Human myeloma cell‐ and plasma‐derived extracellular vesicles contribute to functional regulation of stromal cells

Abstract

Circulating small extracellular vesicles (sEV) represent promising non-invasive biomarkers that may aid in the diagnosis and risk-stratification of multiple myeloma (MM), an incurable blood cancer. Here, we comprehensively isolated and characterized sEV from human MM cell lines (HMCL) and patient-derived plasma (psEV) by specific EV-marker enrichment and morphology. Importantly, we demonstrate that HMCL-sEV are readily internalised by stromal cells to functionally modulate proliferation. psEV were isolated using various commercial approaches and pre-analytical conditions (collection tube types, storage conditions) assessed for sEV yield and marker enrichment. Functionally, MM-psEV was shown to regulate stromal cell proliferation and migration. In turn, pre-educated stromal cells favour HMCL adhesion. psEV isolated from patients with both pre-malignant plasma cell disorders (monoclonal gammopathy of undetermined significance [MGUS]; smouldering MM [SMM]) and MM have a similar ability to promote cell migration and adhesion, suggesting a role for both malignant and pre-malignant sEV in disease progression. Proteomic profiling of MM-psEV (305 proteins) revealed enrichment of oncogenic factors implicated in cell migration and adhesion, in comparison to non-disease psEV. This study describes a protocol to generate

Optimizing extracellular vesicles’ isolation from chronic lymphocytic leukemia patient plasma and cell line supernatant

“…Primary cells have the disadvantages of limited life span, the need for special supplements for their maintenance, and their limited expansion capacity ( 50 ). On the other hand, many biological in vitro experiments have also utilized standardized cell lines like the stromal cell line HS5 where EV doses are incubated with these cells and proliferation is assayed by addition of MTS tetrazolium compound after 72 or 96 hours ( 26 , 54 ). MTS assays can assess cell proliferation, cell viability, and cytotoxicity ( 88 ).…”

“…Investigating the potential downstream effects of any reagent utilized during the EV isolation protocol has proved essential in light of the recent work by Santucci et al (42) demonstrating that the use of DTT can promote EV interaction with surrounding macromolecules via disulfide bridges and cause protein aggregations above 37°C. Furthermore, in this work, we optimize proliferation assays with stroma cells (26,54) and report the benefit of using HS5-GFP instead of HS-5 to set up these assays. The use of this fluorescent cell line allows monitoring the effect of EVs on the cells at various time points while minimizing the amount of EVs needed.…”

“…Furthermore, in this work, we optimize proliferation assays with stroma cells ( 26 , 54 ) and report the benefit of using HS5-GFP instead of HS-5 to set up these assays. The use of this fluorescent cell line allows monitoring the effect of EVs on the cells at various time points while minimizing the amount of EVs needed.…”

“…Functional assays offer a means to help the EV global community to compare research and assess the quality, efficacy, and therapeutic dose of EVs. In this work, we report an optimized proliferation assay ( 26 , 54 ) where we have utilized a stromal cell line with GFP fluorescence (HS5-GFP) that can allow us to monitor the effect of EVs at several time points with minimal sample requirements.…”

Translational Potential of RNA Derived From Extracellular Vesicles in Multiple Myeloma

“…Furthermore, EVs share common surface markers with their cell of origin, designating them as a promising target for biomarker discovery, diagnostics and therapeutics in cancer (14,28,30). Importantly, EVs protect their content (cargo) including RNAs from degradation in the extracellular environment and can be successfully collected for downstream analyses from biofluids including blood, making them ideal candidates for liquid biopsy (14,30,31). In this review, we discuss the current knowledge on the role of EVs in MM with a particular focus on EV-RNA and their potential translational application in MM.…”