Lung tumor exosomes induce a pro-inflammatory phenotype in mesenchymal stem cells via NFκB-TLR signaling pathway

Exosomes were isolated from serum-free culture medium of A549 cells through a series of centrifugation and filtration steps. Under transmission electron microscope, these exosomes were observed to be cup-shaped vesicles of approximately 30–120 nm in diameter (Fig. 1a). In addition, CD63 and HSP70, the protein markers of exosomes, were also detectable (Fig. 1b). Since exosomes could be labeled by lipophilic cell tracking dyes, DiO-labeled exosomes were added to MSCs serum-free culture medium. Exosomes uptake was observed 2 h after application (Fig. 1c) and accumulated over time (data not shown). These data demonstrate that MSCs can efficiently internalize A549 cell-derived exosomes.

In our previous report about mRNA expression microarray [19], we noticed a remarkable elevation of genes associated with inflammation. Given the important link between inflammation and cancer progression, we began to explore whether A549 exosomes could change inflammatory response in MSCs. MSCs were exposed to 200 ug/ml A549 exosomes for 24 h, and the cytokine secretion profile of exosome-treated MSCs were detected by ELISA. Enhanced release of IL-6, IL-8, and MCP-1 was shown (Fig. 2a). To examine whether enhanced release of cytokines in exosome-treated MSCs was time- or concentration-dependent; we treated MSCs with different concentrations of A549 exosomes for 24 and 48 h. As shown in Fig. 2b for cytokine mRNA expression or Fig. 2c for cytokine protein release, no obvious time- or concentration-dependent manner was observed. To further confirm that the enhanced release of these cytokines was caused by exosome, we treated MSCs with exosome-depleted culture medium. As expected, enhanced expression of IL-6, IL-8, and MCP-1 was not observed (Fig. 2d). These data suggest that after exposure to cancer cell-derived exosomes, a new kind of pro-inflammatory MSCs (P-MSC) was generated.

We next determined whether the altered cytokine expression in P-MSCs is accompanied by changes in tumor-promoting capacity in vivo. Using a nude mouse model, we subcutaneously injected A549 cells with P-MSCs, MSCs or PBS.As shown in Fig. 3a, the size of tumors significantly increased in the presence of P-MSCs in respect to tumor alone or tumor co-injected with unstimulated MSCs. Such stimulation in tumor growth was confirmed by measurement of tumor diameter (Fig. 3b). Since MCP-1-mediated macrophage recruitment was relevant with tumor growth and angiogenesis [20], the tumors were excised for examination of immune cell infiltration. We observed that F4/80 macrophages were much more abundant in tumors receiving P-MSCs than those receiving un-stimulated MSCs (Fig. 3c, d). This tumor-promoting effect and the macrophage-recruiting function of P-MSCs corresponded well to their high expression levels of MCP-1 (Fig. 2a), the major macrophage chemokines. In addition, Ki67 staining also showed elevated cell proliferation rate of A549 cells co-administered with PMSCs (Fig. 3e, f). Thus, MSCs educated by A549 exosomes gained an enhanced capacity in recruiting macrophages and in promoting tumor growth in vivo.

To further investigate the signaling pathways involved in induction of PMSCs by A549 exosomes, we focused on TLR pathway as it has been reported to be closely related to inflammation. We analyzed gene-expression profiles of MSCs activated by or not by A549 exosomes through the use of a human toll-like receptor signaling pathway PCR array (see the Gene list at Table 1). According to the PCR array analysis (Fig. 4a), inflammation-related pathways including NFκB, JNK, and p38 signaling, were actually activated in P-MSCs. We then investigated the signaling events triggered by exosomes through Western blotting. As shown in Fig. 4b, a rapid activation of NFκB was detected by phosphorylation of Ikkα/β and the p65 subunit that peaked at 0.5–2 h after exosome exposure. Rapid activation of JNK, ERK, and p38 signaling was not detected (Fig. 4b). NFκB signaling activation involves phosphorylation of the p65 protein and translocation of the p65 protein to the cell nucleus. Therefore, we determined the localization of p65 in MSCs before and after exposure to A549 exosomes. As expected, A549 exosome stimulation resulted in a significant translocation of p65 from a cytoplasmic to a nuclear localization in Fig. 4c. Moreover, we demonstrated that NFκB inhibitor PDTC remarkably suppressed the enhanced expression of IL-6, IL-8, and MCP-1 induced by A549 exosomes in MSCs (Fig. 4d). Together, these results suggest that lung tumor-derived exosomes induce the generation of P-MSCs through activation of NFκB signaling pathway.

