The potential role of serum extracellular vesicle derived small RNAs in AML research as non-invasive biomarker.

BACKGROUND: Extracellular vesicles (EV) are cell-derived vesicles released by all cells in health and disease. Accordingly, EVs are also released by cells in acute myeloid leukemia (AML), a hematologic malignancy characterized by uncontrolled growth of immature myeloid cells, and these EVs likely carry markers and molecular cargo reflecting the malignant transformation occurring in diseased cells. Monitoring antileukemic or proleukemic processes during disease development and treatment is essential. Therefore, EVs and EV-derived microRNA (miRNA) from AML samples were explored as biomarkers to distinguish disease-related patterns ex vivo or in vivo. METHODOLOGY: EVs were purified from serum of healthy (H) volunteers and AML patients by immunoaffinity. EV surface protein profiles were analyzed by multiplex bead-based flow cytometry (MBFCM) and total RNA was isolated from EVs prior to miRNA profiling via small RNA sequencing. RESULTS: MBFCM revealed different surface protein patterns in H versus AML EVs. miRNA analysis showed individual as well as highly dysregulated patterns in H and AML samples. CONCLUSIONS: In this study, we provide a proof-of-concept for the discriminative potential of EV derived miRNA profiles as biomarkers in H versus AML samples.

Leukemia derived dendritic cell (DCleu) mediated immune response goes along with reduced (leukemia-specific) regulatory T-cells.

The blastmodulatory Kit-M, composed of granulocyte-macrophage colony-stimulating-factor (GM-CSF) and Prostaglandin E1 (PGE1), is known to convert myeloid leukaemic blasts (from AML patients) into leukaemia derived dendritic cells (DCleu), which activate immunoreactive cells to gain antileukemic/leukaemia-specific activity. In this study we had a special focus on the influence of Kit-M treated, DC/DCleu containing patients'whole blood (WB, n = 16) on the provision of immunosuppressive regulatory T-cells. We could confirm that Kit-M significantly increased frequencies of (mature) dendritic cells (DC) and DCleu from leukemic whole blood (WB) without induction of blast proliferation. After mixed lymphocyte culture (MLC) with patients' T-cells we confirmed that DCleu mediated leukemia-specific responses- going along with activated and leukemia-specific T- and NK-cells in an intracellular cytokine staining assay (ICS) and a degranulation assay (Deg)- resulted in an increased anti-leukemic cytotoxicity (Cytotoxicity Fluorolysis Assay = CTX). We could demonstrate that (leukemia-specific) CD4+ and CD8+ regulatory T-cell population (Treg) decreased significantly after MLC compared to controls. We found significant positive correlations of leukemia-specific CD3+CD4+ cells with frequencies of (mature) DCleu. Achieved anti-leukemic cytotoxicity correlated significantly positive with leukemia-specific CD3+CD8+ cells and significantly negatively with (leukemia-specific) Treg. In summary we demonstrate that immunesuppressive (leukemia-specific) regulatory T-cells are significantly downregulated after Kit-M triggered MLC- going along with a (reinstalled) antileukemic reactivity of the immune system (as demonstrated with functional assays ICS, Deg, CTX).

Description and optimization of a multiplex bead-based flow cytometry method (MBFCM) to characterize extracellular vesicles in serum samples from patients with hematological malignancies.

Extracellular Vesicles (EVs) are membranous vesicles produced by all cells under physiological and pathological conditions. In hematological malignancies, tumor-derived EVs might reprogram the bone marrow environment, suppress antileukemic immunity, mediate drug resistance and interfere with immunotherapies. EVs collected from the serum of leukemic samples might correlate with disease stage, drug-/immunological resistance, or might correlate with antileukemic immunity/immune response. Special EV surface protein patterns in serum have the potential as noninvasive biomarker candidates to distinguish several disease-related patterns ex vivo or in vivo. EVs were isolated from the serum of acute myeloid leukemia (AML), acute lymphoid leukemia (ALL), chronic lymphoid leukemia (CLL) patients, and healthy volunteers. EVs were characterized by transmission electron microscopy and fluorescence nanoparticle tracking analysis, and EV surface protein profiles were analyzed by multiplex bead-based flow cytometry to identify tumor- or immune system-related EVs of AML, ALL, CLL, and healthy samples. Aiming to provide proof-of-concept evidence and methodology for the potential role of serum-derived EVs as biomarkers in leukemic versus healthy samples in this study, we hope to pave the way for future detection of promising biomarkers for imminent disease progression and the identification of potential targets to be used in a therapeutic strategy.

Dendritic Cell-Triggered Immune Activation Goes along with Provision of (Leukemia-Specific) Integrin Beta 7-Expressing Immune Cells and Improved Antileukemic Processes.

Integrin beta 7 (ß7), a subunit of the integrin receptor, is expressed on the surface of immune cells and mediates cell-cell adhesions and interactions, e.g., antitumor or autoimmune reactions. Here, we analyzed, whether the stimulation of immune cells by dendritic cells (of leukemic derivation in AML patients or of monocyte derivation in healthy donors) leads to increased/leukemia-specific ß7 expression in immune cells after T-cell-enriched mixed lymphocyte culture-finally leading to improved antileukemic cytotoxicity. Healthy, as well as AML and MDS patients' whole blood (WB) was treated with Kit-M (granulocyte-macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) in order to generate DCs (DCleu or monocyte-derived DC), which were then used as stimulator cells in MLC. To quantify antigen/leukemia-specific/antileukemic functionality, a degranulation assay (DEG), an intracellular cytokine assay (INTCYT) and a cytotoxicity fluorolysis assay (CTX) were used. (Leukemia-specific) cell subtypes were quantified via flow cytometry. The Kit treatment of WB (compared to the control) resulted in the generation of DC/DCleu, which induced increased activation of innate and adaptive cells after MLC. Kit-pretreated WB (vs. the control) led to significantly increased frequencies of ß7-expressing T-cells, degranulating and intracellular cytokine-producing ß7-expressing immune cells and, in patients' samples, increased blast lysis. Positive correlations were found between the Kit-M-mediated improvement of blast lysis (vs. the control) and frequencies of ß7-expressing T-cells. Our findings indicate that DC-based immune therapies might be able to specifically activate the immune system against blasts going along with increased frequencies of (leukemia-specific) ß7-expressing immune cells. Furthermore, ß7 might qualify as a predictor for the efficiency and the success of AML and/or MDS therapies.