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Ferroptosis is a recently discovered form of cell death that plays an important role in tumor growth and holds promise as a target for antitumor therapy. However, evidence in the regulation of ferroptosis in lung adenocarcinoma (LUAD) remains elusive. Here, we show that retinoic acid receptor alpha (RARA) is upregulated with the treatment of ferroptosis inducers (FINs). Pharmacological activation of RARA increases the resistance of LUAD to ferroptosis according to cell viability and lipid peroxidation assays, while RARA inhibitor or knockdown (KD) does the opposite. Through transcriptome sequencing in RARA-KD cells and chromatin immunoprecipitation (CHIP)-Seq data, we identify thioredoxin (TXN) and protein phosphatase 1 F (PPM1F) as downstream targets of RARA, both of which inhibit ferroptosis. We confirm that RARA binds to the promotor region of TXN and PPM1F and promotes their transcription by CHIP-qPCR and dual-luciferase assays. Overexpression of TXN and PPM1F reverses the effects of RARA knockdown on ferroptosis in vitro and vivo. Clinically, RARA knockdown or inhibitor increases cells' sensitivity to pemetrexed and cisplatin (CDDP). Immunohistochemistry (IHC) of LUAD from our cohort shows the same expression tendency of RARA and the downstream targets. Our study uncovers that RARA inhibits ferroptosis in LUAD by promoting TXN and PPM1F, and inhibiting RARA-TXN/PPM1F axis represents a promising strategy for improving the efficacy of FINs or chemotherapy in the treatment of LUAD patients., (© 2024. The Author(s).)

Background: Lung cancer is one of the most common tumors in the world, and metastasis is one of the major causes of tumor-related death in lung cancer patients. Tumor-associated macrophages (TAMs) are a major component of the tumor microenvironment (TME) and are frequently associated with tumor metastasis in human cancers. However, the regulatory mechanisms of TAMs in lung cancer metastasis remain unclear., Methods: Single-cell sequencing analysis of lung cancer and normal tissues from public databases and from 14 patients who underwent surgery at Zhongshan Hospital was performed. In vitro co-culture experiments were performed to evaluate the effects of TAMs on lung cancer migration and invasion. Changes in the expression of IL-6, STAT3, C/EBPΒ, and EMT pathway were verified using RT-qPCR, western blotting, and immunofluorescence. Dual luciferase reporter assays and ChIP were used to reveal potential regulatory sites on the transcription factor sets. In addition, the effects of TAMs on lung cancer progression and metastasis were confirmed by in vivo models., Results: TAM infiltration is associated with tumor progression and poor prognosis. IL-6 secreted by TAMs can activate the JAK2/STAT3 pathway through autocrine secretion, and STAT3 acts as a transcription factor to activate the expression of C/EBPβ, which further promotes the transcription and expression of IL-6, forming positive feedback loops for IL6-STAT3-C/EBPβ-IL6 in TAMs. IL-6 secreted by TAMs promotes lung cancer progression and metastasis in vivo and in vitro by activating the EMT pathway, which can be attenuated by the use of JAK2/STAT3 pathway inhibitors or IL-6 monoclonal antibodies., Conclusions: Our data suggest that TAMs promote IL-6 expression by forming an IL6-STAT3-C/EBPβ-IL6 positive feedback loop. Released IL-6 can induce the EMT pathway in lung cancer to enhance migration, invasion, and metastasis. The use of IL-6-neutralizing antibody can partially counteract the promotion of LUAD by TAMs. A novel mechanism of macrophage-promoted tumor progression was revealed, and the IL6-STAT3-C/EBPβ-IL6 signaling cascade may be a potential therapeutic target against lung cancer., (© 2024. The Author(s).)

