PI resistance in MM cells. (A) MM.1S cells were cultured with 500 ng/mL of HA under shear stress for 24 hours in the presence of GSI-XII or an MMP9/13 inhibitor at the indicated concentrations. Nuclear extracts were subjected to immunoblotting for the expression of Notch2-ICD, CD44-ICD, and histone H1 (internal control). (B) MM.1S cells were cultured with 500 ng/mL of HA under shear stress and treated with bortezomib in combination with GSI-XII or an MMP9/13 inhibitor for 72 hours. Dose-response curves of each combination were generated to make nonconstant normalized isobolograms at IC50 using CompuSyn software.15 The isobolograms shown are representative of at least 3 independent experiments. A combination index (CI) <1.0 indicates the synergism of the 2 drugs. (C) We subcutaneously inoculated 5 × 106 luciferase-expressing KMM.1-CD44v9 cells into the right thigh of NOD/SCID mice. When inoculated tumors were palpable (Day 1), mice were randomly assigned to 4 arms of treatment with intraperitoneal injection of vehicle alone (Control), 2 mg/kg of GSI-XII, 2 mg/kg of bortezomib, or GSI-XII plus bortezomib (GSI-XII first, followed by bortezomib) twice a week for 3 weeks. Left panel: Representative photographs of mice on Day 1 and Day 29 (original magnification, ×2). Right panel: Quantitative data of in vivo bioluminescence imaging on Day 1 and Day 29 expressed as photon units (photons/s). The means ± SD (bars) are shown. ∗P < .05 by one-way ANOVA with Tukey’s multiple comparison test (n = 4). (D) MOPC cells were cultured with bortezomib (20 nM) in the presence of vehicle (PBS) or 500 ng/mL of HA under shear stress along with the indicated concentrations of anti-CD44 antibody. Cell proliferation was assessed after 72 hours and is expressed as a percentage of the value for corresponding untreated cells. ∗P < .05 determined by one-way ANOVA with the Student-Newman-Keuls multiple comparison test (n = 3). (E) We intravenously injected 5 × 105 luciferase-expressing MOPC315.BM.Luc cells into BALB/c mice. Four weeks after transplantation, mice were injected with 50 μg/kg of HA and treated with intraperitoneal administration of isotype-matched immunoglobulin (IgG), anti-CD44 antibody (anti-CD44), IgG plus bortezomib (IgG + BTZ), or anti-CD44 antibody plus bortezomib (anti-CD44 + BTZ) (IgG or anti-CD44 antibody first, followed by bortezomib) twice a week for an additional 4 weeks. Kaplan-Meier survival curves are shown. P value was determined between the anti-CD44 + BTZ group and the IgG + BTZ group by the log-rank test. (F) Graphic abstract. See Results and Discussion for details.

Targeting CD44 signaling overcomes cluster-mediated PI resistance in MM cells. (A) MM.1S cells were cultured with 500 ng/mL of HA under shear stress for 24 hours in the presence of GSI-XII or an MMP9/13 inhibitor at the indicated concentrations. Nuclear extracts were subjected to immunoblotting for the expression of Notch2-ICD, CD44-ICD, and histone H1 (internal control). (B) MM.1S cells were cultured with 500 ng/mL of HA under shear stress and treated with bortezomib in combination with GSI-XII or an MMP9/13 inhibitor for 72 hours. Dose-response curves of each combination were generated to make nonconstant normalized isobolograms at IC50 using CompuSyn software.15 The isobolograms shown are representative of at least 3 independent experiments. A combination index (CI) <1.0 indicates the synergism of the 2 drugs. (C) We subcutaneously inoculated 5 × 106 luciferase-expressing KMM.1-CD44v9 cells into the right thigh of NOD/SCID mice. When inoculated tumors were palpable (Day 1), mice were randomly assigned to 4 arms of treatment with intraperitoneal injection of vehicle alone (Control), 2 mg/kg of GSI-XII, 2 mg/kg of bortezomib, or GSI-XII plus bortezomib (GSI-XII first, followed by bortezomib) twice a week for 3 weeks. Left panel: Representative photographs of mice on Day 1 and Day 29 (original magnification, ×2). Right panel: Quantitative data of in vivo bioluminescence imaging on Day 1 and Day 29 expressed as photon units (photons/s). The means ± SD (bars) are shown. ∗P < .05 by one-way ANOVA with Tukey’s multiple comparison test (n = 4). (D) MOPC cells were cultured with bortezomib (20 nM) in the presence of vehicle (PBS) or 500 ng/mL of HA under shear stress along with the indicated concentrations of anti-CD44 antibody. Cell proliferation was assessed after 72 hours and is expressed as a percentage of the value for corresponding untreated cells. ∗P < .05 determined by one-way ANOVA with the Student-Newman-Keuls multiple comparison test (n = 3). (E) We intravenously injected 5 × 105 luciferase-expressing MOPC315.BM.Luc cells into BALB/c mice. Four weeks after transplantation, mice were injected with 50 μg/kg of HA and treated with intraperitoneal administration of isotype-matched immunoglobulin (IgG), anti-CD44 antibody (anti-CD44), IgG plus bortezomib (IgG + BTZ), or anti-CD44 antibody plus bortezomib (anti-CD44 + BTZ) (IgG or anti-CD44 antibody first, followed by bortezomib) twice a week for an additional 4 weeks. Kaplan-Meier survival curves are shown. P value was determined between the anti-CD44 + BTZ group and the IgG + BTZ group by the log-rank test. (F) Graphic abstract. See Results and Discussion for details.

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