Single-cell and bulk RNA-sequencing reveal SPP1 and CXCL12 as cell-to-cell communication markers to predict prognosis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) generally presents as an immunosuppressive microenvironment. The characteristics of cell-to-cell communication in the LUAD microenvironment has been unclear. In this study, the LUAD bulk RNA-seq data and single-cell RNA-seq data were retrieved from public dataset. Differential expression genes (DEGs) between LUAD tumor and adjacent non-tumor tissues were calculated by limma algorithm, and then detected by PPI, KEGG, and GO analysis. Cell-cell interactions were explored using the single-cell RNA-seq data. Finally, the first 15 CytoHubba genes were used to establish related pathways and these pathways were used to characterize the immune-related ligands and their receptors in LUAD. Our analyses showed that monocytes or macrophages interact with tissue stem cells and NK cells via SPP1 signaling pathway and tissue stem cells interact with T and B cells via CXCL signaling pathway in different states. Hub genes of SPP1 participated in SPP1 signaling pathway, which was negatively correlated with CD4+ T cell and CD8+ T cell. The expression of SPP1 in LUAD tumor tissues was negatively correlated with the prognosis. While CXCL12 participated in CXCL signaling pathway, which was positively correlated with CD4+ T cell and CD8+ T cell. The role of CXCL12 in LUAD tumor tissues exhibits an opposite effect to that of SPP1. This study reveals that tumor-associated monocytes or macrophages may affect tumor progression. Moreover, the SPP1 and CXCL12 may be the critic genes of cell-to-cell communication in LUAD, and targeting these pathways may provide a new molecular mechanism for the treatment of LUAD.

Integrated analysis highlights the significance role of ITGAL in lung adenocarcinoma.

Integrin alpha L (ITGAL), a member of the integrin family, is associated with carcinogenesis and immune regulation. However, the biological functions of ITGAL in lung adenocarcinoma (LUAD) remain poorly understood. In this study, we utilized the TCGA dataset to analyse ITGAL mRNA expression in LUAD and examined its correlation with clinical prognosis. Three-dimensional (3D) Matrigel culture, 5-bromodeoxyuridine (BrdU) ELISA, wound-healing migration and cell adherence assays were used to demonstrate the potential role of ITGAL in LUAD progression. Additionally, we analysed single-cell sequencing data of LUAD to determine the expression and biological function of ITGAL. Our research revealed that the expression of ITGAL in LUAD samples is an independent predictor of prognosis. Patients with high expression of ITGAL had significantly better overall survival (OS), progression-free survival (PFS) and disease-specific survival (DSS) compared to the low-expression group. Meanwhile, the expression of ITGAL suppressed malignant progression in LUAD cells. Functional enrichment analyses showed that ITGAL was significantly correlated with cell immune response and immune checkpoint, consistent with the analysis of single-cell sequencing in paired samples of normal and tumour. Furthermore, we confirmed that ITGAL expression affect the tumour microenvironment (TME) through regulation of the expression of cytokines in NK cells of LUAD. In summary, ITGAL is a prognostic biomarker for LUAD patients, and it repressed malignant progression in LUAD cells. Moreover, ITGAL expression also enhanced the effect of immunotherapy and may be an important target in LUAD therapy.

Different Endotracheal Intubations for Non-Small Cell Lung Cancer Surgery: A Retrospective-Case-Matched Study on Postoperative Complications and Quality of Life.

