Tumor Microenvironment Of Lung Adenocarcinoma Research Articles

Knockdown of heat shock protein family D member 1 (HSPD1) in lung cancer cell altered secretome profile and cancer-associated fibroblast induction

The crosstalk between lung cancer cells and cancer-associated fibroblast (CAF) is pivotal in cancer progression. Heat shock protein family D member 1 (HSPD1) is a potential prognostic biomarker associated with the tumor microenvironment in lung adenocarcinoma (LUAD). However, the role of HSPD1 in CAF activation remains unclear. This study established stable HSPD1-knockdown A549 lung cancer cells using a lentivirus-mediated shRNA transduction. A targeted label-free proteomic analysis identified six significantly altered secretory proteins in the shHSPD1-A549 secretome compared to shControl-A549. Functional enrichment analysis highlighted their involvement in cell-to-cell communication and immune responses within the tumor microenvironment. Additionally, most altered proteins exhibited positive correlations and significant prognostic impacts on LUAD patient survival. Investigations on the effects of lung cancer secretomes on lung fibroblast WI-38 cells revealed that the shControl-A549 secretome stimulated fibroblast proliferation, migration, and CAF marker expression. These effects were reversed upon the knockdown of HSPD1 in A549 cells. Altogether, our findings illustrate the role of HSPD1 in mediating CAF induction through secretory proteins, potentially contributing to the progression and aggressiveness of lung cancer.

Analysis of cancer-associated fibroblasts related genes identifies COL11A1 associated with lung adenocarcinoma prognosis

BackgroundThe treatment of lung adenocarcinoma is difficult due to the limited therapeutic options. Cancer-associated fibroblasts play an important role in the development of cancers. This study aimed to identify a promising molecular target associated with cancer-associated fibroblasts for the treatment of lung adenocarcinoma.MethodsThe Cancer Genome Atlas lung adenocarcinoma dataset was used to screen hub genes associated with cancer-associated fibroblasts via the EPIC algorithm and Weighted Gene Co-expression Network Analysis. Multiple databases were used together with our data to verify the differential expression and survival of COL11A1. Functional enrichment analysis and the single-cell TISCH database were used to elucidate the mechanisms underlying COL11A1 expression. The correlation between COL11A1 and immune checkpoint genes in human cancers was also evaluated.ResultsUsing the EPIC algorithm and Weighted Gene Co-expression Network Analysis, 13 hub genes associated with cancer-associated fibroblasts in lung adenocarcinoma were screened. Using the GEPIA database, Kaplan-Meier Plotter database, GSE72094, GSE75037, GSE32863, and our immunohistochemistry experiment data, we confirmed that COL11A1 overexpresses in lung adenocarcinoma and that high expression of COL11A1 is associated with a poor prognosis. COL11A1 has a genetic alteration frequency of 22% in patients with lung adenocarcinoma. COL11A1 is involved in the extracellular matrix activities of lung adenocarcinoma. Using the TISCH database, we found that COL11A1 is mainly expressed by cancer-associated fibroblasts in the tumor microenvironment rather than by lung adenocarcinoma cells. Finally, we found that COL11A1 is positively correlated with HAVCR2(TIM3), CD274 (PD-L1), CTLA4, and LAG3 in lung adenocarcinoma.ConclusionCOL11A1 may be expressed and secreted by cancer-associated fibroblasts, and a high expression of COL11A1 may result in T cell exhaustion in the tumor microenvironment of lung adenocarcinoma. COL11A1 may serve as an attractive biomarker to provide new insights into cancer therapeutics.

Identifying an immunogenic cell death-related gene signature contributes to predicting prognosis, immunotherapy efficacy, and tumor microenvironment of lung adenocarcinoma.