Metformin's effect on tumor treatment was complex, because it significantly reduced cancer cell proliferation in vitro, but made no difference in prognosis in several clinical cohorts. Our transcriptome sequencing results revealed that tumor-associated macrophage (TAM) infiltration significantly increased in active lung adenocarcinoma (LUAD) patients with long-term metformin use. We further identified that the tumor suppressive effect of metformin was more significant in mice after the depletion of macrophages, suggesting that TAMs might play an important role in metformin's effects in LUAD. Combining 10X Genomics single-cell sequencing of tumor samples, transcriptome sequencing of metformin-treated TAMs, and the ChIP-Seq data of the Encode database, we identified and validated that metformin significantly increased the expression and secretion of S100A9 of TAMs through AMPK-CEBP/β pathway. For the downstream, S100A9 binds to RAGE receptors on the surface of LUAD cells, and then activates the NF-κB pathway to promote EMT and progression of LUAD, counteracting the inhibitory effect of metformin on LUAD cells. In cell-derived xenograft models (CDX) and patient-derived xenograft models (PDX) models, our results showed that neutralizing antibodies targeting TAM-secreted S100A9 effectively enhanced the tumor suppressive effect of metformin in treating LUAD. Our results will enable us to better comprehend the complex role of metformin in LUAD, and advance its clinical application in cancer treatment., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ferroptosis is a type of regulated cell death characterized by iron accumulation and lipid peroxidation. The molecular mechanisms underlying ferroptosis regulation in non-small cell lung cancer (NSCLC) are poorly understood. In this study, we found that protein kinase A (PKA) inhibition enhanced ferroptosis susceptibility in NSCLC cells, as evidenced by reduced cell viability and increased lipid peroxidation. We further identified cAMP-responsive element protein 1 (CREB1), a transcription factor and a substrate of PKA, as a key regulator of ferroptosis. Knockdown of CREB1 sensitized NSCLC cells to ferroptosis inducers (FINs) and abolished the effects of PKA inhibitor and agonist, revealing the pivotal role of CREB1 in ferroptosis regulation. Using a high-throughput screening approach and subsequent validation by chromatin immunoprecipitation (ChIP) and dual-luciferase assays, we discovered that CREB1 transcriptionally activated stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the conversion of saturated fatty acids to monounsaturated fatty acids. SCD conferred ferroptosis resistance by decreasing the availability of polyunsaturated fatty acids for lipid peroxidation, and its overexpression rescued the effect of CREB1 knockdown on ferroptosis in vitro. Besides, CREB1 knockdown suppressed xenograft tumor growth in the presence of Imidazole Ketone Erastin (IKE), a potent FIN, and this effect was reversed by SCD. Finally, we showed that high expression of CREB1 was associated with poor prognosis in NSCLC patients from public datasets and our institution. Collectively, this study illustrates the effect of PKA/CREB1/SCD axis in regulating ferroptosis of NSCLC, targeting this pathway may provide new strategies for treating NSCLC patients., (© 2023. The Author(s).)

Purpose: The tumor immune microenvironment (TME) plays a vital role in tumorigenesis, progression, and treatment. Macrophages, as an important component of the tumor microenvironment, play an essential role in antitumor immunity and TME remodeling. In this study, we aimed to explore the different functions of different origins macrophages in TME and their value as potential predictive markers of prognosis and treatment., Methods: We performed single-cell analysis using 21 lung adenocarcinoma (LUAD), 12 normal, and four peripheral blood samples from our data and public databases. A prognostic prediction model was then constructed using 502 TCGA patients and explored the potential factors affecting prognosis. The model was validated using data from 4 different GEO datasets with 544 patients after integration., Results: According to the source of macrophages, we classified macrophages into alveolar macrophages (AMs) and interstitial macrophages (IMs). AMs mainly infiltrated in normal lung tissue and expressed proliferative, antigen-presenting, scavenger receptors genes, while IMs occupied the majority in TME and expressed anti-inflammatory, lipid metabolism-related genes. Trajectory analysis revealed that AMs rely on self-renew, whereas IMs originated from monocytes in the blood. Cell-to-cell communication showed that AMs interacted mainly with T cells through the MHC I/II signaling pathway, while IMs mostly interacted with tumor-associated fibrocytes and tumor cells. We then constructed a risk model based on macrophage infiltration and showed an excellent predictive power. We further revealed the possible reasons for its potential prognosis prediction by differential genes, immune cell infiltration, and mutational differences., Conclusion: In conclusion, we investigated the composition, expression differences, and phenotypic changes of macrophages from different origins in lung adenocarcinoma. In addition, we developed a prognostic prediction model based on different macrophage subtype infiltration, which can be used as a valid prognostic biomarker. New insights were provided into the role of macrophages in the prognosis and potential treatment of LUAD patients., (© 2023. Springer Nature Switzerland AG.)

