Coordinate regulation of residual bone marrow function by paracrine trafficking of AML exosomes

Abstract

We recently demonstrated that acute myeloid leukemia (AML) cell lines and patient-derived blasts release exosomes that carry RNA and protein; following an in vitro transfer, AML exosomes produce proangiogenic changes in bystander cells. We reasoned that paracrine exosome trafficking may have a broader role in shaping the leukemic niche. In a series of in vitro studies and murine xenografts, we demonstrate that AML exosomes downregulate critical retention factors (Scf, Cxcl12) in stromal cells, leading to hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow. Exosome trafficking also regulates HSPC directly, and we demonstrate declining clonogenicity, loss of CXCR4 and c-Kit expression, and the consistent repression of several hematopoietic transcription factors, including c-Myb, Cebp-β and Hoxa-9. Additional experiments using a model of extramedullary AML or direct intrafemoral injection of purified exosomes reveal that the erosion of HSPC function can occur independent of direct cell-cell contact with leukemia cells. Finally, using a novel multiplex proteomics technique, we identified candidate pathways involved in the direct exosome-mediated modulation of HSPC function. In aggregate, this work suggests that AML exosomes participate in the suppression of residual hematopoietic function that precedes widespread leukemic invasion of the bone marrow directly and indirectly via stromal components.

Figure 1

AML xenografts and exosomes modulate BM compartmental signaling. (a) BM and PB chimerism were measured by flow cytometry for human CD45+ cells. Data from 6 independent experiments with total 67 animals engrafted with either hypoxia- or normoxia-conditioned Molm-14. The results are presented as mean ± s.e.m. (b) Femur sections of engrafted mice were examined by IHC. Low chimerism: < 5% huCD45+; high chimerism: > 50% huCD45+ in the BM. (C-G) NSG mice were engrafted with Molm-14 (Xenograft) (n = 10) or (H-K) exposed to Molm-14-derived exosomes through IF injection (Intrafemoral) (n = 8). (c and d) CFU-C assays were performed on c-Kit+ progenitor cells and PBMC isolated from xenografted animals. (e) The stromal regulatory gene profile in xenografted recipient BM stroma was evaluated by qRT-PCR. (f and g) Transfers of human FLT3 and CXCR4 in AC220-treated c-Kit+ cells and recipient BM stroma were determined by RT-PCR. N1 to N6 and H1 to H6 represent the individual animal. (H-I) CFU-C assays were performed on c-Kit+ progenitor cells and PBMC from IF-injected animals. (j and k) The presence of human transcripts (FLT3 and CXCR4) in c-Kit+ progenitor and BM stromal cells from IF-injected animals was examined by RT-PCR. Vehicle medium was used as the control. Data are representative of 6 independent xenograft experiments and four independent IF experiments. The results are presented as mean ± s.d. *P < 0.01.

Figure 2

AML cells release more exosomes under physiological oxygen condition and AML exosomes attenuate stromal cell expression of HSC maintenance factors. (a) Exosome production from Molm-14 and HL60 under normoxic and hypoxic conditions was compared by nanoparticle tracking analysis. Data represents 6 independent experiments. *P < 0.01. (b) The exosome RNA concentration was determined by spectrophotometry. Data represents nine independent experiments *P < 0.05. (c) Transfer of transcripts from (human) Molm- 14 exosomes to (murine) OP9 cells was detected by qRT-PCR. N-ECM, H-ECM, N-Exo, H-Exo represent exosome-containing media (ECM) or exosomes (Exo) produced under normoxic (N) or hypoxic (H) conditions. (d) GFP-expressing OP-9 stromal cells were exposed to PKH-26- labeled Molm-14 exosomes for 3 h. (e and f) The stromal regulatory gene profile in OP9 under normoxia (red bar) or hypoxia (blue bar) after exposure to exosomes or ECM from Molm-14 cultured under hypoxia was evaluated by qRT-PCR normalized to GAPDH. Data represent three independent experiments.