The effects of exosomes on NFκB prompted us to study more closely the role of TLRs in exosome-mediated signaling. RT-PCR results showed that all TLRs were detectable within 33 cycles in MSCs (Fig. 5a). To determine which TLR was specifically responsible for tumor exosome-mediated cytokine up-regulation in MSCs, we first detected TLRs changes after exosome stimulation. We found that TLR2, TLR7, and TLR8 mRNA expression was significantly increased after exosomes stimulation (Fig. 5b). Here, we focused on TLR2 for three reasons. Firstly, MSCs have higher expression of TLR2 than TLR7 or TLR8 as demonstrated in Fig. 5a, although up-regulation of TLR7 and TLR8 is more significant (Fig. 5b). Secondly, TLR2 is localized on cell plasma membrane whereas TLR7 or TLR8 resides within endosomal compartments. Lastly, TLR2 can recognize lipopeptides while both TLR7 and TLR8 were shown to sense single-stranded viral RNA [21‐23]. Using a TLR2 neutralizing antibody, we found that blockade of TLR2 in MSCs decreased the expression of inflammatory factors induced by lung tumor-derived exosomes and inhibited activation of NFκB signaling pathway (Fig. 5c, d).

Furthermore, we knocked down TLR2 with three pairs of designed siRNAs, and we chose siRNA3 as it had the highest interference efficiency of nearly 75 % (Fig. 5e).

SiRNA knockdown of TLR2 also attenuated the expression of IL-6, IL-8 and MCP-1 (Fig. 5f, Additional file 1). Collectively, these data implicated that TLR2 was relevant for exosomes-mediated increase of cytokines IL-6, IL-8, and MCP-1 in MSCs.

To find out whether proteins on exosomes were responsible for PMSCs induction, we treated exosomes with proteinase K before exposure to MSCs. Interestingly, diminished expression of IL-6, IL-8, andMCP-1 could be observed after treatments with proteinase K (Fig. 6a), suggesting that proteins on exosomes were related with MSCs cytokine production. A variety of molecules have been classified as TLR2 ligands including Versican, HMGB1, and heat shock proteins (HSP). HSP70 is highly expressed on A549 exosomes and it was reported to be a TLR2 ligand [24]. We then evaluated whether A549 exosomes induced generation of P-MSCs through HSP70 or not. Neutralizing antibody of HSP70 could partially suppress the enhanced expression of IL-6, IL-8, and MCP-1 induced by A549 exosomes in MSCs (Fig. 6b). Recombinant human HSP70 (rHSP70) added to MSCs culture mimicked the effect of A549 exosomes in a dose-dependent manner (Fig. 6c). Moreover, interfering TLR2 expression with siRNA could attenuate the effects caused by rHSP70 (Fig. 6d). Altogether, these results indicate that Hsp70 expressed on the surface of exosomes could trigger TLR2 signaling in MSCs.

Exosome extraction was performed as previously described [41]. Briefly, A549 cells were cultured in serum-free DF12 medium for 24 h. Then, the culture medium was collected and centrifuged at 800g for 5 min and additional 2000g 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 cutoff membrane (CentriPlus-70, Millipore). The volume of supernatant was reduced from approximately 250–500 mL to less than 5 mL. The supernatant was then ultracentrifuged at 100,000g 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 exosomes were fixed with 1 % glutaraldehyde in PBS (pH 7.4). After rinsing, a 20-uL 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 microscope.