BACKGROUND: Lung isolation and separation is still controversial in thoracic surgery. Preferences of the surgeon can drive the decision to use single- vs. double-lumen endotracheal intubation. We aimed to compare complications and quality of life (QOL) after radical lung cancer resection with a single-lumen tube (ST) and a double-lumen tube (DT) for patients with non-small cell lung cancer (NSCLC). METHODS: A total of 309 patients who underwent radical lung cancer resection with video-assisted thoracoscopy-lobectomy were subsequently included in the study. Based on the type of endotracheal intubation tube used during surgery, we divided all the patients into a single-lumen tube group (ST-G) and double-lumen tube group (DT-G). Then, we applied propensity score matching (1:1) to balance the baseline characteristics between the two groups. The Analysis of Variance (ANOVA) of two-factor repeated measures data was performed to compare postoperative complications at three and six months after surgery and postsurgical QOL at baseline at one month, three months, six months, and twelve months. RESULTS: Within three months after surgery, patients in the ST-G presented less cough symptoms in Lung Cancer Symptom Scale (LCSS), lower cough symptom scores (CSS) (one month and three months, p < 0.05) and better performance of Leicester Cough Questionnaire (LCQ) scores in physical part (one month, three months and six months, p < 0.05) with better overall QOL (one month and three months, p < 0.05) than those in the DT-G. CONCLUSIONS: Patients with STs displayed less postoperative cough symptoms and higher overall QOL than those with DTs. Although DT is the gold standard for thoracic surgeries, we suggest that postoperative cough symptoms should be given sufficient attention by surgeons.

Quality of life after lung cancer surgery: sublobar resection versus lobectomy.

BACKGROUND: This study aimed to compare the postoperative quality of life (PQOL) between non-small-cell lung cancer (NSCLC) patients who underwent video-assisted thoracoscopic sublobar resection (subsegment, segment, or wedge) and lobectomy. Meanwhile, we developed a PQOL scale for patients with NSCLC after optimization. METHODS: Developing and evaluating the postoperative quality-of-life scale of non-small-cell lung cancer (NSCLC-PQOL) followed by the international principles for developing quality-of-life scale. Therefore, we used the NSCLC-PQOL scale to evaluate the PQOL of patients who underwent different surgeries. RESULTS: The overall PQOL of patients who underwent video-assisted thoracoscopic lobectomy and sublobar resection gradually worsened from discharge to 3 months postoperatively and progressively improved from three to 6 months postoperatively. And the sublobar resection group showed better PQOL in chest tightness, breath shortness, breathlessness, cough and expectoration than the lobectomy group, and the differences were statistically significant (P < 0.05). The final version of the NSCLC-PQOL contained three dimensions: "signs-symptoms", "psychological and psychiatric", and "social-life" dimensions. CONCLUSIONS: The sublobar resection group showed better PQOL in "chest tightness", "breath shortness", "breathlessness", "cough", and "expectoration" than the lobectomy group. Twenty-two items formed a well-behaved PQOL scale after being validated satisfactorily. The scale was a suitable rating tool for evaluating the NSCLC-PQOL of patients. TRIAL REGISTRATION: As this study was a retrospective study and not a clinical trial, we did not register this study in the Chinese Clinical Trial Registry.

A cuproptosis-related lncRNA signature-based prognostic model featuring on metastasis and drug selection strategy for patients with lung adenocarcinoma.

Introduction: Lung adenocarcinoma is a common cause of mortality in patients with cancer. Recent studies have indicated that copper-related cell death may not occur in the same way as previously described. Long non-coding RNAs (lncRNAs) play a key role in the occurrence and development of tumors; however, the relationship between cuproptosis and lncRNAs in tumorigenesis and lung adenocarcinoma (LUAD) treatment has not been well established. Our study aimed to construct a model to analyze the prognosis of lung adenocarcinoma in patients using a carcinogenesis-related lncRNA (CR) signature. Methods: The transcriptional profiles of 507 samples from The Cancer Genome Atlas were assessed. Cox regression and co-expression analyses, and the least absolute shrinkage and selection operator (LASSO) were used to filter the CR and develop the model. The expression status of the six prognostic CRs was used to classify all samples into high- and low-risk groups. The overall disease-free survival rate was compared between the two groups. The Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes were used to identify the pathways and mechanisms involved in this model. Subsequently, immunotherapy response, sensitivity, and correlation analyses for several anti-tumor medications were performed. In vitro experiments, including qPCR, were conducted in nine lung adenocarcinoma cell lines and 16 pairs of lung adenocarcinoma and para-carcinoma tissues. Results: After confirmation using the ROC curve, patients in the low-risk category benefited from both overall and disease-free survival. Gene Ontology analysis highlighted cell movement in the model. In the in vitro experiments, qPCR results showed the expression levels of six CRs in 16 pairs of carcinoma and para-carcinoma tissues, which were in accordance with the results of the model. AL138778.1 is a protective factor that can weaken the invasion and migration of A549 cells, and AL360270.1 is a hazardous factor that promotes the invasion and migration of A549 cells. According to this model, targeted treatments such as axitinib, gefitinib, linsitinib, pazopanib, and sorafenib may be more appropriate for low-risk patients. Conclusion: Six CR profiles (AL360270.1, AL138778.1, CDKN2A-DT, AP003778.1, LINC02718, and AC034102.8) with predictive values may be used to evaluate the prognosis of patients with lung adenocarcinoma undergoing therapy.

