Scalable Enrichment of Immunomodulatory Human Acute Myeloid Leukemia Cell Line-Derived Extracellular Vesicles.

Heide-Marie Binder,Katharina Muigg,Patricia Ebner-Peking,Rodolphe Poupardin,Christian G Huber,Katharina Schallmoser,Constantin Blöchl,Martin Wolf,Richard Greil,Balazs Vari,Thomas Heuser,Anna M Raninger,Nicole Maeding,André Cronemberger Andrade,Fausto Gueths Gomes,Lisa Pleyer,Dirk Strunk,Linda Krisch,Astrid Obermayer

doi:10.3390/cells10123321

Abstract

Acute myeloid leukemia (AML) cells can secrete trophic factors, including extracellular vesicles (EVs), instructing the stromal leukemic niche. Here, we introduce a scalable workflow for purification of immunomodulatory AML-EVs to compare their phenotype and function to the parental AML cells and their secreted soluble factors. AML cell lines HL-60, KG-1, OCI-AML3, and MOLM-14 released EVs with a peak diameter of approximately 80 nm in serum-free particle-reduced medium. We enriched EVs >100x using tangential flow filtration (TFF) and separated AML-derived soluble factors and cells in parallel. EVs were characterized by electron microscopy, immunoblotting, and flow cytometry, confirming the double-membrane morphology, purity and identity. AML-EVs showed significant enrichment of immune response and leukemia-related pathways in tandem mass-tag proteomics and a significant dose-dependent inhibition of T cell proliferation, which was not observed with AML cells or their soluble factors. Furthermore, AML-EVs dose-dependently reduced NK cell lysis of third-party K-562 leukemia targets. This emphasizes the peculiar role of AML-EVs in leukemia immune escape and indicates novel EV-based targets for therapeutic interventions.

Highlights

Acute myeloid leukemia is a genetically heterogeneous disease that originates after sequential acquisition of mutations and genomic aberrations resulting in clonal expansion of leukemia-initiating cells [1]
Common mutations target signaling and kinase pathway members, epigenetic modifiers, transcription factors, and tumor suppressors, including nucleophosmin 1 (NPM1), fms-like tyrosine kinase 3 (FLT3), rat sarcoma virus (RAS) protein family members, tumor protein p53 (TP53), and DNA methyltransferase 3 alpha (DNMT3A), representing prognostic markers involved in therapy stratification [2,3,4]
Acute myeloid leukemia (AML) cell lines cultured with this serum replacement showed viability and apoptosis rates comparable to conventional fetal bovine serum (FBS)-supplemented cultures

Summary

Introduction

Acute myeloid leukemia is a genetically heterogeneous disease that originates after sequential acquisition of mutations and genomic aberrations resulting in clonal expansion of leukemia-initiating cells [1]. New forms of therapy, including small molecules, adoptive immune cell therapy and mutation-targeting agents are being applied, AML remains a disease with a high mortality due to a high rate of relapse, in the elderly [5,6]. Secretion of EVs has been recognized as an additional major mechanism for cell-to-cell and cell-environment communication [8]. Studies indicated already a key role of EVs in orchestrating immune responses [9,10]. The contribution of EVs to tumor progression and anti-tumor immunity is well accepted [11,12]. Hypoxia can induce elevated secretion of proangiogenic EVs by pluripotent stem cells indicating a more general mechanism [15]. Minimal information for studies of EVs (MISEV) guidelines were established in 2014 and updated in 2018, recommending stringent reporting of EV isolation and characterization parameters, and encouraging the use of the ‘umbrella term’ EV for the various species of exomeres, exosomes, microvesicles, ectosomes, oncosomes, and apoptotic bodies [16,17]

Methods

Results

Conclusion