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Additional file 1. Table S1. Summary of clinicopathological data and CT findings of 15 patients with METex14 skipping-positive NSCLC. Table S2. CT findings of the primary tumor in adenocarcinoma. Table S3. CT imaging features of the primary tumor compared to the tumor size. Table S4. CT findings of lymph nodes and distant metastases. (a) CT findings of lymph node metastases in patients with stage III/IV NSCLC (n=10). (b) Patterns of distant metastasis in patients with stage IV NSCLC (n=6). (c) CT findings of distant metastases in patients with stage IV NSCLC (n=6). Figure S1. CT images of stage I/II NSCLC with METex14 skipping.

Additional file 3: Cytokine levels in BALF before BLM OA or at 7 or 14 days after BLM OA (n = 5–8 per group). Data are presented as the mean ± SD for normal distribution (IL-13), or as the median with interquartile range for non-normal distribution (TNF-α, IL-4, IL-5, IL-10).

Additional file 5. ALDH1a1 immunostaining in lung tissue. Representative images of ALDH1a1 immunostaining in PBS- or BLM-treated lung tissue sections on day 14.

Additional file 9: Lung homogenate was generated from the left lung of mice at 7 days after PBS OA (DAMP- group) or BLM OA (DAMP+ group). Upper inserts (pore size, 0.4 μm; Corning) with cultured lung homogenates were dipped into the basal plate of MSCs (1.0 × 105 cells/well) cultured in DMEM with 10% FBS (S-MSCs) or in serum-free STK2 media (SF-MSCs). After 72 hours, the MSCs were harvested, and intracellular proteins were extracted for TGF-β1 measurement. TGF-β1 was measured using an ELISA kit. Data were calculated as a TGF-β1 per each number of live MSCs in the culture media, and expressed as a percentage of the mean in DAMP- group. Data are presented as means ± SD (n = 3 per group).

Additional file 7. CD45−/ALDHbr cell therapy ameliorates BLM-induced pulmonary fibrosis. Masson’s trichrome staining of lung tissue sections obtained on day 14 from BLM-treated mice transferred with CD45−/ALDHdim and CD45−/ALDHbr cells.

Additional file 1: Representative flow cytometry analysis related to the defined positive and negative MSC surface markers on MSCs cultured in DMEM with 10% FBS (S-MSC) or in serum-free STK2 medium (SF-MSC).

Additional file 4: Cytokine levels in serum before BLM OA or at 4, 7, 14, or 21 days after BLM OA (n = 3–7 per group). Data are presented as the median with interquartile range.

Additional file 2: Fluorescence microscopic ex vivo images of engrafted DiI-labeled SF-MSCs (yellow arrows) in murine lungs on the day after injection.

Additional file 1. Antibodies used in flow cytometric experiments.

Additional file 6. Double staining of ALDHbr cells and side population cells in lung tissue. (A) Representative image of lung CD45−/CD31−/ALDHbr live cells. (B) Representative image of lung CD45−/CD31−/Hoechstdim (side population, SP) live cells. (C) The gating of SP cells was confirmed using verapamil, which is an inhibitor for Hoechst staining. (D) SP cells in lung CD45−/CD31−/ALDHbr live cells. (E) ALDHbr cells in SP cells.

Additional file 4. Expression of cell surface proteins in CD45−/ALDHdim cells and CD45−/ALDHbr cells. Sorted CD45−/ALDHdim cells and CD45−/ALDHbr cells were examined for cell surface markers associated with mesenchymal stem cells (MSCs), fibroblasts, and stem cells.

Additional file 8. Discrimination of mCherry-heterozygotic mouse. DNA extracted from the tails of mice was amplified by PCR using primers shown in Table S2. The expression of the mCherry-heterozygotic band was evaluated using agarose gel electrophoresis.

Additional file 10: Representative flow cytometry analysis of the Tregs fraction (red box) in murine blood or lung CD4+ T cells at 6, 10, or 14 days after BLM OA. BLM-administered mice were treated with or without SF-MSC plus Treg depletion Ab (PC61) as shown in Figure 6A.

Additional file 3. Effect of magnetic-activated cell sorting for the enrichment of CD45−/ALDHbr cells. Representative image of lung CD45−/ALDHbr cells before and after magnetic-activated cell sorting using the Tissue Stem Cell Pre-Enrichment Kit. ALDHbr gating was determined by comparing samples stained with ALDEFLUOR alone and with ALDEFLUOR and diethylaminobenzaldehyde (DEAB), an ALDH inhibitor.

