Cell culture and reagents

95C and 95D cells were obtained from the Shanghai Institute of Cell Biology (Shanghai, China). The paired cells are human giant cell lung carcinoma cell lines, and have the same genetic background and varied metastatic capacity. 95D cells had the higher metastatic capacity [17]. The cells were cultured in DMEM (Gibco BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA). A549 (CRM-CCL-185) and H1299 (CRL-5803) cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). A549 was cultured in DMEM/F12 (Gibco BRL) supplemented with 10% FBS, and H1299 was cultured in DMEM supplemented with 10% FBS at 37 °C in a 5% CO2 incubator.

Primary NFs and CAFs were cultured in our laboratory [18]. For the preparation of ρ0 cells which was depleted of mitochondrial DNA, parental tumor cell lines A549 and H1299 cells were cultured for 30 or 60 days in low-dose Ethidium bromide (EtBr, 50 ng/mL) supplemented with 100 μg/mL pyruvate and 50 μg/mL uridine. After 60 days of culture, ρ0 cells were transfer to medium lacking EtBr [19]. EtBr (E1510), pyruvate (P5280), uridine (U3003), DNaseI (D7291), Carbonyl cyanide 3-chlorophenylhydrazone (CCCP, C2759) and hydrogen peroxide (H2O2, 516813) were purchased from Merck Millipore (Billerica, MA, USA).

EVs isolation

EVs-depleted FBS was purchased from SBI (Palo Alto, CA, USA). Cells were cultured in DMEM with 10% EVs-depleted FBS at 37 °C for 48 h, and their culture media were collected for EVs isolation. The isolation of EVs was performed using ExoQuick-TC™ kit (EXOTC10A-1, SBI). BCA Assay reagent (23225, Pierce™ Thermo Fisher, Waltham, MA, USA) was used to measure the concentration of isolated EVs. The purified EVs resuspended in PBS were stored at − 80 °C.

Transmission electron microscopy (TEM)

The morphology of EVs was evaluated using a TEM (H-600, Hitachi, Tokyo, Japan) at a voltage of 75 kV. EVs sample was diluted with PBS and dropped on a carbon-coated copper grid. To remove unattached EVs, the grid was washed with ddH2O. After staining with a 2% phosphotungstic acid, the grids were air-dried and subjected to TEM assay.

Nanoparticle tracking analysis (NTA)

NTA of EVs samples was carried out using a Zeta View PMX 120 (Particle Metrix, Meerbusch, Germany) as previously described [14]. Briefly, EVs were diluted (v/v, 1:4000) with ddH2O, and their size distributions and particle concentrations were analyzed using the parameters (min area 5, max area 1000, min brightness 20, and camera 0.713 μm/px) at 25 °C.

Intracellular uptake of EVs

PKH67 (MINI67, Sigma-Aldrich, St. Louis MO, USA) fluorescent staining was performed to label EVs. The PKH67-labeled EVs were cocultured with NFs for 24 h in FBS-free DMEM medium at 37 °C. The cells were fixed with 4% paraformaldehyde solution for 10 min, and then the cells were incubated with phalloidin (C2207S, Beyotime, Shanghai, China) for 30 min. DAPI staining solution (E607303, BBI Life Sciences, Shanghai, China) was used to stain the cell nucleus. The stained cells were observed under a laser scanning confocal microscopy (LSM900, Zeiss, Oberkochen, Germany).

Transfection

The sequences of the mimics (miR-1290, miR-652, miR-222), inhibitors (miR-1290), and negative control were all synthesized from Ribobio Company (Guangzhou, China). The pDONR233-MT1G plasmid and empty plasmid were purchased from Youbio (G152804, Hunan, China). For the transient transfection of plasmid and miRNA-mimic/inhibitor, Lipofectamine3000 reagent (L3000015, Invitrogen, Carlsbad, CA, USA) was used to according to the manufacturer's instructions.

Quantitative PCR (qPCR)

Trizol reagent was purchased from Thermo Fisher Scientific (15596026) to extract total RNA from cells and EVs according to the manufacturer’s instructions, and cel-miR-39 (Ribobio) was added into each EVs sample at a final concentration of 10 pmol/μL acting as external reference. Reverse transcription was performed using Mir-X™ miRNA First-Strand Synthesis Kit for miRNAs (638315, Clontech, Mountain View, CA, USA) or RevertAid First Strand cDNA Synthesis Kit (K1621, Thermo Fisher Scientific) for general genes. For mitochondrial DNA isolation, DNA was extracted from cells or cell-free supernatants with QIAamp DNA Blood Mini Kit (Qiagen, Duesseldorf, Germany). DNA was extracted from formaldehyde-fixed mouse tissue using the TIANamp FFPE DNA Kit (TIANGEN, DP331, Beijing, China) according to the manufacturer’s instructions. qPCR was conducted using SYBR Green (4309155, Life Technologies Corporation, Gaithersburg, MD, USA) and performed on ABI 7500 PCR detection system (Foster City, CA, USA). The sequences of all indicated primers were listed in Table S1-S2. The relative expression levels of mRNAs, miRNA and mtDNA were calculated with 2–ΔΔCt method. Actin was selected as the housekeeping gene. U6 and miR-39 were selected as the housekeeping gene of miRNA for cells and EVs, respectively.