Immunogenic cell death (ICD) is a regulated form of cell death that triggers an adaptive immune response. The objective of this study was to investigate the correlation between ICD-related genes (ICDGs) and the prognosis and the immune microenvironment of patients with lung adenocarcinoma (LUAD). ICD-associated molecular subtypes were identified through consensus clustering. Subsequently, a prognostic risk model comprising 5 ICDGs was constructed using Lasso-Cox regression in the TCGA training cohort and further tested in the GEO cohort. Enriched pathways among the subtypes were analyzed using GO, KEGG, and GSVA. Furthermore, the immune microenvironment was assessed using ESTIMATE, CIBERSORT, and ssGSEA analyses. Consensus clustering divided LUAD patients into three ICDG subtypes with significant differences in prognosis and the immune microenvironment. A prognostic risk model was constructed based on 5 ICDGs and it was used to classify the patients into two risk groups; the high-risk group had poorer prognosis and an immunosuppressive microenvironment characterized by low immune score, low immune status, high abundance of immunosuppressive cells, and high expression of tumor purity. Cox regression, ROC curve analysis, and a nomogram indicated that the risk model was an independent prognostic factor. The five hub genes were verified by TCGA database, cell sublocalization immunofluorescence analysis, IHC images and qRT-PCR, which were consistent with bioinformatics analysis. The molecular subtypes and a risk model based on ICDGs proposed in our study are both promising prognostic classifications in LUAD, which may provide novel insights for developing accurate targeted cancer therapies.

Identification of CNKSR1 as a biomarker for “cold” tumor microenvironment in lung adenocarcinoma: An integrative analysis based on a novel workflow

BackgroundTherapies targeting PD1/PD-L1 pathway have revolutionized the treatment of lung cancer. However, anti-PD1/PD-L1 therapies have proven beneficial for only a select group of lung adenocarcinoma (LUAD) patients and generally do not work for immuno-cold tumors characterized by a lack of immune cell infiltration. Identifying novel biomarkers is vital to broad therapeutic options for LUAD patients with no response to anti-PD1/PD-L1 immunotherapies. MethodsOur study has developed a novel strategy to identify a promising biomarker that addresses the limitations of anti-PD1/PD-L1 immunotherapy in treating immunological cold tumors. We exacted LUAD RNA-seq data from the Cancer Genome Atlas database (TCGA). Using several machine learning methods, we identified the candidate biomarker. Based on the expression level of PD-L1 and the identified biomarker, samples were categorized into four groups. We further used ESTIMATE, ssGSEA, and CIBERSORT algorithms to calculate the immune infiltration level of each group. The results were validated in three independent bulk datasets and one scRNA-seq dataset. Immunohistochemistry (IHC) assessments were performed in clinical samples to further evaluate the coexpression of CNKSR1 and PD-L1, and to compare CD8 + T cell infiltration among groups. ResultsAfter comprehensive analyses, CNKSR1 was identified as a novel promising biomarker for immuno-cold LUAD. CNKSR1 mRNA expression levels exhibited a negative correlation with both PD-L1 mRNA expression and the extent of immune cell infiltration in LUAD. Besides, in contrast to the significant association between the expression of PD-L1 and the majority of other well-established or widely studied immune checkpoint molecules, a mutually exclusive expression pattern is observed between CNKSR1 and these molecules. The aforementioned results were consistent in validation datasets. The prognostic model built based on the CNKSR1 coexpression module also showed robust predictive performance. Additionally, IHC assessments have confirmed that the coexpression of CNKSR1 and PD-L1 is rare in LUAD samples. Notably, LUADs in the high-CNKSR1 group, characterized by high CNKSR1 but low PD- L1 expression, demonstrated reduced infiltration of CD8+ T cells. ConclusionsIn summary, CNKSR1 emerges as a promising biomarker for immune-cold LUADs, and the study into CNKSR1 modulating T-cell infiltration may lead to the identification of compensatory molecules to enhance the effectiveness of current immunotherapy for LUAD.

Heterogeneity of Lipid Metabolism and its Clinical and Immune Correlation in Lung Adenocarcinoma.

