Understanding the cellular processes that underlie early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies 1 . Here we studied 246,102 single epithelial cells from 16 early-stage LUADs and 47 matched normal lung samples. Epithelial cells comprised diverse normal and cancer cell states, and diversity among cancer cells was strongly linked to LUAD-specific oncogenic drivers. KRAS mutant cancer cells showed distinct transcriptional features, reduced differentiation and low levels of aneuploidy. Non-malignant areas surrounding human LUAD samples were enriched with alveolar intermediate cells that displayed elevated KRT8 expression (termed KRT8 + alveolar intermediate cells (KACs) here), reduced differentiation, increased plasticity and driver KRAS mutations. Expression profiles of KACs were enriched in lung precancer cells and in LUAD cells and signified poor survival. In mice exposed to tobacco carcinogen, KACs emerged before lung tumours and persisted for months after cessation of carcinogen exposure. Moreover, they acquired Kras mutations and conveyed sensitivity to targeted KRAS inhibition in KAC-enriched organoids derived from alveolar type 2 (AT2) cells. Last, lineage-labelling of AT2 cells or KRT8 + cells following carcinogen exposure showed that KACs are possible intermediates in AT2-to-tumour cell transformation. This study provides new insights into epithelial cell states at the root of LUAD development, and such states could harbour potential targets for prevention or intervention.

Background Early and accurate identification of acute exacerbations of COPD may lead to earlier treatment and prevent hospital admission. Electronic diaries have been developed for symptom monitoring and accelerometers to monitor activity. However, it is unclear whether this technology is usable in the COPD population. This study aimed to assess the feasibility of an electronic diary (eDiary) for symptom reporting using the MoreCare app and activity monitoring with the Garmin Vivofit 2 in COPD. Methods Participants were recruited from the London COPD Cohort. Participants were provided a Garmin Vivofit 2 activity monitor and an android tablet with the MoreCare app for a period of 3 months. Results 25 COPD patients were recruited (mean±sd age 70.8±7.1 years, forced expiratory volume in 1 s (FEV1) 49.8±14.8% predicted). Age, gender, disease severity and exacerbation frequency had no impact on eDiary compliance. There was a moderate positive correlation between median daily very active minutes and FEV1 % pred (ρ=0.62, p=0.005). Daily step counts decreased during the initial 7 days of exacerbation and recovery compared to a pre-exacerbation baseline. A decision-tree model identified change in sputum colour, change in step count, severity of cold, exacerbation history and use of rescue medication as the most important predictors of acute exacerbations of COPD in this cohort. Conclusions Symptom and activity monitoring using digital technology is feasible in COPD. Further large-scale digital health studies are needed to assess whether eDiaries can be used to identify patients at risk of exacerbation and guide early intervention.

Early and accurate identification of acute exacerbations of COPD may lead to earlier treatment and prevent hospital admission. Electronic diaries have been developed for symptom monitoring and accelerometers to monitor activity. However, it is unclear whether this technology is usable in the COPD population. This study aimed to assess the feasibility of an electronic diary (eDiary) for symptom reporting using the MoreCare app and activity monitoring with the Garmin Vivofit 2 in COPD.Participants were recruited from the London COPD Cohort. Participants were provided a Garmin Vivofit 2 activity monitor and an android tablet with the MoreCare app for a period of 3 months.25 COPD patients were recruited (mean±sd age 70.8±7.1 years, forced expiratory volume in 1 s (FEV1) 49.8±14.8% predicted). Age, gender, disease severity and exacerbation frequency had no impact on eDiary compliance. There was a moderate positive correlation between median daily very active minutes and FEV1 % pred (ρ=0.62, p=0.005). Daily step counts decreased during the initial 7 days of exacerbation and recovery compared to a pre-exacerbation baseline. A decision-tree model identified change in sputum colour, change in step count, severity of cold, exacerbation history and use of rescue medication as the most important predictors of acute exacerbations of COPD in this cohort.Symptom and activity monitoring using digital technology is feasible in COPD. Further large-scale digital health studies are needed to assess whether eDiaries can be used to identify patients at risk of exacerbation and guide early intervention.

