The institutional review board approved data collection and analysis, and the requirement for written informed consent from each patient was waived. All animal experiments were performed in accordance with the protocols approved by the Institutional Animal Care and Use Committee. All efforts were made to minimize the number of animals used and their discomfort.

To confirm that patients with non-small cell lung cancer (NSCLC) and ILD had lymph node or lung metastases more frequently than those without ILD, we examined 516 consecutive patients clinically diagnosed with stage I NSCLC who underwent complete resection with systematic lymph node dissection between January 2011 and December 2020 at our institution.

Chest computed tomography (CT) was performed to detect ILD at the initial diagnosis, classify the stages of all patients, and measure the tumor size before surgery. ILD was considered present if there was subpleural or peribronchovascular reticulation characterized by traction bronchiectasis or bronchiolectasis with or without surrounding ground-glass opacities and honeycombing (20). Regional lymph node metastasis was clinically defined when the shorter diameter of a given lesion was ≥1.0 cm. Additional diagnostic testing, including brain magnetic resonance imaging, bone scintigraphy, and positron emission tomography combined with CT, was performed at the individual physician’s discretion according to the patient’s symptoms and clinical findings. Histological type was determined according to the World Health Organization classification (21). Disease stages were based on the tumor–node–metastasis classification of the International Union Against Cancer, eighth edition (22). Complete resection was defined as a gross and histological cancer-free surgical margin.

Six-week-old female C57BL/6 mice were obtained from CLEA Japan (Tokyo, Japan). The mice were maintained in a pathogen-free facility. Female C57BL/6 mice aged 8 weeks were intraperitoneally anesthetized using pentobarbital sodium (Kyoritsu Seiyaku Co., Tokyo, Japan), followed by a single intratracheal injection of 3 mg/kg of bleomycin (BLM) sulfate (Wako, Osaka, Japan) in 50 μl of sterile phosphate-buffered saline (PBS), as described previously (23).

The mouse Lewis lung cancer (LLC) cell line was purchased from the Health Science Research Resources Bank (Osaka, Japan) and maintained in Roswell Park Memorial Institute 1640 medium supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Yokohama, Japan). Mouse pulmonary fibroblasts were extracted from C57BL/6 mouse lungs, as previously described (24).

LLC-luciferase-expressing cells (4 × 10⁴) were suspended in PBS containing 15% Matrigel (BD Biosciences, San Jose, CA, USA) and injected into the parenchyma of the left lung lobe through the rib cage using a 29 G needle. To directly visualize the lung, a 4- to 5-mm incision was made in the skin under the left shoulder, which was then closed (25). Next, 7, 10, and 14 days after the injection of LLC cells, mice were intraperitoneally injected with 200 μl of d-luciferin (15 mg/ml, ViviGlo Luciferin; Promega, Madison, WI, USA) under anesthesia with 2% isoflurane. Bioluminescence images were then obtained using the In Vivo Imaging System(IVIS)Lumina II with Living Image ver. 3.2 (Xenogen, Alameda, CA, USA), following the manufacturer’s protocol.

Lung tissues were fixed in 4% formalin for 12–16 h, dehydrated, and embedded in paraffin. The sample blocks were sliced into 4-μm-thick sections and stained with hematoxylin and eosin (HE). The Masson’s trichrome (MT) assay was used to assess lung fibrosis. The Ashcroft score (26) was used to evaluate the degree of pulmonary fibrosis.

Lung tissue sections were incubated in blocking solution (10% goat serum in PBS) for 30 min at room temperature, followed by incubation with anti-mouse α-smooth muscle actin (α-SMA) – Cy3 (Sigma-Aldrich, St. Louis, MO, USA) monoclonal antibody.

Matrigel invasion activity was assayed using the FluoroBlok Cancer Cell Invasion Assay System (BD Biosciences) in accordance with the manufacturer’s instructions. Briefly, tdTomato-labeled cells (5 × 10⁴) were seeded in serum-free culture medium onto Matrigel-coated filters with or without fibroblasts isolated from mouse lungs. Culture medium supplemented with 10% FBS was added to the lower chambers. After incubation in 5% CO₂ at 37 °C for 16–20 h, the fluorescence intensity of the invaded cells was determined using a GENios microplate reader (Tecan, Männedorf, Switzerland).

Collagen levels in the lung tissues were determined using the SIRCOL collagen assay (Biocolor Ltd., Carrickfergus, UK), according to the manufacturer’s instructions. Briefly, left lung lobes were homogenized, and collagen was solubilized in 0.5 M acetic acid. The extracts were incubated with Sirius red dye, and the absorbance was determined at 540 nm. The results were expressed as milligrams of collagen per left lung.

We examined whether pharmacological treatment with pirfenidone (PFD) blocks lung cancer progression promoted by the IP lung environment. PFD was the first anti-fibrotic agent approved for the treatment of idiopathic pulmonary fibrosis (IPF) in Japan. PFD (Shionogi & Co., Osaka, Japan) was suspended in a 0.5% carboxymethylcellulose (Nacalai Tesque, Kyoto, Japan) solution. PFD (300 mg/kg/day) was administered orally twice daily (7:00–10:00 and 19:00–22:00) and continued until dissection, as previously described (27, 28).

We intratracheally administered PBS or BLM with daily oral PFD. Two weeks after intratracheal administration, we injected LLC cells into the left lungs. Two weeks after the LLC cell injection, we evaluated the lungs of the four groups (PBS-vehicle, PBS-PFD, BLM-vehicle, and BLM-PFD).

