Healthy Lung Tissue Research Articles

Abstract 14698: Identifying Sources of Prostacyclin Outside the Vascular Wall and Their Contribution to Cardiovascular Protection

Introduction: Prostacyclin is a key anti-thrombotic hormone synthesised through cyclooxygenase (COX) enzymes. Historically, its production has been almost universally associated with the endothelium however the role of non-vascular sources has not been systematically evaluated due to a lack of suitable tools. This has resulted in an underappreciation of its cardiovascular biology and role in other contexts. Hypothesis: We hypothesised that cells outside the vascular wall contribute to generation of prostacyclin which can enter the circulation to provide anti-thrombotic protection. Methods: Global, endothelial and fibroblast COX1 or COX2 knockout mice were studied alongside freshly isolated healthy human lung tissue and primary cell cultures. Prostacyclin levels in plasma and released from cells/tissue ex vivo were measured by ELISA. Individual cell populations were isolated by FACS. Thrombosis in vivo was measured using a FeCl 3 -carotid artery injury model. Results: Vascular endothelial COX1 deletion strongly reduced prostacyclin release from mouse aorta and heart, but not from isolated lung, colon, kidney or spleen (Fig A). This non-vascular prostacyclin required COX1 as it was abolished by global COX1 and not COX2 deletion. Freshly isolated lung fibroblasts exhibited greater prostacyclin synthesis than endothelial or other cell types (Fig B) and this was confirmed in primary human lung cell cultures. Deletion of COX1 from fibroblasts in mice reduced lung prostacyclin release (Fig C) and plasma prostacyclin levels (Fig D) and this was associated with accelerated thrombotic occlusion after carotid artery injury (Fig E). Conclusions: These findings define a new paradigm in prostacyclin biology in which fibroblast/non-vascular-derived prostacyclin works in parallel with endothelium-derived prostanoids to control thrombosis. This may have particular relevance to understanding how local diseases of the lung result in cardiovascular risk.

Basal Cell-derived WNT7A Promotes Fibrogenesis at the Fibrotic Niche in Idiopathic Pulmonary Fibrosis.

Loss of epithelial integrity, bronchiolarization, and fibroblast activation are key characteristics of idiopathic pulmonary fibrosis (IPF). Prolonged accumulation of basal-like cells in IPF may impact the fibrotic niche to promote fibrogenesis. To investigate their role in IPF, basal cells were isolated from IPF explant and healthy donor lung tissues. Single-cell RNA sequencing was used to assess differentially expressed genes in basal cells. Basal cell and niche interaction was demonstrated with the sLP-mCherry niche labeling system. Luminex assays were used to assess cytokines secreted by basal cells. The role of basal cells in fibroblast activation was studied. Three-dimensional organoid culture assays were used to interrogate basal cell effects on AEC2 (type 2 alveolar epithelial cell) renewal capacity. Perturbation was used to investigate WNT7A function invitro and in a repetitive bleomycin model invivo. We found that WNT7A is highly and specifically expressed in basal-like cells. Proteins secreted by basal cells can be captured by neighboring fibroblasts and AEC2s. Basal cells or basal cell-conditioned media activate fibroblasts through WNT7A. Basal cell-derived WNT7A inhibits AEC2 progenitor cell renewal in three-dimensional organoid cultures. Neutralizing antibodies against WNT7A or a small molecule inhibitor of Frizzled signaling abolished basal cell-induced fibroblast activation and attenuated lung fibrosis in mice. In summary, basal cells and basal cell-derived WNT7A are key components of the fibrotic niche, providing a unique non-stem cell function of basal cells in IPF progression and a novel targeting strategy for IPF.

Impact of TMPRSS2 Expression, Mutation Prognostics, and Small Molecule (CD, AD, TQ, and TQFL12) Inhibition on Pan-Cancer Tumors and Susceptibility to SARS-CoV-2.