Background: This study identified the expression and prognosis significance of secretory or membrane-associated proteins in KRAS lung adenocarcinoma (LUAD) and depicted the characteristics between the immune cell infiltration and the expression of these genes., Methods: Gene expression data of LUAD samples ( n = 563) were accessed from The Cancer Genome Atlas (TCGA). The expression of secretory or membrane-associated proteins was compared among the KRAS-mutant, wild-type, and normal groups, as well as the subgroup of the KRAS-mutant group. We identified the survival-related differentially expressed secretory or membrane-associated proteins and conducted the functional enrichment analysis. Then, the characterization and association between their expression and the 24 immune cell subsets were investigated. We also constructed a scoring model to predict KRAS mutation by LASSO and logistic regression analysis., Results: Secretory or membrane-associated genes with differential expression ( n = 74) across three groups (137 KRAS LUAD, 368 wild-type LUAD, and 58 normal groups) were identified, and the results of GO and KEGG indicated that they were strongly associated with immune cell infiltrations. Among them, ten genes were significantly related to the survival of patients with KRAS LUAD. The expression of IL37, KIF2, INSR, and AQP3 had the most significant correlations with immune cell infiltration. In addition, eight DEGs from the KRAS subgroups were highly correlated with immune infiltrations, especially TNFSF13B. Using LASSO-logistic regression, a KRAS mutation prediction model based on the 74 differentially expressed secretory or membrane-associated genes was built, and the accuracy was 0.79., Conclusion: The research investigated the relationship between the expression of KRAS-related secretory or membrane-associated proteins in LUAD patients with prognostic prediction and immune infiltration characterization. Our study demonstrated that secretory or membrane-associated genes were closely associated with the survival of KRAS LUAD patients and were strongly correlated to immune cell infiltration., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 Yunyi Bian et al.)

Purpose: Macrophages (MΦs) play a dual role in the promotion and suppression of lung adenocarcinoma (LUAD), the function of which is influenced by the metabolic status. The role of protein tyrosine phosphatase receptor type F (PTPRF) in cancer has not been elucidated, and its role in MΦs remains to be seen., Methods: The Seahorse XFe 96 Cell Flow Analyzer detected glucose metabolism in tumor cells and macrophages. The expressions of FSCN1, M-CSF, IL4, PTPRF and IGF1 in macrophages were detected by Western blotting and qRT-PCR. Binding of FSCN1 and IGF1R was detected by co-immunoprecipitation. The tumor status in animals was observed using the IVIS Lumina III imaging system., Results: We found that Fascin Actin-Bundling Protein 1 (FSCN1) activates the PI3K-AKT and JAK-STAT signaling pathways in LUAD cells via binding to IGF-1R, thereby promoting the secretion of cytokines such as IL4 and M-CSF. IL4 and M-CSF promote the expression of PTPRF in MΦs, leading to M2 polarization of MΦs by increasing glucose intake and lactate production. In return, M2-type MΦs act on LUAD cells by secreting cytokines such as IGF-1, CCL2, and IL10, which ultimately promote tumor progression. In vivo experiments proved that the knockdown of FSCN1 in A549 cells and PTPRF in MΦs greatly reduced LUAD proliferative and metastatic capacity, which was consistent with the in vitro findings., Conclusions: This study investigated the reprogramming effects of FSCN1 and PTPRF on inflammatory cytokines in the LUAD microenvironment, revealing potential mechanisms by which FSCN1 and PTPRF promote tumor progression and providing a new experimental basis for LUAD treatment., (© 2022. Springer Nature Switzerland AG.)

