Collection of patient samples and tissue specimens

We collected pulmonary tissue samples from patients with a confirmed diagnosis of IPF during lung transplantation surgery at Wuxi People’s Hospital. The inclusion criteria for selecting IPF patients were as follows: Male patients aged over 50 years with diagnostic imaging of the chest via high-resolution computed tomography (HRCT) showing usual interstitial pneumonia (UIP) patterns. These patterns were primarily characterized by subpleural and basal predominant reticular abnormalities and honeycombing, with or without associated traction bronchiectasis, and minimal ground-glass opacities. Additionally, post-operative histopathological confirmation of UIP was required, including evidence of patchy pulmonary fibrosis and visible foci of fibroblast proliferation. Patients with other known causes of interstitial lung disease (ILD), such as familial or occupational exposure, connective tissue disease, or drug toxicity, were excluded. For comparison, we obtained normal lung tissues from two sources: donor lungs during transplant procedures and non-tumorous regions of lungs excised during lung cancer surgeries. The study was approved by the Ethics Committee of Wuxi People’s Hospital (No. KY22060) and all participants provided their informed consent.

Cell culture and primary fibroblasts isolation

A549 cells and the HFL1 (human fetal lung fibroblast 1) cell lines were obtained from iCell Bioscience in Shanghai. We isolated primary fibroblasts from both IPF and healthy lung tissues. These tissues were finely chopped into segments of 1–2 mm, homogenized, and then cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, USA), enriched with 10% fetal bovine serum (FBS) and 1% antibiotic mix. Following cell adhesion, we removed the non-adherent cells. The HFL1 cells were grown in F12K medium (Meilunbio, Dalian, China), fortified with 10% FBS (Gibco), all within an environment maintained at 37 °C with a 5% CO2 atmosphere.

Reagents

Transforming Growth Factor-beta (TGF-β) was purchased from Abcam (UK), preserved at − 20 °C within a 10 mM citrate buffer. The PI3K inhibitor, LY294002, was acquired from Sigma and similarly conserved at − 20 °C, but in dimethylsulfoxide (DMSO). Bortezomib, sourced from MedChemExpress (MCE, USA), was also stored at − 20 °C, but dissolved in 0.9% saline solution.

Quantitative RT-PCR analysis

The total RNA was extracted from both lung tissue and fibroblast samples using the Animal RNA Isolation Kit (Beyotime, China) and then reverse transcribed into cDNA with the PrimeScript RT Master Mix (Takara, Japan). Quantitative real-time PCR was performed in the Applied Biosystems 7500 Real-Time PCR System (ABI, USA) using the SYBR qPCR master mix (Vazyme, Nanjing, China). In this process, GAPDH served as the reference gene, and the data were analyzed using the 2−△△Ct method.

Western blotting

Total protein from lung tissues and fibroblasts were prepared using a lysis buffer. These proteins were then separated via gel electrophoresis and subsequently transferred to membranes. After blocking these membranes with milk, they were incubated overnight with primary antibodies at 4 °C. These antibodies targeted SPP1, α-SMA, COL1A1, Akt, pAkt, mTOR, pmTOR, and GAPDH. Post-incubation, the membranes were treated with secondary antibodies and the protein bands were visualized using enhanced chemiluminescence in a gel imaging system.

Tissue histological analysis

Lung tissue samples were processed through fixation, embedding, and sectioning. These sections underwent deparaffinization and rehydration, followed by staining with hematoxylin and eosin (H&E) for lung pathology assessment. Masson’s trichrome staining was used for assessing the degree of fibrosis, employing a specific staining kit (Sigma). Immunohistochemical staining on these samples was performed to identify target proteins, using 3,3’-diaminobenzidine and subsequent microscopic examination for colorimetric analysis.

Immunofluorescence staining

For immunofluorescence staining, cells and tissue sections were prepared through fixation, permeabilization, and blocking processes. These were incubated with primary antibodies targeting α-SMA and SPP1 at 4 °C overnight. Subsequently, the samples were washed and treated with secondary antibodies, specifically goat anti-rabbit Alexa Fluor 488 and goat anti-mouse Alexa Fluor 550, for 1 h in a light-shielded environment. DAPI staining was performed for nuclear visualization for 10 min. The final step involved capturing the stained samples’ images using a Leica confocal microscope.