As a cellular protease, transmembrane serine protease 2 (TMPRSS2) plays roles in various physiological and pathological processes, including cancer and viral entry, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, we conducted expression, mutation, and prognostic analyses for the TMPRSS2 gene in pan-cancers as well as in COVID-19-infected lung tissues. The results indicate that TMPRSS2 expression was highest in prostate cancer. A high expression of TMPRSS2 was significantly associated with a short overall survival in breast invasive carcinoma (BRCA), sarcoma (SARC), and uveal melanoma (UVM), while a low expression of TMPRSS2 was significantly associated with a short overall survival in lung adenocarcinoma (LUAD), demonstrating TMPRSS2 roles in cancer patient susceptibility and severity. Additionally, TMPRSS2 expression in COVID-19-infected lung tissues was significantly reduced compared to healthy lung tissues, indicating that a low TMPRSS2 expression may result in COVID-19 severity and death. Importantly, TMPRSS2 mutation frequency was significantly higher in prostate adenocarcinoma (PRAD), and the mutant TMPRSS2 pan-cancer group was significantly associated with long overall, progression-free, disease-specific, and disease-free survival rates compared to the wild-type (WT) TMPRSS2 pan-cancer group, demonstrating loss of functional roles due to mutation. Cancer cell lines were treated with small molecules, including cordycepin (CD), adenosine (AD), thymoquinone (TQ), and TQFL12, to mediate TMPRSS2 expression. Notably, CD, AD, TQ, and TQFL12 inhibited TMPRSS2 expression in cancer cell lines, including the PC3 prostate cancer cell line, implying a therapeutic role for preventing COVID-19 in cancer patients. Together, these findings are the first to demonstrate that small molecules, such as CD, AD, TQ, and TQFL12, inhibit TMPRSS2 expression, providing novel therapeutic strategies for preventing COVID-19 and cancers.

Autoreactive napsin A-specific T cells are enriched in lung tumors and inflammatory lung lesions during immune checkpoint blockade.

Cancer treatment with immune checkpoint blockade (ICB) often induces immune-related adverse events (irAEs). We hypothesized that proteins coexpressed in tumors and normal cells could be antigenic targets in irAEs and herein described DITAS (discovery of tumor-associated self-antigens) for their identification. DITAS computed transcriptional similarity between lung tumors and healthy lung tissue based on single-sample gene set enrichment analysis. This identified 10 lung tissue-specific genes highly expressed in the lung tumors. Computational analysis was combined with functional T cell assays and single-cell RNA sequencing of the antigen-specific T cells to validate the lung tumor self-antigens. In patients with non-small cell lung cancer (NSCLC) treated with ICB, napsin A was a self-antigen that elicited strong CD8+ T cell responses, with ICB responders harboring higher frequencies of these CD8+ T cells compared with nonresponders. Human leukocyte antigen (HLA) class I ligands derived from napsin A were present in human lung tumors and in nontumor lung tissues, and napsin A tetramers confirmed the presence of napsin A-specific CD8+ T cells in blood and tumors of patients with NSCLC. Napsin A-specific T cell clonotypes were enriched in lung tumors and ICB-induced inflammatory lung lesions and could kill immortalized HLA-matched NSCLC cells ex vivo. Single-cell RNA sequencing revealed that these T cell clonotypes expressed proinflammatory cytokines and cytotoxic markers. Thus, DITAS successfully identified self-antigens, including napsin A, that likely mediate effective antitumor T cell responses in NSCLC and may simultaneously underpin lung irAEs.

Unsupervised machine learning identifies predictive progression markers of IPF

ObjectivesTo identify and evaluate predictive lung imaging markers and their pathways of change during progression of idiopathic pulmonary fibrosis (IPF) from sequential data of an IPF cohort. To test if these imaging markers predict outcome.MethodsWe studied radiological disease progression in 76 patients with IPF, including overall 190 computed tomography (CT) examinations of the chest. An algorithm identified candidates for imaging patterns marking progression by computationally clustering visual CT features. A classification algorithm selected clusters associated with radiological disease progression by testing their value for recognizing the temporal sequence of examinations. This resulted in radiological disease progression signatures, and pathways of lung tissue change accompanying progression observed across the cohort. Finally, we tested if the dynamics of marker patterns predict outcome, and performed an external validation study on a cohort from a different center.ResultsProgression marker patterns were identified and exhibited high stability in a repeatability experiment with 20 random sub-cohorts of the overall cohort. The 4 top-ranked progression markers were consistently selected as most informative for progression across all random sub-cohorts. After spatial image registration, local tracking of lung pattern transitions revealed a network of tissue transition pathways from healthy to a sequence of disease tissues. The progression markers were predictive for outcome, and the model achieved comparable results on a replication cohort.ConclusionsUnsupervised learning can identify radiological disease progression markers that predict outcome. Local tracking of pattern transitions reveals pathways of radiological disease progression from healthy lung tissue through a sequence of diseased tissue types.Key Points• Unsupervised learning can identify radiological disease progression markers that predict outcome in patients with idiopathic pulmonary fibrosis.• Local tracking of pattern transitions reveals pathways of radiological disease progression from healthy lung tissue through a sequence of diseased tissue types.• The progression markers achieved comparable results on a replication cohort.