Background: Lung adenocarcinoma is one of the most commonly diagnosed malignancies worldwide. Macrophage plays crucial roles in the tumor microenvironment, but its autocrine network and communications with tumor cell are still unclear., Methods: We acquired single-cell RNA sequencing (scRNA-seq) ( n = 30) and bulk RNA sequencing ( n = 1480) samples of lung adenocarcinoma patients from previous literatures and publicly available databases. Various cell subtypes were identified, including macrophages. Differentially expressed ligand-receptor gene pairs were obtained to explore cell-to-cell communications between macrophages and tumor cells. Furthermore, a machine-learning predictive model based on ligand-receptor interactions was built and validated., Results: A total of 159,219 single cells (18,248 tumor cells and 29,520 macrophages) were selected in this study. We identified significantly correlated autocrine ligand-receptor gene pairs in tumor cells and macrophages, respectively. Furthermore, we explored the cell-to-cell communications between macrophages and tumor cells and detected significantly correlated ligand-receptor signaling pairs. We determined that some of the hub gene pairs were associated with patient prognosis and constructed a machine-learning model based on the intercellular interaction network., Conclusion: We revealed significant cell-to-cell communications (both autocrine and paracrine network) within macrophages and tumor cells in lung adenocarcinoma. Hub genes with prognostic significance in the network were also identified., Competing Interests: All authors have no conflicts of interest to declare., (Copyright © 2022 Xiaodong Yang et al.)

Background: The tumor immune microenvironment (TIME) played an important role in immunotherapy prognosis and treatment response. Immune cells constitute a large part of the tumor microenvironment and regulate tumor progression. Our research is dedicated to studying the infiltrating immune cell in lung adenocarcinoma (LUAD) and seeking potential targets., Methods: The scRNA-seq data were collected from our FDZSH and two public datasets. The code for cell-type mapping algorithms was downloaded from the CIBERSORTx portal. The bioinformatics data of LUAD patients could be approached from The Cancer Genome Atlas (TCGA) portal. Weighted gene coexpression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) analyses were performed to construct a risk model. TIMER2 and TIDE helped with the immune infiltration estimation, while PROGENy helped the cancer-related pathways' enrichment analysis. GSE31210 dataset and IMVigor ICB therapy cohort validated our findings as the external validation datasets., Results: We clustered the scRNA-seq dataset (integrating our FDZSH datasets and other public datasets) into 23 subpopulations. After curated cell annotation, we implemented Cibersort and WGCNA analysis to anchor the brown module and natural killer cell cluster1 due to the most relationship with tumor trait. The overlap of the brown module gene, natural killer cell signature, and DEGs of tumor and adjacent normal samples was screened by LASSO Cox regression. The obtained 5-gene risk model showed an excellent prognostic performance in the validation dataset. Furthermore, there was a correlation between risk score and tumor-infiltrating immune cells and tumor genomics abnormity. Patients with higher risk scores had a significantly lower immunotherapy response rate., Conclusion: Our observations implied that immune cells played a pivotal role in TIME and established a 5-gene signature (including IDH2, ADRB2, SFTPC, CCDC69, and CCND2) on the basement of nature killer markers targeted by WGCNA analysis. The significance of clinical outcome and immunotherapy response prediction was validated robustly., Competing Interests: The authors declare no competing interests in this work., (Copyright © 2022 Xing Jin et al.)

Objectives: Platinum-based chemotherapies are currently the first-line treatment of non-small cell lung cancer. This study will improve our understanding of the causes of resistance to cisplatin, especially in lung adenocarcinoma (LUAD) and provide a reference for therapeutic decisions in clinical practice., Methods: Cancer Cell Line Encyclopedia (CCLE), The Cancer Genome Atlas (TCGA) and Zhongshan hospital affiliated to Fudan University (zs-cohort) were used to identify the multi-omics differences related to platinum chemotherapy. Cisplatin-resistant mRNA and miRNA models were constructed by Logistic regression, classification and regression tree and C4.5 decision tree classification algorithm with previous feature selection performed via least absolute shrinkage and selection operator (LASSO). qRT-PCR and western-blotting of A549 and H358 cells, as well as single-cell Seq data of tumor samples were applied to verify the tendency of certain genes., Results: 661 cell lines were divided into three groups according to the IC50 value of cisplatin, and the top 1/3 (220) with a small IC50 value were defined as the sensitive group while the last 1/3 (220) were enrolled in the insensitive group. TP53 was the most common mutation in the insensitive group, in contrast to TTN in the sensitive group. 1348 mRNA, 80 miRNA, and 15 metabolites were differentially expressed between 2 groups (P < 0.05). According to the LASSO penalized logistic modeling, 6 of the 1348 mRNAs, FOXA2, BATF3, SIX1, HOXA1, ZBTB38, IRF5, were selected as the associated features with cisplatin resistance and for the contribution of predictive mRNA model (all of adjusted P-values < 0.001). Three of 6 (BATF3, IRF5, ZBTB38) genes were finally verified in cell level and patients in zs-cohort., Conclusions: Somatic mutations, mRNA expressions, miRNA expressions, metabolites and methylation were related to the resistance of cisplatin. The models we created could help in the prediction of the reaction and prognosis of patients given platinum-based chemotherapies., (© 2022. The Author(s).)

