Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promote non-small cell lung cancer cell growth and mobility as well as macrophage M2 polarization via miR-21-5p delivery
To investigate the lung cancer-promoting mechanism of mesenchymal stem cell-secreted extracellular vesicles (MSC-EV).
EV were isolated from culture media of human bone marrow-derived MSCs that were pre-challenged with or without hypoxia (referred to as H-EV and N-EV, respectively). After treatment with N-EV or H-EV, A549 and H23 cell proliferation, apoptosis, trans-well invasion and epithelial-to-mesenchymal transition (EMT) were examined. Polarization of human primary monocytes-derived macrophages with or without N-EV or H-EV induction were analyzed by flow cytometry and ELISA. PTEN, PDCD4 or RECK gene was overexpressed in A549 cells, while miR-21-5p was knocked down in MSCs, A549 or H23 lung cancer cells or primary monocytes by miR-21-5p inhibitor transfection. Protein level of PTEN, PDCD4, RECK, AKT or STAT3 as well as phosphorylation level of AKT or STAT3 protein were assayed by western blot. Tumorigenicity of A549 and H23 cells with or without MSC-EV co-injection was assayed on immunocompromised mice. The xenograft tumor were examined for cell proliferation, angiogenesis, apoptosis and intra-tumoral M1/M2 macrophage polarization.
Comparing to N-EV, H-EV treatment significantly increased A549 and H23 cell proliferation, survival, invasiveness and EMT as well as macrophage M2 polarization. MiR-21-5p knocked down significantly abrogated the cancer-promoting and macrophage M2 polarizing effects of H-EV treatment. H-EV treatment downregulated PTEN, PDCD4 and RECK gene expression largely through miR-21-5p. Overexpressing PTEN, PDCD4 and RECK in A549 cells significantly reduced the miR-21-5p-mediated anti-apoptotic and pro-metastatic effect of H-EV, while overexpressing PTEN in monocytes significantly reduced macrophage M2 polarization after induction with the presence of H-EV. H-EV co-injection significantly increased tumor growth, cancer cell proliferation, intra-tumoral angiogenesis and M2 polarization of macrophages in vivo partially through miR-21-5p.
Increased miR-21-5p delivery by MSC-EV after hypoxia pre-challenge can promote lung cancer development by reducing apoptosis and promoting macrophage M2 polarization.
KeywordsMesenchymal stem cells Extracellular vesicles Hypoxia miR-21-5p Lung cancer Macrophage polarization
Human bone marrow derived MSCs
Mesenchymal stem cells
Peripheral blood mononuclear cells
Mesenchymal stem cells (MSCs, also known as mesenchymal stromal cells) are progenitor cells known for their inflammation resolving, wound healing and homeostasis maintaining properties with high heterogeneity . The therapeutic value of MSCs against various inflammatory diseases and disorders is being tested in clinical trials and preclinical studies, and multiple researches have demonstrated that these anti-inflammatory and immunoregulatory effects of MSCs is generally mediated in a non-contact fashion, mostly via extracellular vesicle secretion (EV) [2, 3]. EV is composed of secreted small particles including exosome and microvesicles with diameter less than 1000 nm, mediating intercellular communications by transferring proteins and nucleotides from donor cells to recipient cells, thus regulating gene expression and intracellular signal transduction in the latter [4, 5]. Previous researches have demonstrated that EV secreted by MSCs (MSC-EV) mediate the anti-inflammatory and cytoprotective effect of MSCs against ischemia-reperfusion injury or other acute injuries in different tissues by inhibiting apoptosis, immune cell-induced cell death and pro-inflammatory response via microRNA delivery . A recent research by Luther et al. showed that MSC-EV protect cardiomyocytes from apoptosis during myocardial infarction via miR-21-5p transfer, which inhibits the protein expressions of several pro-apoptotic genes such as PTEN, PDCD4 and Fas ligand, while Song et al. demonstrated that IL-1β-treated MSCs secrete exosomes with increased miR-146a package, which is transferred into macrophages and promote their M2 polarization [7, 8].