A pulmonologist's guide to perform and analyse cross-species single lung cell transcriptomics.

Single-cell ribonucleic acid sequencing is becoming widely employed to study biological processes at a novel resolution depth. The ability to analyse transcriptomes of multiple heterogeneous cell types in parallel is especially valuable for cell-focused lung research where a variety of resident and recruited cells are essential for maintaining organ functionality. We compared the single-cell transcriptomes from publicly available and unpublished datasets of the lungs in six different species: human (Homo sapiens), African green monkey (Chlorocebus sabaeus), pig (Sus domesticus), hamster (Mesocricetus auratus), rat (Rattus norvegicus) and mouse (Mus musculus) by employing RNA velocity and intercellular communication based on ligand-receptor co-expression, among other techniques. Specifically, we demonstrated a workflow for interspecies data integration, applied a single unified gene nomenclature, performed cell-specific clustering and identified marker genes for each species. Overall, integrative approaches combining newly sequenced as well as publicly available datasets could help identify species-specific transcriptomic signatures in both healthy and diseased lung tissue and select appropriate models for future respiratory research.

Novel Harmonization Method for Multi-Centric Radiomic Studies in Non-Small Cell Lung Cancer.

The purpose of this multi-centric work was to investigate the relationship between radiomic features extracted from pre-treatment computed tomography (CT), positron emission tomography (PET) imaging, and clinical outcomes for stereotactic body radiation therapy (SBRT) in early-stage non-small cell lung cancer (NSCLC). One-hundred and seventeen patients who received SBRT for early-stage NSCLC were retrospectively identified from seven Italian centers. The tumor was identified on pre-treatment free-breathing CT and PET images, from which we extracted 3004 quantitative radiomic features. The primary outcome was 24-month progression-free-survival (PFS) based on cancer recurrence (local/non-local) following SBRT. A harmonization technique was proposed for CT features considering lesion and contralateral healthy lung tissues using the LASSO algorithm as a feature selector. Models with harmonized CT features (B models) demonstrated better performances compared to the ones using only original CT features (C models). A linear support vector machine (SVM) with harmonized CT and PET features (A1 model) showed an area under the curve (AUC) of 0.77 (0.63–0.85) for predicting the primary outcome in an external validation cohort. The addition of clinical features did not enhance the model performance. This study provided the basis for validating our novel CT data harmonization strategy, involving delta radiomics. The harmonized radiomic models demonstrated the capability to properly predict patient prognosis.

Prevalence and Antimicrobial Susceptibility of Bovine Mycoplasma Species in Egypt.

