Metabolic Reprogramming of Bone Marrow Stromal Cells By Leukemic Extracellular Vesicles in Acute Lymphoblastic Leukemia (ALL)
Authors: Johnson Suzanne M. , Dempsey Clare , Chadwick Amy , Harrison Stephanie , Liu Jizhong , Di Yujun , McGinn Owen , Parihar Mayur , Sotgia Federica , Lisanti Michael , Krishnan Shekhar , Saha Vaskar 
Published: October 2019

Abstract
In ALL, leukemic cells have been shown to modulate the bone marrow microenvironment through aberrant cytokine production favoring leukemic cell survival. Extracellular vesicles have also been recognised as key mediators of non-paracrine cellular interactions in cancer. This mechanism of tumor-stromal interaction has been described in chronic lymphocytic leukemia where recipient stromal cells undergo phenotypic changes.
We now report a similar phenomenon in ALL. Light microscopy showed that primary ALL blasts and cell lines, released anucleate extracellular vesicles into extracellular fluids. On transmission electron microscopy, leukemic extracellular vesicles (LEVs) were observed to be heterogeneous, ranging from 100nm exosome-like particles to large 6µm particles. Larger LEVs were enclosed in lipid-rich membranes and contained several organelles including ribosomes, lysosomes, golgi bodies and mitochondria. On fluorescent immunostaining, LEVs demonstrated an organized cytoskeleton with expression of actin, vinculin and talin. On imaging flow cytometry, a relative excess of circulating CD19-positive LEVs were observed in patient samples at diagnosis compared to post-treatment; these were readily distinguished from CD61-expressing platelets. On time-lapse microscopy, LEVs generated by green fluorescent labeled ALL cells, appeared as dynamic particles and were internalized by both leukemic and bone marrow stromal cells. Confocal microscopy revealed internalized labeled LEVs located in the perinuclear region of recipient cells for up to a week. Lipophilic tracer labeled LEVs, ALL cell lines and primary cells were transplanted intrafemorally in NSG mice as independent experiments. Transplanted LEVs were observed in peripheral blood at day 9 of transplantation and in marrow stromal cells in contralateral femurs at day 14 of injection. Bilateral femoral flushes at day 14 in both LEV and ALL xenografts, showed free LEVs in extracellular spaces as well as internalization of LEVs by murine mesenchymal cells. While internalization of LEVs by heterogeneous leukemic cell lines led to phenotypic transformation to the cell of origin, recipient marrow stromal cells did not demonstrate change in phenotype, viability or proliferation. In keeping with this, both control and LEV internalized stromal cells had similar ATP levels. Instead, metabolic analyses using an extracellular flux analyzer indicated that recipient stromal cells demonstrated altered normoxic metabolism, with decreased mitochondrial respiration, and increased extracellular acidification associated with raised lactate production. Thus indicating aerobic glycolysis as the main source of energy. In concordance with this, megakaryocytes, granulocytes and endothelial cells but not lymphoblasts in leukemic murine and human bone marrow demonstrated perimembranous expression of the lactate export protein MCT4 (monocarboxylate transporter 4). In contrast, in normal and remission marrow, while granulocytes express membranous MCT4, endothelial cells do not express MCT4 and megakaryocytes showed a predominant cytoplasmic expression.
Thus internalized LEVs triggered a metabolic switch from oxidative phosphorylation to aerobic glycolysis in recipient stromal cells resulting in extracellular lactate generation. We speculate that extracellular lactate in the microenvironment serves as the preferred energy substrate for ALL cells, a phenomenon reported in other cancers and termed the reverse Warburg effect. Targeting lactate-dependent metabolism may therefore represent a novel common therapeutic strategy in ALL and other cancers.

Disclosures
No relevant conflicts of interest to declare.