All data are presented as mean ± standard deviation. Differences in categorical outcomes were evaluated using the χ² test. Normally distributed continuous variables were compared using an unpaired t-test for two groups or one-way analysis of variance for multiple groups. All reported P-values were two-sided, and the significance level was P < 0.05.

Correlations between patients with ILD and postoperative pathological characteristics are shown in Table 1 . Patients with ILD had a significantly greater number of moderately or poorly differentiated carcinomas and carcinomas with vascular invasion, lymphatic permeation, pleural invasion, lymph node metastases, and intrapulmonary metastases than those without ILD ( Table 1 ).

We established a murine BLM-induced IP model. BLM was administered intratracheally to C57BL/6 mice. At 2 weeks after BLM administration, HE and MT staining of collagen revealed severe pulmonary damage and collagen deposition, which continued for at least 4 weeks after administration ( Supplementary Figure 1 ).

We then established an orthotopic lung cancer model. We directly injected 4 × 10⁴ LLC cells into the left lungs of C57BL/6 mice. Fourteen days after implantation, the primary tumor was visible to the naked eye, and metastases to the mediastinal lymph nodes were observed ( Supplementary Figures 2A-C ). Although these mice lived 18–23 days after injection, no metastatic nodules were observed in distant organs, including the contralateral lung ( Supplementary Figure 2A ).

Using these models, we examined whether the BLM-induced IP lung environment affected the biological behavior of lung cancer. First, we intratracheally administered PBS or BLM. We injected LLC cells into the left lung 2 weeks later. The lungs of both groups were evaluated 2 weeks after LLC cell injection ( Figure 1A ). The difference in the primary tumor size did not significantly differ between the PBS and BLM groups; however, the weight of the mediastinal lymph nodes was significantly higher in the BLM group than in the PBS group ( Figures 1B-D ). Surprisingly, contralateral lung metastases were also observed in the BLM group ( Figure 1E ).

α-SMA-positive lung fibroblasts or myofibroblasts have been reported to play an important role in the pathogenesis of IPF (29). Indeed, α-SMA-positive cells were more frequently observed in the BLM-treated lungs than in control lungs ( Supplementary Figure 3A ). In addition, α-SMA-positive spindle-shaped cells were identified more often in the tumors of the BLM-induced IP model than in control PBS tumors ( Supplementary Figure 3B ). Therefore, we considered that α-SMA-positive fibroblasts or myofibroblasts are associated with lung cancer progression.

Next, we isolated fibroblasts from both lungs 2 weeks after the intratracheal administration of PBS or BLM. Fibroblasts from BLM-induced IP lung environment showed a more spindle-shaped morphology and α-SMA-positive cells were more frequently detected than in control lungs ( Supplementary Figure 4 ). As shown in Figure 2A , we directly co-injected 4 × 10⁴ LLC cells into the left lung of C57BL6 mice with medium alone, 4 × 10⁴ fibroblasts from the control PBS lung, and 4 × 10⁴ fibroblasts from the BLM-induced IP lung environment after dividing the mice into three groups (LLC-only, PBS-fibroblast, and BLM-fibroblast groups). Two weeks after the co-injection, the difference in the size of the primary tumor was not statistically significant among the three groups ( Figures 2B, C ); however, a significant increase in the weight of the mediastinal lymph nodes was observed in the BLM-fibroblast group ( Figure 2D ). In addition, contralateral lung metastases were observed only in the BLM-fibroblast group ( Figures 2B, E ). α-SMA-positive cells were more frequently detected in the tumors of the BLM-fibroblast group than in those of other groups ( Supplementary Figure 5 ).

Since fibroblasts from BLM-induced IP lung environment promote lymph node metastasis of cancer cells, we examined whether fibroblasts affected the invasive capacity of LLC cells. After transfecting LLC cells with the fluorescent protein tdTomato ( Figure 2F ), we assessed the invasive capacity of cancer cells using a Matrigel invasion assay. LLC cells were seeded into the upper chamber after the addition of medium alone, in combination with fibroblasts from the PBS control lung, or in combination with fibroblasts from the BLM-induced IP lung environment ( Figure 2G ). A significantly increased number of invading LLC cells was detected in the BLM-fibroblast group compared with that in the other groups ( Figure 2G ).

HE and MT staining revealed that severe pulmonary damage and BLM-induced collagen deposition were suppressed by the administration of PFD (300 mg/kg/day) ( Supplementary Figure 6A ). Furthermore, the BLM-induced elevation of pulmonary hydroxyproline (an indicator of collagen levels) was significantly suppressed ( Supplementary Figure 6B ). According to Ashcroft’s method, quantitative histology also showed that PFD significantly attenuated the score when administered to BLM-treated mice ( Supplementary Figure 6C ).

The intratracheal administration of PBS or BLM with daily oral PFD, injection of LLC cells into the left lung 2 weeks after intratracheal administration, and evaluation of the lungs of the animals in the four groups (PBS-vehicle, PBS-PFD, BLM-vehicle, and BLM-PFD) 2 weeks after LLC cell injection is shown in Figure 3A . The difference in the size of the primary tumor was not statistically significant between the groups ( Figures 3B, C ); however, a significant decrease in the weight of mediastinal lymph nodes was observed in the BLM-PFD group compared to that in the BLM-vehicle group ( Figure 3D ). Contralateral lung metastases were only observed in the BLM-vehicle group ( Figure 3E ).