Simple SummaryBovine Mycoplasma species, particularly antimicrobial resistant Mycoplasma bovis are important causes of bovine respiratory disease (BRD) in cattle, which causes major economic losses worldwide. Thus, the current study aimed to determine the prevalence and antimicrobial resistance profiles of bovine Mycoplasma spp. isolated from cattle’s respiratory tracts, in addition to evaluating the fluoroquinolone resistance in the recovered isolates using broth microdilution and conventional PCR techniques in Egypt. Our result showed that M. bovis was the most common spp. (61%), followed by M. bovirhinis (15%). In total, mycoplasma isolates were more prevalent among all examined lung tissues (38%), followed by nasal swabs (35%), tracheal tissues (28%), and tracheal swabs (27%). All the examined mycoplasma isolates (n = 76) were 100% susceptible to spectinomycin, tulathromycin, spiramycin, and tylosin, but high doxycycline and enrofloxacin minimum inhibitory concentrations (MICs) values were observed among 43.4% and 60.5% of the tested isolates, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis, respectively, by PCR assays. All molecularly confirmed mycoplasma isolates (n = 5) were positive for the gyrA gene (100%), meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, understanding antimicrobial resistance mechanisms is a significant tool for the future development of genetic-based diagnostic techniques for the rapid detection of resistant mycoplasma strains.Among many bovine Mycoplasma species (spp.), Mycoplasma bovis is recognized as a significant causative agent of respiratory diseases in cattle. In recent years, resistant M. bovis isolates, especially to fluoroquinolones, have been reported globally as a result of the extensive usage of antimicrobials in the treatment of bovine pneumonia. Therefore, the aim of this study is to investigate the prevalence and antimicrobial susceptibility patterns of bovine Mycoplasma spp. isolated from the respiratory tracts of cattle in Egypt and to assess the fluoroquinolones resistance in the recovered mycoplasma isolates via broth microdilution and conventional PCR techniques. Conventional phenotypic methods identified 128 mycoplasma isolates (32%) from 400 different samples, with M. bovis being the predominant spp. (61%), followed by M. bovirhinis (15%). Of note, mycoplasma isolates were rarely isolated from total healthy lung tissues (7/55, 12.7%), but they were frequently isolated from pneumonic lungs (31/45, 68.9%). All the examined mycoplasma isolates (n = 76) were sensitive to tilmicosin, tylosin, tulathromycin, spiramycin, and spectinomycin (100% each), while 60.5% and 43.4% of the examined isolates had high minimum inhibitory concentration (MIC) values to enrofloxacin and doxycycline, respectively. Three and two mycoplasma isolates with high enrofloxacin MICs were confirmed to be M. bovis and M. bovirhinis, respectively, by PCR assays. All molecularly confirmed mycoplasma isolates (n = 5) were positive for the gyrA gene (100%); meanwhile, three isolates (60%) were positive for the parC gene. In conclusion, our findings revealed alarming resistance to enrofloxacin and doxycycline antibiotics; thus, antimicrobial usage must be restricted and molecular techniques can help in the rapid detection of the resistant strains.

Intratumoral Heterogeneity and Immune Modulation in Lung Adenocarcinoma in Female Smokers and Never Smokers.

Single-cell analysis of healthy lung tissue and lung cancer reveals distinct tumor cell populations, including cells with differential immune modulating capacity between smokers and never smokers, which could guide future therapeutic strategies.

Comparative Profiling of Circulating Exosomal Small RNAs Derived From Peruvian Patients With Tuberculosis and Pulmonary Adenocarcinoma.

Tuberculosis (TB) is one of the most fatal infectious diseases, caused by the aerobic bacteria Mycobacterium tuberculosis. It is estimated that one-third of the world’s population is infected with the latent (LTB) version of this disease, with only 5-10% of infected individuals developing its active (ATB) form. Pulmonary adenocarcinoma (PA) is the most common and diverse form of primary lung carcinoma. The simultaneous or sequential occurrence of TB and lung cancer in patients has been widely reported and is known to be an issue for diagnosis and surgical treatment. Raising evidence shows that patients cured of TB represent a group at risk for developing PA. In this work, using sRNA-sequencing, we evaluated the expression patterns of circulating small RNAs available in exosomes extracted from blood samples of Peruvian patients affected by latent tuberculosis, active tuberculosis, or pulmonary adenocarcinoma. Differential expression analysis revealed a set of 24 microRNAs perturbed in these diseases, revealing potential biomarker candidates for the Peruvian population. Most of these miRNAs are normally expressed in healthy lung tissue and are potential regulators of different shared and unique KEGG pathways related to cancers, infectious diseases, and immunology.

In Vitro Effect of Δ9-Tetrahydrocannabinol and Cannabidiol on Cancer-Associated Fibroblasts Isolated from Lung Cancer.

