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Introduction: Patient-derived organoids have emerged as a promising in vitro model for precision medicine, particularly in cancer, but also in noncancer-related diseases. However, the optimal culture medium for culturing patient-derived lung organoids has not yet been agreed upon. This study aimed to shed light on the optimal selection of a culture media for developing studies using patient-derived lung organoids., Methods: Tumor and normal paired tissue from 71 resected non-small cell lung cancer patients were processed for organoid culture. Lung cancer organoids (LCOs) were derived from tumor tissue and normal lung organoids (LNOs) from nonneoplastic lung tissue. Three different culture media were compared: permissive culture medium (PCM), limited culture medium (LCM), and minimum basal medium (MBM). We assessed their effectiveness in establishing organoid cultures, promoting organoid growth and viability, and compared their differential phenotypic characteristics., Results: While PCM was associated with the highest success rate and useful for long-term expansion, MBM was the best option to avoid normal organoid overgrowth in the organoid culture. The density, size, and viability of LNOs were reduced using LCM and severely affected with MBM. LNOs cultured in PCM tend to differentiate to bronchospheres, while alveolosphere differentiation can be observed in those cultured with LCM. The morphological phenotype of LCO was influenced by the culture media of election. Mesenchymal cell overgrowth was observed when LCM was used., Conclusion: This work highlights the importance of considering the research objectives when selecting the most suitable culture medium for growing patient-derived lung organoids., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Background: During pseudoglandular stage of the human lung development the primitive bronchial buds are initially conformed by simple tubules lined by endoderm-derived epithelium surrounded by mesenchyme, which will progressively branch into airways and start to form distal epithelial saculles. For first time alveolar type II (AT2) pneumocytes appears. This study aims to characterize the genes and microRNAs involved in this differentiation process and decipher its role in the starting alveolar differentiation., Methods: Gene and microRNA profiling was performed in human embryonic lungs from 7 to 12 post conception weeks (pcw). Protein expression location of candidate genes were analyzed by immunofluorescense in embryonic lung tissue sections. mRNA/miRNA target pairs were identified using computational approaches and their expression was studied in purified epithelial/mesenchymal cell populations and in isolated tips and stalks from the bronchial tree. Additionally, silencing experiments in human embryonic lung mesenchymal cells and in human embryonic tip-derived lung organoids were performed, as well as organoid differentiation studies. AT2 cell markers were studied by qRT-PCR and by immunofluorescence. The TGFB-β phosphorylated pathways was analyzed with membrane protein arrays. Lung explants were cultured in air/liquid interface with/without peptides., Results: We identified 88 differentially expressed genes, including IGFBP3. Although IGFBP3 mRNA was detected in both epithelial and mesenchymal populations, the protein was restricted to the epithelium, indicating post-transcriptional regulation preventing IGFBP3 protein expression in the mesenchyme. MicroRNA profiling identified miR-34a as an IGFBP3 regulator. miR-34a was up-regulated in mesenchymal cells, and its silencing in human embryonic lung mesenchymal cells increased IGFBP3 levels. Additionally, IGFBP3 expression showed a marked downregulation from 7 to 12 pcw, suggesting its involvement in the differentiation process. The differentiation of human tip-derived lung embryonic organoids showed a drastic reduction in IGFBP3, supported by the scRNAseq data. IGFBP3 silencing in organoids activated an alveolar-like differentiation process characterized by stem cell markers downregulation and upregulation of AT2 markers. This process was mediated by TGFβ signalling inhibition and BMP pathway activation., Conclusions: The IGFBP3/miR-34a axis restricts IGFBP3 expression in the embryonic undifferentiated lung epithelium, and the progressive downregulation of IGFBP3 during the pseudoglandular stage is required for alveolar differentiation., (© 2024. The Author(s).)

Purpose: In patients with extrathoracic malignancies (EM) the role of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) for the assessment of abnormal mediastinal lymph nodes (MLN) is controversial. The aim of this study was to assess the diagnostic yield and prognostic significance of EBUS-TBNA in these patients., Methods: Retrospective analysis of patients with EM and abnormal MLN detected by Computed Tomography (CT) and/or Positron Emission Tomography (PET)., Results: A total of 161 patients with EM and abnormal MLN were included (93 males, 58%). The most common EM was melanoma (19%) and gastrointestinal cancer (17%). Assessed lymph nodes were mediastinal in 70% of cases and hilar in 30%. The most frequently sampled lymph nodes were subcarinal (45%) and lower right paratracheal (21%). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of EBUS-TBNA for the diagnosis of malignancy were 88%, 100%, 100% and 87%, respectively. These values were similar regardless the type of EM except for head and neck tumors where the NPV was particularly low (67%). The diagnosis of neoplastic involvement by EBUS-TBNA implied a worse prognosis in terms of overall survival (p < 0.02) and cancer-specific survival (p < 0.001)., Conclusions: In patients with EM and abnormal MLN, EBUS-TBNA has a high diagnostic yield. However, the NPV decrease in patients with head and neck tumors. Neoplastic MLN detected by EBUS-TBNA has prognostic implications in these patients., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In resected non-small cell lung cancer (NSCLC), post-surgical recurrence occurs in around 40% of patients, highlighting the necessity to identify relapse biomarkers. An analysis of the extracellular vesicle (EV) cargo from a pulmonary tumor-draining vein (TDV) can grant biomarker identification. We studied the pulmonary TDV EV-miRNAome to identify relapse biomarkers in a two-phase study (screening and validation). In the screening phase, a 17-miRNA relapse signature was identified in 18 selected patients by small RNAseq. The most expressed miRNA from the signature (EV-miR-203a-3p) was chosen for further validation. Pulmonary TDV EV-miR-203a-3p was studied by qRT-PCR in a validation cohort of 70 patients, where it was found to be upregulated in relapsed patients ( p = 0.0194) and in patients with cancer spread to nearby lymph nodes (N+ patients) ( p = 0.0396). The ROC curve analysis showed that TDV EV-miR-203a-3p was able to predict relapses with a sensitivity of 88% (AUC: 0.67; p = 0.022). Moreover, patients with high TDV EV-miR-203a-3p had a shorter time to relapse than patients with low levels (43.6 vs. 97.6 months; p = 0.00703). The multivariate analysis showed that EV-miR-203a-3p was an independent, predictive and prognostic post-surgical relapse biomarker. In conclusion, pulmonary TDV EV-miR-203a-3p is a promising new relapse biomarker for resected NSCLC patients.