As a distinct type of inflammatory disease, tumors are often considered as “wounds that never heal” on the immunological prospective. In general, MSCs, as they do to the site of other tissue wound, are found to migrate to the tumor microenvironment, where they transformed into tumor associated MSCs and potently inhibit anti-tumor immune attack via EV secretion [9, 10]. However, researchers might draw different conclusions on the participation of MSCs in oncogenesis and development of specific type of cancers. For example, a recent study by Pan et al. suggested that treatment of human bone marrow derived MSCs (hBM-MSC) secretome could inhibit proliferation and metastatic capacity of A549 non-small cell lung cancer cells, and Kalimuthu et al. reported that EV secreted by mouse BM-MSC could inhibit lewis lung cancer development in vivo, while the lung cancer-promoting role of MSCs or EV secreted by MSCs is also demonstrated [11, 12, 13, 14]. We believe that these controversies might result from the high heterogeneity of MSCs of different tissue origin, different experimental settings and the complexity of the tumor microenvironment (TME), where a variety types of non-cancer cells were found, such as cancer-associated fibroblasts, tumor infiltrating lymphocytes and, most abundantly, the “M2” like tumor associated macrophages, which is currently the best appreciated immunosuppressive and cancer promoting immune cell in the TME.
The present research was inspired by a recent publication by Lo Sicco et al., who reported that MSC-EV treatment could polarize macrophage towards the M2 phenotype, and this effect could be enhanced when MSCs were challenged with hypoxic culture condition (1% O2 atmosphere) before EV isolation . TME is often composed of hypoxic area due to the distorted angiogenesis, and hypoxia plays pivotal role in the establishment of the immunosuppression in the TME . Several other researchers also reported the enhanced cytoprotective and anti-inflammatory effect of EV secreted by MSCs after hypoxia pre-challenge in different tissue damage models [17, 18, 19]. Particularly, Cui et al. compared the expression level of several miRNA known as hypoxia inducible in EV secreted from hypoxia pre-challenged and un-challenged MSCs, showing that miR-21-5p is the most significantly upregulated miRNA in EV after hypoxia challenge . MiR-21-5p has been demonstrated as a powerful anti-apoptotic and cancer-promoting miRNA, indicated as prognostic marker in lung cancer and other cancer types [20, 21, 22]. Canfrán et al. and Xi et al. both reported that miR-21-5p depletion favors the macrophage M1 polarization, enhancing the macrophage-mediated pro-inflammatory and tumoricidal response [23, 24].
Based on these previous findings we speculated that MSCs might be reshaped by hypoxia, secreting EV that contain increased level of hypoxia inducible miR-21-5p, which can be transferred to recipient cells such as cancer cells and macrophages, thus promoting cancer cell survival and immunosuppression in the TME. In the present research we compared the influence of treatment with EV secreted by MSCs cultured in normal conditions or challenged with hypoxia on A549 and H23 cancer cell growth, metastatic activity as well as macrophage polarization. Our data indicated that encountering with hypoxic environment confers the cancer promoting effect on MSC-EV; EV secreted by MSCs after hypoxia pre-challenge could significantly promote cancer cell survival and metastasis in vitro, tumor development in vivo and macrophage M2 polarization via miR-21-5p delivery.
Materials and methods
Cell culture and preparation
A549 and H23 human NSCLC cells as well as human bone-marrow derived MSCs (hBM-MSCs) were purchased from American type culture collection (Manassas, USA). hBM-MSCs were cultured using a human MSCs proliferation kit (STEMCELL Technologies, Vancouver, Canada) following manufacturer’s instructions. A549 or H23 cells were cultured in DMEM with 1000 mg/L D-Glucose (STEMCELL Technologies) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, USA) and 100 U/ml of penicillin/streptomycin (Thermo Fisher Scientific) in a humidified incubator. MiR-21-5p inhibition in A549, H23, hBM-MSCs or isolated monocytes were performed by stable transfection of miR-21-5p inhibitor (GeneCopoeia, Rockville, USA). A non-targeting miRNA inhibitor control (i-NC) was also transfected as negative control for miR-21-5p inhibition.
