Adipose tissues were obtained from patients undergoing liposuction according to procedures approved by the Ethics Committee at the Chinese Academy of Medical Sciences and Peking Union Medical College. MSCs were isolated and culture-expanded as previously reported (17). Passage 3 cells were used for following experiments. Lung cancer cell line A549 was purchased from cell bank at the Chinese Academy of Medical Sciences. A549 cells were cultured in DF12 containing 10% fetal bovine serum (FBS), penicillin (100 U/ml) and streptomycin (100 lg/ml) at 37°C in humidified air with 5% CO₂.

Exosome extraction was performed as previously described (18). Briefly, A549 culture medium was collected and centrifuged at 800 × g for 5 min and additional 2,000 × g for 10 min to remove lifted cells. The supernatant was subjected to filtration on a 0.1-mm pore polyethersulfone membrane filter (Corning) to remove cell debris and large vesicles, followed by concentration by a 100,000 MW cut-off membrane (CentriPlus-70, Millipore). The volume of supernatant was reduced from approximately 250–500 ml to approximately 30 ml. The supernatant was then ultracentrifuged at 100,000 × g for 1 h at 4°C using 70Ti rotor (Beckman Coulter). The resulting pellets were resuspended in 6 ml PBS and ultracentrifuged at 100,000 × g for 1 h at 4°C using 100Ti rotor (Beckman Coulter).

Purified A549-exosomes were labeled with 1 μM Dil (Invitrogen) as previously described (19). Pelleted exosomes were washed to remove unbound PKH67, resuspended in PBS/5% BSA and then added to AD-MSC medium for 4 h. AD-MSCs were then washed in PBS, fixed in 4% paraformaldehyde, and imaged by microscopy.

Purified exosomes were fixed with 1% glutaraldehyde in PBS (pH 7.4). After rinsing, a 20 μl drop of the suspension was loaded onto a formvar/carbon-coated grid, negatively stained with 3% (w/v) aqueous phosphotungstic acid for 1 min, and observed by transmission electron microscopy.

Microarray and data analysis were performed by KangChen Biotechnology, Shanghai, China. The company used Arraystar Human LncRNA Microarray V3.0 which is designed for the global profiling of human lncRNAs and protein-coding transcripts. Approximately 30,586 lncRNAs and 26,109 coding transcripts can be detected by this third-generation lncRNA microarray.

Exosomes were isolated from the culture supernatants of lung tumor cell line A549 through a series of centrifugation and filtration steps. Fig. 1A shows the morphology of A549 cells under a light microscope. The exosome concentration in A549 culture medium was 21.2±3.2 μg/ml. Under transmission electron microscopy, the exosomes were observed to be round vesicles of approximately 30–100 nm in size (Fig. 1B). CD63 and HSP70, typical protein markers of exosomes, were detectable in the A549-derived exosomes (Fig. 1C). To examine whether A549-exosomes could be transferred into AD-MSCs, the exosomes were fluorescently labeled with PKH67 and incubated with MSCs for 4 h. After treatment, over 80% of the MSCs cells exhibited green fluorescence (Fig. 1D).

To investigate the biological function of A549 exosomes on adipogenic and osteogenic differentiation of AD-MSCs, 200 μg/ml A549-derived exosomes were added into AD-MSC culture medium. AD-MSCs treated with A549 exosomes were then cultured in osteogenic induction medium or adipogenic induction medium. A549 exosome treatment inhibited osteogenic differentiation, which was indicated by the decrease of ALP activity (Fig. 2A), as well as decreased mineral deposition detected by Alizarin red staining (Fig. 2B). Moreover, lower mRNA expression levels of osteo-specific markers were detected in exosome treated AD-MSCs (Fig. 2D). According to the results of Oil red O staining by day 10 of differentiation, the accumulation of lipid droplets decreased significantly after treatment with A549 exosomes (Fig. 2C). In addition, mRNA expression levels of adipogenic transcription factors and adipocyte-specific markers PPARγ and LPL decreased remarkably in cells treated with A549 exosomes compared with control group (Fig. 2E). The results above suggested that A549 acted as a negative regulator in osteogenic and adipogenic differentiation of AD-MSCs.