Understanding cellular processes underlying early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies. While most if not all single-cell RNA sequencing (scRNA-seq) studies of lung cancer provided details on immune and stromal states, little insight is drawn to epithelial cells given their paucity (~4%) when performing unbiased scRNA-seq analysis without prior enrichment. Here, we performed in-depth scRNA-seq of enriched (by sorting for EPCAM+) epithelial cell subsets from 16 early-stage LUADs and 47 matching normal lung (NL) tissues. We also studied tissues from the same LUADs and adjacent NL by whole exome sequencing and a subset by high-resolution spatial protein and transcriptome analysis. We also performed scRNA-seq analysis of murine lungs from a human-relevant model of LUAD development following exposure to tobacco carcinogen, including strains with an alveolar type II (AT2) cell-specific lineage reporter. After extensive quality control, we retained 246,102 high quality human epithelial cells which comprised diverse normal alveolar and airway lineages as well as cancer cell populations. Diversity among cancer cells was strongly linked to LUAD oncogenic drivers. KRAS-mutant cancer cells were unique in their transcriptional features, strikingly reduced differentiation, low levels of copy number changes, and increased variability amongst the cells themselves. The local epithelial niche of LUADs, relative to that of NL, was enriched with intermediary cells in lung alveolar differentiation. A subset of these cells displayed elevated KRT8 expression (KRT8+ alveolar cells; KACs), increased plasticity and frequency of KRASG12D mutations, and its gene expression profiles were enriched in lung precancer and LUAD and signified poor survival. Notably, KACs harboring KRAS mutations were only found in the ecosystem of KRAS-mutant LUADs. Murine KACs were evident in lungs of tobacco carcinogen-exposed mice that develop KRAS-mutant LUADs but not in the saline-treated control group. While murine KACs emerged prior to tumor onset, they persisted for months after carcinogen cessation, and like their human counterparts, acquired driver Kras mutations, were poorly differentiated, and harbored KRAS-specific transcriptional programs. Spatial transcriptomics analysis showed that KAC and KRAS signatures were elevated in both murine and human tumors as well as in KACs that were in the local spatial vicinity of the LUADs. Organoids derived from lungs of tumor-bearing reporter mice were markedly enriched with KACs and were conspicuously sensitive to targeted inhibition of KRAS-G12D. This study provides new insights into the landscape of normal epithelial and malignant cells in LUAD, the role of alveolar intermediate subsets in development of the malignancy, particularly that driven by mutant KRAS, and, thus, potential targets for early interception. Citation Format: Guangchun Han, Ansam Sinjab, Warapen Treekitkarnmongkol, Zahraa Rahal, Yuejiang Liu, Alejandra G. Serrano, Jiping Feng, Ke Liang, Khaja Khan, Wei Lu, Sharia Hernandez, Xuanye Cao, Enyu Dai, Yunhe Liu, Guangsheng Pei, Jian Hu, Lorena I. Gomez Bolanos, Edwin R. Parra, Tina Cascone, Boris Sepesi, Seyed Javad Moghaddam, Paul Scheet, Marcelo V. Negrao, John V. Heymach, Mingyao Li, Jichao Chen, Steven M. Dubinett, Junya Fujimoto, Luisa M. Solis, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum Spira, Linghua Wang, Humam Kadara. An atlas of epithelial cell states and plasticity in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 113.

Supplementary Data from The Single-Cell Immunogenomic Landscape of B and Plasma Cells in Early-Stage Lung Adenocarcinoma

Introduction Visually normal areas of the lung with high attenuation on computed tomography (CT) imaging, termed CT lung injury, may represent injured but not yet remodelled lung parenchyma. This prospective cohort study examined if CT lung injury is associated with future interstitial features on CT and restrictive spirometry abnormality among participants from the Coronary Artery Risk Development in Young Adults (CARDIA) study. Methods CARDIA is a population-based cohort study. CT scans obtained at two time points were assessed objectively for amount of lung tissue characterised as CT lung injury and interstitial features. Restrictive spirometry was defined as having a forced vital capacity (FVC) <80% predicted with forced expiratory volume in 1 s/FVC ratio >70%. Results Among 2213 participants, the median percentage of lung tissue characterised as CT lung injury at a mean age of 40 years was 3.4% (interquartile range 0.8–18.0%). After adjustment for covariates, a 10% higher amount of CT lung injury at mean age 40 years was associated with a 4.37% (95% CI 3.99–4.74%) higher amount of lung tissue characterised as interstitial features at mean age 50 years. Compared to those with the lowest quartile of CT lung injury at mean age 40 years, there were higher odds of incident restrictive spirometry at mean age 55 years in quartile 2 (OR 2.05, 95% CI 1.20–3.48), quartile 3 (OR 2.80, 95% CI 1.66–4.72) and quartile 4 (OR 3.77, 95% CI 2.24–6.33). Conclusions CT lung injury is an early objective measure that indicates risk of future lung impairment.

