Extracellular vesicles activated cancer-associated fibroblasts promote lung cancer metastasis through mitophagy and mtDNA transfer

Abstract

Background: Studies have shown that oxidative stress and its resistance plays important roles in the process of tumor metastasis, and mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage is an important molecular event in oxidative stress. In lung cancer, the normal fibroblasts (NFs) are activated as cancer-associated fibroblasts (CAFs), and act in the realms of the tumor microenvironment (TME) with consequences for tumor growth and metastasis. However, its activation mechanism and whether it participates in tumor metastasis through antioxidative stress remain unclear.

Methods: The role and signaling pathways of tumor cell derived extracellular vesicles (EVs) activating NFs and the characteristic of induced CAFs (iCAFs) were measured by the transmission electron microscopy, nanoparticle tracking analysis, immunofluorescence, collagen contraction assay, quantitative PCR, immunoblotting, luciferase reporter assay and mitochondrial membrane potential detection. Mitochondrial genome and single nucleotide polymorphism sequencing were used to investigate the transport of mtDNA from iCAFs to ρ⁰ cells, which were tumor cells with mitochondrial dysfunction caused by depletion of mtDNA. Further, the effects of iCAFs on mitochondrial function, growth and metastasis of tumor cells were analysed in co-culture models both in vitro and in vivo, using succinate dehydrogenase, glutathione and oxygen consumption rate measurements, CCK-8 assay, transwell assay, xenotransplantation and metastasis experiments as well as in situ hybridization and immunohistochemistry.

Results: Our findings revealed that EVs derived from high-metastatic lung cancer cells packaged miR-1290 that directly targets MT1G, leading to activation of AKT signaling in NFs and inducing NFs conversion to CAFs. The iCAFs exhibit higher levels of autophagy and mitophagy and more mtDNA release, and reactive oxygen species (ROS) could further promote this process. After cocultured with the conditioned medium (CM) of iCAFs, the ρ⁰ cells may restore its mitochondrial function by acquisition of mtDNA from CAFs, and further promotes tumor metastasis.

Conclusions: These results elucidate a novel mechanism that CAFs activated by tumor-derived EVs can promote metastasis by transferring mtDNA and restoring mitochondrial function of tumor cells which result in resistance of oxidative stress, and provide a new therapeutic target for lung cancer metastasis.

Keywords: Cancer-associated fibroblasts; Extracellular vesicles; Lung cancer; Metastasis; Mitophagy; mtDNA.

Fig. 1

95D-derived EVs packaged miR-1290 activated NFs to CAFs. A The representative images of EVs derived from 95C and 95D were analyzed by TEM. Scale bar, 100 nm. B EVs were detected by NTA analysis. C Immunoblotting analyses for EVs markers CD63, TSG101 and negative control Calnexin. D NFs were pre-treated with 95C or 95D derived EVs for 24 h. Confocal imaging showed the delivery of PKH67-labeled EVs (green) to Phalloidin labelled NFs (red) and the localization of DAPI-stained nuclei (blue). Yellow arrows represented delivered EVs. Scale bar, 20 μm. E After NFs were incubated with 95C or 95D derived EVs for 48 h, the protein levels of α-SMA and FAP were analyzed by immunoblotting. F Representative images of the contraction ability were analyzed by collagen contraction assay. Isolated primary CAFs were used as the positive control group. G-H After NFs transfected with indicated mimics, the mRNA levels of α-SMA and FAP were analyzed by qPCR. Negative control (NC), Untreated (UN). I The protein levels of α-SMA and FAP were detected by immunoblotting. J Immunoblotting analysis of α-SMA and FAP in NFs treated with blank control (PBS) or EVs derived from 95C/vec and 95C/miR-1290 or 95D/control and 95D/anti-miR-1290. K Representative images and quantification analysis of IF of α-SMA in NFs treated as indicated. Scale bar, 20 μm. L Collagen contraction assay was assessed for the contraction ability of NFs treated as indicated. Data were shown as the mean ± SD of at least three independent experiments. ***P < 0.001, ns: not significant

Fig. 2

EVs packaged miR-1290 activates NFs to CAFs via MT1G/AKT and promotes lung cancer metastasis. A The binding sites and corresponding mutation sites between miR-1290 and the target gene MT1G. B Luciferase reporter assay in 293 T cells cotransfected with the wild or mutant-type MTIG 3' UTR and miR-1290 mimics. Untreated (UN). Blank Vector (Control). C qPCR analysis of MT1G, α-SMA and FAP expression in NFs treated with miR-1290 mimics. Negative control (NC). D Immunoblotting analysis of MT1G, p-AKT, AKT, α-SMA and FAP in NFs transfected with miR-1290 mimics. E IF analysis and quantification data of α-SMA in NFs treated as indicated. Scale bar, 20 μm. F–H pDONR233-MT1G plasmid was transfected in NFs treated with miR-1290 mimics and 95C/miR-1290-EVs, respectively, F qPCR assays for MT1G, α-SMA and FAP, G Immunoblotting analysis of MT1G, p-AKT, AKT, α-SMA and FAP, H IF analysis for α-SMA. Scale bar, 20 μm. I-J H1299 was cocultured with conditional medium (CM) from different groups indicated, I CCK8 assay for cell proliferation, and J Transwell assays for the cell migration analysis. Scale bar, 100 μm. K Schematic representation of the establishment process of the EVs-educated tumor metastasis model. L The representative tissue images and HE images of lung foci. Scale bar, 200 μm. M Quantitative metastatic area in lung metastatic foci. N ISH images of miR-1290 and IHC images of MT1G, p-AKT and α-SMA in lung foci of different experimental groups. Red arrow: positive signal in the stroma; Black arrow: positive signal in the tumor. Data were shown as the mean ± SD of at least three independent experiments.**P < 0.01,  ***P < 0.001, ns: not significant