There is evidence that demonstrates the effect of cannabinoid agonists inhibiting relevant aspects in lung cancer, such as proliferation or epithelial-to-mesenchymal transition (EMT). Most of these studies are based on evidence observed in in vitro models developed on cancer cell lines. These studies do not consider the complexity of the tumor microenvironment (TME). One of the main components of the TME is cancer-associated fibroblasts (CAFs), cells that are relevant in the control of proliferation and metastasis in lung cancer. In this work, we evaluated the direct effects of two cannabinoid agonists, tetrahydrocannabinol (THC) and cannabidiol (CBD), used alone or in combination, on CAFs and non-tumor normal fibroblasts (NFs) isolated from adenocarcinoma or from healthy lung tissue from the same patients. We observed that these compounds decrease cell density in vitro and inhibit the increase in the relative expression of type 1 collagen (COL1A1) and fibroblast-specific protein 1 (FSP1) induced by transforming growth factor beta (TGFβ). On the other hand, we studied whether THC and CBD could modulate the interactions between CAFs or NFs and cancer cells. We conditioned the culture medium with stromal cells treated or not with THC and/or CBD and cultured A549 cells with them. We found that culture media conditioned with CAFs or NFs increased cell density, induced morphological changes consistent with EMT, inhibited cadherin-1 (CDH1) gene expression, and induced an increase in the relative expression of cadherin-2 (CDH2) and vimentin (VIM) genes in A549 cells. These changes were inhibited or decreased by THC and CBD administered alone or in combination. In another series of experiments, we conditioned culture media with A549 cells treated or not with THC and/or CBD, in the presence or absence of TGFβ. We observed that culture media conditioned with A549 in the presence of TGFβ induced an increase in the expression of COL1A1 and VIM, both in CAFs and in non-tumor NFs. Both THC and CBD ameliorated these effects. In summary, the results presented here reinforce the usefulness of cannabinoid agonists for the treatment of some relevant aspects of lung cancer pathology, and demonstrate in a novel way their possible effects on CAFs as a result of their relationship with cancer cells. Likewise, the results reinforce the usefulness of the combined use of THC and CBD, which has important advantages in relation to the possibility of using lower doses, thus minimizing the psychoactive effects of THC.

Abstract 1344: Immune transcriptional profiling in a large animal model of histiocytic sarcoma reveals putative drivers and tumor suppressors

Abstract Histiocytic sarcoma (HS) is a rare, yet lethal malignancy with a median overall survival of six months and no universally agreed standard of care therapy. Due to the paucity of human cases, there is a drive to develop model systems to improve our understanding of the pathology underlying this disease and screen new treatment strategies. Spontaneous canine HS exhibits increased prevalence in specific breeds, shares key genetic and biologic similarities with the human disease, and occurs in an immunocompetent setting. Our previous studies revealed a spectrum of hot and cold immune tumor microenvironments (TME) based on tumor infiltrating lymphocyte (TIL) quantification in 18 canine and 5 human HS cases. In dogs, increased TIL densities were positively associated with longer survival times. Despite this association, the disease course of HS in our veterinary patient cohort was nearly uniformly fatal. To evaluate for potential inhibitors of T cell immunity in the TME and identify putative drivers and suppressors of HS, we performed immune transcription profiling. Tumors were selected from 3 short-lived cases of splenic HS that were sparsely infiltrated with TIL and 3 long-lived cases of pulmonary HS that were T cell inflamed. Tumors were compared to corresponding grossly healthy splenic and lung tissues collected from 3 dogs without neoplasia undergoing routine post-mortem examination. RNA was extracted from formalin-fixed paraffin embedded tissues and immune transcription profiling was performed using the NanoString nCounter Canine IO Panel. Analyses of all tumors compared to normal tissues, and subset analyses of tissues stratified based on tissue origin site, revealed enrichment of ITGAX (CD11c) expression in the HS tumor samples consistent with histiocytic origin. In each subset analysis, SPP1 (osteopontin) was highly expressed in HS tumors compared with normal tissues. Osteopontin has been shown to be protumorigenic in a variety of cancers, is a potent chemokine for macrophages, and can suppress T cell activation through ligation of CD44, thus acting as a T cell checkpoint. Additionally, we found decreased expression of TXNIP in HS tumors compared to healthy tissues. TXNIP is an established tumor suppressor gene in various tumor types, including acute myeloid leukemia. Future studies will interrogate the functional implications of these transcriptional changes with the aim of developing novel therapeutic strategies for HS in both species. Citation Format: Jennifer A. Lenz, Charles-Antoine Assenmacher, Enrico Radaelli, Matthew J. Atherton. Immune transcriptional profiling in a large animal model of histiocytic sarcoma reveals putative drivers and tumor suppressors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1344.