The role of lipid metabolism in lung adenocarcinoma (LUAD) is not completely researched. Lipid metabolism reprogramming is a characteristic of malignancies and contributes to carcinogenesis and progression. The transcriptome and scRNA- seq data and clinical information were downloaded from the public databases. Lipid metabolism pathways were collected from the MSigDB database, and molecular subtypes were classified based on lipid metabolism features via consensus clustering. The bidirectional crosstalk between immune cells and malignant cells was analyzed. Differences in lipid metabolism at the single-cell level and their correlation with the tumor microenvironment (TME) were also studied. LUAD patients were classified into two subtypes, showing distinct mutation and lipid metabolism features based on lipid metabolism characteristics. Meanwhile, significant differences in the overall survival, clinical characteristics, and immune landscape were observed between the two subtypes. We also found that clust1 had higher oxidative stress status. There were 116 differentially expressed genes between the two subtypes, which were significantly associated with cell cycle progression. We identified 4001 immune cells, including 483 malignant cells and 3518 normal cells, and found active intercellular communication and significant differences in lipid metabolism characteristics between the malignant cells and normal cells. Furthermore, several lipid metabolism pathways were found to be associated with TME factors, including hypoxia and angiogenesis. The current findings indicated that lipid metabolism was involved in the development and cellular heterogeneity of LUAD and revealed widespread reprogramming across multiple cellular elements in the TME of LUAD. This characterization improved the current understanding of tumor biology and enabled the identification of novel targets for immunotherapy.

Abstract 171: Chemotherapeutic agent, cisplatin, influences the proportion of TREM2 (+) M2 macrophages in the TME of LUAD

Abstract Background: Triggering receptor expressed on myeloid cells (TREM2) serves as an immune signaling hub that senses and interacts with a wide range of ligands arising from damaged tissues in pathologic conditions. Despite TREM2’s diverse role in casting immune suppressive environment, the effects of chemotherapy on tumor microenvironment (TME), TREM2 and their interactions are not well established. In this study, we aimed to investigate the characteristics of TREM2 (+) Mφ within the lung TME (Tu), focusing on the impact of chemotherapy on the immune dynamics of lung adenocarcinoma (LUAD). Method: Changes in the number and distribution of Mφ subclusters in normal appearing lung tissue (NL) and Tu were examined using open-source single cell RNA sequencing (scRNA-seq) datasets and immunofluorescent staining of lung cancer-induced Kras-G12D mouse tissue. Clinical measures affecting the expression of TREM2 were explored using the TCGA-LUAD and -LUSC datasets, and the effects of anticancer drugs and the apoptotic cells on Mφ were observed by differentiating THP1 cells into M0, M1, and M2. Results: scRNA-seq analysis on LUAD datasets showed that Mφ were the second most abundant cell population that constitutes the lung TME, and the proportion of Mφ in the Tu decreases compared to NL. Among the Mφ subclusters in Tu, a subcluster which specifically increased in Tu shows enrichment of apoptotic cell clearance gene sets, including TREM2. In lung cancer induced Kras-G12D mouse, compared to NL the Tu has an increase in M2 proportion, showing the characteristics of interstitial Mφ in addition with overexpression of TREM2 and enrichment of monocyte chemotaxis gene set enrichment. Among TCGA-LUAD, the high TMB group has low TREM2 expression, and the proportion of TREM2 (+) M2 in the Tu decreases in cisplatin-treated Kras-G12D mouse Tu, suggesting the expression of TREM2 in M2 is suppressed by either cisplatin treatment itself or active immunity in Tu. Additionally, we are going to observe the effect on TREM2 expression by adding cisplatin treated A549-GFP cells or cisplatin alone, to the THP-1 cells that were differentiated into M0, M1, or M2 state. Conclusion: In addition to changes in the lung immune environment due to the tumor, anticancer treatment changes in the characteristics of M2 in TME, in terms of decrease in TREM2 (+) Mφ fraction. Considering that TREM2 plays a central inhibitory role in the tumor immune environment, the effect of anticancer chemotherapeutic agents should be considered in the development TREM2 targeting agents. Citation Format: Yoon Jin Cha, Min Kyung Park, Yoon Soo Chang. Chemotherapeutic agent, cisplatin, influences the proportion of TREM2 (+) M2 macrophages in the TME of LUAD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 171.

GPX8+ cancer-associated fibroblast, as a cancer-promoting factor in lung adenocarcinoma, is related to the immunosuppressive microenvironment

