Exosomes Mediate Communication Between the Microenvironment and Leukemic Cells in Acute Myeloid Leukemia
Authors: Ruvolo Peter P. , Jacamo Rodrigo , Ruvolo Vivian R , Burks Jared K , Battula V Lokesh , McQueen Teresa , Konopleva Marina , Kornblau Steven M. , Garzon Ramiro , Croce Carlo M. , Andreeff Michael 
Published: October 2019

Abstract
Abstract 3469
It is becoming clear that the microenvironment plays a critical role in tumorigenenesis and drug resistance in cancers including Acute Myeloid Leukemia (AML). Mesenchymal stromal cells (MSC) are a key component of the bone marrow (BM) niche where AML cells reside. Our group and others have reported that MSC are critical mediators of leukemic cell engraftment and survival. Much of the emphasis on studies of the role of MSC in the microenvironment has focused on chemokines and cytokine effects especially those involving the SDF-1/CXCR4 axis. While paracrine mechanisms of MSC regulation of leukemic cells are important, in this report we suggest a novel role for MSC in altering AML cells by exosome-mediated transfer of genetic material. It is only within the last 10 years that microvesicles such as exosomes have been implicated in mammalian cell-to-cell communication. In blood biology, this is perhaps best evidenced in the regulation of antigen presenting cells by exosome containing miRs from T cells (Mittelbrunn et al Nat Commun. 2011; 2:282). At present, the role for exosomes in leukemia biology is unknown. Exosomes are derived from late endosomal processing, are enriched in ceramides, and contain proteins that are critical in membrane trafficking such as the tetraspanin CD63. To determine if MSC could transfer exosomes to AML cells, we introduced GFP-tagged CD63 into MSC. Live confocal imaging of MSC revealed that MSC can indeed secrete exosomes. The secreted exosomes are often observed in clusters, likely due to the high ceramide content of the particles. GFP-CD63 containing exosomes from MSC were purified using the Exoquick kit from System Biosciences (Mountain View, CA) and introduced to OCI-AML3 and THP-1 cells. Confocal imaging revealed exosomal uptake by only few leukemia cells; however, cells that contained the microvesicles appeared to contain clusters of these particles. Co-culture of GFP-CD63+ containing MSC with AML derived OCI-AML3 cells or KG-1 cells for 48 hours resulted in transfer of exosomes to leukemic cells as determined by analytical flow cytometry. Roughly 1.5 % of OCI-AML3 and 0.8 % of KG1 cells were found to have incorporated GFP-CD63. Though exosome transfer from MSC to AML cells appears to occur at low frequency, at least under the conditions employed here, the possibility that even a small number of leukemic cells could be altered by this mechanism could have significant ramifications. Introduction of miRs or other non-coding RNAs could profoundly modify the leukemic cells. If the primary site of residence of leukemic stem cells (LSC) is in the BM niche, then MSC could alter LSC thus creating a heterogeneous population of leukemic cells. The question arises how the BM microenvironment of a healthy individual might differ from that of an AML patient. We conducted miR profiling on MSC from normal healthy donors (N = 15) and AML derived MSC (N = 28). Eighteen miRs showed significant differences in expression between the two groups: AML derived MSC were found to express > 2 fold more miR-450b-5p, mir-382, and miR-539. On the other hand, healthy donor MSC express > 2 fold higher levels of mir-19a, miR-93, and miR-542-5p compared to MSC from AML patients. While it is not known how the exosomes of AML derived MSC differ from those of healthy donors, the difference in miR profiles between the two groups suggests that the miR content of their exosomes could vary and that exosomes from each group could have different effects. Determination of the miR content in the exosomes from each group will shed light on possible mechanisms and analysis is underway. Finally, it is also possible that MSC could use exosomes to alter the microenvironment itself. Preliminary data obtained by confocal microscopy revealed transfer of CD63+ exosomes from GFP-CD63+ MSC to MSC expressing red fluorescent protein (RFP) during co-culture conditions. At this point, the mechanism of transfer is not clear as it is possible that transfer occurs via cell-to cell contact between MSCs and/or incorporation of secreted exosomes by recipient MSC. Still, this data suggests that MSC can exchange genetic materials and that this novel mechanism could act as a plausible means of modifying the microenvironment. In conclusion, exosomal transfer represents a new mechanism in cell-to-cell communication and is active in the leukemia microenvironment with transfer from normal MSC to AML cells or other component cells of the leukemic bone marrow niche.

Disclosures:
No relevant conflicts of interest to declare.