Isolation of EV from hBM-MSCs culture media
Before EV isolation, hBM-MSCs at about 85% confluent were thoroughly washed with complete culture medium and then cultured under 20% O2 (normoxic) OR 1% O2 (hypoxic) conditions for 24 h, followed by EV isolation as described by Lo Sicco et al.  with minor modifications. Briefly the cell culture media was centrifuged at 300×g for 5 min at room temperature, 2000×g for 30 min at 4 °C and 10,000×g for 30 min at 4 °C to remove cells and cell debris. The supernatant was centrifuged at 100,000×g for 60 min at 4 °C, and the pellet was resuspended in sterile PBS, followed by another centrifugation at 100,000×g for 60 min at 4 °C to isolate MSC-EV. MSC-EV were re-suspended in sterile PBS before use. MSC-EV aliquots were equilibrated for EV density based on their total protein concentration measured using a BCA protein assay kit (GeneCopoeia) following manufacturer’s instructions. EV precipitation from cell culture media was confirmed by western blot detecting EV marker CD9 and CD81 (Additional file 1: Figure S1).
Demonstration of EV uptake by A549 or PBMCs
To demonstrate the uptake of EV by monocytes or A549 cells, we incubated the EV secreted by hypoxia-challenged or naïve MSCs with 40 μM of CFSE dye (Thermo Fisher Scientific) at 30 °C for 1 h, and the EV were pelleted down by centrifugation at 100,000×g for 60 min at 4 °C and washing with PBS. Recipient cells were incubated with re-suspended EV for 24 h under cell culture condition, and cells with or without EV incubation were subject to flow cytometry detecting cellular fluorescence at 492/517 nm (Additional file 2: Figure S2).
Cell functional assays
Cell proliferation assay was performed using a CCK-8 cell counting kit (Dojindo, Kumamoto, Japan) following manufacturer’s instructions. Briefly, A549 or H23 cells were seeded on 96 well plate in combination with different MSC-EV, and viable cells were measured by incubation with CCK-8 solution for 1 h under cell culture condition followed by measuring the optimal density at 450 nm using a microplate reader. Cell apoptosis was analyzed by flow cytometry detecting FITC-Annexin V/PI positive staining rate on cell surface of A549 or H23 cells after indicated treatment. For hypoxia or cisplatin challenge, A549 or H23 cells were pre-treated with different MSC-EV for 24 h, followed by incubation under 1% O2 (hypoxic) conditions or with 5 μM of cisplatin (Tocris Bioscience, Minneapolis, USA) for 24 h, respectively. Cells with Annexin V positive staining were considered apoptotic cells and those with Annexin V−PI+ being necrotic cells. Trans-well invasion assay was performed using matrigel invasion chambers (Thermo Fisher Scientific). After treatment with different MSC-EV for 24 h in a serum-free environment, A549 or H23 cells were lifted by trypsin (Raybiotech, Norcross, USA), and equal amount of A549 or H23 cells from each treatment group were seeded in the invasion chamber, which was inserted in complete culture medium. Cells were incubated for another 12 h before A549 or H23 cells migrated to the bottom of the chamber being stained with 0.5% methylrosaniline chloride.
Macrophage induction and analysis
Human monocytes were isolated from peripheral blood donated by healthy individuals. Briefly, peripheral blood mononuclear cells (PBMC) were first isolated by ficoll 400 gradient (Sigma-Aldrich, St. Louis, USA) centrifugation, and monocytes from PBMC were isolated using human monocyte enrichment kit (STEMCELL Technologies) following manufacturer’s instructions. Isolated monocytes were induced with 20 ng/mL of macrophage colony stimulating factor (R&D Systems, Minneapolis, USA) with the presence of different MSC-EV in RPMI-1640 medium supplemented with 10% FBS (STEMCELL Technologies) and 100 U/ml of penicillin/streptomycin (Thermo Fisher Scientific). After 5 days of induction with fresh media and induction environment replaced on day 3, cells were subject to flow cytometry detecting their surface expression level of CD68 and CD163/CD206 or CD40/CD86 using fluorescent antibody purchased from BD Biosciences (San Jose, USA). After induction, macrophages were cultured in fresh medium for 2 days, and protein expression level of IL-10, TGF-β, CCL18 and VEGF-α in macrophage culture medium were determined by using a customized ELISA assay kit (MultiSciences, Hangzhou, China) following manufacturer’s instructions.