Distance between tumor and bronchial resection margin is an independent predictor of recurrence-free survival and overall survival in primary endobronchial neoplasm.

BACKGROUND: The distance between tumor and bronchial resection margin (DBTM) had no clear standard in lung cancer surgery. We aimed to select the optimal cut-off value to provide a standard for surgery of the patients with primary endobronchial neoplasm. METHODS: We retrospectively analyzed patients with primary endobronchial neoplasm who underwent surgical resection between 2005 and 2012. The receiver operating characteristic curves and the Youden index were used to calculate the optimal cut-off value of the DBTM. Propensity score matching was applied to reduce selection bias. Survival was assessed with Kaplan-Meier analysis, log-rank test and Cox proportional hazards model. RESULTS: A total of 1048 patients comprised in the study cohort and 1.7 cm was determined the optimal cut-off value, including 531 grouped in DBTM ≤ 1.7 cm and 517 grouped in DBTM > 1.7 cm. Before propensity score matching, the 5-year recurrence-free survival was 38.7% in DBTM ≤ 1.7 cm group and 67.1% in DBTM > 1.7 cm (hazard ratio 0.48, P < 0.001), while the 5-year overall survival was 30.1% and 50.7%, respectively (hazard ratio 0.64, P < 0.001). After propensity score matching, the 5-year recurrence-free survival was 38.8% in DBTM ≤ 1.7 cm group and 66.1% in DBTM > 1.7 cm (hazard ratio 0.51, P < 0.001), while the 5-year overall survival was 34.7% and 50%, respectively (hazard ratio 0.81, P = 0.012). Multivariable cox model showed that the DBTM was an independent predictor for recurrence-free survival (hazard ratio 0.51, P = 0.001) and overall survival (hazard ratio 0.84, P = 0.026). CONCLUSIONS: The DBTM was an independent predictor for outcomes in primary endobronchial neoplasm, and patients with the DBTM ≤ 1.7 cm should actively receive adjuvant therapy after surgery. The distance between tumor and bronchial resection margin (DBTM), the distance between the most proximal border of the macroscopic tumor and bronchial resection margin was measured by pathologist in the fresh specimens before formalin fixation. The DBTM less than 1.7cm had higher recurrence-free survival and overall survival and was an independent prognostic factor for patients with primary endobronchial neoplasm.

Noncoding SNP at rs1663689 represses ADGRG6 via interchromosomal interaction and reduces lung cancer progression.

A previous genome-wide association study (GWAS) revealed an association of the noncoding SNP rs1663689 with susceptibility to lung cancer in the Chinese population. However, the underlying mechanism is unknown. In this study, using allele-specific 4C-seq in heterozygous lung cancer cells combined with epigenetic information from CRISPR/Cas9-edited cell lines, we show that the rs1663689 C/C variant represses the expression of ADGRG6, a gene located on a separate chromosome, through an interchromosomal interaction of the rs1663689 bearing region with the ADGRG6 promoter. This reduces downstream cAMP-PKA signaling and subsequently tumor growth both in vitro and in xenograft models. Using patient-derived organoids, we show that rs1663689 T/T-but not C/C-bearing lung tumors are sensitive to the PKA inhibitor H89, potentially informing therapeutic strategies. Our study identifies a genetic variant-mediated interchromosomal interaction underlying ADGRG6 regulation and suggests that targeting the cAMP-PKA signaling pathway may be beneficial in lung cancer patients bearing the homozygous risk genotype at rs1663689.