Background: Lung adenocarcinoma (LUAD), the major subtype of lung cancer, is among the leading cause of cancer-related death worldwide. Energy-related metabolic reprogramming metabolism is a hallmark of cancer shared by numerous cancer types, including LUAD. Nevertheless, the functional pathways and molecular mechanism by which FAM83A-AS1 acts in metabolic reprogramming in lung adenocarcinoma have not been fully elucidated. Methods: We used transwell, wound-healing scratch assay, and metabolic assays to explore the effect of FAM83A-AS1 in LUAD cell lines. Western blotting, Co-IP assays, and ubiquitination assays were used to detect the effects of FAM83A-AS1 on HIF-1α expression, degradation, and its binding to VHL. Moreover, an in vivo subcutaneous tumor formation assay was used to detect the effect of FAM83A-AS1 on LUAD. Results: Herein, we identified FAM83A-AS1 as a metabolism-related lncRNA, which was highly correlated with glycolysis, hypoxia, and OXPHOS pathways in LUAD patients using bioinformatics analysis. In addition, we uncovered that FAM83A-AS1 could promote the migration and invasion of LUAD cells, as well as influence the stemness of LUAD cells in vivo and vitro. Moreover, FAM83A-AS1 was shown to promote glycolysis in LUAD cell lines in vitro and in vivo , and was found to influence the expression of genes related to glucose metabolism. Besides, we revealed that FAM83A-AS1 could affect glycolysis by regulating HIF-1α degradation. Finally, we found that FAM83A-AS1 knockdown could inhibit tumor growth and suppress the expression of HIF-1α and glycolysis-related genes in vivo . Conclusion: Our study demonstrates that FAM83A-AS1 contributes to LUAD proliferation and stemness via the HIF-1α/glycolysis axis, making it a potential biomarker and therapeutic target in LUAD patients., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Most cancer cells have fundamentally different metabolic characteristics, particularly much higher glycolysis rates than normal tissues, which support the increased demand for biosynthesis and promote tumor progression. We found that transforming growth factor (TGF)-β plays a dual function in regulating glycolysis and cell proliferation in non-small cell lung cancer., Methods: We used the PET/MRI imaging system to observe the glucose metabolism of subcutaneous tumors in nude mice. Energy metabolism of non-small cell lung cancer cell lines detected by the Seahorse XFe96 cell outflow analyzer. Co-immunoprecipitation assays were used to detect the binding of Smads and HIF-1α. Western blotting and qRT-PCR were used to detect the regulatory effects of TGF-β and HIF-1α on c-MYC, PKM1/2, and cell cycle-related genes., Results: We discovered that TGF-β could inhibit glycolysis under normoxia while significantly promoting tumor cells' glycolysis under hypoxia in vitro and in vivo. The binding of hypoxia-inducible factor (HIF)-1α to the MH2 domain of phosphorylated Smad3 switched TGF-β function to glycolysis by changing Smad partners under hypoxia. The Smad-p107-E2F4/5 complex that initially inhibited c-Myc expression was transformed into a Smad-HIF-1α complex that promoted the expression of c-Myc. The increased expression of c-Myc promoted alternative splicing of PKM to PKM2, resulting in the metabolic reprogramming of tumor cells. In addition, the TGF-β/Smad signal lost its effect on cell cycle regulatory protein p15/p21. Furthermore, high expression of c-Myc inhibited p15/p21 and promoted the proliferation of tumor cells under hypoxia., Conclusions: Our results indicated that HIF-1α functions as a critical factor in the dual role of TGF-β in tumor cells, and may be used as a biomarker or therapeutic target for TGF-β mediated cancer progression., (© 2021. The Author(s).)