RT-qPCR and western blot
MiR-21-5p expression in cells or isolated EV as well as pre-miR-21 expression in cells were analyzed by RT-qPCR using U6 spliceosomal RNA as internal reference. Briefly, total RNAs extracted by TRIsol were reversely transcribed into cDNA using TaqMan Small RNA Assays kits with pre-miR-21-, hsa-miR-21-5p- or RUN6B-specific RT primers (Invitrogen, Waltham, USA). Primers for pre-miR-21 are as follows: forward, 5’-TACCTCGAGTGTCTGCTTGTTTTGCCT-3′; reverse, 5’-TACGAATTCTGTTTAAATGAGAACATT-3′. Primers for miR-21-5p are as follows: Forward: 5’-TAGCTTATCAGACTGATGTTGA-3′; reverse: 5’-TGCGTGTCGTGGAGT-3′. Primers for U6 are as follows: forward: 5’-GCTTCGGCAGCACATATACTAAAAT-3′; Reverse: 5’-CGCTTCACGAATTTGCGTGTCAT-3′. Semi-quantification of miR-21-5p or pre-miR-21 expression level was performed using 2-ΔΔCt method. Protein level of N-cadherin, E-cadherin, and Vimentin (NBP1–48309, NBP2–19051 and NBP1–31327, respectively, Novus Biologicals), CD9 and CD81 (NBP2–22187 and NB100–65805, Novus Biologicals) Arginase-1 and iNOS (P05089 and MAB9502, R&D Systems), PTEN (4C11A11, BioLegend, San Diego, USA), PDCD4 (NBP2–26138, Novus Biologicals, Littleton, USA), RECK (MA5–14781, Invitrogen), AKT (ab8805, Abcam, Cambridge, USA), STAT3 (NBP2–22471, Novus Biologicals), GAPDH (NB300–221, Novus Biologicals) as well as Ser473 phosphorylation of Akt protein (649,001, BioLegend) and Tyr705 phosphorylation of STAT3 (AF4607, R&D Systems) in cell lysate was analyzed by western blot. Protein expression level of each gene was normalized to that of GAPDH by gray scale analysis using ImageJ software (Ver. 1.52b).
In vivo assay using xenograft model
Animal use and experimental methods were approved by the Ethics review committee of Luoyang Central Hospital Affiliated to Zhengzhou University. Xenograft model was established by injecting A549 or H23 cells subcutaneously on the back of NU/J nude mice (Jackson laboratory, Bar Harbor, USA). Briefly, 48 male mice at age of 4–6 weeks with roughly equal body weight were randomly assigned to 8 groups. MSC-EV from differentially treated MSCs were re-suspended in sterile PBS and aliquoted with the same total protein concentration (as described above) and were intravenously injected in each experimental mouse every 2 days after A549 or H23 cell injection, and tumor volume on each animal was monitored using a Vernier caliper. Mice were euthanized at day 30 after A549 or H23 cell transplantation, and tumors were dissected and weighted. Cell proliferation and intra-tumoral angiogenesis was determined by immunohistochemical staining of Ki67 (ab15580, Abcam) and CD31 (ab28364, Abcam), respectively, in xenograft tumor derived tissue section. Cell apoptosis in tumor tissue was detected using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Andy Fluor™ 488 apoptosis detection kit (Genecopoeia) following manufacturer’s instructions with minor modifications. Briefly, dissected tumor tissue buffered in pre-chilled PBS was grinded through a cell strainer to make single cell suspension; after centrifugation, cell pellet was resuspended in fixation buffer, and same number of cells were loaded on 96-well plate for fluorescent TUNEL staining and assessment with a microplate reader. Murine macrophages within the xenograft was isolated from the single cell suspension using anti-mouse F4/80 microbeads (Miltenyi Biotec, Auburn, USA) following the product manual. Evaluation of M1/M2 state of these macrophages was performed by detecting Arginase-1 and iNOS protein level by western blot.