Lentivirus-mediated transfection

Lentiviruses was purchased from Heyuan Company (Shanghai, China), designed to express shRNA targeting SPP1 or vector-SPP1 plasmids. Fibroblasts were cultured in 24-well plates, subsequently introducing a complete medium enriched with 5 µg/mL polybrene and the respective viruses. After a period of 12–24 h, the medium was replaced and the transfection efficiency was assessed through microscopic examination of cellular fluorescence at 72 h. Following this, the infected cells were selected using puromycin in the culture medium. The success of SPP1 knockdown or overexpression in these lines was verified by qRT-PCR and western blot analysis.

Cell counting kit-8 (CCK-8) assay

The CCK-8 assay provided by Vazyme (China) was used to measure the growth rate of fibroblasts and A549 cells. In this assay, the fibroblasts and A549 cells were seeded into a 96-well plate, with seeding densities of 4,000 and 1,000 cells per well, respectively. The assay was conducted daily from day 1 to day 5. The old culture medium was removed, and 90 µL of fresh medium along with 10 µL of CCK-8 solution was added to each well. After incubation, the absorbance was measured in each well to create proliferation curves, using the absorbance data as a reference.

EdU cell proliferation assay

To further analyze cell growth, an EdU (5-ethynyl-2’-deoxyuridine) cell proliferation assay was conducted using a kit provided by RiboBio (China). Fibroblasts and A549 cells were seeded in 96-well plates and allowed to adhere. After adhesion, the old culture medium was replaced with 100 µL of EdU solution in each well and the plates were incubated for 2 h. Subsequent to this incubation, the cells were fixed and permeabilized. The cells were then stained with Apollo dye for 30 min in a light-protected environment and nuclei were highlighted using DAPI staining. Fluorescence microscope was used to examine and photograph the stained cells.

Bleomycin-induced mouse pulmonary fibrosis model

Male C57BL/6 mice, aged between 6 and 8 weeks, were procured from Changzhou Animal Company (China) for this experiment, which was approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. These mice were randomly divided into two groups: a control group (n = 7) and a bleomycin-treated group (n = 7). The bleomycin group received a tracheal injection of 2.5 mg/kg of bleomycin via a microinjector, whereas the control group received a similar volume of saline. On the 21st day post-injection, the mice were euthanized to collect their lung tissues for analysis.

Data collection and functional analysis

This investigation utilized four datasets: two focused on non-small cell lung cancer (NSCLC) (GSE18842 and GSE74706) and two on idiopathic pulmonary fibrosis (IPF) (GSE110147 and GSE53845). We applied R (4.1.3) and the limma (3.50.3) package to analyze these datasets for genes showing more than a twofold change in expression, either up or down, with a significance threshold of p < 0.01. To identify genes commonly affected across all datasets, an online tool for Venn diagrams (http://bioinformatics.psb.ugent.be/webtools/Venn/) was utilized. Both Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed using the clusterProfiler (4.2.2) package in R, setting a significance cutoff at p < 0.05. A protein–protein interaction (PPI) network was conducted through the STRING database, visualized using Cytoscape software (https://cn.string-db.org). Additionally, we sourced expression and clinical data on NSCLC from The Cancer Genome Atlas (TCGA), including 999 NSCLC and 103 normal samples, to conduct Kaplan–Meier survival analysis using the R package ‘survival’ (3.2.13) and to determine statistical significance at p < 0.05. Analysis of immune cell infiltration was conducted using the TIMER database.

Statistical analysis

GraphPad Prism 5.0 was used for our statistical analysis. Our data were presented as the mean ± standard error of the mean (SEM). Student’s t-test (2-tailed) was used to determine differences for experiments between two groups. For experiments involving more than two groups, we applied one-way ANOVA followed by Dunnett’s test to adjust for multiple comparisons. A threshold for statistical significance was established at a two-tailed p-value of less than 0.05. All in vitro experiments were independently replicated at least three times.