Patrick Zardo

Background: Alveolar type II (AT-II) epithelial cells are essential for alveolar repair, immune regulation, and surfactant secretion. Despite their promise for pulmonary disease modeling, limited access and culture methods hinder translational use. We established a patient-derived 3D AT-II organoid system from fibrotic and non-fibrotic lung tissue to maintain AT-II-associated features, enable cryopreservation, and capture disease-associated metabolic alterations. Methods: HT-II-280 ⁺ AT-II cells were isolated by magnetic bead sorting from 63 lung tissues (15 idiopathic pulmonary fibrosis, 26 secondary fibrosis, 22 tumor-distant controls). Cells were expanded as organoids in 3D culture from initial passage 0 up to passage 3. AT-II-associated features were assessed by immunofluorescence, flow cytometry, and transmission electron microscopy. Cryopreserved cells were recovered after ≥ 28 days and tested for viability and organoid-forming capacity. Metabolic profiling was performed using extracellular flux assays. Results: AT-II cells were successfully (~ 80%) isolated and combined with a serum- free feeder-free culturing approach to reproducibly generated alveolospheres with highly efficient colony formation (> 90% in P1), especially in AT-II cells from fibrotic explants. Primary tissue-derived lung organoids display heterogeneous morphologies and sizes, most prominently in fibrotic-derived cultures, as indicated by histology and microcomputed tomography. Culture conditions were optimized to minimize differentiation towards AT-I cells or dedifferentiated epithelial states with partial basaloid features. Expression of key AT-II-associated markers (proSP-C, HT-II-280), and the presence of lamellar bodies were maintained across passages at the population level. Cryopreservation maintained high viability, organoid-forming capacity, and metabolic activity, enabling long-term storage. Fibrotic organoids exhibited disease-associated metabolic reprogramming characterized by a pronounced glycolytic shift with increased ATP production. Conclusion: We established a reproducible cell-line-free 3D culture system from primary human AT-II cells of end-stage ILD lungs to generate patient-derived lung organoids. These organoids maintain AT-II-associated features across passages, remain viable after cryostorage, and capture disease-associated metabolic reprogramming. Fibrotic-derived AT-II cells consistently demonstrated a Warburg-like glycolytic phenotype, reflecting increased energy demand. This scalable model in vitro provides a defined resource for mechanistic studies of epithelial dysfunction in pulmonary diseases and supports biobanking for future precision medicine applications.

Pulmonary Fibrosis (PF) is a life‐threatening illness that is characterized by progressive scarring in the lung interstitium. There is an urgent need for new PF therapies because current treatments only slow down the progression of fibrosis, and the median life expectancy post‐diagnosis is only 4–6 years. Since PF patients frequently exhibit telomere attrition, overexpressing telomerase, the enzyme responsible for synthesizing telomeres, represents a compelling therapeutic option. In this study, we in vitro transcribed human telomerase reverse transcriptase (hTERT) mRNA using modified nucleosides (modRNA). ModRNA hTERT treatment led to transient activation of telomerase activity in a dose‐dependent manner in MRC‐5 cells and, importantly, in primary human alveolar type II pneumocytes. Consequently, the proliferative capacity was increased, concomitant with reduced DNA damage and elongated telomere length. Notably, the induction of cellular immune response was only detectable at the highest modRNA concentration and returned to normal levels within 48 h. Next, we demonstrated that circularized, exonuclease‐resistant modRNA hTERT extended the transient expression profile, which may be clinically advantageous. Finally, we provided therapeutic proof of concept in organotypic 3D ex vivo human precision‐cut lung slices derived from end‐stage PF patients. Intriguingly, a single modRNA hTERT treatment inhibited senescence, as indicated by significantly lower levels of senescence‐associated β‐galactosidase. Pro‐inflammatory markers (IL6 and IL8) and, concurrently, the key fibrosis mediators TGFβ and COL1A1 were markedly reduced after modRNA and circular RNA hTERT treatment. In conclusion, the data presented herein provide initial evidence for the potential of RNA‐based hTERT therapy for treating human lung fibrosis.