BackgroundCancer-associated fibroblasts (CAFs) play a crucial role in the tumor microenvironment of lung adenocarcinoma (LUAD) and are often associated with poorer clinical outcomes. This study aimed to screen for CAF-specific genes that could serve as promising therapeutic targets for LUAD.MethodsWe established a single-cell transcriptional profile of LUAD, focusing on genetic changes in fibroblasts. Next, we identified key genes associated with fibroblasts through weighted gene co-expression network analysis (WGCNA) and univariate Cox analysis. Then, we evaluated the relationship between glutathione peroxidase 8 (GPX8) and clinical features in multiple independent LUAD cohorts. Furthermore, we analyzed immune infiltration to shed light on the relationship between GPX8 immune microenvironment remodeling. For clinical treatment, we used the tumor immune dysfunction and exclusion (TIDE) algorithm to assess the immunotherapy prediction efficiency of GPX8. After that, we screened potential therapeutic drugs for LUAD by the connectivity map (cMAP). Finally, we conducted a cell trajectory analysis of GPX8+ CAFs to show their unique function.ResultsFibroblasts were found to be enriched in tumor tissues. Then we identified GPX8 as a key gene associated with CAFs through comprehensive bioinformatics analysis. Further analysis across multiple LUAD cohorts demonstrated the relationship between GPX8 and poor prognosis. Additionally, we found that GPX8 played a role in inducing the formation of an immunosuppressive microenvironment. The TIDE method indicated that patients with low GPX8 expression were more likely to be responsive to immunotherapy. Using the cMAP, we identified beta-CCP as a potential drug-related to GPX8. Finally, cell trajectory analysis provided insights into the dynamic process of GPX8+ CAFs formation.ConclusionsThis study elucidates the association between GPX8+ CAFs and poor prognosis, as well as the induction of immunosuppressive formation in LUAD. These findings suggest that targeting GPX8+ CAFs could potentially serve as a therapeutic strategy for the treatment of LUAD.

Abstract C116: Oncogenic Kras signaling shapes the tumor microenvironment in lung adenocarcinoma

Abstract Despite advancements in targeted therapy and immunotherapy, lung cancer remains the leading cause of cancer-related deaths in both men and women worldwide, with an estimated 2.2 million deaths each year. At 85%, non-small cell lung cancer (NSCLC) is by far the most common subtype, comprising adenocarcinomas and lung squamous cell carcinoma. Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR) and anaplastic lymphoma receptor tyrosine kinase (ALK) genes are common, with the worst overall survival in KRAS mutant adenocarcinoma patients. About 40% of lung tumors with KRAS mutations also harbor mutations in tumor protein p53 gene (TP53), a tumor suppressor gene. KrasG12C and KrasG12D are both prevalent mutations in lung cancer. Sotorasib, a KrasG12C inhibitor, was recently approved for treatment of NSCLC. However, only 37% of patients have an objective response to Sotorasib, with a median duration of just 11 months. The KrasG12D inhibitor MRTX1133 is entering clinical trials; elucidating the mechanism of action and potential resistance is imperative to design combination treatment approaches. While cell-intrinsic mechanisms of resistance in epithelial oncogenic KRASG12D (Kras*) are well studied, our group and others have shown that KRASG12D also extrinsically regulates the tumor microenvironment (TME) in pancreatic cancer. Whether and how this oncogene regulates the TME of lung cancer is less understood. To study the crosstalk between epithelial lung cells expressing oncogenic KRAS and the surrounding TME, we have established a triple transgenic murine model of Kras*-driven NSCLC, wherein both lung epithelial-specific expression of Kras* and expression of mutant TP53R172H (Tp53*) can be controlled in an inducible and reversible manner. Our preliminary data show that expression of Kras* drives tumor growth, while Kras* depletion results in regression, even in the presence of Tp53*. Here we interrogate how Kras* modulates the TME in lung adenocarcinomas. Harnessing scRNA sequencing, immunohistochemistry, and multiple biochemical approaches, we will provide insight into changes in stromal and immune contexture upon Kras* depletion in mice. Citation Format: Rachael Baliira, Emily Lasse Opsahl, Ivana Barravecchia, Marina Pasca di Magliano, Stefanie Galban. Oncogenic Kras signaling shapes the tumor microenvironment in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C116.