Immunoblotting

The cells were lysed in IP buffer (87787, Thermo Fisher Scientific) containing inhibitors cocktail (4693116001, Roche, Basel, Switzerland). The protein concentration was measured by BCA Assay reagent (23225, Pierce Chemical). The protein was separated by SDS-PAGE and then transferred to PVDF membrane (Merck Millipore). The membrane was incubated with first antibody at 4 °C overnight, and then incubated with the second antibody 1 h. The bands were observed with an enhanced chemiluminescence detection kit (36208-A, Yeasen, Shanghai, China) and analyzed by ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).

The antibodies were used as follows, α-SMA (14395–1-AP), Vimentin (10366–1-AP), PINK1 (23274–1-AP), AKT (60203–2-IG), p-AKT (66444–1-IG), Calnexin (10427–2-AP), E-cadherin (20874–1-AP) and Snail (13099–1-AP) were purchased from Proteintech (Wuhan, China). Actin (AC026), GAPDH (AC002), and FAP (A6349) were purchased from Abclonal (Wuhan, China). MT1G (CSB-PA17384A0Rb) was purchased from Cusabio (Wuhan, China). LC3 (L7543) was purchased from Sigma-Aldrich. BNIP3 (Ab10433), CD63 (Ab134045) and TSG101 (Ab125011) were purchased from Abcam (Cambridge, MA, USA).

Immunofluorescence (IF)

The cells were fixed with 4% paraformaldehyde solution for 10 min, and then blocked in 5% donkey serum in PBS for 1 h and incubated with the primary antibody (α-SMA, 55135–1-AP, Proteintech) at 4˚C overnight. The cells were incubated with fluorochrome-conjugated secondary antibody (Anti-Rabbit, SAB4600234, Sigma-Aldrich) for 45 min. DAPI staining solution (E607303, BBI Life Sciences) was used to stain the cell nucleus. For fluorescence analysis, cell samples were visualized on a laser scanning confocal microscopy (LSM900, Zeiss).

Collagen contraction assay

A total of 2 × 105 NFs were suspended in 100 μL DMEM. Then the cell suspension was mixed with 400 μL of cold collagen gel working solution (Cell Contraction Assay, CBA-201, Cell Biolabs, San Diego, CA, USA), added to one well of 24-well plates and allowed to solidify for 60 min at 37 °C. After collagen polymerization, 1.0 mL of culture medium containing EVs is added atop each collagen gel lattice. After incubation two days with medium, gently release collagen gels from the sides of the culture dishes with a sterile spatula, the gels were photographed by digital camera.

Luciferase reporter assay

The 3′ UTR segments of the MT1G genes were amplified by PCR and inserted into the vector pmiR-RB-REPORT™ (Ribobio). Co-transfections of MT1G 3′ UTR plasmids with miR-1290 mimic into the cells were accomplished by using Lipofectamine3000 (L3000075, Invitrogen). Luciferase activity was measured 48 h after transfection by the Dual-Luciferase Reporter Assay System (E1910, Promega, Madison, WI, USA).

Mitochondrial membrane potential (MMP) detection

After 2 × 104 cells were treated in a 6-well plate, JC-1 dye (J6004L, Uelandy, Suzhou, China) was added for 20 min at 37 °C. MMP was detected and analyzed using relative fluorescence ratio staining. The red to green fluorescence ratio was lower in damaged mitochondria cells than in normal cells. The fluorescence intensity was measured by BioTek Cytaiton1 (Hercules, CA, USA).

Succinate dehydrogenase (SDH) and Glutathione (GSH) assay

SDH activity was measured using Succinate Dehydrogenase Activity Assay Kit (D799375, Sangon, Shanghai, China). GSH concentration was measured using GSH Content Assay Kit (D799614, Sangon). All assays were performed following the manufacturer’s instruction. SDH activity was measured by absorbance at 600 nm and GSH was detected by absorbance at 412 nm using BioTek Cytaiton1 (Hercules).

Oxygen consumption rate (OCR) assay

Cells were seeded in XFe 96-well microplates (6000 cells/well) (Agilent Technologies, Sana Clara, USA), followed by indicated stimulation for a further 48 h. Cells were washed and incubated in base medium (Agilent Technologies) at 37 °C for 1 h. OCR was measured in real-time with Mito Stress Test Kit (103015–100, Agilent Technologies) using the Seahorse XFe96 Analyser (Agilent Technologies) following manufacturer’s instructions.

