Cell lines and human tissues

HEK293T cell line, human SCLC cell lines H128, H69, H446, H82 and human hepatic stellate cell line LX-2 were obtained from American Type Culture Collection (ATCC). HEK293T were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin. H128, H69, H446, H82, and LX-2 were cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin. All cells were cultured in 37 ℃ incubator containing 5% CO2. Lung cancer tissues were purchased from Xi’an bioaitech Co., Ltd (Xi’an, China). The samples were analyzed by IHC staining using anti-POSTN antibody according to the standard method.

EVs purification and characterization

When cells reached about 80% confluence, the supernatants of cells were collected. Extracellular vesicles (EVs) were extracted by sequential centrifugation steps. The collected supernatants were centrifuged at 10,000 × g for 30 min and 110,000 × g for 70 min. The pellets were then washed with PBS and diluted with 50 μL PBS. The size of the purified vesicles was characterized by DynaPro Plate Reader II (WYATT technology, USA). Western blot was used to determine the positive staining of exosome markers CD63 and TSG101 after detecting the protein concentration.

Construction of the expression vectors

Human POSTN gene (NM_006475) and truncations were amplified by reverse transcription polymerase chain reaction (RT-PCR) and inserted into the expression vector pEB-3 × flag-Puro. They were eventually named pEB-POSTN-3 × flag-Puro, pEB-EMI-3 × flag-Puro, pEB-FAS1-3 × flag-Puro, and pEB-CVD-3 × flag-Puro, respectively. The pLV3-CMV-NOTCH1-NECD (1-1426aa)-HA-Puro plasmid was constructed by MiaoLing Biology. To clarify the confirmation of successful insertion of nucleotide sequences of POSTN into expression vectors, we listed our sequencing results of POSTN in supplementary file.

POSTN-Forward Primer:5’-CGGCTAGCATGATTCCCTTTTTACCCATG-3’.

POSTN-Reverse Primer:5’-CCGCTCGAGCTGAGAACGACCTTCCCTTAA-3’.

EMI-Forward Primer:5’-CGGCTAGCATGATTCCCTTTTTACCCATGTTTT-3’.

EMI-Reverse Primer:5’-CCGCTCGAGCACGATGCCCAGAGTGCC-3’.

FAS1-Forward Primer:5’-CGGCTAGCATGGGAGCCACCACAACGCAG-3’.

FAS1-Reverse Primer:5’-CCGCTCGAGTGGATAGAGGAGTTTATCTACAACATGA-3’.

CVD-Forward Primer:5’-CGGCTAGCATGGCAGACACACCTGTTGGAAA-3’.

CVD-Reverse Primer:5’-CCGCTCGAGCTGAGAACGACCTTCCCTTAA-3’.

The shRNA sequences targeting POSTN were obtained from Millipore Sigma and are synthesized by Sangon Biotech (Shanghai, China). Annealed oligos were ligated into pLKO.1 vector or Tet-pLKO-Hygro vector according to Addgene protocols [50].

shPOSTN#1:

Forward Primer: 5’-CCGGCGGTGACAGTATAACAGTAAACTCGAGTTTACTGTTATACTGTCACCGTTTTTG-3’.

Reverse Primer: 5’-AATTCAAAAACGGTGACAGTATAACAGTAAACTCGAGTTTACTGTTATACTGTCACCG-3’.

shPOSTN#2:

Forward Primer: 5’-CCGGCACTTGTAAGAACTGGTATAACTCGAGTTATACCAGTTCTTACAAGTGTTTTTG-3’.

Reverse Primer: 5’-AATTCAAAAACACTTGTAAGAACTGGTATAACTCGAGTTATACCAGTTCTTACAAGTG-3’.

DOX-shPOSTN:

Forward Primer: 5’-CTAGCCGGTGACAGTATAACAGTAAATACTAGTTTTACTGTTATACTGTCACCGTTTTTG-3’.

Reverse Primer: 5’-AATTCAAAAACGGTGACAGTATAACAGTAAAACTAGTATTTACTGTTATACTGTCACCGG-3’.

RNA interference

Briefly, hepatic stellate cells with 80% confluence were transiently transfected with siNOTCH1 or control siRNA using DharmaFECT (T-2001–03, Dharmacon, USA) according to the manufacturer’s instructions. After 6-h transfection, the cells were cultured in suitable medium for another 48–72 h. The following siRNA sequences for targeting genes were synthesized by Sangon Biotech (Shanghai, China).

siNOTCH1:

sense: 5'-CCCUUUGAGUCUUCAUACA-3';

antisense: 5'-UGUAUGAAGACUCAAAGGG-3'

Cell transfection and lentivirus infection

HEK293T cells were seeded into 6-well plates and cultured until about 80% confluence. Then, cells were transfected with pLKO.1 vector and the packaging plasmid psPAX2 and pMD2.G, using the Lipofectamine 2000 kit (Invitrogen) according to the manufacturer’s instructions. The transfecting fluid was replaced with DMEM medium supplemented with 10% FBS after 10–12 h incubation. After another 48 h, the recombinant lentivirus was filtered with 0.22 μm membrane (Merck Millipore) and collected.