Identification and prognostic biomarkers among ZDHHC4/12/18/24, and APT2 in lung adenocarcinoma

S-palmitoylases and S-depalmitoylases are differentially expressed in various cancers and several malignant tumors and show a strong prognostic ability. Notwithstanding, the potential clinical impact of S-palmitoylases and S-depalmitoylases, particularly in the prognosis and progression of lung adenocarcinoma (LUAD), has not been clarified. Expression levels of S-palmitoylases and S-depalmitoylases in LUAD were investigated using TCGA. GEPIA was used to evaluate the mRNA levels of S-palmitoylases and S-depalmitoylases at different pathological stages. Metascape was used to investigate the biological significance of S-palmitoylases and S-depalmitoylases. The Kaplan–Meier plotter was used to analyze the prognostic value of S-palmitoylases and S-depalmitoylases. CBioportal was used to analyze gene alterations in S-palmitoylases and S-depalmitoylases. UALCAN was used to examine DNA promoter methylation levels of S-palmitoylases and S-depalmitoylases. Finally, we investigated the relationship between S-palmitoylases, S-depalmitoylases, and tumor-infiltrating immune cells using TIMER. Correlations with immune checkpoint-related genes were determined using the R packages reshape2, ggpubr, ggplot2, and corrplot. PCR was also performed to assess the degree of ZDHHC4/12/18/24 and APT2 transcript expression in lung adenocarcinoma and adjacent normal lung tissues. HPA was utilized to investigate protein levels of S-palmitoylases and S-depalmitoylases in LUAD and normal lung tissue. Our study found that ZDHHC2/3/4/5/6/7/9/12/13/16/18/20/21/23/24, APT1/2, PPT1, LYPLAL1, ABHD4/10/11/12/13 and ABHD17C mRNA expression was significantly upregulated in LUAD, whereas ZDHHC1/8/11/11B/14/15/17/19/22, ABHD6/16A and ABHD17A mRNA expression was significantly downregulated. The functions of the differentially expressed S-palmitoylases and S-depalmitoylases were mainly associated with protein-cysteine S-palmitoyltransferase and protein-cysteine S-acyltransferase activities. Patients with high expression of ZDHHC4/12/18/24, APT2, ABHD4, ABHD11 and ABHD12 had a shorter overall survival. Infiltration of six immune cells (B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells) was closely associated with the expression of ZDHHC4/12/18/24 and APT2. ZDHHC4/12/18/24 and APT2 positively correlated with the immune checkpoint-related gene CD276. We assessed the mRNA levels of ZDHHC4/12/18/24 and APT2 using qRT-PCR and found increased expression of ZDHHC4/12/18/24 in LUAD compared with healty control lung tissues. ZDHHC4/12/18/24, and APT2 are potential prognostic biomarkers of LUAD. Their expression levels could be related to the tumor microenvironment in LUAD.

Discovery of a novel ROS-based signature for predicting prognosis and immunosuppressive tumor microenvironment in lung adenocarcinoma.

The role of reactive oxygen species (ROS) is critical in the emergence and progression of lung adenocarcinoma (LUAD), affecting cell survival, proliferation, angiogenesis, and metastasis. Further investigations are needed to elucidate these effects' precise pathways and devise therapeutic approaches targeting ROS. Moreover, the expression pattern and clinical significance of the ROS-related genes in LUAD remain elusive. Through comprehensive analysis incorporating 1494 LUAD cases from The Cancer Genome Atlas, six Gene Expression Omnibus series, and a Chinese LUAD cohort, we identified a ROS-related signature with substantial predictive value in various LUAD patient cohorts. The ROS-related signature has demonstrated a significant negative relationship with antitumor immunity and dendritic cell maturation and activation. Moreover, The ROS-related signature showed predictive value on immunotherapy outcomes across multiple types of solid tumors, including LUAD. These findings reinforce the ROS-related signature as a predictive tool for LUAD and provide new insights into its link with antitumor immunity and immunotherapy efficacy. These results have implications for refining clinical assessments and tailoring immunotherapeutic strategies for patients with LUAD.

Identification and validation of a 4-extracellular matrix gene signature associated with prognosis and immune infiltration in lung adenocarcinoma