Additional file 8: S-MSCs or SF-MSCs were seeded into 24-well plates at a density of 5 × 103 cells/well. These MSCs were cultured in serum-free DMEM for 48 h. The supernatants of the culture medium were collected at 6, 12, 24, or 48 h, and the TGF-β1 concentration in the supernatants was measured using an ELISA kit. Data are expressed as concentration of TGF-β1 per each live 100,000 MSCs in the cell culture media. Data are presented as means ± SD (n = 4 per group). On the indicated hours, there was no statistically significant difference in TGF-β1 between the two MSC groups.

Additional file 6: Hydroxyproline levels in the murine left lung at 14 days after BLM OA. At 4 days after BLM OA, mice were further subjected to subcutaneous (sc) or intraperitoneal (ip) administration of SF-MSCs at a dose of 1 × 105 in 100 µL of PBS. Mice without MSC administration were used as controls. Data are presented as the means ± SD (n = 5–8 per group). NS, not significant.

Additional file 2. Primers used in this study.

Additional file 1: Fig. S1. Frequency distribution of BALF lymphocyte nuclear area. The dark gray bars represent the frequency distribution of the BALF lymphocyte nuclear area in sarcoidosis patients, while the light gray bars represent those in the other ILD patients. The vertical dotted line represents the sarcoidosis-diagnostic threshold of the nuclear area (428.1 μm2). Representative lymphocytes with small nuclear areas, those near the threshold value, and those with large nuclear areas are shown in the lower left panel, lower middle panel, and lower right panel, respectively. Fig. S2. Frequency distribution of BALF lymphocyte nuclear perimeters. The dark gray bars represent the frequency distribution of the BALF lymphocyte nuclear perimeters in sarcoidosis patients, while the light gray bars represent those in the other ILD patients. The vertical dotted line represents the sarcoidosis-diagnostic threshold of the nuclear perimeter (71.3 μm). Representative lymphocytes with short nuclear perimeters, those near the threshold value, and those with large nuclear perimeters are shown in the lower left panel, lower middle panel, and lower right panel, respectively. Fig. S3. Frequency distribution of BALF lymphocyte nuclear radius ratios. The dark gray bars represent the frequency distribution of the BALF lymphocyte nuclear radius ratios in sarcoidosis patients, while the light gray bars represent those in the other ILD patients. The vertical dotted line represents the sarcoidosis diagnostic threshold of the nuclear radius ratio (1.346). Representative lymphocytes with small nuclear radius ratios, those near the threshold value, and those with large nuclear radius ratios are shown in the lower left panel, lower middle panel, and lower right panel, respectively. Fig. S4. Frequency distribution of BALF lymphocyte nuclear roundness. The dark gray bars represent the frequency distribution of BALF lymphocyte nuclear roundness in sarcoidosis patients, while the light gray bars represent those in the other ILD patients. The vertical dotted line represents the sarcoidosis diagnostic threshold for nuclear roundness (1.0057). Representative lymphocytes with small nuclear roundness, those near the threshold value, and those with large nuclear roundness are shown in the lower left panel, lower middle panel, and lower right panel, respectively. Fig. S5. The four morphological parameters of BALF lymphocyte nuclei. The figures are representing the definition of area, perimeter, radius ratio and roundness of lymphocyte nucleus

Additional file 7: Representative flow cytometry analysis on engrafted DiI-labeled MSCs in the murine lung. DiI-labeled SF-MSCs were injected into mice subcutaneously (sc) or intraperitoneally (ip) at a dose of 2.0 × 105 cells/mouse in 100 µL of PBS at 4 days after BLM OA. On the day after DiI-labeled SF-MSC injection, MSCs engrafted in murine lungs were measured using flow cytometry.