Ion channel gene GJB2 influences the intercellular communication by Up-regulating the SPP1 signaling pathway identified by the single-cell RNA sequencing in lung adenocarcinoma.

Objective: Firstly, observe the prognostic significance and the biological functional effects of gap junction protein beta 2 (GJB2 or Cx26) in lung adenocarcinoma (LUAD). Subsequently, explore the role played by GJB2 in intercellular communication by single-cell RNA sequencing. Method: We made a differential analysis of GJB2 expression through public databases and investigated the clinical characteristics and prognostic significance. ESTIMATE analysis and Tumor Immune Estimation Resource (TIMER) database were utilized to illustrate the association of GJB2 with immune infiltration and components of the tumor microenvironment. Gene Ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and Gene set enrichment analysis (GSEA) were performed to study the biological function of GJB2. Cell-cell communication was analyzed using the CellChat R package through sc-RNA data. Results: GJB2 has an outstanding prognosis value in LUAD and a close relationship was found between GJB2 and immune infiltration in LUAD. GJB2 could participate in several tumor biological processes, including extracellular matrix remodeling and upregulation of multiple cancer-related active pathways. GJB2 related hub-genes influence intercellular communication through the SPP1 signaling pathway. Conclusion: Our study illustrates one mechanism by which GJB2 exerts its cancer-specific relevant effects, that is, causing changes in intercellular communication through the SPP1 signaling pathway. Blockade of this pathway may limit the functional role of GJB2 and provide us with promising new perceptions for LUAD treatment.

Molecular features and evolutionary trajectory of ASCL1+ and NEUROD1+ SCLC cells.

BACKGROUND: Small cell lung cancer (SCLC) is the most aggressive subtype of lung cancer without recognised morphologic or genetic heterogeneity. Based on the expression of four transcription factors, ASCL1, NEUROD1, POU2F3, and YAP1, SCLCs are classified into four subtypes. However, biological functions of these different subtypes are largely uncharacterised. METHODS: We studied intratumoural heterogeneity of resected human primary SCLC tissues using single-cell RNA-Seq. In addition, we undertook a series of in vitro and in vivo functional studies to reveal the distinct features of SCLC subtypes. RESULTS: We identify the coexistence of ASCL1+ and NEUROD1+ SCLC cells within the same human primary SCLC tissue. Compared with ASCL1+ SCLC cells, NEUROD1+ SCLC cells show reduced epithelial features and lack EPCAM expression. Thus, EPCAM can be considered as a cell surface marker to distinguish ASCL1+ SCLC cells from NEUROD1+ SCLC cells. We further demonstrate that NEUROD1+ SCLC cells exhibit higher metastatic capability than ASCL1+ SCLC cells and can be derived from ASCL1+ SCLC cells. CONCLUSIONS: Our studies unveil the biology and evolutionary trajectory of ASCL1+ and NEUROD1+ SCLC cells, shedding light on SCLC tumourigenesis and progression.

Hematopoietic transcription factor GFI1 promotes anchorage independence by sustaining ERK activity in cancer cells.

The switch from anchorage-dependent to anchorage-independent growth is essential for epithelial metastasis. The underlying mechanism, however, is not fully understood. In this study, we identified growth factor independent-1 (GFI1), a transcription factor that drives the transition from adherent endothelial cells to suspended hematopoietic cells during hematopoiesis, as a critical regulator of anchorage independence in lung cancer cells. GFI1 elevated the numbers of circulating and lung-infiltrating tumor cells in xenograft models and predicted poor prognosis of patients with lung cancer. Mechanistically, GFI1 inhibited the expression of multiple adhesion molecules and facilitated substrate detachment. Concomitantly, GFI1 reconfigured the chromatin structure of the RASGRP2 gene and increased its expression, causing Rap1 activation and subsequent sustained ERK activation upon detachment, and this led to ERK signaling dependency in tumor cells. Our studies unveiled a mechanism by which carcinoma cells hijacked a hematopoietic factor to gain anchorage independence and suggested that the intervention of ERK signaling may suppress metastasis and improve the therapeutic outcome of patients with GFI1-positive lung cancer.