Abstract 1374: Discovery of MHC class I and class II neoantigens in lung cancer in needle biopsy tissue samples using an optimized high-throughput workflow

Abstract Human leukocyte antigen-associated peptides, known as immunopeptides, play an essential role in adaptive immunity by activating and ensuring the specificity of T-cells. The identification and quantification of the immunopeptidome bear the potential to enable personalized treatments, especially in cancers, vaccines, infectious, and autoimmune diseases. Mass spectrometry is currently the only technology that can reliably measure and identify immunopeptide profiles of biological samples on a large scale. However, the usually high sample input amount and poor scalability are limiting. Here, we introduce a semi-automated workflow to robustly identify immunopeptides from low amounts of cultured cells and tissue samples by systematically optimizing each step of the sample preparation and acquisition. We optimized the native lysis and immunoprecipitation workflow while ensuring scalability and reproducibility. Leveraging the magnetic properties of the beads, 1,000 samples can be processed within a week by a single operator. The established sample preparation offers high reproducibility and identifications of good quality. For class-I immunopeptides, &gt60% of the peptides identified are 9-mers, &gt80% predicted strong binders, and the expected amino acids are enriched at the anchor positions. For class-II, &gt50% of the peptides identified are 14-to-16-mers, and &gt50% are predicted strong binders. Furthermore, the pipeline is highly sensitive as we could still identify over 2,800 class-I immunopeptides when processing as little as 2.5 mg fresh frozen tissue and &gt9,000 class-I and &gt12,000 class-II immunopeptides when preparing 10 million JY cells. Overall, the pipeline is scalable, highly reproducible, and results in high-quality identifications while supporting very limited sample input. Finally, we measured a cohort of 12 cancerous and matched healthy lung tissues from as little as 15 mg tissue, whereby we could identify &gt11,000 class-I immunopeptides and &gt9,000 class-II on average. For class-I, matched samples clustered together, while &gt3,000 immunopeptides were upregulated in the cancer tissues, with a significant enrichment for proteins related to lung cancer. Overall, we established a scalable, efficient pipeline for cell line and tissues immunopeptidomics for class-I and II that generates high-quality identifications and that only requires small amounts of input material and is ready to shed light into immunopeptidomics heterogeneity through large-scale profiling of patients. Citation Format: Ilja E. Shapiro, Luca Raess, Marco Tognetti, Tikira Temu, Oliver M. Bernhardt, Yuehan Feng, Roland Bruderer, Lukas Reiter. Discovery of MHC class I and class II neoantigens in lung cancer in needle biopsy tissue samples using an optimized high-throughput workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1374.

Circ_CSPP1 Regulates the Development of Non-small Cell Lung Cancer via the miR-486-3p/BRD9 Axis.

In this study, we explored the role of circ_CSPP1 in non-small cell lung cancer (NSCLC) using NSCLC cell lines (A549 and H1299) and human bronchial epithelioid cells (16HBE). The differential expression of circ_CSPP1, miR-486-3p and BRD9 in NSCLC by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot in A549 cells, H1299 cells, 16HBE cells, NSCLC tissues and healthy lung tissues. Dual-luciferase reporter assay was conducted to verify the interaction between circ_CSPP1 and miR-486-3p or miR-486-3p and BRD9. The effect of circ_CSPP1/miR-486-3p/BRD9 axis on NSCLC cell proliferation was evaluated using cell counting kit-8 assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine assay. Additionally, transwell assays were performed to evaluate the effect of circ_CSPP1/miR-486-3p/BRD9 axis on A549 and H1299 cell migration and invasion. The effect of circ_CSPP1 on tumor tumorigenesis of A549 cells in vivo was determined by xenograft tumor model and immunohistochemistry assay. Circ_CSPP1 and BRD9 expression were upregulated, while miR-486-3p expression was downregulated in tumor tissues of NSCCL patients and A549 and H1299 cells. Circ_CSPP1 specifically bound miR-486-3p, and miR-486-3p could target BRD9. Circ_CSPP1 upregulation promoted proliferation, invasion and migration of NSCLC cells, circ_CSPP1 knockdown or miR-486-3p upregulation had the opposite effects. Circ_CSPP1 knockdown-induced effects were reverted by miR-486-3p inhibition. Similarly, the effects of miR-486-3p upregulation on NSCLC cell proliferation, invasion and migration were reversed by BRD9 overexpression. In addition, circ_CSPP1 silencing inhibited tumor growth in nude mice. Circ_CSPP1 promoted A549 and H1299 cell malignancy by competitively inhibiting BRD9 and binding to miR-486-3p.

InVivo Visualization and Quantification of Neutrophil Elastase in Lungs of COVID-19 Patients: A First-in-Humans PET Study with 11C-NES.