Rationale: Some patients with pulmonary arteriovenous malformation (PAVM) present with hypoxemia and life-threatening complications, including stroke and cerebral abscess. Catheter embolization is currently the preferred treatment for PAVM. However, previous studies have revealed that the incidence of PAVM recanalization is approximately 10% 5 to 7 years after embolization. In contrast, there are no studies where recanalization has occurred over 10 years after embolization. Patient concerns: Herein, we report 2 cases diagnosed with cerebral embolism due to PAVM recanalization 13 years and 30 years after catheter treatment, in case I and II, respectively. Diagnoses: Both cases were diagnosed with PAVM recanalization on chest computed tomography (CT) examination performed after cerebral embolism development. Furthermore, pulmonary artery angiography revealed blood flow from the pulmonary artery to the vein in the PAVM, confirming PAVM recanalization. Interventions: Coil re-embolization was performed for the all recanalized PAVM. Outcomes: All the target lesions were successfully re-embolized in both cases. However, in case I, the second recanalization of embolized PAVM was confirmed 1 year after coil re-embolization. Consequently, the third embolization was performed in case I. In contrast to case I, the patient in case II was followed up without recanalization for 2 years after embolization. Lessons: We described the first 2 cases diagnosed with PAVM recanalization >10 years after the first catheter embolization. These cases suggest that patients with PAVMs should undergo life-long follow-up after catheter embolization.

Additional file 1.

Rationale: The efficacy of direct oral anticoagulants (DOACs) in the treatment and prophylaxis of cancer-related venous thromboembolism (VTE) is reportedly similar to that of heparin. However, the effect of DOACs on the prophylaxis of cancer-related arterial thromboembolism (ATE) remains unclear. To our knowledge, we present the 1st case where cerebral ATE was encountered during edoxaban administration for VTE in a patient with lung adenocarcinoma. Patient concerns: In March 2017, a 63-year-old female was diagnosed with lung adenocarcinoma (cT2aN3M1b stage IVa) along with having asymptomatic VTE; thus, 60 mg/day edoxaban administration was initiated. In addition, 1st-line chemotherapy generated a partial antitumoral response. However, owing to lung cancer progression, a secondary treatment with pembrolizumab administration was initiated. The patient suddenly experienced aphasia 11 days after pembrolizumab administration. Diagnosis: The patient was diagnosed as multiple cerebral ATE using brain magnetic resonance imaging. However, VTE recurrence was not observed. Based on the findings of lung cancer progression and increased coagulation, cerebral ATE was diagnosed as Trousseau syndrome. Interventions: DOAC administration was switched to heparin administration. Outcomes: Coagulation profile normalized and aphasia improved without any further disease symptoms. Lessons: We considered that DOACs are effective for the treatment and prophylaxis of VTE but may be insufficient for ATE prevention. Therefore, DOACs should be replaced with heparin to prevent ATE when cancer and coagulation become uncontrollable with DOAC.

Stem cell factor (SCF) and its receptor c-Kit have been implicated in tissue remodelling and fibrosis. Alveolar fibroblasts from patients with diffuse interstitial fibrosis secrete more SCF. However, its precise role remains unclear. In this study the potential role of the SCF-c-Kit axis in pulmonary fibrosis was examined. Fibrosis was induced by intratracheal instillation of bleomycin (BLM), which caused increased SCF levels in plasma, bronchoalveolar lavage fluid (BALF) and lung tissue, as well as increased expression by lung fibroblasts. These changes were accompanied by increased numbers of bone marrow-derived c-Kit(+) cells in the lung, with corresponding depletion in bone marrow. Both recombinant SCF and lung extracts from BLM-treated animals induced bone-marrow cell migration, which was blocked by c-Kit inhibitor. The migrated cells promoted myofibroblast differentiation when co-cultured with fibroblasts, suggesting a paracrine pathogenic role. Interestingly, lung fibroblast cultures contained a subpopulation of cells that expressed functionally active c-Kit, which were significantly greater and more responsive to SCF induction when isolated from fibrotic lungs, including those from patients with idiopathic pulmonary fibrosis (IPF). This c-Kit(+) subpopulation was αSMA-negative and expressed lower levels of collagen I but significantly higher levels of TGFβ than c-Kit-negative cells. SCF deficiency achieved by intratracheal treatment with neutralizing anti-SCF antibody or by use of Kitl(Sl)/Kitl(Sl-d) mutant mice in vivo resulted in significant reduction in pulmonary fibrosis. Taken together, the SCF-c-Kit pathway was activated in BLM-injured lung and might play a direct role in pulmonary fibrosis by the recruitment of bone marrow progenitor cells capable of promoting lung myofibroblast differentiation.