Supplementary Figures S1-S7 and figure legends

Early immunotherapy with inhibitors of immune checkpoints such as PD-1 has revolutionized lung adenocarcinoma (LUAD) treatment. Still, many patients do not respond or relapse following PD-1 blockade. Tumor-promoting inflammation, such as that mediated by the pleiotropic cytokine interleukin 1 beta (IL-1B), fosters immunosuppression in the tumor immune microenvironment (TIME). Our group and others showed that IL-1B blockade inhibits lung cancer development. We thus hypothesized that addition of IL-1B blockade to anti-PD-1 treatment may enhance outcomes against LUAD. Using a human-relevant, tobacco-associated, mouse model of LUAD development, we compared the effects of combined PD-1 and IL-1B blockade relative to treatment with single-agents (anti-IL-1B or anti-PD-1) and control antibody on early lung tumor development and the TIME. Drugs (anti-PD-1 + anti-IL-1B, anti-PD-1, anti-IL-1B, control IgG) were administered at end of exposure to the tobacco-specific carcinogen NNK, to evaluate effects on formation of early lesions (preventive), or at 3 months post-NNK (prophylactic) to interrogate LUAD development (8 groups). Comprehensive interrogation of the lung ecosystem and the TIME was performed using deep single-cell RNA-sequencing (scRNA-seq) analysis in a subset of the mice (n = 3 to 4) from each of the 8 groups (n = 31 total; 143,897 cells after stringent quality control). Mice treated with combined PD-1 and IL-1B blockade displayed reduced development of lung tumors when compared to animals treated with anti-IL-1B, anti-PD-1, or control antibodies. Fractions of cytotoxic Cd8+ T cells were conspicuously higher and those of tumor cells and exhausted Cd8+ T cells evidently lower in lungs of mice treated with combined PD-1 and IL-1B blockade relative to monotherapy- or control antibody-treated animals. Igha+ plasma cells were strikingly highest in lungs of mice treated with combined PD-1 and IL-1B blockade and nearly absent in monotherapy- and control-treated groups. Lungs of mice treated with combined PD-1 and IL-1B blockade showed higher fractions of Cd80+/Cd86+ memory B cells and, consistently, T follicular helper T cells, while exhibiting reduced fractions of naïve B and Cd24a+/Tgfb1+ B cells suggestive of enhanced activation of B cell responses by the combinatorial treatment. These effects were, overall, present, or much more pronounced, in animals that were prophylactically treated. Flow cytometry analysis of lung tissues and immune profiling of bronchioalveolar lavage fluid overall confirmed augmented immune cell responses by combined PD-1 and IL-1B blockade. Our findings show that blocking IL-1B synergizes with anti-PD-1 in regression of early tumor cells and reversal of immunosuppression. Combined blockade of PD-1 and IL-1B may be a promising strategy for early treatment of lung cancer that warrants further clinical studies. Citation Format: Warapen Treekitkarnmongkol, Guangchun Han, Zahraa Rahal, Jiping Feng, Ansam Sinjab, Tina Cascone, Christopher S. Stevenson, Cheryl Sweeney, Matt Edwards, Avrum Spira, Junya Fujimoto, Seyed Javad Moghaddam, Linghua Wang, Humam Kadara. Targeting IL-1B synergizes with PD-1 blockade for enhanced T and B cell immune responses and inhibition of early lung cancer development. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5104.