Coronavirus disease 2019 (COVID-19) can cause life-threatening lung inflammation that is thought to be mediated by neutrophils. The aim of the present work was to evaluate a novel PET tracer for neutrophil elastase (NE). Methods: In this first-in-humans study, 4 patients with hypoxia due to COVID-19 and 2 healthy controls were investigated with PET using 11C-NES and 15O-water for visualization and quantification of NE and perfusion in the lungs, respectively. Results: 11C-NES accumulated selectively in lung areas with COVID-19 opacities on CT scans, suggesting high levels of NE there. In the same areas, perfusion was severely reduced in comparison to healthy lung tissue as measured with 15O-water. Conclusion: The data suggest that NE is associated with severe lung inflammation in COVID-19 patients and that inhibition of NE could potentially reduce the acute inflammatory process and improve the condition.

Long Non-Coding RNA AP000695.2 Acts as a Novel Prognostic Biomarker and Regulates the Cell Growth and Migration of Lung Adenocarcinoma.

Long non-coding RNAs (lncRNAs) are tumor-associated biological molecules and have been found to be implicated in the progression of lung adenocarcinoma (LUAD). LncRNA-AP000695.2 (ENSG00000248538) is a long non-coding RNA (lncRNA) that is widely increased in many tumor types including lung adenocarcinoma (LUAD). However, the aberrant expression profile, clinical significance, and biological function of AP000695.2 in human lung adenocarcinoma (LUAD) need to be further investigated. This study mines key prognostic AP000695.2 and elucidates its potential role and molecular mechanism in regulating the proliferation and metastasis of LUAD. Here, we discovered that AP000695.2 was significantly upregulated in lung adenocarcinoma tissues compared with healthy adjacent lung tissue and higher in LUAD cell lines than in normal human bronchial epithelial cell lines. A higher expression of AP000695.2 was positively correlated with aggressive clinicopathological characteristics, and AP000695.2 served as an independent prognostic indicator for the overall survival, disease-free survival, and progression-free survival in patients with LUAD. Receiver operating curve (ROC) analysis revealed the significant diagnostic ability of AP000695.2 (AUC = 0.838). Our in vivo data confirmed that AP000695.2 promotes the proliferation, migration, and invasion of LUAD cells. GSEA results suggested that AP000695.2 co-expressed genes were mainly enriched in immune-related biological processes such as JAK-STAT signaling pathway and toll-like receptor signaling pathway. Single-sample GSEA analysis showed that AP000695.2 is correlated with tumor-infiltrating immune cells in lung adenocarcinoma. Our findings confirmed that AP000695.2 was involved in the progression of lung adenocarcinoma, providing a novel prognostic indicator and promising diagnostic biomarker in the future.

The prognostic value of N6-methyladenosine RBM15 regulators in lung adenocarcinoma.

N6-methyladenosine (m6A) is the most common internal modification in mammalian mRNAs while RNA-binding motif protein 15 (RBM15) is an important methyltransferase in m6A modification. Increasing evidences have shown that RBM15 has a close correlation with lung cancer. However, specific functions of RBM15 in lung adenocarcinoma (LUAD) are limited. RBM15 expression was analyzed in human LUAD tissues and matched healthy lung tissue. RBM15 was knocked down via siRNA in A549 and H1734 cells. The relationships between RBM15 with cellular functions characteristics and mRNA m6A levels were explored. We performed functional characterization in A549 and H1734 cells lines to elucidate the molecular role of RBM15. Results found that RBM15 was up-regulated in the LUAD tissue and cells, which was linked to poor survival of LUAD patients. RBM15 can be knocked down via siRNA in A549, which leads to the exploration of the associations between RBM15 with cell characteristics. In vivo, RBM15 knockdown could decrease the methylation level, reduce proliferation, accelerate apoptosis and inhibit tumor growth. Our research shows that RBM15 facilitates LUAC cell progression by m6A demethylation. However, it is necessary to conduct further researches on potential downstream molecular mechanisms and m6A modification of RBM15 activity in LUAC.

