P-209: Multiple myeloma-derived circulating extracellular vesicles affect normal human stromal cell behaviour and promote tumor progression: a multi-omic approach

Introduction: We showed that human stromal cells (HS5) treated with extracellular vesicles (EV) derived from plasma of myeloma (MM) patients (MM-EV) promoted adhesion of human MM cell lines (HMCL), with preliminary proteomic profiling of MM- vs healthy donors HD-EV revealing enrichment of factors implicated in cell migration and adhesion.

Aims: demonstrate that MM-EV induce the formation of a tumor microenvironment (TME) favouring MM progression; identify the protein content of MM-EV promoting this; discover signaling drivers of EV-mediated functional remodelling of HS5 towards a pre-metastatic phenotype.

Methods: EV were enriched from 1mL plasma using a commercial kit. We performed: proteomic profiling of EV [x10 HD, x8 MM, x4 asymptomatic MM, x10 premalignant stage MGUS]; phosphoproteomic profiling and gene expression analysis (RNA sequencing) of HS5 cells pre-treated with MM- vs HD/MGUS-EV; in vitro (co-cultures) and in vivo (NSG MM-bearing mice) studies.

Results: HS5 cells treated with MM-EV induced HMCL proliferation (p =.0026) and drug resistance (p =.0013) to anti-MM drugs when compared to untreated HS5-cells. Preconditioning mice with MM-EV significantly enhanced tumour growth vs control mice. Importantly, a higher number of disseminated HMCL was observed in liver, kidneys, heart in the preconditioned mice, indicating an augmented (extramedullary) metastatic potential.
412 proteins were quantified by proteomic profiling of EV with 8/13 corresponding to universal cancer EV markers. Gene ontology analysis of identified proteins (G:Profiler; p <.05) revealed enrichment for cellular component terms, eg “extracellular vesicles/exosomes”, and for biological processes, eg “cell communication”, “endocytosis”. Comparative analysis between our dataset and publicly available datasets revealed EV-markers with potential discriminatory specificity for MM. Comparative analysis revealed 40 proteins differentially regulated between HD- and MM-EV (p <.05; log2 fold change ≥2). A specific protein signature was found in ≥30% of MM-EV vs ≤30% HD-EV. These proteins were not found in human whole plasma (Lehallier et al, Nat Med 2019) or solid tumors-EV (Hoshino et al, Cell 2020; Vinik et al, Science Advances 2020).
120 phosphosites were differentially expressed between HS5 pre-treated with MM-EV vs HD-EV (>1.5-fold change, p<.05). Among the differentially expressed proteins were kinases, phosphatases, translation and transcription regulators. 624 gene terms were differentially expressed between HS5 pre-treated with MM- vs HD-EV (GSEA, FDR < 0.05), including epidermal growth factor (EGF), tumor necrosis factor alpha (TNFA), epithelial to mesenchymal transition (EMT) signaling.

Conclusions: In this first of its kind studies in MM we show that MM-EV may play a key role in disease progression by re-programming the TME. Ongoing studies will indicate: the value of MM-EV as biomarkers; whether targeting interactions between MM-EV and the (pre)metastatic niche could enforce current therapeutic strategies.