BackgroundThe extracellular matrix (ECM) plays a crucial role in the development and tumor microenvironment of lung adenocarcinoma (LUAD). This study aimed to establish a risk score of ECM-related genes in LUAD and explore the association between the risk score and patient survival as well as immune cell infiltration, somatic mutations, and therapy response. MethodsGene expression data from The Cancer Genome Atlas (TGCA) and eight Gene Expression Omnibus (GEO) databases were used to analyze and identify differentially expressed genes (DEGs). Prognostic ECM-related genes were identified and utilized to formulate a prognostic signature. A nomogram was constructed using TCGA dataset and validated in two GEO datasets. Differences between high- and low-risk patients were analyzed for function enrichment, immune cell infiltration, somatic mutations, and therapy response. Finally, Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of DEGs in LUAD. ResultsA risk score based on four ECM-related genes, ANOS1, CD36, COL11A1, and HMMR, was identified as an independent prognostic factor for overall survival (OS) compared to other clinical variables. Subsequently, a nomogram incorporating the risk score and TNM staging was developed using the TCGA dataset. Internal and external validation of the nomogram, conducted through calibration plots, C-index, time-dependent receiver operating characteristics (ROC), integrated discrimination improvement (IDI), and decision curve analyses (DCA), demonstrated the excellent discriminatory ability and clinical practicability of this nomogram. The risk score correlated with the distribution of function enrichment, immune cell infiltration, and immune checkpoint expression. More somatic mutations occurred in the high-risk group. The risk score also demonstrated a favorable ability to predict immunotherapy response and drug sensitivity. ConclusionA novel signature based on four ECM-related genes is developed to help predict LUAD prognosis. This signature correlates with tumor immune microenvironment and can predict the response to different therapies in LUAD patients.

Link between m6A modification and infiltration characterization of tumor microenvironment in lung adenocarcinoma.

N6-methyladenosine (m6A) RNA methylation plays a pivotal role in immune responses and the onset and advancement of cancer. Nonetheless, the precise impact of m6A modification in lung adenocarcinoma (LUAD) and its associated tumor microenvironment (TME) remains to be fully elucidated. Here, we distinguished distinct m6A modification patterns within two separate LUAD cohorts using a set of 21 m6A regulators. The TME characteristics associated with these two patterns align with the immune-inflamed and immune-excluded phenotypes, respectively. We identified 2064 m6A-related genes, which were used as a basis to divide all LUAD samples into three distinct m6A gene clusters. We applied a scoring system to evaluate the m6A gene signature of the m6A modification pattern in individual patients. To authenticate the categorization significance of m6A modification patterns, we established a correlation between m6A score and TME infiltration profiling, tumor somatic mutations, and responses to immunotherapy. A high level of m6A modification may be associated with the aggressiveness and poor prognosis of LUAD. Further studies should investigate the mechanism of action of m6A regulators and m6A-related genes to improve the diagnosis and treatment of patients with LUAD.

RUNX1 as a Potential Target for Combined Radioimmunotherapy of Lung Adenocarcinoma

Radioimmunotherapy for non-small cell lung cancer has good clinical application prospects. The role and mechanism of RUNX1 in DNA damage repair were explored for its potential role in lung adenocarcinoma radioimmunotherapy. To study the effect of RUNX1 expression level on the expression of DNA damage repair system related factors and radiation sensitivity of lung adenocarcinoma cells. As an important nuclear transcription factor, RUNX1 was explored whether directly regulating the expression of Nrf2, Rad51, BRCA1, and verifying their respective DNA binding sites in the promoter region through relevant databases. To observe the effect of RUNX1 knockout and overexpression on the expression level of PD-L1 in tumor cells at the cell level; The effect of RUNX1 expression level on the sorting and presentation of PD-L1 cells was investigated by the method of nucleocytoplasmic separation. According to literature reports, CMTM6 and ALIX play a key role in the process of PD-L1 cell sorting and presentation, and explore whether RUNX1 plays a role through this factor. The effect of phosphorylation level of different splicing bodies of RUNX1 (RUNX1a/b/c) on the expression level and DNA damage repair system related factors on tumor radiosensitivity were also explored. According to TCGA database, RUNX1 is highly expressed and phosphorylated in lung adenocarcinoma. Through gene comparison with the database, it was found that RUNX1 binding sites existed in the promoter region of several factors related to this study, including ALIX, Nrf2, BRCA1, RAD51, ATM, H2AX, etc. After being activated by MAPKp38 phosphorylation, RUNX1a can positively regulate Nrf2 signal pathway. The expression of RUNX1 and p-RUNX1 is time-dependent on ionizing radiation. At the same time, it was found that the expression of RUNX1 and p-RUNX1 was dose-dependent on ionizing radiation, and the expression trend of Nrf2 signal pathway related factors was consistent with RUNX1. RUNX1 regulates the expression of PD-L1, BRCA1, ALIX and Nrf2. Bioinformatics analysis and flow cytometry data show that RUNX1 has inhibitory effect on tumor microenvironment of lung adenocarcinoma. RUNX1 regulates DNA damage repair system and has inhibitory effect on tumor immunity. Inhibiting the expression of RUNX1 in lung adenocarcinoma cells can enhance the effect of radioimmunotherapy.