Lung adenocarcinomas (LUADs) constitute the most frequently diagnosed lung cancer subtype and are typified by poor clinical outcome and inferior response to therapy. Strategies to treat LUAD at its earliest stages heavily rely on improved understanding of its pathogenesis. Accumulating evidence shows that progression of cancers, including LUADs, is tightly linked to a dynamic interplay between cancer cells and the tumor immune microenvironment (TIME), which can block activation of tumor-infiltrating T cells (TILs), thereby hindering an effective anti-tumor immune response. Yet, how the TIME is shaped and the mechanisms mediating immune evasion in early-stage LUAD remain poorly understood. To fill this void, we dissected the phenotypic and clonotypic states as well as interaction networks of TILs by performing deep cellular and molecular profiling of 225,641 T cell transcriptomes and their T cell receptor (TCR) repertoire in 16 early-stage LUADs and matched normal lung (NL) samples using simultaneous single-cell RNA and TCR sequencing (scRNA/TCR-seq). Clustering analysis revealed a highly heterogeneous T cell landscape with distinct cell states identified including naïve, effector, regulatory, helper and exhausted cell subsets, as well as cellular phenotypes indicating stress and interferon response. The landscape of TILs in LUAD tissues was far more heterogeneous when compared to NLs, and the compositions and phenotypes of TILs differed significantly across patients and in association with clinicopathological variables. Relative to NLs, LUADs were specifically enriched with regulatory and stress response subsets, whereas fractions of cytotoxic T cell subsets (e.g., CD4+ CTLs, CD8+ tissue-resident memory/TRMs, and CD8+ natural killer T/NKT cells) were decreased. scTCR-seq analysis revealed significantly increased TCR clonotype diversity and reduced clonality in LUADs compared to NLs. We then tracked phenotype transitions of TILs by integrative analysis of TCR clonotypes and transcriptional states. Intriguingly, expanded TCR clonotypes were mostly TILs with effector phenotypes that were largely shared between LUADs and NLs, suggesting that their clonal expansion may not be tumor-specific. Of note, expanded clones were also enriched with immunosuppressive phenotypes (e.g., regulatory, exhausted CD8, and stress response T cells). In contrast, we found that contracted TCR clonotypes were characterized by memory/effector phenotypes. Together, our results reveal and characterize a diverse TIL landscape in early-stage LUAD encompassing a pre-existing and expanded T cell compartment that is likely tumor-unspecific, as well as switching in T cell phenotypes, and that together comprise viable targets for early immunotherapeutic interception of this malignancy. Citation Format: Guangchun Han, Ansam Sinjab, Dapeng Hao, Lorena Gomez Bolanos, Enyu Dai, Luisa Maren Solis, Edwin Parra, Stephen Swisher, Tina Cascone, Boris Sepesi, Junya Fujimoto, Steven Dubinett, Ignacio Wistuba, Christopher Stevenson, Avrum Spira, Humam Kadara, Linghua Wang. Immune suppressive phenotypes and pre-existing bystander T cell repertoire in early-stage lung adenocarcinoma revealed by integrative single-cell profiling [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 4077.

Lung adenocarcinomas (LUAD) represent the most common lung cancer subtype and frequently harbor somatic mutations in the KRAS oncogene (KM-LUADs). While enhanced screening has improved early diagnosis of KM-LUAD, patient prognosis remains moderate to poor. Decoding the earliest events driving KM-LUADs can inform of ideal targets for its interception. Previous work showed that tobacco carcinogen (NNK) exposure leads to a pervasive field of injury comprised of molecular (e.g., KRAS mutations) and inflammatory changes that are shared between LUADs and their adjacent normal-appearing ecosystem. We and others have also shown that early immune and inflammatory alterations are implicated in the progression of normal lung (NL) epithelia and premalignant lesions (PMLs) to KM-LUAD. Yet, we still do not know the identities of specific epithelial subsets or how they promote a field of injury and inspire KM-LUAD pathogenesis. Here, we performed single-cell RNA-sequencing (scRNA-seq) of lungs from a human-relevant mouse model that develops PMLs and somatic KM-LUADs following NNK exposure. Analysis of 203,991 cells including 19,513 epithelial subsets after NNK cessation and at the onset of KM-LUADs revealed a unique population of alveolar cells that closely associated with tumor inception. These cells were highly evident in NNK- but not in control saline-exposed animals. Trajectory analysis showed that tumor clones developed through these transitionary cells, henceforth referred to as alveolar intermediate cells (AICs). Notably, AICs persisted for months after NNK cessation and acquired the same driver Kras mutations found in the resultant LUADs, thus supporting a role for AICs as KM-LUAD progenitors. Intriguingly, AICs harbored elevated expression of key components of p53 signaling (Trp53, Cdkn2a) and pro-inflammatory responses (IL-1β receptor Il1r1, NF-κB), and augmented cell-cell communication with Il1b+ macrophages which were enriched in LUAD-bearing lungs. Indeed, targeting IL-1β attenuated KM-LUAD development and increased anti-tumor immunity. In parallel, murine AIC expression profiles were significantly enriched in transcriptomes of human PMLs and LUADs. We thus probed our in-house and expanding scRNA-seq cohort of enriched (by sorting) epithelial subsets from human LUADs and NL, including 191,491 alveolar cells. AICs were not only evident in human lung tissues, but their fractions were also significantly increased in LUADs relative to NL. In conclusion, we identified a unique alveolar cell state that typified KM-LUAD progenitors, associated with inflammatory cues, and progressed along the pathologic continuum of damaged epithelium to KM-LUADs. Ongoing studies are evaluating whether AICs, in concert with tumor-initiating inflammation, trigger a field of injury that may underlie early phenotypic initiation and development of KM-LUAD. Citation Format: Ansam Sinjab, Guangchun Han, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M. Solis, Seyed Javad Moghaddam, Junya Fujimoto, Jichao Chen, Matthew Edwards, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. An emerging role for inflammation-associated alveolar intermediate cells in early phenotypic development of KRAS-mutant lung adenocarcinoma [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 1586.

