Leukemia-Derived Exosomes Reorganize Bone Marrow Microenvironment In AML

Increasing evidence suggests that leukemia cells take shelter in the bone marrow (BM) microenvironment (niche), where they hide from chemotherapy and continue to divide. As yet, the identity of niche cells and secreted factors that facilitate leukemia cell growth and assist them in evading chemotherapy is unclear. Further, how leukemia cells alter the bone marrow microenvironment is not known yet. In this study, we provide compelling evidences of a novel role of leukemia-derived exosomes in altering the microenvironment constituents by paracrine mechanisms.As proof-of-concept, we analyzed the cytokines mRNA profiles of primary human and mouse stromal cell co-cultured with primary CD34+CD38- cells from AML patients. Stromal cells co-cultured with leukemia showed increased levels of IL-6, IL-1β, VEGFα, TNF and reduced SDF1 mRNA expression. Similar pattern of gene expression changes were observed from stroma cells co-cultured with leukemia-derived exosomes.By using CFSE labeled exosomes, we observed that leukemia-derived exosomes target marrow stromal and endothelial cells both in-vitro and in-vivo directly. In our in vivo AML model, established using xenografted AML cell lines or primary AML patient samples in Rag2^-/^- γc^-/^-mice, we observed expansion of LT-HSC and hematopoietic progenitors compartment. The leukemia animals also showed cellular composition changes in the stromal compartment suggesting osteoblast differentiation was blocked. Interestingly, milder but similar changes were observed in mice treated with leukemia-derived exosomes. Exosomes derived from normal human peripheral blood did not induce significant changes in either hematopoietic or stromal compartments in recipient mice. These data indicate that leukemia cells secrete specialized exosomes to modulate the BM microenvironment.

Fluidigm dynamic array analysis of BM stromal cells from leukemic mice revealed that the cell adhesion molecules (NCAM1, VCAM1, CD44, OPN & ICAM1) and factors important for angiogenesis (Angpt1, Angpt 2 &VEGF) were all upregulated in leukemia-modified stromal cells whereas genes important for osteoblast (OCN, OSX), chondrocyte (SOX9) development and HSC maintenance (SDF1 and SCF) were down regulated. These results suggest that leukemia cells can remodel the BM microenvironment by changing the stromal cell composition and influencing expression of important molecular regulators.

To evaluate the HSC functions in exosomes-treated mice, we used 5-fluorouracil (5-FU) to suppress hematopoiesis and induce myeloablative stress. Leukemia-derived exosome-pretreated mice succumbed to death earlier compared to the control group (p=0.0001) suggesting that HSCs from leukemia-derived exosome-treated mice may have lower stem cell activity than their counterparts from normal mice. Furthermore, more LT-HSC and hematopoietic progenitors from leukemia-derived exosome-pretreated mice were in active cell cycle (p=0.004 and p=0.01 respectively). These findings support our hypothesis that leukemia cells/exosomes directly or indirectly through leukemia-modified niche, altered the HSCs physiological and quiescence properties.

Next we analyzed the ability of leukemia-modified niche to support the normal hematopoiesis. We co-cultured freshly sorted normal CD45.2 LT-HSCs (LSK CD150+CD48-Flk2-) with leukemia cells/exosomes pre-treated stroma cells for 48 hours and transplanted the co-cultured HSC into irradiated CD45.1 mice. 18 weeks after transplantation, we observed a significantly decreased engraftment of the HSCs co-cultured with leukemic cells/exosomes stroma compared with the HSCs co-cultured with normal stroma (p=0.003). Finally, leukemia engrafted better and developed more rapidly (p=0.0026) in mice that received leukemia-derived exosomes pre-treatment. These data suggest that changes induced by leukemia-derived exosomes in the BM niche accelerate leukemia progression and decrease their ability to support HSCs. Collectively, our data demonstrate that the leukemia cells manipulate the bone marrow microenvironment, partly through leukemia-derived exosomes, to suppress the normal hematopoiesis and facilitate growth of the leukemic progeny.