Supplementary Figure 1. Principle and quantification of the V-well adhesion assay of fluorescently labeled myeloma cells adapted by Weetall et al. 2001.
Supplementary Figure 2. Validation of image cytometric analysis of cell cycle in four INA-6 cultures.
Supplementary Figure 3. Cell cycle analysis of INA-6 pellets gained from V-Well Adhesion assay (Fig. 3).
Supplementary Figure 4. Representative (one of the four independent sample sets as seen in Supplementary Figure 3) curve fitting analysis of cell cycle profiles generated by Image Cytometry.
Supplementary Figure 5. Correlation of RNAseq with qPCR Left: Validation of RNAseq results (Fig. 4) with qPCR showing the log2(foldchange expression) of 18 genes.
Supplementary Figure 6. Functional enrichment analysis by Metascape using genes that are differentially expressed between MSC-interacting subpopulations.
Supplementary Figure 7. Expression levels of adhesion genes that are downregulated and associated with survival (p < 0.01). Bone Marrow Plasma Cell (BMPC), Monoclonal Gammopathy of Undetermined Significance (MGUS), Smoldering Multiple Myeloma (sMM), Multiple Myeloma (MM), Multiple Myeloma Relapse (MMR).
Supplementary Figure 8. Expression levels of adhesion genes that are not downregulated and associated with survival (p < 0.01).
Funding
Deutsche Forschungsgemeinschaft (DFG)
Deutsche Krebshilfe (German Cancer Aid)
Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)
Interdisziplinäres Zentrum für Klinische Forschung, Universitätsklinikum Würzburg (IZKF Würzburg)
ARTICLE ABSTRACT
Multiple myeloma involves early dissemination of malignant plasma cells across the bone marrow; however, the initial steps of dissemination remain unclear. Human bone marrow–derived mesenchymal stromal cells (hMSC) stimulate myeloma cell expansion (e.g., IL6) and simultaneously retain myeloma cells via chemokines (e.g., CXCL12) and adhesion factors. Hence, we hypothesized that the imbalance between cell division and retention drives dissemination. We present an in vitro model using primary hMSCs cocultured with INA-6 myeloma cells. Time-lapse microscopy revealed proliferation and attachment/detachment dynamics. Separation techniques (V-well adhesion assay and well plate sandwich centrifugation) were established to isolate MSC-interacting myeloma subpopulations that were characterized by RNA sequencing, cell viability, and apoptosis. Results were correlated with gene expression data (n = 837) and survival of patients with myeloma (n = 536). On dispersed hMSCs, INA-6 saturate hMSC surface before proliferating into large homotypic aggregates, from which single cells detached completely. On confluent hMSCs, aggregates were replaced by strong heterotypic hMSC–INA-6 interactions, which modulated apoptosis time dependently. Only INA-6 daughter cells (nMA-INA6) detached from hMSCs by cell division but sustained adherence to hMSC-adhering mother cells (MA-INA6). Isolated nMA-INA6 indicated hMSC autonomy through superior viability after IL6 withdrawal and upregulation of proliferation-related genes. MA-INA6 upregulated adhesion and retention factors (CXCL12), that, intriguingly, were highly expressed in myeloma samples from patients with longer overall and progression-free survival, but their expression decreased in relapsed myeloma samples. Altogether, in vitro dissemination of INA-6 is driven by detaching daughter cells after a cycle of hMSC-(re)attachment and proliferation, involving adhesion factors that represent a bone marrow–retentive phenotype with potential clinical relevance.
Novel methods describe in vitro dissemination of myeloma cells as detachment of daughter cells after cell division. Myeloma adhesion genes were identified that counteract in vitro detachment with potential clinical relevance.