The Percepta Genomic Sequencing Classifier (GSC) was developed to up-classify as well as down-classify the risk of malignancy for lung lesions when bronchoscopy is non-diagnostic. We evaluated the performance of Percepta GSC in risk re-classification of indeterminate lung lesions. This multicenter study included individuals who currently or formerly smoked undergoing bronchoscopy for suspected lung cancer from the AEGIS I/ II cohorts and the Percepta Registry. The classifier was measured in normal-appearing bronchial epithelium from bronchial brushings. The sensitivity, specificity, and predictive values were calculated using predefined thresholds. The ability of the classifier to decrease unnecessary invasive procedures was estimated. A set of 412 patients were included in the validation (prevalence of malignancy was 39.6%). Overall, 29% of intermediate-risk lung lesions were down-classified to low-risk with a 91.0% negative predictive value (NPV) and 12.2% of intermediate-risk lesions were up-classified to high-risk with a 65.4% positive predictive value (PPV). In addition, 54.5% of low-risk lesions were down-classified to very low risk with >99% NPV and 27.3% of high-risk lesions were up-classified to very high risk with a 91.5% PPV. If the classifier results were used in nodule management, 50% of patients with benign lesions and 29% of patients with malignant lesions undergoing additional invasive procedures could have avoided these procedures. The Percepta GSC is highly accurate as both a rule-out and rule-in test. This high accuracy of risk re-classification may lead to improved management of lung lesions.

Decoding the complex molecular and cellular processes during lung adenocarcinoma (LUAD) development is needed to devise early intervention strategies. To comprehensively capture LUAD neoplastic heterogeneity and cellular plasticity, we performed single-cell RNA-sequencing (scRNA-seq) of 257,481 enriched epithelial cells (EPCAM+ sorting) from 16 early-stage LUADs, each with 3 matched normal lung (NL) samples at defined spatial proximities to the tumor (n=47). 29,076 LUAD-derived cells clustered by patient and harbored distinct gene expression features (e.g., oxidative stress response), signifying interpatient LUAD heterogeneity. We also identified, using whole exome sequencing (WES) of matching lung and germline control samples, recurrent oncogenic driver alterations (e.g., EGFR, TP53, KRAS). Transcriptomic features of malignant cells were shared between LUADs (e.g., loss of lineage-specific gene expression) or private such as those associated with driver mutation status (e.g., KRAS). Indeed, clusters of malignant cells were overall segregated based on driver mutations (e.g., KRAS, EGFR). Malignant cells from KRAS-mutant LUADs (KM-LUADs) had increased activation of NF-kB, estrogen and hypoxia signaling, comprising a unique gene module (GM) that correlated with a less differentiated state. We also found hallmark pathways (cholesterol metabolism, DNA replication, cell fate decision) specific to EGFR-mutant LUADs (EM-LUADs). Notably, cells from one EM-LUAD and its 3 multiregion NL tissues clustered closely and had activated pro-tumor lymphoid signatures (CD4 naïve, Treg). Mutation burden increased with tumor proximity and intriguingly, EGFR exon20 mutation was evident in the tumor (VAF = 0.29) and its most proximal NL (VAF = 0.05), signifying a mutational field effect. Copy number variations (CNVs) derived from WES of all samples were overall consistent with those inferred from scRNA-seq data. Relative to EM-LUADs, malignant cells from KM-LUADs displayed lower CNV burdens. Interpatient CNV heterogeneity was prominent even among LUADs harboring the same oncogenic drivers. Notably, intratumor heterogeneity (ITH) was high among epithelial cells within single regions from the same LUAD. Among LUADs, malignant cell clades with KRAS mutations and lower CNV scores displayed less differentiated states. To investigate biological pathways driving ITH, we derived 6 GMs with tumor-relevant functional features, including a transcription/translation regulation GM that consistently correlated with reduced differentiation. Our analysis of a large number of lung epithelial cells from LUAD patients reveals in-depth insights into LUAD taxonomy which can help identify epithelial heterotypes, unravel the continuum of early differentiation events and expand our understanding of early LUAD pathogenesis. Citation Format: Guangchun Han, Ansam Sinjab, Warapen Treekitkarnmongkol, Dapeng Hao, Enyu Dai, Luisa M. Solis, Edwin R. Parra, Stephen Swisher, Tina Cascone, Boris Sepesi, Jichao Chen, Steven Dubinett, Junya Fujimoto, Ignacio I. Wistuba, Christopher S. Stevenson, Avrum E. Spira, Linghua Wang, Humam Kadara. Single-cell sequencing of early-stage lung adenocarcinomas reveals prominent intratumoral heterogeneity and epithelial plasticity programs [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 2126.

