FIGURE 3 from Modeling Myeloma Dissemination <i>In Vitro</i> with hMSC-interacting Subpopulations of INA-6 Cells and Their Aggregation/Detachment Dynamics

Detachment of INA-6 daughter cells after cell division. A–D, INA-6 divisions in interaction with confluent hMSCs. Seeding ratio INA-6:MSC = 4:20. A, Three examples of dividing INA-6 cells generating either two MA, or one MA and one nMA daughter cells as described in G. Dashed circles mark mother cells (white), MA cell (blue), and first position of nMA cell (green). Scale bar: 20 µm. B, Cell division of MSC-adhering (MA) mother cell can yield one mobile non–MSC-adhering (nMA) daughter cell. C, Frequencies of INA-6 pairs defined in A and B per observed cell division. A total of 65 divisions were evaluated for each of three independent time-lapse recordings. D, Rolling duration of nMA cells after division did not depend on hMSC donor [H(2) = 5.250, P-unc = 0y.072]. Datapoints represent single nMA cells after division. E–G, Adhesive and cell cycle assessment of MSC-interacting INA-6 subpopulations using the V-Well assay. E, Schematic of V-Well Assay (see Supplementary Fig. S1 for detailed analysis). MSC-interacting subpopulations were separated by subsequent centrifugation and removal of the pellet. The pellet size was quantified by its total fluorescence brightness. Adhering subpopulations were resuspended by rough pipetting. F, Relative cell pellet sizes of adhesive INA-6 subpopulations that cycle either asynchronously or were synchronized at mitosis. Gray lines in-between points connect dependent measurements of cocultures (n = 9) that shared the same hMSC-donor and INA-6 culture. Cocultures were incubated for three different durations (1, 2, and 3 hours after INA-6 addition). Timepoints were pooled, since time did not show an effect on cell adhesion [F(2,4) = 1.414, P-unc = 0.343]. Factorial RM-ANOVA shows an interaction between cell cycle and the kind of adhesive subpopulation [F(1, 8) = 42.67, P-unc = 1.82e-04]. Technical replicates = 4 per datapoint. G, Cell cycles were profiled in cells gathered from the pellets of four independent cocultures (n = 4) and the frequency of G₀–G₁ cells are displayed depending on coculture duration (see Supplementary Fig. S3 for cell cycle profiles). Four technical replicates were pooled after pelleting. Statistics: D: Kruskal–Wallis H-test. F: Paired t test. G: Paired t test, two-factor RM-ANOVA. Datapoints represent INA-6 from independent cocultures with hMSCs from 3 unique donors.