Autophagic flux in cancer cells at the invasive front in the tumor-stroma border.

Cancer cells at the invasive front directly interact with stromal tissue that provides a microenvironment with mechanical, nutrient, and oxygen supply characteristics distinct from those of intratumoral tissues. It has long been known that cancer cells at the invasive front and cancer cells inside the tumor body exhibit highly differentiated functions and behaviors. However, it is unknown whether cancer cells at different locations exhibit a variety of autophagic flux, an important catabolic process to maintain cellular homeostasis in response to environmental changes. Here, using transmission electron microscopy (TEM), we found that invading cancer cells at the invasive front, which show mesenchymal transcriptomic traits, exhibit higher autophagic flux than cancer cells inside the tumor body in human primary non-small cell lung cancer (NSCLC) tissues. This autophagic feature was further confirmed by a live cell autophagic flux monitoring system combined with a 3D organotypic invasion coculture system. Additionally, the increased autophagic flux endows cancer cells with invasive behavior and positively correlates with the advanced tumor stages and the reduced survival period of lung cancer patients. These findings expand the understanding of autophagic dynamics during cancer invasion.

Spi-B Promotes the Recruitment of Tumor-Associated Macrophages via Enhancing CCL4 Expression in Lung Cancer.

Tumor immune escape plays a critical role in malignant tumor progression and leads to the failure of anticancer immunotherapy. Spi-B, a lymphocyte lineage-specific Ets transcription factor, participates in mesenchymal invasion and favors metastasis in human lung cancer. However, the mechanism through which Spi-B regulates the tumor immune environment has not been elucidated. In this study, we demonstrated that Spi-B enhanced the infiltration of tumor-associated macrophages (TAMs) in the tumor microenvironment using subcutaneous mouse models and clinical samples of human lung cancer. Spi-B overexpression increased the expression of TAM polarization- and recruitment-related genes, including CCL4. Moreover, deleting CCL4 inhibited the ability of Spi-B promoting macrophage infiltration. These data suggest that Spi-B promotes the recruitment of TAMs to the tumor microenvironment via upregulating CCL4 expression, which contributes to the progression of lung cancer.

Risk factors and survival analysis of arrhythmia following lung cancer surgery: a retrospective study.

BACKGROUND: Surgical treatment of lung cancer is one of the important treatments for early-stage non-small cell lung cancer (NSCLC). However, arrhythmia, especially atrial fibrillation (AF) and supraventricular arrhythmia, are quite common among patients after surgical treatment of lung cancer. The impact of postoperative arrhythmia (PA) on survival is rarely reported. Our aim was to evaluate the risk factors of PA and its impact on overall survival (OS) after lung cancer surgery. METHODS: A total of 344 patients diagnosed with NSCLC who underwent lung cancer surgery were enrolled in this study. These patients were divided into two groups based on the occurrence of PA. Univariate and multivariate logistic regression analyses were conducted to identify the risk factors of PA. The Kaplan-Meier method was applied to show the OS differences between the two groups. RESULTS: The incidence of PA was 16% (55/344). Among these 55 patients, 20 had AF, 30 had sinus tachycardia, and 5 had premature beats. A total of 332 patients underwent lung cancer radical resection. Operation type (P<0.001), preoperative abnormal ECG (P=0.032), transfusion (P=0.016), postoperative serum potassium concentration (P=0.001) and clinical stage (P<0.05) were risk factors for PA. PA (HR 2.083, 95% CI, 1.334-3.253; P=0.001), age (HR 1.543, 95% CI, 1.063-2.239; P=0.025) and mediastinal lymph node metastasis (HR 2.655, 95% CI, 1.809-3.897; P<0.001) were independent prognostic risk factors for OS by multivariate cox analysis. CONCLUSIONS: We identified PA as an independent prognostic risk factor to predict poor OS in patients who underwent lung cancer surgery and had risk factors for PA. We therefore provides guidance for PA in improving the prognosis of lung cancer patients.

