Cell lines and mice
The used cell lines and their respective culture methods are detailed in Supplementary Table 1. B16F10, MRC5, THP1 cell lines were sourced from Korean Cell Line Bank (Seoul, Korea). The B16F10 cells were transfected with lentiviral particles containing CMV-luciferase (firefly) gene (#LVP009, AMSBIO, Cambridge, MA, USA) to establish B16F10-luc cell line. The 4T1 and 4T1-luc cell lines were obtained from Dr. Byun Y (Seoul National University). The BEAS-2B cell line was obtained from Dr. Chung KH (Sungkyunkwan University), and the LX2 cell line from Dr. Friedman SL (Icahn School of Medicine at Mount Sinai). All cell lines were maintained in a humidified incubator at 37℃ with 5% CO2.
In all animal experiments, 7–9 weeks old male C57BL/6 and BALB/c mice (Raonbio, Seoul, Korea) were maintained in the specific-pathogen-free facility of Seoul National University Institute of Laboratory Animal Resources (Seoul, South Korea).
Irradiation
For in vivo radiation model, mice received two fractions of 22 Gy on chest or abdomen, with a targeting area of 1.5 cm × 1 cm, utilizing a beam-guided collimator. For the left lung radiation model, a single dose of 40 Gy was delivered to the middle part of the left lung, focusing on an area of 0.2 cm × 0.2 cm. The irradiation procedures were conducted using the X-RAD 320 (Precision, Madison, CT, USA) equipped with a Dynamic Collimator (Precision) and an F2 filter (1.5 mm Al + 0.25 mm Cu + 0.75 mm Sn) at 320 KV, 12.5 mA, and a source-to-shelf distance (SSD) of 33 cm. Cell irradiation was conducted using the X-RAD 160 (Precision). The procedure involved the use of an F1 filter (2 mm Al) at 160 KV, 19 mA, and a SSD of 50 cm.
Evaluation of lung metastasis after irradiation
Fourteen days after chest irradiation, either 6×105 4T1-luc cells (BALB/c) or 4×105 B16F10-luc cells (C57BL/6) were intravenously injected via the tail vein. BALB/c-nude mice were injected with 1.5×105 4T1-luc cells. Bioluminescence intensity in the lung was detected using IVIS Spectrum In Vivo Imaging System (PerkinElmer, Waltham, MA, USA). 150 mg/kg D-Luciferin Potassium Salt (#122799, PerkinElmer) was injected intraperitoneally into the mice 10 min before detection. The survival rate was determined by documenting the date of death of the mice. Ethical approval for all procedures was obtained from the Institutional Animal Care and Use Committee of Seoul National University (#SNU-230228-3, #SNU-230624-3, #SNU-230916-3, #SNU-231111-2).
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and small interfering RNA transfection
Total RNA extraction was carried out using Trizol reagent (#15596018, ThermoFisher, Waltham, MA, USA), chloroform, and absolute ethanol following the manufacturer's protocol. cDNA synthesis was accomplished by using 1 µg of total RNA with the Maxime RT PreMix kit (#25081, IntronBio, Seongnam, South Korea), followed by qPCR using SYBR green supermix (#1708884, Bio-Rad laboratories, Hercules, CA, USA) on the CFX Connect Real-Time PCR System (Bio-Rad laboratories). The specific primer sequences employed are detailed in Supplementary Table 2.
The knockdown of YES1 and SRC was accomplished using Lipofectamine 2000 transfection reagents (#11668027, ThermoFisher) according to the manufacturer’s protocol. Briefly, to start the transfection process, Lipofectamine 2000 and TOM media (transfection optimized medium, #TR004-01, WELGENE, Gyeongsan, South Korea) were mixed and incubated for 5 min. Subsequently, 100 pmol siRNA was added to the mixture and incubated for an additional 20 min, and cells were incubated with the preincubated mixture for 24 h. Predesigned siYES1 pools (No. 7525-1,2,3), siSRC pools (No. 6714-1,2,3), and negative control siRNA (#SN-1001) were purchased from Bioneer (Daejeon, South Korea).
In vivo fibrosis evaluation: micro-CT and immunohistochemistry (IHC)
One month after irradiation, chest CT images were obtained using a Quantum GX2 Micro-CT Imaging System (PerkinElmer). The Micro-CT utilized an X-ray filter (0.06 mm Cu + 0.5 mm Al) at 90 KV, 0.088 mA, within a dose of approximately 77 mGy per mouse. Subsequent analysis of mouse lung volume was carried out using Caliper Micro-CT analysis software (Caliper LifeSciences, Hopkinton, MA, USA).
For IHC, the lung and liver tissues were dissected from sacrificed mice. Paraffin blocks were prepared and subsequent staining was conducted using Hematoxylin and Eosin (H&E), Masson’s trichrome, Sirius Red, or specific F4/80 antibody. Stained slides were photographed using a Vectra instrument (PerkinElmer). 10× magnification images were analyzed using ImageJ software (Bethesda, MD, USA). Ethical approval for all procedures was obtained from the Institutional Animal Care and Use Committee of Seoul National University (#SNU-230325-1, #SNU-231029-1). The used IHC antibodies are listed in Supplementary Table 3.