Rationale Peripheral airway obstruction is a key feature of chronic obstructive pulmonary disease (COPD), but the mechanisms of airway loss are unknown. This study aims to identify the molecular and cellular mechanisms associated with peripheral airway obstruction in COPD. Methods Ten explanted lung specimens donated by patients with very severe COPD treated by lung transplantation and five unused donor control lungs were sampled using systematic uniform random sampling (SURS), resulting in 240 samples. These samples were further examined by micro-computed tomography (CT), quantitative histology and gene expression profiling. Results Micro-CT analysis showed that the loss of terminal bronchioles in COPD occurs in regions of microscopic emphysematous destruction with an average airspace size of >= 500 and <1000 mu m, which we have termed a "hot spot". Based on microarray gene expression profiling, the hot spot was associated with an 11-gene signature, with upregulation of pro-inflammatory genes and downregulation of inhibitory immune checkpoint genes, indicating immune response activation. Results from both quantitative histology and the bioinformatics computational tool CIBERSORT, which predicts the percentage of immune cells in tissues from transcriptomic data, showed that the hot spot regions were associated with increased infiltration of CD4 and CD8 T-cell and B-cell lymphocytes. Interpretation The reduction in terminal bronchioles observed in lungs from patients with COPD occurs in a hot spot of microscopic emphysema, where there is upregulation of IFNG signalling, co-stimulatory immune checkpoint genes and genes related to the inflammasome pathway, and increased infiltration of immune cells. These could be potential targets for therapeutic interventions in COPD.

The molecular events involved in the development of bronchial premalignant lesions (PMLs), and their progression to lung squamous cell carcinoma, are not well understood. Prior work characterized lung PML molecular subtypes by identifying co-expressed gene modules associated with histologic severity and progression/persistence. The proliferative subtype was enriched for PMLs with dysplasia. Genes related to interferon signaling and antigen processing/presentation (Module 9) were decreased in progressive/persistent lesions within this subtype, suggesting early immune suppression is related to PML progression. However, the mechanisms that drive these alterations are unclear. We investigated the role of microRNAs (miRNAs) in regulating gene expression associated with PML outcomes. mRNA and miRNA were extracted and sequenced from longitudinally collected endobronchial biopsies from patients with PMLs (148 samples, 30 patients). miRNAs targeting each gene co-expression module were identified based on target gene enrichment and the degree of negative correlation between the miRNA expression and its targets. Expression association with outcome within the proliferative subtype was tested with a mixed effects model adjusting for batch and patient as random effect. Cell type specificity of miRNA expression was tested based on cell type specific sequencing data from FANTOM5 and cell type marker correlation analysis. Genes regulated by NLRC5 were identified with ChIPseq data from Ludigs et al. Target gene suppression was confirmed by transfecting SW900 cells with miR-149-5p. miR-149-5p level in PML biopsies was examined by miRNA in situ hybridization (miR-ISH). miR-149-5p is identified as potential regulator of Module 9 gene expression and is significantly up-regulated in progressive/persistent PMLs. Its expression is highly enriched in epithelial cells in FANTOM5 and positively correlates with basal cell markers within PMLs. Predicted targets of miR-149-5p are down-regulated in the progressive PMLs in both our and data from Merrick et al. MHC-I and related gene expressions are down-regulated in progressing/persistent PMLs. These genes are regulated by the transcriptional coactivator NLRC5 which is a predicted target gene of miR-149-5p. We find that overexpressing miR-149-5p in SW900 cells decreases the expression levels of both NLRC5 and NLRC5 regulated genes. Additionally, miR-ISH targeting miR-149-5p in proliferative biopsy samples confirm its expression in epithelium compartment and association with outcome. Our data suggest epithelial miR-149-5p might be a key regulator of gene expression contributing to PML progression. We hypothesize that by suppressing NLRC5, miR-149-5p inhibits MHC-I gene expression of epithelial cells, promoting early immune depletion and lesion progression. miR-149-5p might therefore be a therapeutic target for preventing PML progression. Citation Format: Boting Ning, Roxana M. Pfefferkorn, Gang Liu, Sherry Zhang, Hanqiao Liu, Christopher Stevenson, Sarah A. Mazzilli, Avrum E. Spira, Marc E. Lenburg, Jennifer E. Beane. The role of epithelial miR-149 in immune modulation and progression of bronchial premalignant lesions [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 1488.

