An investigation into Extracellular Vesicles content and function within the Multiple Myeloma tumour microenvironment

Multiple myeloma (MM) is an incurable blood cancer that results from the monoclonal expansion of plasma cells in the bone marrow (BM). The progression of MM requires complex interactions between the cells and the bone marrow microenvironment (BME). These interactions are facilitated by extracellular vesicles (EVs) that transfer proteins and RNA between cells as a form of communication. The following study analysed the effects of MM EVs on monocyte cell function and tumour niche formation using an in vitro co-culture model to mimic the BME. In addition, a comprehensive multi-omic ex vivo analysis of MM patient-derived EVs isolated from BM and peripheral blood (PB) was performed. Overall, this research is critical for an improved understanding of MM EV function in the pathogenesis of MM as well as the identification of new therapeutic targets and disease biomarkers.
A novel in vitro co-culture model was established to study MM-EV uptake and function on immature monocyte cells and revealed differential effects that are dependent on the EV cell line of origin. Notably, H929 EVs were taken up by THP1 cells with greater efficiency than U266 and MM1s EVs. Furthermore, H929 EV uptake by THP1 cells correlated with a significant increase in the secretion of pro-inflammatory mediators IL-6 and MMP9 into the TME, which was only marginally detected following U266 EVs uptake and not detected following the uptake of MM1s EVs. Moreover, the conditioned media collected from THP1 cells following the uptake of H929 EVs enhanced the migration and proliferation of myeloma cells suggesting the formation of a metastatic niche. Proteomic analysis of MM-EVs derived from the three cell lines revealed significant differences in their protein cargo that could be correlated with phenotypic alterations within the BME. Specifically, pathway analysis highlighted potential pathophysiological mechanisms associated with pro-metastatic MM EVs, including multiple components of the spliceosome and its regulators that were highly enriched in H929 EVs compared to U266 and MM1s EVs.
Comprehensive multi-omic protein and miRNA analysis of plasma EVs isolated from the BM and PB of patients at various stages of the disease, from the pre-malignant MGUS to various stages of MM including active disease, remission and relapse was performed by mass spectrometry and RNA sequencing, respectively. Bioinformatic analysis of the data revealed significant dysregulation in MM EV protein and miRNA content compared to EVs from control and MGUS BM and PB EVs. The MM BM and PB EVs exhibit enrichment for several pro-tumourigenic proteins and miRNAs previously associated with MM or MM EVs, supporting their involvement in disease development and potential as diagnostic biomarkers. Furthermore, the unique enrichment of proteins and miRNAs could be correlated with the disease stage, highlighting their potential for disease monitoring. Finally, findings from the protein and miRNA analysis informed the development of MM PB EV protein and miRNA biomarker signatures with diagnostic and predictive power, and potential for clinical application as a non-invasive liquid biopsy. Overall, the results from this study contribute to our understanding of the role of EVs in MM cell communication and disease progression and provide novel EV biomarker signatures for MM diagnosis and monitoring.