Extracellular Microvesicle Cytokines Secreted from Bone Marrow Mesenchymal Cells Mediate Proliferative and Survival Advantage in Acute Myeloid Leukaemia

Background Haematopoietic stem cell transplantation is still the most effective anti-leukaemic therapy for AML treatment for a large number of patients, but a significant proportion of these will relapse post-transplant and the probability of long term survival is low. The bone marrow microenvironment has been implicated as a major contributor to chemotherapy resistance and relapse through mediating interactions between residual haematopoietic stem cells (HSC), leukaemic stem cells (LSC) and mesenchymal stem cells (MSC) which have been shown to support and maintain the leukaemic niche. Interactions within this malignant niche can be facilitated by exosomes, microvesicles secreted by multiple cell types that function as delivery vehicles of cargo consisting of mRNA, DNA, miRNA, enzymes and cytokines. Exosomes have become the focus of much interest in recent years as there is increasing evidence that they are involved in cancer progression and resistance to therapy, however the role of secreted exosomes in mediating cell communication in the post-transplant microenvironment is relatively unknown. Results Stromal MSC cultures were derived from diagnostic, post-allogeneic bone marrow transplant (BMT) AML patients (AML-MSC, n=20) and normal bone marrow donors (NBM-MSC, n=5). MSC supernatants were collected from monolayers at passage 3 and exosome preparations were extracted and quantified using NanoSight analysis and western blotting for CD81 and CD63 exosome membrane proteins. Results show that exosome particle number and protein content was significantly increased in diagnostic AML-MSCs samples compared to normal and post-BMT samples (p=0.0028). miRNA yield was also found to be significantly higher in diagnostic samples compared to normal and post-BMT marrow MSC production (p=0.0017). Ex vivo co-culture assays using sucrose cushion derived functional exosome preparations from primary AML-MSCs revealed an exosome induced proliferative effect when cultured with primary AML blasts (p