To determine whether or not lung tumor-derived exosomes could contribute to generation of TAFs from MSCs, we treated AD-MSCs with A549-derived exosomes. MSCs were cultured in medium containing 200 μg/ml A549-derived exosomes for 6 days. No morphological changes such as elongated cellular processes were observed (Fig. 3A). Additionally, we tested the expression of myofibroblastic/fibroblastic markers such as α-SMA and FAP. FAP was increased in MSCs incubated with A549-derived exosomes compared to controls incubated in medium without exosomes after 6 days (Fig. 3B), but expression of α-SMA was not changed. These results suggested that A549-derived exosomes did not induce fibroblastic differentiation of AD-MSCs, at least within 6 days.

To examine the lncRNA expression profiles in AD-MSCs treated with or without A549 exosomes, we used Arraystar Human LncRNA Microarray V3.0 which contains 30,586 lncRNA probes collected from Ref Seq, UCSC known genes and Gencode and 26,109 mRNA probes. Total RNA was extracted and examined for quality control before array. The OD260/OD280 ratios were approximately 2.0, and the OD260/OD230 ratios were >1.8. The overview of lncRNA expression profiles is summarized in Table I and Fig. 4A and B. Overall, we found that 9.1% of lncRNAs (2775 out of 30586) and 9.3% of protein-coding mRNA (2439 out of 26109) were differentially expressed (fold-change ≥2; P-value ≤0.05) between A549 exosome treated AD-MSCs and control AD-MSCs.

Fig. 4C shows the hierarchical cluster of lncRNAs expression between A549 exosome treated AD-MSCs and control AD-MSCs. Among the 2775 differentially expressed lncRNAs, 1263 lncRNAs were upregulated in experimental group compared to the control group, while 1512 lncRNAs were downregulated. The top 10 up- and down-regulated lncRNAs in A549 exosome treated AD-MSCs compared to control AD-MSCs are shown in Fig. 4D and Table II. We classified these differentially expressed lncRNAs into 5 groups: ‘sense-overlapping’, the lncRNA exon is overlapping a coding transcript exon on the same genomic strand; ‘intronic’, the lncRNA is overlapping the intron of a coding transcript on the same genomic strand; ‘antisense’, the lncRNA is transcribed from the antisense strand; ‘bidirectional’, the lncRNA is oriented head to head to a coding transcript within 1000 bp; ‘intergenic’: there are no overlapping or bidirectional coding transcripts near the lncRNA.

Fig. 5A shows the distribution of the five classes of lncRNAs with changed expression in A549 exosome treated AD-MSCs. The lncRNAs are mainly between 200 and 3000 bp in length. Fig. 5B shows the length distribution of differentially expressed lncRNAs. The majority of the differentially expressed lncRNAs have a length between 500 and 1000 bp.

The microarray also detected 2439 differentially expressed mRNA (absolute fold-change ≥2; P-value ≤0.05). Among them, 1940 mRNAs were upregulated in AD-MSCs treated with A549 exosomes compared to the control group, while 499 mRNAs were downregulated. Fig. 6A showed the hierarchical cluster of mRNA expression between A549 exosome treated AD-MSCs and control AD-MSCs. Specifically, we picked out the top 10 up- and down-regulated mRNAs. (Fig. 6B). Moreover, we performed GO analysis to determine the gene and gene product enrichment in biological processes, cellular components and molecular functions. We found that the highest enriched GOs targeted by upregulated mRNAs in A549 exosome treated AD-MSCs were mRNA metabolic process (ontology: biological process) (Fig. 7A), intracellular part (ontology: cellular component) (Fig. 7B) and structural constituent of ribosome (ontology: molecular function) (Fig. 7C). The highest enriched GOs targeted by the downregulated transcripts in A549 exosome treated AD-MSCs were detection of mechanical stimulus involved in sensory perception (ontology: biological process) (Fig. 7D), primary cilium (ontology: cellular component) (Fig. 7E) and acetylgalactosaminyltransferase activity (ontology: molecular function) (Fig. 7F). Pathway analysis indicated that 32 pathways were upregulated and 7 were downregulated in A549 exosome treated MSCs. Fig. 7G and H show the top 10 of the upregulated and 7 of the down-regulated pathways, respectively.