MM cell clusters acquire PI resistance via activation of CD44 signaling. (A) We cultured MM.1S, KMM.1-mock and KMM.1-CD44v9 cells in the presence of 500 ng/mL of HA under shear stress for the indicated periods and isolated nuclear extracts for immunoblot analysis for the expression of Notch2-ICD, CD44-ICD and histone H1 (internal control). (B) Cytospin specimens were prepared during the experiments described in A for immunocytochemical staining with anti-CD44 and anti-Notch2 antibodies, followed by the staining with Alexa Fluor 488-conjugated anti-mouse IgG (green) and Alexa Fluor 594-conjugated anti-rabbit IgG (red). (C) Tumor sections were obtained from mice described in Figure 5D and stained with FITC-conjugated anti-CD138 (green) and anti-CD44 or anti-Notch2, followed by the staining with Alexa Fluor 594-conjugated anti-rabbit IgG (red). (D) Histological sections were prepared from soft tissue tumors and livers of MOPC-transplanted BALB/c mice (Figure 2F) and stained with FITC-conjugated anti-mouse CD138 (green) and anti-mouse CD44 or anti-mouse Notch2, followed by the staining with Alexa Fluor 594-conjugated anti-rabbit IgG (red). Nuclei were counterstained with DAPI (blue). Bar represents 15 μm. (E) We examined the correlation between the expression of CD44 and its effectors and the prognostic impact of CD44 using DNA microarray data from patients with MM treated with Total Therapy 2/3 (GEO accession number GSE4581) or bortezomib monotherapy⁵⁴ (BTZ monotherapy) with the GenomicScape tool (www.genomicscape.com). Upper and middle panels: Correlation between CD44 and NFkB1 or between Notch2 and HES1. Lower panels: Kaplan-Meier curves of patients with MM with high (red) and low (blue) CD44 expression. P values and the hazard ratio (HR) were determined by the log-rank test. (F) KMS12-BM and MM.1S cells were cultured with HA under shear stress for the indicated periods and subjected to quantitative reverse transcription-PCR for downstream CD44 effectors. Data were quantified by the 2^–ΔΔCt method using GAPDH as a reference gene and are shown as the fold increase against the values for Day 0. (G) Mock- or CD44-ICD-transfected KMM.1 cells were cultured with the indicated concentrations of bortezomib or carfilzomib. 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 = 6-10).