Pulmonary Fibrosis (PF) is a life-threatening illness that is characterized by progressive scarring in the lung interstitium. There is an urgent need for new PF therapies because current treatments only slow down the progression of fibrosis and the median life expectancy post-diagnosis is only 4 to 6 years. Since PF patients frequently exhibit telomere attrition, overexpressing telomerase, the enzyme responsible for synthesizing telomeres represents a compelling therapeutic option. In this study, we in vitro transcribed human telomerase reverse transcriptase (hTERT) mRNA using modified nucleosides (modRNA). ModRNA hTERT treatment led to transient activation of telomerase activity in a dose-dependent manner in MRC-5 cells and, importantly, in primary human alveolar type II pneumocytes (ATII cells). Consequently, the proliferative capacity was increased, concomitant with reduced DNA damage and elongated telomere length. Notably, induction of cellular immune response was only detectable at the highest modRNA concentration, and returned to normal levels within 48 hours. Next, we demonstrated that circularized, exonuclease-resistant modRNA hTERT extended the transient expression profile which may be clinically advantageous. Finally, we provided therapeutic proof of concept in organotypic 3D ex vivo human precision-cut lung slices derived from end-stage PF patients. Intriguingly, a single modRNA hTERT treatment inhibited senescence as indicated by significantly lower levels of senescence-associated β-galactosidase, and pro-inflammatory IL6 and IL8. Concurrently, the key fibrosis mediators TGFβ and COL1A1 were markedly reduced. In conclusion, the data presented herein provide initial evidence for the potential of RNA-based hTERT therapy for treating human lung fibrosis.

Background: Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancers, and is the leading cause of tumor-related death. Lung adenocarcinoma (LUAD) is the most prevalent subtype of NSCLC. Although significant progress of LUAD treatment has been made under multimodal strategies, the prognosis of advanced LUAD is still poor due to recurrence and metastasis. There is still a lack of reliable markers to evaluate the LUAD prognosis. This study aims to explore novel biomarkers and construct a prognostic model to predict the prognosis of LUAD patients. Methods: The Genomic Data Commons-The Cancer Genome Atlas-Lung Adenocarcinoma (GDC-TCGA-LUAD) dataset was downloaded from the University of California, Santa Cruz (UCSC) Xena browser. The GSE72094 and GSE13213 datasets and corresponding clinical information were downloaded from the Gene Expression Omnibus (GEO) database. By analyzing these datasets using DESeq2 R package and Limma R package, differentially expressed genes (DEGs) were found. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used to analyze possible enrichment pathways. A protein-protein interaction (PPI) network was constructed to explore possible relationship among DEGs by using the STRING database. A survival analysis was performed to identify reliable prognostic genes using the Kaplan-Meier method. A multi-omics analysis was performed using the Gene Set Cancer Analysis (GSCA). The Tumor Immune Estimation Score (TIMER) database was used to analyze the association between prognostic genes and immune infiltration. A Spearman correlation analysis was conducted to examine the correlation between prognostic genes and drug sensitivity. A multivariate Cox regression was used to identify independent prognostic factors. Next, a nomogram was constructed using the rms R package. Finally, the expressions of aspartyl-tRNA synthetase 2 (DARS2) and phosphoribosyl aminoimidazole carboxylase (PAICS) were detected using immunohistochemistry (IHC). Results: We screened out 30 DEGs prior to functional enrichment and PPI network analysis revealing potential enrichment pathways and interactions of these DEGs. Then survival analysis revealed the CENPL, DARS2, and PAICS expression was negatively correlated with LUAD prognosis. Additionally, multi-omics analysis showed CENPL, DARS2, and PAICS expressions were significantly higher in LUAD tissues than normal tissues. CENPL, DARS2, and PAICS were all up-regulated in late stage and M1 stage. Correlation analysis indicated CENPL, DARS2, and PAICS may not be associated with activation or suppression of immune cells. Drug sensitivity analysis revealed many potentially effective drugs and small molecule compounds. Moreover, we successfully constructed a robust and stable nomogram by combining the DARS2 and PAICS expression with other clinicopathological variables. Finally, IHC results showed DARS2 and PAICS were significantly up-regulated in LUAD. Conclusions: The CENPL, DARS2, and PAICS expression was negatively correlated with LUAD prognosis. A prognostic model, which integrated DARS2, PAICS, and other clinicopathological variables, was able to effectively predict LUAD patients prognosis.