Figure 3

AML exosomes modulate the function of HSPC. CFU-C assay for murine c-Kit+ progenitor cells after in vitro exposure to exosomes from Molm-14 (a) or HL-60 cells (b) cultured under hypoxia for 48 h. 10% VF-FBS medium was used as the control. (c) CFU-C for murine c-Kit+ progenitor cells after 48-hr in vitro exposure to exosomes from AML primary cells. (d) CFU-C for human CD34+ cells after 48 h in vitro exposure to exosomes from Molm-14. (e) Murine lin- cells were exposed to N-Rh-PE labeled Molm-14 exosomes overnight, labeled for Sca-1 and c-Kit, and imaged using deconvolution microscopy. (f) Expression of CXCR4 on lineage-depleted BM cells after 24-h exposure to Molm-14 exosomes. (g) Migration of lineage-depleted bone marrow cells along a CXCL12 gradient after 24-h culture with Molm-14 ECM or media alone. (h and i) Hematopoietic gene profile of c-Kit+ cells was examined by qRT-PCR after exposure to exosomes from Molm-14 or HL-60. (j) CFU-C for murine c-Kit+ progenitor cells collected from animals engrafted with Molm-14 (n = 6) or human CD34+ cells (n = 4). (k) Gene expression in c-Kit+ progenitor cells from xenografted animals, normalized to Gapdh. Data are representative of three independent experiments. *P < 0.01.

Figure 4

AML exosomes from extramedullary myeloid tumors dysregulate BM niche signaling. HL-60 cells were conditioned at 1% O₂ before xenografting. (a) Bone marrow, peripheral blood and extramedullary tumor chimerism were measured by flow cytometry for human CD45. Data are representative of three independent experiments (n = 23). (b) Chloroma sections were examined by IHC and flow cytometry for human CD45. (c and d) CFU-C for murine c-Kit+ progenitor cells and PBMC after HL-60 xenograft. (e) Transfer of human FLT3 and CXCR4 into NSG c-Kit+ progenitor cells and stroma was detected by RT-PCR. Gene expression profiles in c-Kit+ progenitor cells (f) and stroma (g) from xenografted animals were examined by qRT-PCR. Data are representative of three independent experiments. (h) CFU-C on c-Kit+ progenitor cells from animals exposed to HL-60-derived exosomes through intrafemoral (IF) injection (n = 8). Vehicle medium was used as the control. (i and j) Expression of hematopoietic and stromal regulatory genes was quantified by qRT-PCR.

Figure 5

Differentially expressed proteins and gene ontology. We performed two TMT experiments to compare proteins in exosome-treated HSPC, compared with controls. From five control and eight exosome-treated samples across 2 experiments, we obtained 282 overlapping proteins differentially expressed (P ≤ 0.0001 for Exp. 1, P ≤ 0.10 for Exp. 2) between exosome- treated and control samples. Because of higher sampling of peptides, Exp. 1 yielded the highest quality data and was thus subjected to more stringent statistical criteria. (a and b) Averaged summed reporter ion intensities for proteins in exosome-treated versus control samples used for a normalization check of data from Exp. 1 and Exp. 2, respectively, showing differentially abundant proteins in color with indicated P-values. (c) Heat map of differentially expressed proteins in Molm-14 exosome-treated versus control HSPC from Exp. 1. (d) Relative protein expression of differentially expressed transcription factors. (e) Up- and downregulated gene ontology processes from DAVID (P < 0.001; see Supplementary File 1) for differently expressed proteins in exosome-treated versus control HSPC.

Figure 6

Network interactions for differentially expressed proteins in exosome-treated HSPC. (a) Upregulated protein interaction network (generated on String DB 9.1, with highest confidence setting). (b) Fold change (exosome-treated vs control) in upregulated candidate proteins by TMT and verification by qRT-PCR on cDNA from exosome-treated HSPC. (c) Downregulated protein interaction network. (d) Fold change (exosome-treated vs the control) in downregulated candidate proteins by TMT and verification by qRT-PCR on cDNA from exosome-treated HSPC. Graphs reflect means ± s.e.m.