Single-cell analyis of tobacco-associated lung adenocarcinoma development
Ontology highlight
ABSTRACT: Understanding cellular processes underlying early lung adenocarcinoma (LUAD) development is needed to devise intervention strategies. Here, we performed single-cell RNA sequencing (scRNA-seq) of mouse lungs from Gprc5a-/- mice during lung tumor development. We coupled scRNA-seq analysis with spatial transcriptomics of tumor-bearing lungs.
Project description:This study aims to elucidate the impact of gut microbiota alterations on tumorigenesis and immune response in lung adenocarcinoma (LUAD). Using Gprc5a-/- mice as a model, we performed fecal microbiota transfer (FMT) from Gprc5a-/- and Gprc5a-/-; Lcn2-/- donors to investigate the role of gut microbiome changes in modulating tumor growth and the immune microenvironment. Single-cell RNA sequencing (scRNA-seq) was conducted on colonic lamina propria and subcutaneous tumor tissues. Our findings demonstrate that gut microbiota from Lcn2-deficient mice promotes systemic inflammation and immunosuppression, enhancing tumor progression. This study provides insights into the microbiome's influence on LUAD and potential therapeutic strategies targeting microbiome-related pathways.
Project description:Mechanisms underlying cancer stemness in Kras-mutant lung adenocarcinoma (KM-LUAD) are poorly understood. We previously found that mice with knockout of Gprc5a develop LUADs with somatic Kras mutations. We also previously derived Gprc5a-/- KM-LUAD cells (MDA-F471 cells) from a mouse exposed to the tobacco-specific carcinogen NNK. We also derived cancer stem cells (CSCs; grown and cultured as spheres in 3D cultures) from MDA-F471 cells. To better understand the biology of these CSCs, we compared the transcriptomes of MDA-F471 cells and their CSC counterparts by bulk RNA-sequencing.
Project description:Smoking perpetuates in cytologically-normal airways a molecular “field of injury” that is pertinent to lung cancer and early detection. The evolution of airway field changes prior to lung cancer onset is poorly understood largely due to the long latency of lung malignancy in smokers. Here we studied airway expression changes prior to lung cancer onset in mice with knockout of the Gprc5a gene and tobacco carcinogen (nicotine-specific nitrosamine ketone; NNK) exposure and that develop the most common type of lung cancer, lung adenocarcinoma (LUAD). Cytologically-normal airway epithelial brushings were collected before exposure and at multiple times following NNK exposure until time of LUAD development and then analyzed by RNA-sequencing (RNA-Seq).
Project description:We have shown that Gprc5a-/- mice form Kras-mutant lung tumors spontaneously which is accelerated by tobacco carcinogen (NNK) exposure. We found in these mice that Lcn2 was distinctively up-regulated along the spectrum of Kras-mutant lung cancer development. To understand the role of Lcn2 in lung cancer pathogenesis, we generated Gprc5a-/-/Lcn2-/- mice and found that these animals have increased lung tumor devleopment following NNK compared to Gprc5a-/- animals with intact Lcn2. To understand these effects, we performed RNA-sequencing (RNA-Seq) of lung tissues from Gprc5a-/-/Lcn2-/- and Gprc5a-/- mice at baseline (prior to NNK exposure) and of tumor-bearing lungs from both groups at seven months post-NNK exposure.
Project description:Brain metastases are common in lung adenocarcinoma (LUAD) patients, and by far, the metastasis mechanisms are not fully understood. We performed a comprehensive single-cell level transcriptomic analysis on one LUAD patient with CTC, primary tumor tissue and metastatic tumor tissue using scRNA-seq approach to identify metastasis related biomarkers. Further scRNA-seq were performed on 7 patients to validate the cancer metastatic hallmark. with single cells collected from either metastatic or primary LUAD tissues. we obtained a more comprehensive picture over lung cancer metastasis in the single-cell level, giving a new perspective to the role of RAC1 in the LUAD brain metastasis, and related pathways to participate in the metastasis process.