Reactive oxygen species (ROS) assay

Cells after treated were collected, washed with PBS twice, and incubated with DCFH-DA (S0033S, Beyotime) at a final concentration of 10 μM in FBS-free DMEM for 15 min at 37 °C in the dark, and then washed three times with FBS-free DMEM. The cells were collected, and ROS levels were analyzed using flow cytometer (FlowSight, Millipore, Billerica, MA, USA).

Mitochondrial genome sequencing

Total DNA was extracted from cells with QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s recommendations. The DNA samples were quantified with NanoDrop 2000 (Thermo Fisher Scientific) and ascertained with electrophoresis. The mitochondria genome was sequenced by Genesky Biotechnologies (Shanghai, China).

Single nucleotide polymorphism (SNP) sequencing

Using H1299ρ0 cells as control, NFs treated with 95D-EVs and H1299ρ0 cells cocultured with conditional medium (CM) from NFs treated with 95D-EVs, DNA of these three groups was extracted with QIAamp DNA Blood Mini Kit (Qiagen) and sequenced by Sangon Biotech (Shanghai, China) to analyze the variation of single nucleotide in the genome.

Mitochondrial function

Cells were incubated with Mito-Red (M7512, Invitrogen) at a final concentration of 25 nM for 15 min at 37 °C, and subsequently stained with DAPI (E607303, BBI). Next, images were captured and analyzed using a laser scanning confocal microscopy (LSM900, Zeiss).

CCK8 assay

According to the manufacturer's instructions, the cell viability was measured through the CCK8 kit (C0005, Targetmol, Boston, MA). The cells of the control group or the treatment group were seeded in a 96-well plate for an appropriate time, the viability was measured OD450 after 2 h incubation with determination solution using Microplate Reader (BioTek ELx800, Winooski, VT).

Transwell assay

1 × 105 lung cancer cells were suspended in serum-free medium and seeded into the transwell chambers with inserts of 8-μm pore size (353097, Falcon, NY, USA). The medium with 10% FBS was placed into the bottom chamber (353,047, Falcon). After 24 h, the cells that had migrated through the membrane and stuck to the lower surface of the membrane were stained with crystal violet solution (V5265, Sigma-Aldrich) and were measured by light microscope (AMEX-1200, AMG).

Animal experiment

H1299 (2 × 106), H1299ρ0 cells (2 × 106), the mixture of 2 × 106 H1299ρ0 cells with 5 × 105 NFs or 5 × 105 induced CAFs (iCAFs, that originated from NFs treatment with 95D-EVs) cells (4:1) was resuspended in 100 μL PBS and injected subcutaneously into 5-week-old female BALB/C nude mice as previously described [18]. Tumor formation was examined every 3 days. The tumor volume was calculated as volume (mm3) = d2 × D/2, where d and D were the shortest and the longest diameters, respectively.

To examine the roles of EVs in metastasis, 5 × 105 H1299 cells were intravenously injected into 5-week-old female BALB/c nude mice through the tail vein. Subsequently, mice were randomly divided into groups and intravenously injected with equal numbers of EVs from different tumor cells twice a week for 1 month [20].

For lung metastasis experiments, the nude mice were randomly divided into four groups, 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 5-week-old female BALB/c nude mice via the tail vein, respectively [21]. The nude mice were sacrificed by cervical dislocation at the indicated timepoint, and lung tissue was removed, imaged and the number of nodules on the surface of the lung was recorded to assess tumor metastasis. Lung tissues were then fixed with 4% paraformaldehyde for hematoxylin–eosin (H&E).

In situ hybridization (ISH) and immunohistochemistry (IHC)

Tissue sections of 41 clinical lung cancer pathological sections from the Department of Pathology of the second Xiangya Hospital (2019–2021) were collected (Table S3). For ISH, the miR-1290 miRCURY LNA detection probe (Exiqon, Denmark) was used by the manufacturer’s protocol. IHC was performed using universal two-step detection kit (PV-9000, ZSGB-Bio, Beijing). The sections were stained with DAB (ZLI-9017, ZSGB-Bio, Beijing) for 1–3 min, and lightly counterstained with Mayer hematoxylin (ZLI-9610, ZSGB-Bio). The expression of each protein was semi-quantitatively evaluated using the method previously described [14]. TOM20 (11802–1-AP, Proteintech) was used in this experiment.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 8 software. Quantitative values of all experiments were expressed as the mean ± SD. Differences among/between sample groups were analyzed by one-way ANOVA or the independent samples T test. Pearson’s correlation coefficient was used to measure the relationship between miR-1290, α-SMA and BNIP3. P < 0.05 was considered to be statistically significant.