Background: Differences in genetics and microenvironment of LUAD patients with or without TP53 mutation were analyzed to illustrate the role of TP53 mutation within the carcinogenesis of LUAD, which will provide new concepts for the treatment of LUAD., Methods: In this study, we used genetics and clinical info from the TCGA database, including somatic mutations data, RNA-seq, miRNA-seq, and clinical data. More than one bioinformatics tools were used to analyze the unique genomic pattern of TP53-related LUAD., Results: According to TP53 gene mutation status, we divided the LUAD patients into two groups, including 265 in the mutant group (MU) and 295 in the wild-type group (WT). 787 significant somatic mutations were detected between the groups, including mutations in titin (TTN), type 2 ryanodine receptor (RYR2) and CUB and Sushi multiple domains 3(CSMD3), which were up-regulated in the MU. However, no significant survival difference was observed. At the RNA level, we obtained 923 significantly differentially expressed genes; in the MU, α-defensin 5(DEFA5), pregnancy-specific glycoprotein 5(PSG5) and neuropeptide Y(NPY) were the most up-regulated genes, glucose-6-phosphatase (G6PC), alpha-fetoprotein (AFP) and carry gametocidal (GC) were the most down-regulated genes. GSVA analysis revealed 30 significant pathways. Compared with the WT, the expression of 12 pathways in the mutant group was up-regulated, most of which pointed to cell division. There were significant differences in tumor immune infiltrating cells, such as Macrophages M1, T cells CD4 memory activated, Mast cells resting, and Dendritic cells resting. In terms of immune genes, a total of 35 immune-related genes were screened, of which VGF (VGF nerve growth factor inducible) and PGC (peroxisome proliferator-activated receptor gamma coactivator) were the most significant up-regulated and down-regulated genes, respectively. Research on the expression pattern of immunomodulators found that 9 immune checkpoint molecules and 6 immune costimulatory molecules were considerably wholly different between the two groups., Conclusions: Taking the mutant group as a reference, LUAD patients in the mutant group had significant differences in somatic mutations, mRNA-seq, miRNA-seq, immune infiltration, and immunomodulators, indicating that TP53 mutation plays a crucial role in the occurrence and development of LUAD., (© 2021. The Author(s).)

Background: Lung adenocarcinoma (LUAD) patients with different American Joint Committee on Cancer stages have different overall 5-year survival rates. The tumor microenvironment (TME) and intra-tumor heterogeneity (ITH) have been shown to play a crucial role in the occurrence and development of tumors. However, the TME and ITH in different lesions of LUAD have not been extensively explored., Methods: We present a 204,157-cell catalog of the TME transcriptome in 29 lung samples to systematically explore the TME and ITH in the different stages of LUAD. Traditional RNA sequencing data and complete clinical information were downloaded from publicly available databases., Results: Based on these high-quality cells, we constructed a single-cell network underlying cellular and molecular features of normal lung, early LUAD, and advanced LUAD cells. In contrast with early malignant cells, we noticed that advanced malignant cells had a remarkably more complex TME and higher ITH level. We also found that compared with other immune cells, more differences in CD8+/CTL T cells, regulatory T cells, and follicular B cells were evident between early and advanced LUAD. Additionally, cell-cell communication analyses, revealed great diversity between different lesions of LUAD at the single-cell level. Flow cytometry and qRT-PCR were used to validate our results., Conclusion: Our results revealed the cellular diversity and molecular complexity of cell lineages in different stages of LUAD. We believe our research, which serves as a basic framework and valuable resource, can facilitate exploration of the pathogenesis of LUAD and identify novel therapeutic targets in the future., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