AbstractTumor-infiltrating B and plasma cells (TIB) are prevalent in lung adenocarcinoma (LUAD); however, they are poorly characterized. We performed paired single-cell RNA and B-cell receptor (BCR) sequencing of 16 early-stage LUADs and 47 matching multiregion normal tissues. By integrative analysis of ∼50,000 TIBs, we define 12 TIB subsets in the LUAD and adjacent normal ecosystems and demonstrate extensive remodeling of TIBs in LUADs. Memory B cells and plasma cells (PC) were highly enriched in tumor tissues with more differentiated states and increased frequencies of somatic hypermutation. Smokers exhibited markedly elevated PCs and PCs with distinct differentiation trajectories. BCR clonotype diversity increased but clonality decreased in LUADs, smokers, and with increasing pathologic stage. TIBs were mostly localized within CXCL13+ lymphoid aggregates, and immune cell sources of CXCL13 production evolved with LUAD progression and included elevated fractions of CD4 regulatory T cells. This study provides a spatial landscape of TIBs in early-stage LUAD.Significance:While TIBs are highly enriched in LUADs, they are poorly characterized. This study provides a much-needed understanding of the transcriptional, clonotypic states and phenotypes of TIBs, unraveling their potential roles in the immunopathology of early-stage LUADs and constituting a road map for the development of TIB-targeted immunotherapies for the treatment of this morbid malignancy.This article is highlighted in the In This Issue feature, p. 2483

Background: Bronchial pre-malignant lesions (PMLs) are the putative precursors for bronchial squamous cell carcinoma. PMLs represent a spectrum of histologies, from low-grade lesions (hyperplasia, metaplasia) to high-grade lesions (dysplasia, carcinoma in situ). The majority of these lesions will regress or remain stable without clinical intervention while a subset of lesions will progress to invasive carcinoma. We performed single-cell RNA sequencing (scRNAseq) of these lesions to elucidate the cross-talk between epithelial, stromal, and immune populations in lesions of increasing histological grade. Methods: Thirty lesions from seventeen participants were biopsied via bronchoscopy. Cells were sorted by CD45+/- FACS gating and sequenced with the Cel-Seq2 protocol. Celda was used to bi-cluster genes into modules and cells into clusters. Cells were filtered by mitochondrial percentage (%mito < 50%), minimum UMI counts (nUMI > 300), and doublet detection. Cell types were labeled by marker gene expression. Results: After filtering low quality cells, we analyzed 4,382 cells. We observed expected smoking related shifts in epithelial cell type proportions, including an increase in secretory cells (χ2 = 31.39, p = 2.11 X 10-8) and a decrease in ciliated cells (χ2 = 4.83, p = 0.028) among current smokers. Distinct differences in expression of transcriptional modules were observed between KRT5+ (basal) cells from different histologic grades. Basal cells from high grade lesions expressed smoking detoxification and cell cycle gene programs, while low grade lesion basal cells expressed differentiation gene programs. We also identified a group of cells from CIS lesions involved in an epithelial-to-mesenchymal transition, marked by an increase in SPARC and COL4A1 expression and a decrease in CDH1 expression. Subpopulations of immune cells identified include macrophages, CD4/8+ T, B, dendritic cells, and natural killer cells. Several clusters of CD4+ and CD8+ T cells displayed an exhausted phenotype, marked by the expression of PD-1, CTLA4, LAG3, and TIGIT. Samples with high grade histology (dysplasia, carcinoma in situ) were enriched in CD4+ Tregs and myeloid cells compared to low grade histology samples (hyperplasia, metaplasia), which were enriched in Natural Killer and cytotoxic CD8+ T cells (χ2 = 298.95, p = 0.001). Discussion: Our results suggest that changes in specific transcriptional programs are associated with the transition of epithelial cells to more invasive states and that changes in immune populations are associated with increasing histological grade. These signatures can suggest novel avenues for chemoprevention and cancer interception. Citation Format: Conor Shea, Lukas Kalinke, Kitty De Jong, Kate Gowers, Diane Ding, Sherry Zhang, Gang Liu, Jack Cunningham, Ipsita Dey-Guha, Mark Hennon, Sai Yendamuri, Christopher Stevenson, Avrum Spira, Mary E. Reid, Marc E. Lenburg, Sam M. Janes, Jennifer E. Beane, Sarah A. Mazzilli, Joshua D. Campbell. Epithelial, stromal, and immune changes associated with lung squamous premalignant lesion severity identified by single-cell RNA-seq [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 2201.

