(A) Immunoblots of GFP-231 transduced with lentivirus control (-shC) or with 2 independent shWNT5A (1 and 2). β-Actin was used as loading control. Numbers below the blots represent the average fold change over GFP-231–shC. See complete unedited blots in the supplemental material. (B) Representative flow cytometry of indicated cells show that WNT5A shRNA reduces the percent of GFP⁺/DsRed⁺ hybrid cells. (C) A single GFP-231 and a single DsRed-MSC were loaded in the microfluidic cell-pairing device and treated with vehicle or anti-WNT5A over time. Bar graph shows percent of engulfment ± SEM (n = 10, in biological triplicate). Representative images illustrating that anti-WNT5A inhibited hybrid cell formation. Scale bar: 20 µm. (D) GFP-231 and MSC loaded in the microchip and, after 3 days, were treated with PTX 1 μM for 48 hours (n = 10 cells/condition, in biological triplicate). Scale bar: 20 µm. (E) ELISA for IL-6 protein secretion in supernatants of cocultures of DsRed-MSCs and GFP-231–shC or –shWNT5A after 72 hours. (F) ELISA for CCL2 protein secretion in the supernatants of DsRed-MSCs treated with vehicle (–) or with IL-6 recombinant protein (rh), 30 ng/mL. (G) Representative pHrodo and merge fluorescence images of GFP-231–shC treated with vehicle, tocilizumab 30 μg/mL, or CCR2 inhibitor (CCR2i) 40 μM, and GFP-231-shWNT5A treated with vehicle, IL-6 rh 30 ng/mL, and WNT5A rh 1 μg/mL before incubation with pHrodo-labeled MSCs for 48 hours. Scale bar: 50 µm. Bars quantify total red fluorescence normalized to cell number. pHrodo signal was quantified in 3 fields/condition using ImageJ. For all panels, data are expressed as individual values with mean ± SEM. In B, 1-way ANOVA with Turkey’s multiple comparison test was employed; in C–F, 2-tailed unpaired Student’s t test; and in G, 2-way ANOVA with Tukey’s multiple comparison test was employed. *P < 0.05, **P < 0.005, ***P < 0.0005; ****P < 0.0001.