For lentivirus infection, the cells were infected by virus together with 8 μg/mL polybrene (Sigma), and screened by 2.5 μg/mL puromycin (MCE). The stable puromycin-resistant cells were collected for subsequent experiments.

EdU assay

The proliferation of POSTN-overexpressed H446 cells was detected by using EdU kit according to the manufacturers’ instructions. Briefly, 8 × 104 H446 cells were seeded into 12-well plates and cultured overnight. Then, cells were labeled by 1 × EdU working solution and incubated for 2 h. After EdU labeling, cells were fixed with 4% paraformaldehyde and permeabilized with 0.3%Triton X-100. The cells was dyed using click reaction solution and captured under a fluorescence microscope. DAPI was used to stain the cell nuclei. Three random fields of each section were captured with EVOS FL Auto Cell Imaging System (Life Technologies, USA).

Cell migration and invasion assays

For cell migration assay, 1–1.5 × 106 H446 cells were seeded into 6-well plates and cultured overnight. Then, scratches were made in the confluent monolayers using a pipette tip, followed by washing the wounds twice with PBS. Images of cells migrating to the wound area at both 0 and 24 h were captured using a phase-contrast microscope (Nikon). Finally, the distance migrated by the cells was measured by using ImageJ software.

The cell invasion assay was performed in 24-well Transwell® plates (Costar, USA). 6 × 104 H446 cells were seeded in the top chamber of the insert with RPIM-1640 culture medium containing 2% FBS, while the bottom chamber was added with 500 μL of RPIM-1640 medium containing 20% FBS. The assay was performed after cultured in 37 ℃ incubator containing 5% CO2 for 24 h. Then, the migrated cells were stained and counted using crystal violet. Three random fields of each section were captured with Nikon microscopy.

Sphere formation assay

The sphere formation assay was performed in Ultra-Low Attachment 6-Well Plates (Crystalgen, Ninbo). 3 × 104 SCLC cells were seeded and clutured with DMEM-F12 medium containing 0.4% BSA, 5 μg/mL insulin, 20 ng/mL EGF, 20 ng/mL FGF, and 1% P.S. The random fields of each well were captured with Nikon microscopy.

RNA sequencing and bioinformatics analysis

Total RNA was extracted by TRizol (YEASEN, China) from LX-2 cells, transfected with POSTN-overexpressed vector, and treated with H128 CM or H69 CM for 48 h and RNase-free DNase I to remove genomic DNA contamination. RNA integrity was evaluated with a 1.0% agarose gel. Thereafter, the quality and quantity of RNA were assessed using a NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). The high-quality RNA samples were subsequently submitted to the Sangon Biotech (Shanghai, China) for library preparation and sequencing. Sequencing libraries were generated using VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina® following the manufacturer's recommendations and index codes were added to attribute sequences to each sample. The libraries were then quantified and pooled. Paired-end sequencing of the library was performed on the HiSeq XTen sequencers (Illumina, San Diego, CA). FastQC (version 0.11.2) was used for evaluating the quality of sequenced data. Clean reads were mapped to the reference genome by HISAT2 (version 2.0) with default parameters. RSeQC (version 2.6.1) was used to analyze the alignment results. Gene expression values of the transcripts were computed by StringTie (version 1.3.3b). Library preparation and high-throughput sequencing were performed by Sangon Biotech (Shanghai, China).

Quantitative real-time PCR

Total RNA was extracted using the Trizol reagent (YEASEN, China). The cDNA was synthesized using HifairTM II 1st Strand cDNA Synthesis SuperMix Kit (YEASEN, China) according to the manufacturer’s protocol. Detection on cDNAs was performed using SYBR Green (11201ES08*, YEASEN, China) with the required primers (Supplementary Table 2). The data were normalized to the expression of β-actin by utilizing the standard 2 − ΔΔCT method. All the primers were synthesized by Sangon Biotech (Shanghai, China).

Western blot and antibodies

Cells or tumor tissues were harvested and lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China) and phosphatase inhibitors (Bimake, USA). The supernatant was collected after centrifugation and protein concentration was evaluated by BCA Protein Assay Kit (23,225, Thermo, USA). 20–50 μg of proteins per sample were loaded and separated by SDS polyacrylamide gel. Subsequently, proteins were transferred to polyvinylidene fluoride membranes (PVDF, IPVH00010, Millipore, USA) and blocked with 5% non-fat milk in TBST. The blocked membranes were incubated with primary antibodies overnight at 4℃ (Supplementary Table 3). Appropriate goat anti-mouse or goat anti-rabbit secondary antibodies were used and the immunoblots were recorded with the Bio-rad chemidoc MP system after incubating with ECL solution.