A prognostic nomogram for lung adenocarcinoma based on immune-infiltrating Treg-related genes: from bench to bedside.

BACKGROUND: Accumulating evidence suggests that lymphocyte infiltration in the tumor microenvironment is positively correlated with tumorigenesis and development, while the role of Tregs (regulatory T cells) has been controversial. Therefore, we attempted to discover the possible value of Tregs for lung adenocarcinoma (LUAD). METHODS: The gene-sequencing data of LUAD were applied from three Gene Expression Omnibus (GEO) datasets-GSE10072, GSE32863 and GSE43458; the corresponding fractions of tumor-infiltrating immune cells were extracted from the CIBERSORTx portal. Weighted gene coexpression network analysis (WGCNA) and protein-protein interaction (PPI) network analysis were conducted to identify the significant module and candidate genes related to Tregs. The role of candidate genes in LUAD was further verified using data from The Cancer Genome Atlas (TCGA) database. Finally, we constructed a nomogram model to predict the prognosis of LUAD by plotting Kaplan-Meier (K-M), receiver operating characteristic (ROC) and calibration curves, which elucidated the performance of the nomogram. RESULTS: In total, 10,047 genes in 333 samples (196 tumor and 137 normal samples) from the GEO database were included. By WGCNA and PPI analysis, we identified a significant black module and 36 candidate genes related to Treg. Next, the candidate genes were verified using TCGA data by Cox regression analysis to screen 13 hub genes that stratified LUAD patients into low- or high-risk groups. Low-risk patients showed a significantly longer overall survival (OS) than high-risk patients (3-year OS: 70.2% vs. 35.2%; 5-year OS: 36.6% vs. 0; P=1.651E-09), and the areas under the ROC curves (AUCs) showed good (3-year AUC: 0.733; 5-year AUC: 0.777). Next, we constructed a survival nomogram combining the hub genes and clinical parameters; the low-risk patients still showed a favorable prognosis compared with that of the high-risk patients (P=7.073E-13), and the AUCs were better (3-year AUC: 0.763; 5-year AUC: 0.873). CONCLUSIONS: We revealed the role of immune-infiltrating Treg-related genes in LUAD and constructed a prognostic nomogram, which may help clinicians make optimal therapeutic decisions and help patients obtain better outcomes.

Development and validation of a survival model for lung adenocarcinoma based on autophagy-associated genes.

BACKGROUND: Given that abnormal autophagy is involved in the pathogenesis of cancers, we sought to explore the potential value of autophagy-associated genes in lung adenocarcinoma (LUAD). METHODS: RNA sequencing and clinical data on tumour and normal samples were acquired from The Cancer Genome Atlas (TCGA) database and randomly assigned to training and testing groups. Differentially expressed autophagy-associated genes (AAGs) were screened. Within the training group, Cox regression and Lasso regression analyses were conducted to screen five prognostic AAGs, which were used to develop a model. Kaplan-Meier (KM) and receiver operating characteristic (ROC) curves were plotted to determine the performance of the model in both groups. Immunohistochemistry was used to demonstrate the differential expression of AAGs in tumour and normal tissues at the protein level. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were utilized to further elucidate the roles of AAGs in LUAD. RESULTS: The data from the TCGA database included 497 tumour and 54 normal samples, within which 30 differentially expressed AAGs were screened. Using Cox regression and Lasso regression analyses for the training group, 5 prognostic AAGs were identified and the prognostic model was constructed. Patients with low risk had better overall survival (OS) in the training group (3-year OS, 73.0% vs 48.0%; 5-year OS, 45.0% vs 33.8%; P = 1.305E-04) and in the testing group (3-year OS, 66.8% vs 41.2%; 5-year OS, 31.7% vs 25.8%; P = 1.027E-03). The areas under the ROC curves (AUC) were significant for both the training and testing groups (3-year AUC, 0.810 vs 0.894; 5-year AUC, 0.792 vs 0.749). CONCLUSIONS: We developed a survival model for LUAD and validated the performance of the model, which may provide superior outcomes for the patients.