Formation of metastases marks the main cause of death in cancer and is characterized by complex processes, which can only partly be reflected in most in-vitro models due to a lacking human microenvironment. Cisplatin, bevacizumab and vemurafenib were used to modulate different aspects of tumorigenesis in co-cultures of patient-derived, disseminated melanoma cells or MDA-MB-231 cells and fresh human lung tissue as well as fresh human tumor tissue slices. Neoangiogenetic biomarker VEGF was elevated 3.5fold in co-cultures and 5.4fold in lung tumor slices after 48h. Treatment with bevacizumab [200 μg/mL] suppressed VEGF-release 24fold in co-cultures and 25fold in lung tumor tissue slices after 48h, also leading to impaired endothelial cell migration of the supernatants (81% in co-cultures, 83% in tumor slices). Cisplatin treatment [50μM] led to a decline of viability in co-cultures by up to 37.5% and in tumor slices up to 48.7% after 72h. Co-cultures and tumor slices were less sensitive in regards to their respective anti-cancer drug efficacy than the 2D culture. To address patient-specific genetic disparities, co-cultures with disseminated melanoma cells were performed with both patient-derived cells carrying BRAF mutation V600E and non-mutated cells. Treatment with vemurafenib reduced V600E cancer cell number (IC50=6µM) after 48 hours while non-mutated cells showed no significant cancer cell decrease. Here we show that tumor cells in solid tumors and seeded into healthy lung tissue are sensitive to tumor treatments ex vivo. This work aims to translate cancer drug efficacy data from animals towards humans to optimize clinical trial outcome.

Cigarette smoke (Cs) inhalation is one of the main reasons to develop chronic obstructive pulmonary disease (COPD), characterised by degradation of alveoli, emphysema development, inflammation and mucus hypersecretion. Mechanisms that underlie various components of COPD can be modelled in vitro, specifically using whole fresh cigarette smoke. We assessed the effects of Cs and Cs condensate (Csc) on fresh human lung tissue. Human Precision-Cut Lung Slices (PCLS) were exposed to Csc or whole Cs in an Air-Liquid Interface (ALI) using the in vitro exposure device P.R.I.T.® ExpoCube®. Tissue viability, release of cytokines and extracellular matrix (ECM) proteins were analysed after single and repeated exposure and cultivation of up to 96h. Inhibitors were applied to suppress inflammatory responses of tissue to Cs. Csc and Cs induced concentration-dependent loss of tissue viability in fresh lung tissue. Smoke exposure induced an increased release of pro-inflammatory cytokines IL-1α (˜ two fold), IL-1β (˜ three fold) and changes in ECM proteins, e.g. MMP-9 and pro-Collagen1α1 (˜ two fold), as well as Receptor for Advanced Glycation Endproducts (RAGE) from human PCLS. Dexamethasone and Roflumilast inhibited Cs-induced increase of pro-inflammatory cytokines. Csc and Cs induced tissue injury, early biomarkers of inflammation and changes in ECM proteins in vital ex vivo human lung tissue. The exposure of the complex mixture of whole cigarette smoke closely reflects the in vivo situation in human lung tissue. Next we will assess the response of human PCLS exposed to Cs and infected with rhinovirus to study exacerbations.