Background: Cancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non-small cell lung cancer (NSCLC) has not been examined., Materials and Methods: Ultracentrifugation-purified exosomes from the A549 cell line were studied by CRYO-TEM, nanoparticle tracking analysis and western blot (TSG101 marker). YKT6 was inhibited using a DsiRNA and selected pre-microRNAs. MicroRNAs targeting YKT6 were validated by Renilla/Luciferase assay and western blot. YKT6 expression and its prognostic impact were analyzed in 98 tissue specimens from resected NSCLC patients., Results: Membranous nanosized vesicles (mode size: 128nm) with TSG101 protein were purified from A549 cells. YKT6 inhibition reduced exosome release by 80.9%. We validated miR-134 and miR-135b as miRNAs targeting YKT6, and transfection with the pre-miRNAs also produced a significant reduction in exosome release. The analysis of YKT6 in tumor samples showed that patients with high levels had shorter disease-free and overall survival., Conclusions: YKT6 is a key molecule in the regulation of exosome release in lung cancer cells and is in turn precisely regulated by miR-134 and miR-135b. Moreover, YKT6 levels impact prognosis of resected NSCLC patients., Competing Interests: All authors declare no conflicts of interest.

Aim: To evaluate the clinical relevance of the presence of lymphocytes in transbronchial needle aspiration (TBNA) samples from pathological mediastinal lymph nodes in patients with suspected lung cancer., Methods: Retrospective observational study evaluating the negative predictive value (NPV) of TBNA samples containing lymphocytes but not malignant cells., Results: A total of 266 TBNA were performed in 252 patients with pathological lymph nodes. One hundred and fifteen TBNA samples had evidence of malignant cells (43%), and 94 (35%) samples were considered as inadequate (absence of adequate cytological material or exclusive presence of bronchial epithelial cells). Out of the 57 TBNA samples remaining (21%), 15 could not be confirmed; in 32, TBNA samples were confirmed with alternative diagnostic techniques and in 10, they were confirmed after clinical and radiological follow-up. The NPV of the 32 samples that were confirmed with alternative diagnostic techniques was 84% decreasing down to 76% when the 10 TBNA samples confirmed after clinical and radiological follow-up were included., Conclusions: The presence of lymphocytes in the TBNA sample does not exclude the neoplasic invasion of the specific lymph node analyzed., (Copyright © 2010 SEPAR. Published by Elsevier Espana. All rights reserved.)

Background and Objective: Exacerbation of chronic obstructive pulmonary disease (COPD) is one of the most frequent causes of hospital admission. We examined the results and feasibility of two programs conducted by a hospital respiratory unit aimed to reduce both the length of hospital stay and the number of hospital admissions for COPD acute exacerbation., Patients and Method: a) Assisted hospital discharge program: Patients admitted for acute exacerbation who met our criteria for early discharge were sent home with the support of a respiratory nurse. Home visits were carried out and direct phone contact with the nurse and physician was provided during a limited period of 6 weeks. Outcome variables studied were length of hospital admission and need for hospital reentry. b) Exacerbation prevention program: A group of patients with severe COPD and at least 3 hospital admissions for exacerbation during the previous year were included. These patients underwent an educational program and were given unlimited direct phone access to the respiratory nurse and physician. When necessary, home visits were carried out. The main outcome variable of this program was the number of hospital admissions., Results: a) Assisted hospital discharge program: A total of 97 patients were included. The mean length of hospital stay was 5.4 1.7 days, which was significantly shorter than the previous average length of stay in our respiratory unit for a diagnosis of COPD exacerbation (8.52 days). The rate of hospital reentry was 17% (within the first 3 months). b) Exacerbation prevention program: 23 patients were enrolled. In this group, the number of hospital admissions decreased significantly from 5.0 1.8 to 1.7 2.4 per year (p = 0.001). Visits to the emergency department were also decreased, from 1.2 1.6 to 0.4 1.6 per patient (p = 0.05). Finally, the length of hospital stay decreased from 38 17 to 16 20 days (p = 0.0001)., Conclusions: A combined use of hospital resources and home care programs which are specifically addressed to severe COPD patients can reduce the need for hospital admission.