Abstract Background: We have shown that human stromal cells (HS5) treated with small 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: 1) Demonstrate that MM-EV induce the formation of a tumour microenvironment (TME) favouring MM progression; 2) identify the protein content of MM-EV promoting this; 3) 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 by RNA sequencing of HS5 cells pre-treated with MM- vs HD/MGUS-EV; functional studies (co-culture HS5:HMCL). Results: HS5 cells treated with MM-EV induced HMCL proliferation (p =.0026) and drug resistance (p =.0013) to anti-MM drugs (proteasome inhibitors) when compared to untreated HS5-cells.412 proteins were quantified by proteomic profiling of EV with 8/13 corresponding to universal cancer EV markers (Hoshino et al, Cell 2020). Gene ontology analysis of identified proteins (G:Profiler; p <.05) revealed enrichment for cellular component terms such as “extracellular vesicles/exosomes” and for biological processes including “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. A specific protein signature was also identified in ≥30% of MGUS-EV vs ≤30% HD/MM/SMM-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. Conclusion: 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 MM-EV:HS5 could enforce current therapeutic strategies. Citation Format: Antonia Reale, Rong Xu, Irena Carmichael, Haoyun Fang, Jaynish S Sha, Tiffany Khong, Nicholas Bingham, Malarmathy Ramachandran, Maoshan Chen, David W Greening, Andrew Spencer. Myeloma-derived circulating extracellular vesicles affect human stromal cell behaviour and promote tumor progression: A multi-omic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2366.