The tumor microenvironment (TME) plays an essential role in the occurrence and progression of malignancy. The potential prognostic TME-related biomarkers of lung adenocarcinoma (LUAD) remained unclear, which were investigated in this research. The RNA-sequencing profiles and corresponding clinical parameters were extracted from TCGA and GEO databases, based on which the stromal and immune scores were calculated through the ESTIMATE algorithm. Overlapping differentially expressed genes between stromal and immune score group were analyzed by the LASSO and Random Forrest algorithms and validated in cases from our center. And a prognostic 8-gene signature was constructed using Cox regression. The infiltration of 22 hematopoietic cell phenotypes was assessed by the CIBERSORT algorithms. We found that female, elder patients, and solid predominant subtype had obviously higher stromal and immune scores. And patients with early stage LUAD received a prominently higher immune score. A high stromal or immune score meant a good prognosis. Subsequently, eight TME-related prognostic genes (ATAD5, CYP4F3, CYP4F12, ESPNL, FXYD2, GPX2, NLGN4Y, and SERPINC1) were identified by both LASSO regression and Radom Forest algorithms. High 8-gene signature group exhibited worse overall survival. Furthermore, B cell naïve, plasma cells, T cell follicular helper, and macrophages M1 were prominently more in high signature group. Nevertheless, fewer T cells CD4 memory resting, monocytes, and dendritic cell resting were identified in the high signature group. The composition of the tumor microenvironment significantly affected the prognosis of LUAD patients. We provided a new strategy for the exploration of prognostic TME-related biomarkers and immunotherapy.

Background: Immunotherapy has achieved excellent results in patients with lung squamous cell carcinoma. However, in which population it can exert the greatest effect is still unknown. Some studies have suggested that its effect is related to the expression level of PD1. Analyzing the relationship between PD1 expression level and genetic differences in lung squamous cell carcinoma patients will be helpful in understanding the underlying causes of this immunotherapy effect and provide a reference for clinical practice., Methods: In this study, we used RNA-seq, miRNA-seq, methylation array, mutation profiles, and copy number variation data from the TCGA database and RNA-seq data from the GEO database to analyze the distinctive genomic patterns associated with PD1 and PDL1 expression. RNA-seq data from 44 LUSC patients who underwent surgery at Zhongshan Hospital were also included in the study., Results: After grouping LUSC patients according to the expression levels of PD1 and PDL1, we found no significant difference in survival between the two groups. However, 178 genes, including IL-21, KLRC3, and KLRC4, were significantly upregulated in both the TCGA and GEO databases in the high expression group, and there was a precise correlation between gene expression and epigenetic changes in the two groups. At the same time, the overall level of somatic mutations was not significantly different between the two groups. It is worth noting that the gene enrichment results showed that the differential pathways were mainly enriched in immune regulation, especially T cell-related immune activities., Conclusion: We found that the differences in gene expression between the two groups were related to immunity, which may affect the effectiveness of immunotherapy. We hope our results can provide a reference for further research and help in finding other targets to improve the efficacy of immunotherapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Lung adenocarcinoma (LUAD) is the leading cause of cancer-related deaths worldwide. Traditional RNA sequencing data fails to detect the exact cellular and molecular changes in tumor cells as they make up only a small proportion of tumor tissue. 10× genomics single-cell RNA sequencing (10× scRNA-seq) and gene expression data of LUAD patients was obtained from the Department of Thoracic Surgery, Zhongshan Hospital, Fudan University, ArrayExpress, TCGA, and GEO databases. Differentially expressed genes (DEGs) were identified in LUAD and alveolar cells (DEGs-scRNA-cancer&#95;cell), tumor- and normal tissue-derived cells (DEGs-scRNA-sample), and normal and LUAD patients (DEGs-Bulk). Flow cytometry and qRT-PCR were performed to validate the significantly differentially expressed ligand-receptor pairs. We selected 159,219 cells and 594 samples in the scRNA-seq data and traditional RNA sequencing, respectively. A total of 1042 DEGs-scRNA-cancer&#95;cell, 788 DEGs-scRNA-sample, and 2510 DEGs-Bulk were identified in this study. We also identified 57 DEGs that were only detected in DEGs-scRNA-cancer&#95;cell (only-DEGs-scRNA-cancer&#95;cell). To explore the relationship between only-DEGs-scRNA-cancer&#95;cell and survival in LUAD, 14 and 22 only-DEGs-scRNA-cancer&#95;cell, which were closely related with survival in TCGA and GEO cohorts were identified. Functional enrichment analyses showed these DEGs-scRNA-cancer&#95;cells were mainly related to cell proliferation and immunoregulation. Our study detected and compared DEGs at different levels and revealed genes that may regulate tumor development. Our results provide a potential new protocol to determine the contribution of DEGs to cancer progression and to help identify potential therapeutic targets.