Project description:Gprc5a is a lung tumor suppressor gene. Gprc5a-knockout (ko) mice can develop spontaneous lung cancer and Gprc5a-ko mouse model is relevant to human lung cancer. Thus, exploration of the mechanisms underlying lung tumorigenesis in Gprc5a-ko mice would be very helpful for revealing those in human lung cancer. We used microarrays to detail the global gene expression profile that underlies oncogenesis by Gprc5a-knockout gene deletion in mouse tracheal epithelial cells. Wild type and gene-knockout mouse tracheal epithelial cells that were divided into two groups were used for RNA extraction.
Project description:Increasing the understanding of the impact of changes in oncogenes and tumor suppressor genes is essential for improving the management of lung cancer. Recently, we identified a new mouse lung-specific tumor suppressor - the G-protein coupled receptor 5A (Gprc5a). We sought to understand the molecular consequences of Gprc5a loss and towards this we performed microarray analysis of the transcriptomes of lung epithelial cells cultured from normal tracheas of Gprc5a knockout and wild-type mice to define a loss-of-Gprc5a gene signature. Gprc5a wild type cells (WT-NLE) and Gprc5a knockout cells (NULL-NLE) were isolated and cultured from trachea of three week old Gprc5a wild type and knockout mice, respectively. Following RNA extraction and purification, the transcriptome of the Gprc5a wild type and knockout cells were analyzed by microarray analysis using the Affymetrix MG-430 2.0 murine array platform.
Project description:Long noncoding RNAs (lncRNAs) are known to regulate the development and progression of various cancers, however, few lncRNAs have been well characterized in lung adenocarcinoma (LUAD). Understanding the expression profile of lncRNAs and protein-coding genes is critical to develop new diagnosis and treatment strategies for LUAD and improving the prognosis of diagnosed patients. Five female LUAD patients with no smoking history were selected to profile lncRNA and protein-coding gene expression with microarrays. Paired tumor tissues and adjacent nontumor tissues were collected and confirmed by pathologists.
Project description:Lung cancer is the most frequent cancer-related cause of death, and adenocarcinoma (LUAD) is the most frequent type. Despite the recent success of immunotherapies, survival of lung cancer patients has not significantly improved in the last decades. New therapies are necessary. We have previously identified sodium-glucose transporter 2 (SGLT2) as the major responsible for glucose uptake in LUAD, and we have showed that treatment with SGLT2 inhibitors significantly delays LUAD development and prolongs survival in murine models. However, our data shows that SGLT2 inhibitors also induce de-differentiation of LUAD cells, leading to a more aggressive phenotype and increased resistance to cisplatin. Glucose deprivation causes reduced αKG levels, leading to reduced activity of αKG-dependent histone demethylases and consequent histone hypermethylation. Supplementation of αKG or inhibition of the histone methyltransferase EZH2 reverse this phenotype, suggesting that this de-differentiated phenotype depends on insufficiency of αKG-dependent histone demethylases and unbalanced EZH2 activity. Consistently, double treatment with an SGLT2 inhibitor and an EZH2 inhibitor significantly reduces the tumor burden in a genetically engineered murine model of LUAD. We further characterized the effect of low glucose-induced tumor de-differentiation, identifying stabilization of hypoxia inducible factor 1α (HIF1α) as a major pathway responsible for the acquisition of a more aggressive phenotype following glucose deprivation. Finally, we identified an HIF1α-dependent transcriptional signature with prognostic significance in human LUAD. Our studies further our knowledge of the relationship between glucose metabolism and cell differentiation in cancer, characterizing the epigenetic adaptation of cancer cells to nutrient deprivation and identifying novel targets to prevent the development of resistance to metabolic therapies.
Project description:Circulating tumor cells (CTCs) represent the molecular characteristics of tumor sites and travel in the blood for seeding distant metastases. "EpCAM+/pan-cytokeratin (CK)+/CD45-/DAPI+" has been widely accepted as a CTC definition, especially in breast cancer, prostate cancer and colorectal cancer. However, reports on CTC detection in non-small cell lung cancer are limited due to a lack of efficient CTC marker. We describe hexokinase 2 (HK2) that assays elevated glycolysis of cancer cells, called Warburg effect, as a new marker for CTC detection in lung adenocarcinoma (LUAD), especially the CK negative CTCs. Single-cell sequencing was used to confirm the malignancy of putative CTCs by detecting genome-wide copy number alternations characteristic of malignant cells. We employed this marker in a variety of liquid biopsies from LUAD patients, including peripheral blood, pleural effusion and cerebrospinal fluid.