Statistical analysis was performed using GraphPad Prism software (Ver. 7.04). Student’s t-test was used to evaluate the differences between two groups. Dunnett’s multiple comparisons test was used for comparing different experimental group to one control group, and Tukey’s multiple comparisons test was used for multigroup comparison. P < 0.05 was considered statistically significant. For in vitro experiments, each group of data represents 3 independent replicates and were presented as mean ± standard deviation (SD) unless otherwise indicated. For in vivo experiments, each group of data represents 6 independent replicates.
Extracellular vesicles secreted by hypoxia pre-challenged mesenchymal stem cells promoted cell proliferation, survival, mobility and EMT of NSCLC cells as well as macrophage M2 polarization in vitro
H-EV treatment promote A549 cell survival and mobility as well as macrophage M2 polarization in vitro by miR-21-5p delivery
H-EV treatment increased tumor cell growth and intratumoral angiogenesis in vivo
EV secreted by MSCs has been suggested as possible anti-cancer drug delivery approach or therapeutic agent by several researches, but different reports on the influence of MSCs or MSC-EV on cancer cell behavior are inconsistent, and the underlying mechanism remains to be discovered. These controversies might be due to the heterogeneity of MSCs of different tissue origins and complexity of the tumor microenvironment. Despite the complicated cross-talk among cancer cells, due to the chaotic angiogenesis in tumor mass, hypoxia in insufficient blood supply area can alter the cell function of cancer cells as well as other cell types residing in the tumor microenvironment. MSCs has been demonstrated to migrate to the tumor microenvironment, where they were reshaped to tumor associated MSCs and exhibit distinct immunosuppressive, angiogenic and cancer promoting functions, which have been suggested to be largely mediated by EV secretion . Previous researches have shown that hypoxia pre-challenge on MSCs increases the promoting effect of MSC-EV on macrophage M2 polarization, and microRNA expression profiles in the MSC-EV can also be significantly altered [15, 18]. Several previously identified hypoxic-inducible miRNAs were found upregulated in MSC-EV after hypoxia pre-challenge, including miR-21-5p, miR-210-3p, miR-23a-3p, etc., and these miRNAs were found to regulate immune cell function and promote cancer development . Caescu et al. found that activation of CSF-1R on murine macrophage facilitated their M2 polarization by inducing miR-21-5p expression, and Xi et al. further demonstrated that miR-21-5p inhibition in macrophages favored their M1 polarization, although the detailed molecular mechanism of miR-21-5p’ action remains to be clarified . Besides, miR-210-3p and miR-23a-3p were also found to reduce CD8+ T cell and natural killer cell cytotoxicity towards cancer cells [26, 27, 28].
MicroRNA delivery via EV from donor cells to regulate intracellular gene expression and cellular behavior of the recipients has been found under various experimental conditions . The present research started with our hypothesis that MSCs pre-challenged by hypoxia might secrete cancer promoting and immunosuppressive EV, resulting in accelerated tumor development; considering that Cui et al. have demonstrated that miR-21-5p was the miRNA most significantly upregulated in MSC-EV after hypoxia pre-challenge, we inferred that the potential cancer promoting and immunosuppressive role of EV secreted by hypoxia pre-challenged MSCs might be at least in part mediated by miR-21-5p delivery. We first found that H-EV, the EV secreted by hypoxia pre-challenged bone marrow-derived MSCs, significantly increased cell proliferation, survival, mobility and EMT of NSCLC cells in vitro. Interestingly, treatment with EV secreted by un-challenged MSCs (N-EV) showed a trend towards inhibiting NSCLC cell proliferation and survival under normal culture conditions, yet moderately increasing cell mobility, which was similar to what Zhao et al. observed in their recent publication . We next investigated the influence of N-EV or H-EV treatment on macrophage polarization, considering that macrophages are often found the most abundant non-cancerous cell types in the tumor microenvironment, who primarily exhibit “M2” like phenotype and immunosuppressive, anti-inflammatory as well as pro-angiogenic function. Our data showed that treatment with N-EV during macrophage induction increased M2 macrophage marker protein expression and M2 macrophage related protein secretion, and treatment with H-EV showed more potent effect comparing to H-EV. These results are similar to Lo Sicco et al’s recent findings .
