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A Minimally-invasive Extracellular Vesicle-based Approach for Monitoring Measurable Residual Disease in Acute Myeloid Leukemia

ABSTRACT: Measurable residual disease (MRD) in acute myeloid leukemia (AML) is monitored through detection of leukemia associated phenotypic protein markers (LAPMs) in bone marrow aspirates, hindering disease real-time monitoring. We explored peripheral blood (PB) extracellular vesicles (EVs)-based methods for MRD monitoring. To confirm that LAPMs are present in AML-derived EVs, EVs were isolated from OCI-AML3 cells by dif-ferential centrifugation, and characterized according to their size (nanoparticle tracking analysis), morphology (transmission electron microscopy) and protein cargo (proteomic analysis and Western blot). CD14 and CD33 were detected in OCI-AML3 cells and their released EVs. To select a method to isolate EVs from the PB of AML patients, three tech-niques were tested: size exclusion chromatography followed by ultrafiltration (SEC-UF), Total Exosome Isolation Kit (Invitrogen) and Exo-spin™ Exosome Purification Kit (Cell Guidance Systems). SEC-UF allowed EVs isolation with higher purity and less aggregates than the other techniques. LAPMs were detected in those EVs but their presence depended on the isolation method. Finally, EVs from seven AML patients’ plasma were isolated by SEC-UF. LAPMs were identified in paired samples at diagnosis and remission, with dif-ferential expression throughout disease evolution. This work highlights the possibility of real-time MRD monitoring through LAPMs’ analysis in AML patient’s circulating EVs.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Extracellular Vesicle, Peripheral Blood, Acute Myeloid Leukemia Cell Line

DISEASE(S): Acute Myeloid Leukemia

SUBMITTER: Hugo Osorio

LAB HEAD: Maria Helena Vasconcelos

PROVIDER: PXD071884 | Pride | 2026-06-29

REPOSITORIES: Pride

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A Minimally Invasive, Extracellular Vesicle-Based Approach for Monitoring Measurable Residual Disease in Acute Myeloid Leukemia: A Proof-of-Concept Study.

Branco Helena H Carreira Joana J Soure Inês I Xavier Cristina P R CPR Rosário Andreia A Amorim Maria M Osório Hugo H Guimarães José E JE Sarmento-Ribeiro Ana Bela AB Sobrinho-Simões Manuel A MA Caires Hugo R HR Vasconcelos M Helena MH

Cells 20260611 12

Measurable residual disease (MRD) in acute myeloid leukemia (AML) is monitored through detection of leukemia-associated phenotypic protein markers (LAPMs) in bone marrow aspirates, hindering disease real-time monitoring. We explored peripheral blood (PB), extracellular vesicle (EV)-based methods for MRD monitoring. To confirm that LAPMs are present in AML-derived EVs, EVs were isolated from OCI-AML3 cells by differential centrifugation and characterized according to their size (nanoparticle trac ...[more]

PMID: 42346095

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Project description:Extracellular vesicles (EVs) exert important functions in the extracellular space and in cell-to-cell communication. Ultracentrifugation, the most frequently used EV isolation technique, requires specialized equipment and provides relatively low purity and yield. A more recently applied and easily accessible EV isolation technique is size exclusion chromatography (SEC). Here, we hypothesized that EVs isolated from dilute cell culture media by concentration on a 10 kDa filter followed by SEC are suitable for proteomic and functional studies. We also hypothesized that the protein-rich SEC fractions are suitable controls to assess biological effects of the non-EV associated secretome. Cell-depleted medium from BEAS-2B bronchial epithelial cells (60 ml) was 0.22 µm filtered, concentrated to 0.5 ml by 10 kDa UF and run over a sepharose CL-4B column. Fractions were assessed for protein and EV content. EV-rich and protein-rich fractions were concentrated by 10 kDa UF. EV concentrates were characterized by tuneable resistive pulse sensing, cryo transmission electron microscopy and nanoLC-MS/MS. Finally, we assessed the effect of EVs or free secreted protein from unexposed cells or cells exposed to 1% (v/v) cigarette smoke extract (CSE) on IL-8 release by naïve BEAS-2B cells or on adhesion of THP-1 monocytes to endothelial cells. UF concentrated the EVs with approximately 100% recovery. Most of EVs eluted in SEC fractions 6-11, while protein became detectable at fraction 12. The final EV recovery was 40 ± 4%, as compared to 27 ± 8% by ultracentrifugation. Mass Spectrometry of the EV fractions identified 388 different proteins, including various EV markers. Only the protein, but not the EV fractions from media of CSE-exposed cells induced IL-8 release by naïve bronchial epithelial cells, whereas only EVs, but not the protein fractions induced THP-1 adhesion to endothelial cells. Thus, UF followed by SEC provides EV isolates with sufficient yield and purity for proteomic studies and allows directly comparing the functional effects of EVs and the non-EV associated secretome.

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