FIGURE 4 from Modeling Myeloma Dissemination <i>In Vitro</i> with hMSC-interacting Subpopulations of INA-6 Cells and Their Aggregation/Detachment Dynamics

Separation and gene expression of INA-6 subpopulations. A, Schematic of WPSC separating nMA-INA6 from MA-INA6. A coculture 96-well plate is turned upside down and attached on top of a “catching plate,” forming a “well-plate sandwich.” nMA-INA6 cells are collected in the catching plate by subsequent rounds of centrifugation and gentle washing. MA-INA6 are enzymatically dissociated from hMSCs or by rough pipetting. Subsequent RNA-seq of MSC-interacting subpopulations reveals distinct expression clusters [right, multidimensional scaling plot (n = 5)]. B, Separation was microscopically tracked after each centrifugation step. C–E, qRT-PCR of genes derived from RNA-seq results. Expression was normalized to the median of CM-INA6. Samples include those used for RNA-seq and six further cocultures (n = 11; non-detects were discarded). C, Adhesion factors, ECM proteins and matrix metalloproteinases. D, Factors involved in bone remodeling and bone homing chemokines. E, Factors involved in (immune) signaling. Statistics: (C–E): Paired t-test. Datapoints represent the mean of three (B–E) technical replicates. INA-6 were isolated from independent cocultures with hMSCs from 5 (A, B), 9 (C--E) unique donors.