Immunofluorescence staining

1–1.5 × 105 cells were seeded into 15 mm glass-bottom dishes and cultured overnight. Then, cells were fixed with 4% paraformaldehyde and permeabilized with 0.3%Triton X-100. Goat serum (Boster, Wuhan, Hubei, China) was used to block the samples for 30 min at room temperature. After that, the cells were incubated with Flag-tag and HA-tag primary antibody overnight at 4℃. The samples were rinsed three times in PBS for 5 min each. Next, cells were incubated with Alexa Fluor 555 Conjugate and Alexa Fluor 647 Conjugate for 1 h at room temperature in the dark. DAPI was used to stain the cell nuclei. Images were captured by Laser Scanning Confocal Microscope FV3000 (Olympus, Japan).

Tumor mouse models

All the mouse experiments were reviewed and approved by the Research Ethics Committee of Sun Yat-sen University (SYSU-IACUC- 2022003356, SYSU-IACUC- 2023000557). BALB/c-nu/nu mice (male, age 4–6 weeks, SPF grade) and NOD-SCID (male, age 4–6 weeks, SPF grade) were purchased from the Experimental Animal Center of Sun Yat-sen University (Guangdong, China).

For the subcutaneous transplantation tumor models, 1 × 106 briefly, H128-DOX-shPOSTN cells were suspended in a total of 100 μL PBS and matrigel (1:1, v/v) and injected into the subcutaneous area of Balb/c-nu/nu mice. The tumor volume was evaluated using a standard caliper and calculated using the formula: V = Length × Width2/2. When the tumor volume reached around 50mm3, mice were randomly divided into two groups (n = 7): Vehicle or Doxycycline. The Dox-shPOSTN group was administered by drinking water dissolved doxycycline at 2 mg/mL concentration in 2% sucrose solution.

For co-injection experiments, H128 cells alone (1 × 106 cells per 100 μL PBS) or together with LX-2 cells (3 × 106 cells per 100 μL PBS; ratio, 1:3) were suspended in a total of 100 μL PBS and matrigel (1:1, v/v) and injected into the subcutaneous area of Balb/c-nu/nu mice. Co-injection mice were randomly divided into four groups and administrated vehicle, DOX supplement, γ-secretase inhibitor DAPT (20 mg/kg, 3 times per week) or combined therapy. Mice were sacrificed and their tumors were harvested, immersed in 4% paraformaldehyde for paraffin sections, which were then used for histological examination.

NOD-SCID (male, age 4–6 weeks, SPF grade) were used for liver metastasis experiments. The experimental liver metastasis model was constructed by intra-splenic injection of a Luc-labeled H128-DOX-shPOSTN single-cell suspension at 3 × 106 cells per 25 μL PBS. Living images were captured with IVIS Lumina II (PerkinElmer). 24 days later, mice were sacrificed, and their livers were harvested for bioluminescence signals analysis.

Immunohistochemistry (IHC) staining

Paraffin-embedded tissue slides were deparaffinated for 45 min at 60 ℃. Then, rehydration was proceeded with following steps, three washes of xylene for 15 min each, two washes of 100% ethanol for 5 min each, one wash of 95% ethanol and 85% ethanol for 5 min, and three times in ddH2O for 1 min each. The slides were heated in 1 × sodium citrate buffer using a microwave and went through the progression of boiling, sub-boiling, and room temperature. After washing once in PBS, 3% hydrogen peroxide was used to incubate slides for another 10 min. Then, the slides were blocked with goat serum and incubated overnight with primary antibodies. Next, IHC staining was performed with HRP-conjugated secondary antibodies and the signal was detected by applying DAB staining. Nuclei were counterstained with Hematoxylin, following dehydration in an ethanol series and mounting. Images were captured with Nikon microscopy.

Masson’s trichrome, and Sirius red staining

The slides were deparaffinized and rehydrated using a standard protocol. For Masson’s trichrome staining, Weigert’s Iron hematoxylin solution was used to stain the cell nuclei. The liver tissue slides were then stained in Ponceau-Acid Fuchsin Solution for 10 min and Aniline Blue Solution for 2 min, respectively. Finally, the slides were dehydrated in an ethanol series rapidly and mounted for examination. Images were captured with Nikon microscopy.

For Sirius Red staining, Iron Hematoxylin Staining Solution was used to stain the cell nuclei. Sirius Red Staining Solution was used to dye collagen fibers for 10 min following by dehydrating and mounting. Images were captured with Nikon microscopy.

Determination of serum ALT and AST levels

Retro-orbital blood was collected for serum ALT and AST activity assay at the Guangdong Engineering & Technology Research Center for Disease-Model Animals at SUN Yat-sen University.