Immune cell analysis
The isolated tissues were processed using a gentleMACS dissociator (Miltenyi Biotec) with dissociation kit (#130-096-730, Miltenyi Biotec, Bergisch Gladbach, Germany). Immune cells were selectively isolated from the dissociated tissues using percoll gradient and then stained with flow cytometry antibodies. The analysis of immune cells was performed using Novocyte flow cytometer (Agilent, Santa Clara, CA, USA). The used flow cytometry antibodies are listed in Supplementary Table 3 and the gating strategies are illustrated in Supplementary Fig. S4 and S6. Ethical approval for all procedures was obtained from the Institutional Animal Care and Use Committee of Seoul National University (#SNU-230513-2, #SNU-230520-1, #SNU-231031-1).
Bone marrow derived monocyte isolation and macrophage differentiation
After trimming the femur and tibia with scissors, the bone marrow was extruded using a syringe. The extruded bone marrow was pipetted thoroughly in RPMI-1640 medium containing 10% FBS and 1% P/S, followed by centrifugation at 500×g for 5 min to collect cells. Red blood cells from the obtained cell pellets were removed by ACK lysis buffer (Gibco, Gaithersburg, MD, USA). The bone marrow cells were seeded into a petri dish, and treated with 30 ng/ml macrophage-colony stimulating factor (M-CSF, Peprotech, Rocky Hill, NJ, USA) for 6 days to induce macrophage differentiation. Subsequently, the differentiated macrophages were detached from the petri dish and utilized for further experiments. As a human macrophage surrogate cells, THP1 cells were treated with 25 ng/ml phorbol myristate acetate (PMA) for 3 days.
Transfusion of Vivotrack680-labeled monocytes and monocyte-derived macrophages
Mouse bone marrow cells were exposed to M-CSF for 3 days, and subsequently labeled with Vivotrack680 (#NEV12000, PerkinElmer). A total of 3×106 stained cells were transfused through the mouse tail vein. Three days after injection, in vivo detection of Vivotrack680 fluorescence was conducted using an IVIS Spectrum (PerkinElmer). After sacrifice, the lungs were dissected to analyze the infiltration of Vivotrack680-labeled cells. Ethical approval for all procedures was obtained from the Institutional Animal Care and Use Committee of Seoul National University (#SNU-230513-2).
Isolation of mouse lung fibroblasts
Mouse lung tissues were dissociated using gentleMACS dissociator (Miltenyi Biotec) in DMEM/F12 medium with 0.14 Wunsch units/ml Liberase Blendzyme 3 and 1× antibiotic/antimycotic. Following dissociation, lung tissues were pipetted thoroughly in DMEM/F12 medium containing 10% FBS and 1% P/S, and then centrifuged at 500g for 5 min to collect cells. The obtained cell pellets were resuspended and seeded into a culture dish. After one week of seeding, cells were detached from the culture dish and used for required experiments.
Western blot analysis
Cell lysis was initiated using a lysis buffer composed of 10 mM Tris-Cl, 1% Triton X-100, 100 mM sodium chloride, 10% glycerol, 1 mM EDTA, 30 mM sodium pyrophosphate, 5 mM glycerol-2-phosphate, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 1% phosphatase inhibitor cocktail 2 (#P5726, Sigma-Aldrich, St. Louis, MO, USA), 1% phosphatase inhibitor cocktail 3 (#P0044, Sigma-Aldrich), and 0.02 tablet/ml protease inhibitor cocktail (#11697498001, Roche, Basel, Switzerland). Bradford assay was employed for quantifying the protein concentrations in the samples. The protein samples were then separated on SDS-polyacrylamide gels and transferred to 0.45 µm nitrocellulose membranes (#10600002, Cytiva, Marlborough, MA, USA). Subsequently, membranes were blocked with 5% skim milk in phosphate-buffered saline with 0.1% Tween 20 (PBST), and incubated with primary antibodies overnight at 4℃. Secondary antibodies were applied for 1 h at room temperature. Membranes were visualized using a chemiluminescent HRP substrate (#WBKLS0500, Merck Millipore, Burlington, MA, USA) and detected by LAS-3000 mini (Fujifilm, Tokyo, Japan). The used antibodies are listed in Supplementary Table 3. All western blot analyses in this study were repeated three times at least independently. All immunoblot full images are presented in Supplementary Materials.
Cytokine array
The mouse chest underwent a single irradiation with 20 Gy, and the mouse lungs were dissected three days later. Isolated lung tissues were processed according to the cytokine array manufacturer's manual (#AAM-BLM-1-2, Raybiotech, Norcross, GA, USA). Briefly, the isolated lungs were lysed using the provided lysis buffer, and supernatant was quantified and biotinylated. The biotinylated sample underwent a reaction with the membrane, and following washing, HRP-conjugated streptavidin was applied. Membranes were visualized and detected by LAS-3000 mini (Fujifilm).