Background: Accounting for tumor heterogeneity, cancer stem cells (CSC) are involved in tumor metastasis, relapse, and drug resistance. Genes regulating CSC characteristics in lung adenocarcinoma (LUAD) were explored and validated in this study., Methods: The mRNA stemness index (mRNAsi) of more than 500 LUAD cases from The Cancer Genome Atlas database were calculated using a one-class logistic regression machine learning algorithm based on the mRNA expression of pluripotent stem cells and their differentiated progeny. mRNAsi-related key genes were identified by weighted correlation network analysis. The expression levels and prognostic roles of key genes were analyzed in Oncomine, PrognoScan, and Kaplan-Meier plotter databases, and validated using data from our center., Results: The mRNAsi was significantly higher in LUAD compared with normal lung tissues. LUAD patients of advanced stage exhibited a higher mRNAsi and worse overall survival (OS). Eight key genes were identified: heat shock 70 kDa protein 4 (HSPA4), cell division cycle associated 7 (CDCA7), cell division cycle 20 (CDC20), cyclin-dependent kinase 1 (CDK1), CAP-GLY domain containing linker protein 1 (CLIP1), cyclin B1 (CCNB1), H2A histone family, member X (H2AFX), and Bloom syndrome, RecQ helicase-like (BLM). These genes were differentially expressed in various types of malignancies and validated in the LUAD cases. LUAD patients with low expression of CDC20, CDK1, CCNB1, H2AFX, or BLM had a significantly better OS, whereas OS was reduced for patients with low expression of CLIP1. In addition, the expression of CDCA7 did not significantly impact the OS of LUAD patients. The protein-protein interaction networks evaluated by STRING demonstrated strong relationships between these key genes, which were validated in our cases., Conclusions: The mRNAsi was significantly higher in LUAD compared with normal samples. Eight mRNAsi-related key genes were associated with prognosis and the cell cycle, and were strongly correlated with each other and differentially expressed in tumor and normal samples. We provide a new strategy for exploring stemness-related genes in LUAD cases.

Purpose : Lung adenocarcinoma (LUAD) is the leading cause of cancer-related deaths worldwide. Although tumor cell-T cell interactions are known to play a fundamental role in promoting tumor progression, these interactions have not been explored in LUAD. Methods : The 10x genomics single-cell RNA sequencing (scRNA-seq) and gene expression data of LUAD patients were obtained from ArrayExpress, TCGA, and GEO databases. scRNA-seq data were analyzed and infiltrating tumor cells, epithelial cells, and T cells were identified in the tumor microenvironment. Differentially expressed ligand-receptor pairs were identified in tumor cells/normal epithelial cells and tumor T cells/non-tumor T cells based on corresponding scRNA-seq and gene expression data, respectively. These important interactions inside/across cancer cells and T cells in LUAD were systematically analyzed. Furthermore, a valid prognostic machine-learning model based on ligand-receptor interactions was built to predict the prognosis of LUAD patients. Flow cytometry and qRT-PCR were performed to validate the significantly differently expressed ligand-receptor pairs. Results : Overall, 39,692 cells in scRNA-seq data were included in our study after quality filtering. A total of 65 ligand-receptor pairs (17 upregulated and 48 downregulated), including LAMB1-ITGB1, CD70-CD27, and HLA-B-LILRB2, and 96 ligand-receptor pairs (41 upregulated and 55 downregulated), including CCL5-CCR5, SELPLG-ITGB2, and CXCL13-CXCR5, were identified in LUAD cancer cells and T cells, respectively. To explore the crosstalk between cancer cells and T cells, 114 ligand-receptor pairs, including 11 ligand-receptor pair genes that could significantly affect survival outcomes, were identified in our research. A machine-learning model was established to accurately predict the prognosis of LUAD patients and ITGB4, CXCR5, and MET were found to play an important role in prognosis in our model. Flow cytometry and qRT-PCR analyses indicated the reliability of our study. Conclusion : Our study revealed functionally significant interactions within and between cancer cells and T cells. We believe these observations will improve our understanding of potential mechanisms of tumor microenvironment contributions to cancer progression and help identify potential targets for immunotherapy in the future., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)