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<0.05) and a significant protection against TKI treated co-cultures (n=7, p<0.001) confirming the pro-leukaemic effects of AML-MSC derived exosomes, these effects were absent in NBM-MSC derived exosome preparations. Within stromal co-cultures the addition of exosomes exerted a significantly increased proliferative effect compared to stroma alone demonstrating the additional support exosomes provide (p=0.0079)

Secreted cytokine profiling was undertaken using a Luminex bead capture array panel of more than 100 secreted targets in paired AML-MSC exosome and total supernatants.

AML-MSC exosome profiles were compared to NBM-MSC fractions and contained significantly higher levels of several inflammatory/angiogenic chemokines including MMP-1 (p=0.0286), CXCL2 (p= 0.0497) and CXCL8 IL-8 (p=0.0284) all of which have been previously associated with poor prognosis in AML. This observation was also reflected in the total supernatant cytokine profile.

Within the exosomal fractions the following targets showed increased levels compared to their corresponding supernatant; MMP-3 involved in chemokine signalling, IL-1ra and TRAIL both involved in immunological response and implicated in AML blast proliferation. Profiles of the post-BMT exosomal fraction followed the signature of diagnostic-MSC fractions, indicating the persistence of a diseased phenotype.

These results demonstrate significant differences in the exosome production and content of AML-MSC compared to NBM-MSC and whilst both NBM and AML derived MSC exosomes are capable of protecting against drug resistance in co-culture, AML-MSC exosomes produce a considerably stronger proliferative drive and this in part may be mediated through a leukaemic specific MMP/pro-angiogenic signalling axis which has the potential to remodel the surrounding microenvironment. Targeting these pathways warrants further investigation to determine whether suppression of the leukaemic microenvironment may influence transplant outcome for these patients.