Identifying distinct T cell subsets in the context of pediatric ARDS

Abstract Acute respiratory distress syndrome (ARDS) is a condition defined by sudden respiratory failure caused by severe lung injury, with disease etiology that can differ across pediatric and adult patients. While T lymphocytes have been implicated in ARDS, the role of T cell subsets in pediatric ARDS (pARDS) is not fully understood. In this study, we sought to characterize the potential role of T cells in pARDS caused by viral lower respiratory tract infection (LRTI). To do this, we performed single cell RNA-Seq on tracheal aspirate samples from three categories of mechanically ventilated pediatric patients: 1) LRTI patients with pARDS, 2) LRTI patients without pARDS, and 3) ventilated patients without underlying LRTI or pARDS. All LRTI patients were diagnosed with respiratory viral infections, 70 percent of which were respiratory syncytial virus (RSV). Within the T lymphocytes, we identified populations of CD8+ T cells, CD4+ regulatory T cells, and γδ T cells that each expressed unique gene signatures with distinct distribution across disease states. We compared these data to T cell subsets within healthy lung tissue at multiple ages, and within the periphery of healthy adults. These analyses identified age, location, and disease-specific differences within T cell subsets, highlighting the unique role of T cells in pARDS. Supported by grants from NIH (R01 AI54470, R01 AI121832)

Localizing the expression of Piezo2 in healthy and diseased lung tissue

Intractable air hunger remains one of the most impactful symptoms of chronic cardiopulmonary disease and current pharmacological management remains limited. However, an endogenous mechanism for air hunger relief exists. Pulmonary stretch receptors (PSRs) responding to lung inflation reduce the brainstem drive to breathe and mitigate air hunger. Harnessing this mechanism could form the basis of a novel therapy. Recent animal studies suggest Piezo2, a mechanically‐gated cation channel, causes PSR depolarization when airway smooth muscle is stretched during inflation. We have assessed whether Piezo2 is associated with PSRs in healthy and diseased human airways to determine whether Piezo2 is a potential pharmaceutical target.MethodsHuman lung biopsies from NIH Lung Tissue Research Consortium were used for identification of Piezo2 receptors in emphysema, fibrosis, bronchiolitis, and normal lung tissue. Localization of Piezo2 was determined with immunofluorescent staining and imaged with confocal microscopy (Zeiss LSM880). As PSR fiber endings are associated with airway smooth muscle, co‐staining for Smooth Muscle Antibody (SMA) and Calcitonin Gene‐Related Peptide (CGRP) was performed to determine whether Piezo2 is associated with PSR.Summary of resultsPiezo2 is associated with airway smooth muscle (as evidenced by SMA, figure 1) and neuroepithelial bodies in normal human lung tissue (evidenced by cGRP, figure 2) and expression persisted in all disease states tested.ConclusionsPiezo2 location is consistent with that of PSR in human lungs, making it a candidate for the mechanoreceptive transducer of human PSRs as has been demonstrated in animal studies. Expression persists in disease, making Piezo2 a potential therapeutic target, but it is still yet to be determined if Piezo2 is more significantly associated with airway or vascular smooth muscle in different disease states.

A Retrospective Study of Effectiveness of Thoracoscopic Lobectomy and Segmentectomy in Patients with Early-Stage Non-Small-Cell Lung Cancer.

Background Surgical treatment is the first choice for non-small-cell lung cancer. To date, there are only few studies on the changes in laboratory indexes in two types of surgery, namely, thoracoscopic lobectomy and segmental pneumonectomy. Aim To investigate the clinical impact of thoracoscopic lobectomy and segmentectomy in patients with early-stage non-small-cell lung cancer. Methods We retrospectively reviewed the medical records of 94 patients with early-stage NSCLC in our hospital from October 2017 to October 2019. The patients were divided into two groups. The patients in control and observation groups received thoracoscopic lobectomy and thoracoscopic segmentectomy, respectively. The perioperative indicators, complications, lung function, T cell subsets, tumor markers, follow-up of tumor recurrence rate, and survival rate were compared between two groups. Results The operation time of the observation group was longer, and the chest drainage volume was less at 24-48 h after the operation, and the chest tube indwelling time and postoperative hospital stay were shorter than those of the control group. No significant differences in complication probability were observed between two groups. The levels of FEV1, FVC, and MVV in the two groups were lower than those before the operation at 3 days after surgery, but the FEV1, FVC, and MVV levels in the observation group were higher than those in the control group. The CD3+, CD4+, and CD4+/CD8+ levels in the two groups were lower than those before the operation at 24 h and 72 h after the operation, but CD3+, CD4+, and CD4+/CD8+ levels in the observation group were higher than those of the control group. Conclusion Thoracoscopic lobectomy and segmental resection have similar clinical effects in the treatment of early-stage NSCLC patients, but segmental resection can preserve healthy lung tissue as much as possible, with less trauma, protect lung function, and promote postoperative recovery.