Bleomycin induces a transient lung fibrosis in mice that has been used to investigate mechanisms related to idiopathic pulmonary fibrosis. Our aim was to determine a sensitive method for assessing lung function in bleomycin treated mice that correlated with the degree of lung fibrosis as measured by collagen immunohistochemistry. Bleomycin (2 U/kg) or saline was intratracheally microsprayed to male C57BL/6 mice under isoflurane anesthesia. Lung function (single compartment model, constant phase model, and work of breathing) was assessed using the flexiVent system, and after euthanasia lungs were inflated with formalin in situ for histological analysis. The lung fibrosis histopathology score for the bleomycin treated animals on day 21 was indicative of mild-to-moderate fibrosis (Saline treated control: 0 ± 0, Bleomycin treated: 4.9 ± 0.4). There were at least three large areas of fibrosis in the peribronchial alveolar regions of the lung, but less than 50% of each lung was affected by fibrosis. Although changes in lung function were less obvious, volume normalized dynamic work of breathing measured at 30 ml/kg tidal volume (Saline treated control: 9.2 ± 0.1 J/l, Bleomycin treated: 10.6 ± 0.3 J/l) and the oscillatory mechanics constant phase model parameter tissue elastance (H; Saline treated control: 31 ± 2 cm H2O/ml, Bleomycin treated: 38 ± 3 cm H2O/ml) were significantly increased on day 21. The work of breathing (r = 0.83) correlated slightly better with fibrosis histopathology score than H (r = 0.64). Work of breathing can detect decrements in lung function due to pulmonary fibrosis, correlates well with the amount of collagen in the lungs, and may be a more sensitive quantitative measure of efficacy for drugs being developed to treat pulmonary fibrosis.

Background The key co-repressor complex components HDAC-2, Mi-2α/β and mSin3a are all critical to the regulation of gene transcription. HDAC-2 function is impaired by oxidative stress in a PI3Kδ dependant manner which may be involved in the chronic glucocorticoid insensitive inflammation in the lungs of COPD patients. However, the impact of cigarette smoke exposure on the expression of mSin3a and Mi2α/β and their role in glucocorticoid responsiveness is unknown. Methods Wild type, PI3Kγ knock-out (PI3Kγ -/- ) and PI3K kinase dead knock-in (PI3Kδ D910/A910 ) transgenic mice were exposed to cigarette smoke for 3 days and the expression levels of the co-repressor complex components HDAC-2, mSin3a, Mi-2α and Mi-2β and HDAC-2 activity in the lungs were assessed. Results Cigarette smoke exposure impaired glucocorticoid function and reduced HDAC-2 activity which was protected in the PI3Kδ D910/A910 mice. Both mSin3a and Mi-2α protein expression was reduced in smoke-exposed mice. Budesonide alone protected mSin3a protein expression with no additional effect seen with abrogation of PI3Kγ/δ activity, however Mi-2α, but not Mi-2β, expression was protected in both PI3Kδ D910/A910 and PI3Kγ -/- budesonide-treated smoke-exposed mice. The restoration of glucocorticoid function coincided with the protection of both HDAC activity and mSin3a and Mi-2α protein expression. Conclusions Cigarette smoke exposure induced glucocorticoid insensitivity and alters co-repressor activity and expression which is prevented by blockade of PI3K signaling with glucocorticoid treatment. Inhibition of PI3Kδ signalling in combination with glucocorticoid treatment may therefore provide a therapeutic strategy for restoring oxidant-induced glucocortiocid unresponsiveness.