Wound healing assay
BEAS-2B, MRC5, and LX2 cells were seeded at 90–95% confluence in the IncuCyte Imagelock 96-well plate (#BA-04856, Sartorius, Gottingen, Germany). The next day, the medium was replaced with serum free medium, and a wound scratch was made using an Incucyte 96-well wound maker (EssenBioscience, Ann Arbor, MI, USA). The extent to which cells migrate and recover from wounds was scanned and analyzed using an Incucyte Zoom device (EssenBioscience).
In vitro proliferation and migration assays
For the proliferation assay, cancer cells were seeded in 96-well plate (2 x 103 cells/well), and after the drug treatment, cell confluence was scanned and analyzed by the IncuCyte Zoom device (EssenBioscience). For the transwell migration assay, cancer cells (3,000 cells/upper well) were seeded in the IncuCyte Clearview 96-well plate (#4582, Sartorius)43. To create a gradient of chemoattractant, 1% FBS was added to the upper well, while 15% FBS was added to the lower well. Drugs were added to both the upper and lower wells at the indicated concentrations. The number of cells above and below was scanned and analyzed by the IncuCyte Zoom device (EssenBioscience).
Lung orthotopic radiotherapy model
6×105 4T1-luc cells or 4×105 B16F10-luc cells were intravenously injected via the tail vein. 6 days after cancer cell injection, mice were randomly separated, and NXP900 treatment or radiotherapy was initiated. Bioluminescence intensity in the lung was detected using the IVIS Spectrum In Vivo Imaging System (PerkinElmer). The survival rate was determined by documenting the date of death of the mice. Ethical approval for all procedures was obtained from the Institutional Animal Care and Use Committee of Seoul National University (#SNU-230823-4).
Spleen-liver metastasis evaluation after irradiation
Fourteen days after abdomen irradiation, 1×105 B16F10 cells were injected into the spleen. Ten days after the spleen injection of cancer cells, the mice were sacrificed, and the liver and spleen were dissected to assess the extent of metastasis through tissue staining. Metastatic nodule areas and numbers were analyzed using ImageJ software. Ethical approval for all procedures was obtained from the Institutional Animal Care and Use Committee of Seoul National University (#SNU-231212-1).
In vivo treatment of drugs
To inhibit SFKs activation, mice were treated with NXP900 (60 mg/kg, dissolved in 3 mM sodium citrate buffer) or dasatinib (30 mg/kg, dissolved in 3 mM sodium citrate buffer) once daily by oral gavage for two weeks. For non-specific macrophage suppression, zoledronate (0.2 mg/kg, dissolved in PBS) was administered once a week through intraperitoneal injection. The administration initiated a week before the irradiation. Anti-mouse F4/80 (10 mg/kg) or anti-mouse PD-1 (10 mg/kg), were administered twice a week through intraperitoneal injection. The administration initiated three days before the irradiation. The used neutralizing antibodies are listed in Supplementary Table 3.
Late-stage lung fibrosis mouse model: bleomycin-induced lung fibrosis
Male C57BL/6 mice aged 7–9 weeks were intratracheally administered with 0.25 mg/kg bleomycin sulfate. The day following bleomycin administration, the groups were randomly divided, and NXP900 was orally administered daily. Three weeks later, chest imaging was performed using the Quantum GX2 micro-CT Imaging System (PerkinElmer). After imaging, mouse lungs were collected, paraffin blocks were prepared, and H&E and Masson’s trichrome staining were conducted. Stained slides were captured using a Vectra instrument (PerkinElmer) and analyzed using ImageJ software (Bethesda, MD, USA). Ethical approval for all procedures was obtained from the Institutional Animal Care and Use Committee of Seoul National University (#SNU-231111-1).
Public data
Survival rate analysis based on gene expression in cancer patients was conducted utilizing the Gene Expression Profiling Interactive Analysis (GEPIA) web server44. Additionally, correlation analysis of multiple genes was carried out using the GEPIA web server. Examination of the expression of various SFKs genes and the results of single-cell sequencing of lung tissues were obtained from the Human Protein Atlas database45. For the analyses of various patient samples, the NCBI GEO database were used: GSE162945, GSE179810, GSE211118, and GSE92592. For heat map analysis using the NCBI GEO dataset, DESeq2 normalization was implemented.
Statistical analysis
Statistical significance was determined using GraphPad Prism 7.0. The significance between experimental groups were analyzed using either one-way ANOVA followed by Tukey's test or an unpaired two-tailed Student t-test. P values less than 0.05 were considered as significant differences. P value presented in this study follows the NEJM style; *p < 0.05, **p < 0.01, ***p < 0.001.