Human adipose tissue was obtained from liposuction aspirates with informed consent of the donors and was performed according to procedures provided by the Ethics Committee at the Chinese Academy of Medical Sciences and Peking Union Medical College. The isolation and culture procedures were described as previously reported [42]. hAMSCs were resuspended in 12 ml culture medium and seeded at a density of 2 × 10⁶ cells in a 75-cm² culture flask. Cell cultures were maintained at 37 °C in a humidified incubator with 5 % CO₂ and passaged with trypsin/EDTA when cells were confluent. Passage 3 cells were used for following experiments.

Cultured cells were lysed by TRIzol (Invitrogen, USA), and RNA was extracted according to the manufacturer’s instruction. One microgram of total RNA from each sample was reverse transcribed using M-MLV (Takara) in a final volume of 20 uL. The polymerase chain reaction (PCR) amplification was carried out using the Step-one System (Bio-Rad) with SYBR Green Mastermix (Takara). All quantitative real-time PCR (qRT-PCR) results were carried out in duplicate and normalized to GAPDH. The primer of the related gene list is found in Table 2.

After washing twice with cold PBS, cells were lysed in RIPA lysis buffer (Beyotime, Shanghai, China) with 1 mM PMSF and protease inhibitor cocktail on ice for 30 min, manually scraped from culture plates and then quantified using the BCA Protein Assay Kit (Beyotime). Proteins were separated on 10 % sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, electroblotted onto a polyvinylidene difluoride (PVDF) membrane (0.22 μm, Millipore, Billerica, MA, USA). The membranes were blocked with 5 % BSA and incubated with specific antibodies overnight at 4 °C and then were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at room temperature. The primary antibodies were as follows: IKKα/β, phosphorus IKKα/β, p65, phosphorus p65, JNK, phosphorus JNK, phosphorus p38 (1/1000, Cell Signaling Technology, USA), CD63, HSP70 (1/1000, Abcam), GAPDH (1/1000, Santa cruz), and β-actin (1/1000, Zhongshan, Beijing China). Secondary (HRP)-conjugated antibodies were purchased from NeoBioscience. Antibody and antigen complexes were detected using chemiluminescent ECL reagent (Millipore, USA).

Three pairs of siRNA of TLR2 were designed and synthesized (Gene Pharma, Inc., Shanghai, China). The synthetics were transfected into AMSCs at the final concentration of 200 nM using lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. The whole transfection process was proceeded in a non-serum medium named opti-mem (Gibco, USA) for 6 h at 37 °C in a humidified environment containing 5 % CO2. After transfection, the medium was changed into DF12 medium with or without cancer exosomes.

Culture supernatants were collected after treatment with or without exosomes for 24 h. The concentrations of all cell cytokines in supernatants were measured using ELISA kits (BD Technologies).

The cultured cells were fixed at 4 °C in ice-cold methanol for 10 min, washed three times in phosphate-buffered saline (PBS), and then permeabilized in 0.1 % Triton X-100/PBS for 10 min at room temperature. Nonspecific binding was blocked with 0.5 % Tween-20/PBS containing 1 % bovine serum albumin (BSA) for 30 min. The primary antibodies were incubated at 4 °C overnight. The secondary antibodies incubated for 1 h at room temperature. The incubated cells were washed in PBS, and Hoechst 33342 (Sigma-Aldrich) was used to visualize nuclei. p65 antibody (10745-1-AP) was purchased from Proteintech.

All nude mice were purchased from the Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). All mice were bred and maintained under specific pathogen-free conditions. Animal use and experimental procedures were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences. All nude mice received a subcutaneous injection of 2 × 10⁶ A549 cells. One group received a subcutaneous injection of 2 × 10⁵ PMSCs. The other group received a subcutaneous injection of 2 × 10⁵ MSCs. The last group only received an injection of A549 cells. The tumor volume was measured after 2 weeks. The tumor tissues were fixed with 10 % PFA and the peripheral blood were collected for C-flow analysis. Each group was treated with HE, Ki67, and F4/80 staining. Ki67 antibody was purchased from Proteintech (19972-1-AP). F4/80 antibody was purchased from Abcam (ab6640).

Data are presented as mean ± SD. Comparisons between groups were analyzed via Student’s t test. Differences were considered statistically significant at *P < 0.05, **P < 0.01, and ***P < 0.001.