Fig. 3

ROS enhances mitophagy and increases release of mtDNA in iCAFs. A-C NFs were treated with EVs derived from 95C or 95D for 48 h. Isolated NFs and CAFs were used as blank control and positive control, respectively. A Immunoblotting analysis of PINK1, BNIP3 and LC3 in the indicated groups. B The fluorescence intensity reflecting the MMP in the different groups. C qPCR analyses for ND1, COX1, D-Loop in the supernatants of indicated cells. D Immunoblotting analysis of PINK1, BNIP3 and LC3 in NFs treated with blank control (PBS) or EVs derived from 95C/vec and 95C/miR-1290 or 95D/control and 95D/anti-miR-1290. E The fluorescence intensity reflecting the MMP in NFs treated as indicated. F qPCR analyses for ND1, COX1, D-Loop in the supernatants of indicated cell. G-I NFs were treated with 95D-derived EVs for 48 h, and then exposed to H₂O₂ (0.25 mM) or CCCP (10 μM), respectively. G Immunoblotting analysis of PINK1, BNIP3 and LC3 in the indicated groups. H The fluorescence intensity reflecting the MMP in the different groups. I qPCR analyses for ND1, COX1, D-Loop in the supernatants of indicated cells. J ISH images of miR-1290 and IHC images of α-SMA and BNIP3 in lung cancer tissue sections. Stromal and tumor are separated by dashed lines (left). The correlation analysis (right) between miR-1290 and α-SMA (upper), and between α-SMA and BNIP3 (lower). Data were shown as the mean ± SD of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001,  ns: not significant

Fig. 4

The differences between normal and mitochondria damaged lung cancer cells. A The representative macrographs of parental cells and cells treated with EtBr for 30 or 60 days. B qPCR analysis of 16 genes (12S, 16S, D-Loop, ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COX1, COX2, COX3, CYTB, ATP6, ATP8) in cells. Untreated (UN). C OCR was detected by seahorse mitochondrial stress in indicated groups. ρ⁰ cells: cells were depleted of mitochondrial DNA after treated with EtBr 60 days. D, E The activity of SDH and the concentration of GSH were detected. F The level of ROS in cells was analyzed by flow cytometry. Data were shown as the mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001

Fig. 5

iCAFs transports mtDNA to tumor cells with mitochondria damaged. A Mitochondrial genome sequencing was performed in H1299, H1299ρ⁰, NFs, iCAFs (NFs treated with 95D-EVs for 48 h) and isolated primary CAFs. B Diagram of experimental procedure for inducing CAFs and coculturing with H1299ρ⁰ cells. C, D PCR and qPCR were used to analyze the ND2 and CYTB in the indicated groups. E SNP sequencing of the ND2 and CTYB was performed. F Confocal imaging showed mitochondria labeled with mito-Red and the localization of DAPI-stained nuclei (blue) in the indicated groups. Mean fluorescence intensity (Mito-Red) data was shown. Scale bar, 10 μm. G The fluorescence intensity reflecting the MMP in the different groups. Data were shown as the mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001

Fig. 6

Mitochondria damaged tumor cells restores mitochondrial function, the ability of cell proliferation, migration and EMT through uptaking mtDNA released by iCAFs. Used H1299, A549, H1299ρ⁰ and A549ρ⁰ cells as control groups, cocultured H1299ρ⁰ and A549ρ⁰ cells with NFs-CM or iCAFs-CM for 48 h, respectively, and the CM was treated with DNaseI enzyme (0.1 mg/mL) in 37 °C for 1 h to hydrolyze mtDNA and followed by heating (70 °C for 10 min) to inactivate enzyme. A OCR was detected by seahorse mitochondrial stress. B, C The activity of SDH and the concentration of GSH were detected. D CCK8 assay for cell proliferation. E Transwell assays for the cell migration analysis. Scale bar, 100 μm. F Immunoblotting analysis of E-cadherin, Vimentin and Snail expression. Data were shown as the mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001

Fig. 7

iCAFs promote the growth and metastasis of mitochondria damaged tumor cells in vivo. A-C After H1299 (2 × 10⁶), H1299ρ⁰ cells (2 × 10⁶), the mixture of 2 × 10⁶ H1299ρ⁰ cells with 5 × 10⁵ NFs or 5 × 10⁵ CAFs cells (4:1) were injected subcutaneously into 5-week-old nude mice, A the tumor growth, B tumor volume and C tumor weight was monitored. D PCR was used to detect the ND2 and CYTB in the indicated groups. E IHC analysis of TOM20 was performed in mouse xenograft tissues. F–H After H1299 (5 × 10⁵), H1299ρ⁰ cells (5 × 10⁵), the mixture of 5 × 10⁵ H1299ρ⁰ cells with 5 × 10⁵ NFs or 5 × 10⁵ iCAFs cells (1:1) were injected into the lateral tail vein of 5-week-old nude mice, F the representative tissue macrographs and HE images of lung foci, and G quantitative analysis of lung metastatic foci. Scale bar, 200 μm. H IHC images of α-SMA and TOM20 in lung foci of different experimental groups. Data were shown as the mean ± SD of at least three independent experiments. **P < 0.01, ***P < 0.001

Fig. 8

Schematic diagram. The role and mechanism of EVs activated CAFs in regulating lung cancer metastasis through mitophagy and mtDNA transfer