Multiple Myeloma-Derived Circulating Extracellular Vesicles Affect Normal Human Stromal Cell Behaviour and Promote Tumor Progression: A Multi-Omic Approach

Background. Extracellular vesicles (EVs) are secreted in the extracellular space, can travel through the blood stream and are transferred to target (stromal) cells. This makes EVs an important means of cell-to-cell communication both at local and distant sites (metastasis). EVs can reprogram signalling pathways in stromal cells promoting the formation of a supportive tumour microenvironment (TME) and pre-metastatic sites/niches. The mechanisms that drive the formation of 'defined’ pre-metastatic sites and promote metastasis formation with associated cancer cells growth and drug resistance is still poorly understood in multiple myeloma (MM), an incurable blood cancer.

Aims. We aimed to: (1) demonstrate that circulating small (sEV) enriched from myeloma (MM) patients (MM-sEV) induce the formation of a TME favouring MM progression; (2) identify the protein content of MM-sEV that promotes this; (3) discover signalling drivers of EV-mediated functional remodelling of human stromal cells (HS5) towards a pre-metastatic phenotype.

Methods. sEV were enriched from blood plasma (1mL) using a commercial kit. We performed: proteomic profiling (nLC and high-resolution mass spectrometry, Orbitrap HF-X) of plasma-sEV derived from healthy donors (HD x10) and patients with MM (x8); proteomic and phosphoproteomic profiling and gene expression analysis by RNA sequencing of HS5-cells pre-treated with MM-sEV vs HD-sEV vs untreated cells; functional studies using a co-culture system (HS5:Human MM cell lines-HMCL).

Results. HS5-cells pre-treated with MM-sEV induced HMCL proliferation (p < 0.05) and drug resistance (p < 0.0001) to anti-MM drugs (proteasome inhibitors) when compared to untreated HS5-cells.

A total of 412 proteins were detected and quantified by proteomic profiling of plasma-sEV with 13 reported as highly enriched in EV marker databases (ExoCarta top 100) and 8/13 corresponding to universal cancer EV-markers proposed by Hoshino et al, Cell 2020. Gene ontology analysis of identified proteins (G:Profiler; p < 0.05) revealed enrichment for cellular component terms such as "extracellular vesicles/exosomes” and for several biological processes including "cell communication", "endocytosis", "cellular response to stimulus". Comparative analysis between our dataset and several publicly available datasets revealed sEV-markers with potential discriminatory specificity for MM. Comparative analysis revealed 40 proteins differentially regulated between HD-sEV and MM-sEV (P < 0.05; log2 fold change ≥2). A specific protein signature identified in MM-sEV was found in ≥30% of MM-sEV but ≤30% HD-sEV. These proteins were not found in human whole plasma (Lehallier et al, Nat medicine 2019) or solid tumors-derived sEV (Hoshino et al, Cell 2020; Vinik et al, Science Advances 2020).

Thousands of proteins and phosphosites were identified and quantified by proteomic profiling of HS5-cells pre-treated with MM-sEV vs HD-sEV. 210 proteins and 120 phosphosites were differentially expressed between MM-sEV and HD-psEV (>1.5-fold change, adj.P.Val < 0.05). Among the differentially expressed proteins were kinases, phosphatases, translation and transcription regulators, transporters, and enzymes.

Gene Set Enrichment Analysis (GSEA, FDR < 0.05) revealed 624 significant differentially expressed terms when comparing HS5-cells pre-treated with MM-sEV vs HD-sEV, including epidermal growth factor (EGF) signaling, tumor necrosis factor alpha (TNFA) signaling, nerve growth factor (NGF) stimulated transcription, epithelial to mesenchymal transition (EMT) signaling, transforming growth factor beta (TGFB) signaling.

Conclusions: In this first of its kind studies in MM we show that MM-sEV may play an important role in disease progression by re-programming the TME. Ongoing studies will indicate the value of MM-sEV as biomarkers and whether targeting the interactions between MM-sEV and stromal cells could enforce the current therapeutic strategies.

Spencer:Amgen: Consultancy, Honoraria; Haemalogix: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; BMS: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria.