 cells treated with LC011-derived exosome. In addition, these N-hMSCs pre-treated with AML-derived exosomes showed enhanced supportability to both primary AML blasts ( Figure 6G) and leukemia cell lines ( Figure 6H) in vitro. Furthermore, a lentiviral shRNA construct was adopted to knock down Rab27a in OCI/AML3 cells, a protein implicated in the exosome release process. The protein level of Rab27a in OCI/AML3-Rab27a-shRNA cells (OCI3-KD) was ≈0.38-fold relative to specific scrambled control cells (referred to herein as OCI3-NS) ( Figure S9A, Supporting Information). There were no significant changes in cell growth of the transduced cells ( Figure S9B, Supporting Information). As expected, exosome secretion was significantly reduced in OCI3-Rab27a-shRNA cells compared with controls ( Figure S9C, Supporting Information). N-hMSCs were cocultured with two types of OCI/AML3 cells in the Transwell system for 10 d before being trypsinized and collected for further analysis. Western blotting analyses indicated an upregulation of pAKT in AML-cocultured N-hMSCs compared with monoculture control, and the activation level was decreased in the OCI3-KD-cocultured group (Figure S9D, Supporting Information). Representative DEGs expression analyses were conducted by qRT-PCR, and the results were Afterward, the separation column was removed from the magnetic field, and these retained cells were flushed out for RNA extraction or further expansion in plastic dishes. B) Morphology of MSCs (P1) at 85%-90% confluence before subculturing. The enlarged inserts suggested that AML-mMSCs were www.advancedsciencenews.com www.advancedscience.com consistent with our previous findings in RNA sequencing array: mRNA levels of genes such as IL6, GDF15, CEBP, PPAR, COL10, MMP8, and ACO2 were upregulated in N-hMSCs cocultured with OCI3-NS cells compared with monoculture controls, while expression levels of SDF1, BMP4, WNT5A, and LDHA2 were downregulated. Although N-hMSCs cocultured with OCI3-KD cells showed a similar trend in regulating the expression of these genes, the degrees were significantly diminished (Figure S9E, Supporting Information). Similar effects were found in both cell energy tests ( Figure S9F, Supporting Information) and adipogenic differentiation assays ( Figure S9G, Supporting Information). These results indicated that the phenotypic changes in AML-educated MSCs were partially reversed due to the reduction of exosome secretion mediated by Rab27a-shRNA in AML cells.

Proteins Are Differentially Expressed in AML-Derived Exosomes
Next, we compared protein abundances in exosomes purified from the culture supernatants of OCI/AML3 cells, healthy donor-derived PBMNCs (peripheral blood-derived mononucleated cells), and immortal lymphocyte cell line LC011 in a highthroughput manner using TMT-based quantitative MS experiments. The raw spectral data were interpreted using Proteome Discoverer 1.4. Eventually, a total of 710 proteins were identified in all three groups of samples ( Figure 7A). Among these, 601 proteins were identified in OCI/AML3-derived exosomes, and the number of proteins identified in PBMNC-derive exosomes and LC011-derive exosomes was 559 and 459, respectively. The overlap of identified proteins from different samples was depicted on the Venn diagram. We found that 456 proteins were both expressed in AML-derived exosomes and PBMNC-derived exosomes, among which 238 were upregulated (OCI/AML3 > PBMNC) and 218 were downregulated (OCI/AML3 < PBMNC). 390 proteins existed in both AMLderived exosomes and LC011-derived exosomes, among which 371 were upregulated (OCI/AML3 > LC011) and 19 were downregulated (OCI/AML3 < LC011). Of note, 127 proteins were identified and quantified only in AML-derived exosomes. Translational initiation (17/127), proteins composing metabolic process (64/127), biological regulation (61/127), RNA binding (48/127), extracellular membrane (54/127), and vesicle (69/127) were overrepresented. Enriched GO terms and KEGG pathways for the 127 specific expressed proteins were identified. The top 30 enriched GO terms and KEGG pathways are shown in Figure 7B,C; Figure S10 and Table S2 (Supporting Information). The results suggested that the most significantly enriched GO terms were focused on the RNA process. Further, we conducted a PPI network analysis of the 127 proteins using the STRING database (Figure 7Ci). The top 20 hub genes were screened according to their MCC values, including SNRPD1, SNRPG,  SNRPE, HNRNRM, RPS14, RPL35A, RPL30, RPS11, PCBP1,  NACA, RPL27A, RPL13, RPL24, RPL4, RPS6, EIF3I, PRPF19, HNRNPR, PRPF19, and FBL. According to the results of the STRING database, we further constructed the PPI network of this top 20 hub genes with a higher degree of connectivity (Figure 7Cii). Based on the GO function analysis, we found that these hub genes were enriched in Translational initiation, Translation, RNA processing, Regulation of gene expression, and Primary metabolic process.

The Pre-Condition of AML-Derived Exosomes Facilitate the Leukemia Engraftment and Growth In Vivo
Given that the uptake of these AML-derived exosomes induces phenotypic variants in MSCs, which in turn enhanced supportability to leukemia cells in vitro, we hypothesized that the remodeling of the stroma microenvironment contributes to leukemia engraftment and development in vivo. A mouse study was conducted to investigate this hypothesis, as illustrated in Figure 8A. Briefly, C57BL/6 mice were pretreated with either AMLderived exosomes or mouse peripheral blood mononuclear cells (PBMNC)-derived exosomes and then transplanted with C1498-GFP mouse AML cells to monitor the leukemia engraftment. As shown in Figure 8Bi, these mice pre-conditioned by AML-derived exosomes showed a significantly higher rate of leukemia cell engraftment by day 12, compared with the other two groups (percentage of C1498-GFP cells in PB: 3.17% vs 1.10% or 1.08%, all p < 0.05). When we looked into the absolute numbers of GFP + cells, significant differences were observed on days 8 and 12 ( Figure 8Bii). As expected, the median survival was significantly shorter in mice of the experimental group versus either control groups or sham group (14.5 d vs 20.5 d in both controls; all p < 0.01; Figure 8C). Moreover, representative mice were sacrificed on day 14, and the spleens were collected, weighted, fixed, and sliced for IHC staining with an anti-GFP antibody. As shown in Figure 8D,E, the analysis further confirmed a higher leukemia burden in mice pre-conditioned by AML-derived exosomes. Taken together, these data suggested that the precondition of AML-derived exosomes facilitates leukemia growth in vivo.

Discussion
MSCs and their lineage differentiated cells, such as osteoblastic cells and adipogenic cells, are essential components of the bone marrow hematopoietic microenvironment and play critical roles to modulate hematopoiesis through both cell-cell interactions and paracrine secretions. [2a,d,20] In recent years, it is becoming increasingly clear that leukemia cells induce various stromal flatter and larger than typical spindle-shaped HC-mMSCs. Bar: 100 μm. C) Following seeding cells at a concentration of 2 × 10 4 per 60 mm TC-treated culture dish, cell counts were determined daily with trypan blue assay. D) Both two types of MSCs differentiated along adipogenic, osteogenic, and chondrogenic lineages with induction of specific medium, including adipocytes (first row), osteoblasts (second row), and chondrocytes (last row). The differentiation degrees were evaluated by qRT-PCR analysis of lineage marker gene expression. Gapdh was used as housekeeping control. The metabolic profile of two MSCs was assessed by measuring the E) OCR and F) ECAR using the Agilent Seahorse XF technology. The black line represents HC-mMSCs, and the gray line represents AML-mMSCs. All measures were performed per triplicate. (Unpaired, n ≥ 3, two-tailed Student's, mean with SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.) Figure 6. The exosome is a crucial mediator of AML cells-mediated MSC education. A) OCI/AML3 cells were cocultured with N-hMSCs for 12 h; then, all cells were harvested and stained for anti-human CD90 and CD45 flow antibodies. 1.5% of the N-hMSCs (Gate2: CD45 − CD90 + ) became positive in the PKH26 dye channel, suggesting material transfer from AML cells to N-hMSCs. B,C) Exosomes were isolated from cultured OCI/AML3 cells, labeled with fluorescent dye CFSE, and added to the N-hMSCs culture. Twenty-four hours later, internalization of the labeled exosomes by N-hMSCs was confirmed using flow analysis and fluorescence imaging. D,E) N-hMSCs pre-treated with AML-exosome or LC011-exosome were induced for lineage differentiation; the differentiations were determined as above-mentioned using Oil Red O staining or