Abstract 3491: New targets and new approaches for multiple myeloma: Extracellular vesicles as functional liquid biomarkers
Authors: Reale Antonia , Khong Tiffany , Xu Rong , Carmichael Irena , Fang Haoyun , Bingham Nicholas , Mithraprabhu Sridurga , Chen Maoshan , Ramachandran Malarmathy , Greening David W. , Spencer Andrew 
Published: June 2022

Abstract
Background: We have previously demonstrated that stromal cells (HS5) pre-treated with small extracellular vesicles (sEV) enriched from blood plasma of myeloma (MM) patients promoted adhesion of human MM cell lines (HMCL), with preliminary proteomic profiling of MM-sEV (vs healthy donors-HD) revealing enrichment of factors implicated in cell migration and adhesion.
Aims: To demonstrate that plasma-derived MM-sEV induce a microenvironment favoring MM progression and identify the protein content of plasma-sEV that promotes this.
Methods: sEV were enriched from plasma (1mL) using a commercial kit. Proteomic profiling (nLC and high-resolution mass spectrometry, Orbitrap HF-X) of plasma-sEV derived from HD (x10) and patients with MM (x8) or pre-malignant conditions (monoclonal gammopathy of undetermined significance - MGUS x10; smouldering/asymptomatic MM - SMM x4), and functional studies (co-culture system HS5:HMCL) were performed.
Results: Stromal cells pre-treated with MM-sEV induced both HMCL proliferation (p &lt; 0.05) and drug resistance (p &lt; 0.0001) to anti-MM drugs (proteasome inhibitors) when compared to untreated stromal cells.The protein concentrations of MM-sEV positively correlated with tumor burden (r0.77; p=0.024).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 &lt; 0.05) revealed enrichment for cellular component terms such as “extracellular vesicles/exosomes” and for several biological processes including “cell communication”, “endocytosis”, “cell migration”, “cellular response to stimulus”, “immune response”. Comparative analysis between our dataset and several publicly available datasets revealed sEV-markers with potential discriminatory specificity for MM, MGUS or SMM. Comparative analysis revealed 40, 40 and 41 proteins differentially regulated between HD-sEV and MM-sEV or MGUS-sEV or SMM-sEV (P &lt; 0.05; log2 fold change ≥2). A specific protein signature identified in MM-sEV was found in ≥30% of MM-sEV but &lt;30% HD-sEV. Specific protein signatures were also identified in MGUS-sEV (≥30% of MGUS-sEV but &lt;30% HD-sEV or MM-sEV or SMM-sEV) and SMM-sEV (≥30% of SMM-sEV but &lt;30% HD-sEV or MM-sEV or MGUS-sEV). These proteins were not found in human whole plasma (Lehallier et al, Nat medicine 2019) or solid tumors-derived sEV described by Hoshino et al (Cell 2020) and Vinik et al (Science Advances 2020).
Conclusions: MM-sEV may play an important role in disease progression by re-programming the tumor microenvironment. The characterization and proteomic profiling of disease-specific circulating sEV as a biomarker discovery strategy may provide translational applications in MM.
Citation Format: Antonia Reale, Tiffany Khong, Rong Xu, Irena Carmichael, Haoyun Fang, Nicholas Bingham, Sridurga Mithraprabhu, Maoshan Chen, Malarmathy Ramachandran, David W. Greening, Andrew Spencer. New targets and new approaches for multiple myeloma: Extracellular vesicles as functional liquid biomarkers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3491.