Cell–cell homophilic interactions confer PI resistance to MM cells. (A) KMS12-BM cells were cultured with the indicated concentrations of bortezomib (BTZ), carfilzomib (CFZ), doxorubicin (ADR), 4-hydroxycyclophosphamide (OHCY), lenalidomide (Lena), or pomalidomide (Poma) in the presence of UBE6T-7 BMSC supernatants in the absence (None) or presence (Cell cluster) of shear stress exposure. Cell proliferation was assessed after 72 hours and is expressed as a percentage of the value for corresponding untreated cells without exposure to shear stress. ∗P < .05 determined by one-way ANOVA with the Student-Newman-Keuls multiple comparison test (n = 6-10). (B) The experiments described in A were carried out with non–cluster-forming RPMI8226 cells. (C) Mock-, CD44s-, CD44v6-, or CD44v9-transfected KMM.1 cells were cultured with suboptimal doses of bortezomib (BTZ) or carfilzomib (CFZ) along with 100 ng/mL of HA under shear stress. Cell proliferation was assessed after 72 hours and is expressed as a percentage of the value for corresponding drug-untreated cells. ∗P < .05 determined by one-way ANOVA with the Student-Newman-Keuls multiple comparison test (n = 6-10). (D) We subcutaneously inoculated 5 × 10⁶ luciferase-expressing KMM.1-mock (Mock) or KMM.1-CD44v9 (CD44v9) cells in the right thigh of NOD/SCID mice. Mice were intraperitoneally administered vehicle alone (PBS) or 2 mg/kg bortezomib (BTZ) twice a week for 3 weeks. Treatments were started when tumor-derived luciferase activity was measurable, defined as Day 1. Left panel: Representative photographs of mice on Day 1 and Day 29 (original magnification, ×2). Right panel: Quantitative data of in vivo bioluminescence 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). (E) MM.1S and KMS12-BM cells were cultured at 1 × 10⁵ cells/mL in the presence of UBE6T-7 supernatants with or without shear stress for 24 hours. Total RNA was isolated and subjected to microarray analysis for global changes in gene expression. The data have been deposited in the MIAME-compliant GEO database under accession number GSE189364. We selected the genes significantly downregulated (blue/sky blue) or upregulated (red/pink) in bortezomib-resistant primary MM cells in a previous study⁴⁰ (BCJ 2017) and compared their expression with that in MM.1S and KM12-BM cell clusters. The numbers indicate fold increases in the expression level of each gene in shear stress-induced cell clusters against nonclustered MM.1S and KM12-BM cells (P = .02 by Spearman's rank correlation).