We further revealed that hypoxia pre-challenge increased miR-21-5p load in MSC-EV, which can be delivered to NSCLC cells. Inhibiting miR-21-5p in either MSCs or NSCLC cells by miR-21-5p inhibitor transfection significantly decreased the promoting effect of H-EV treatment on NSCLC cell proliferation, survival, mobility and EMT, suggesting that these effects of H-EV treatment are largely mediated by miR-21-5p delivery. We further confirmed that the cell proliferation and survival-promoting effect of miR-21-5p on NSCLC cells delivered by H-EV is achieved by PTEN and PDCD4 targeting, and upregulation in NSCLC cell mobility by RECK targeting. Overexpression of these cancer suppressive genes significantly inhibited the effect of H-EV on A549 cells. On macrophage differentiation, our data suggested that increased miR-21-5p delivery by MSC-EV after hypoxia pre-challenge can reduce PTEN expression, thus liberating Akt and STAT3 activation and facilitate macrophage M2 polarization. Using xenograft model, we found that intravenous injection of H-EV significantly increased tumor growth, marked by upregulated cancer cell proliferation and intra-tumoral angiogenesis as well as reduced cell apoptosis. Overall, our data suggested that encountering with hypoxic environment confers the cancer promoting effect on MSC-EV; EV secreted by MSCs after hypoxia pre-challenge could significantly promote tumor development, possibly by increasing cancer cell survival, metastasis and inducing macrophage M2 polarization via miR-21-5p delivery.
Notably, in most of our experiments, miR-21-5p inhibition not completely but only partially reversed the effect of H-EV treatment, especially in our xenograft tumor formation assay, suggesting that other factors loaded in MSC-EV are involved in the cancer promoting and immunoregulatory effect of H-EV as our data suggested. In the present research we also compared the effect of N-EV and H-EV treatment. Besides on macrophage differentiation, N-EV generally showed no statistically significant influence on NSCLC cell behavior or xenograft tumor growth comparing to vehicle treatment. In the present research we found the significant influence of H-EV treatment on macrophage polarization only in in vitro experiments. We also performed IHC staining on M2 macrophage marker protein CD163 and CD206 in tissue sections derived from xenograft tumor and adjacent tissue, but we observed no significant difference in CD163 or CD206 positive staining rate between H-EV treated groups and non-treated counterparts (data not shown). Further verification on the effect of H-EV in promoting macrophage M2 polarization is needed under in vivo conditions.
Collectively, our data suggested that the cancer promoting effect of EV secreted by hypoxia pre-challenged MSCs was mediated at least partially by miR-21-5p delivery. MiR-21-5p delivered into NSCLC cells by these EV reduced PTEN, PDCD4 and RECK gene expression in NSCLC cells, resulting in increased cell proliferation, survival and mobility. MiR-21-5p delivered into monocytes facilitated their M2 polarization during maturation, resulting in the increase in M2 macrophages in the tumor microenvironment.
This work was supported by the Research on Basic and Frontier Technology in Henan Province (Grant No. 152300410007) and Molecular epidemiological study of early syphilis (Grant No. 162102310244).
Availability of data and materials
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
This work was conceived and designed WR,. The experiments were carried out by JH, CL, CY, SW and XZ. The manuscript was prepared by WR, JH, YW, CY and HW. The revised manuscript was managed by WR, YW, XPZ. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Our study was ratified by Ethical and Scientific Committees of Luoyang Central Hospital Affiliated to Zhengzhou University.
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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