Comparative analysis of single-cell transcriptome reveals heterogeneity in the tumor microenvironment of lung adenocarcinoma and brain metastases

PurposeSolid tumors such as lung adenocarcinoma include not only the tumor cells but also the microenvironment in which the tumor cells continuously interact with each other. An in-depth understanding of the oncological features and tumor microenvironment (TME) of lung adenocarcinoma and brain metastases at the single-cell level could provide new therapeutic strategies for brain metastases from lung adenocarcinoma.MethodsTo solve this problem, we performed single-cell RNA sequencing (scRNA-seq) analysis on 15 lung adenocarcinoma samples and 10 brain metastasis samples.ResultsA total of 86,282 single cells were obtained and divided into 8 cell types, including epithelial cells, endothelial cells, fibroblasts, oligodendrocytes, T/NK cells, B cells, mast cells, and macrophages. In brain metastases, we found a significantly lower proportion of T/NK cells and mast cells, and more severe immune dysregulation. In addition, we found a subpopulation of macrophages with high expression of metastasis-promoting-related genes enriched in brain metastatic tissues. Moreover, in brain metastases, we found a significantly increased proportion of myofibroblastic cancer-associated fibroblasts (myCAFs) and a higher angiogenic capacity of endothelial cells. Epithelial cells in brain metastases were more malignant and underwent genomic reprogramming. Next, we found that DNA damage-inducible transcript 4 (DDIT4) expression was upregulated in epithelial cells in brain metastases and was associated with poor prognosis. Finally, we experimentally validated that the downregulation of DDIT4 inhibited the proliferation, migration, and invasion of lung cancer cells.ConclusionsThis study depicts a single-cell atlas of lung adenocarcinoma and brain metastases by scRNA-seq and paves the way for the development of future therapeutic targets for brain metastases from lung cancer.

Revealing the role of regulatory T cells in the tumor microenvironment of lung adenocarcinoma: a novel prognostic and immunotherapeutic signature.

Regulatory T cells (Tregs), are a key class of cell types in the immune system. In the tumor microenvironment (TME), the presence of Tregs has important implications for immune response and tumor development. Relatively little is known about the role of Tregs in lung adenocarcinoma (LUAD). Tregs were identified using but single-cell RNA sequencing (scRNA-seq) analysis and interactions between Tregs and other cells in the TME were investigated. Next, we used multiple bulk RNA-seq datasets to construct risk models based on marker genes of Tregs and explored differences in prognosis, mutational landscape, immune cell infiltration and immunotherapy between high- and low-risk groups, and finally, qRT-PCR and cell function experiments were performed to validate the model genes. The cellchat analysis showed that MIF-(CD74+CXCR4) pairs play a key role in the interaction of Tregs with other cell subpopulations, and the Tregs-associated signatures (TRAS) could well classify multiple LUAD cohorts into high- and low-risk groups. Immunotherapy may offer greater potential benefits to the low-risk group, as indicated by their superior survival, increased infiltration of immune cells, and heightened expression of immune checkpoints. Finally, the experiment verified that the model genes LTB and PTTG1 were relatively highly expressed in cancer tissues, while PTPRC was relatively highly expressed in paracancerous tissues. Colony Formation assay confirmed that knockdown of PTTG1 reduced the proliferation ability of LUAD cells. TRAS were constructed using scRNA-seq and bulk RNA-seq to distinguish patient risk subgroups, which may provide assistance in the clinical management of LUAD patients.

Retracted: PLK1 Is a Potential Prognostic Factor Associated with the Tumor Microenvironment in Lung Adenocarcinoma.

[This retracts the article DOI: 10.1155/2022/7848771.].

NPM3 as a novel oncogenic factor and poor prognostic marker contributes to cell proliferation and migration in lung adenocarcinoma

BackgroundLung cancer is the leading cause of cancer-related deaths worldwide, and despite recent advances in targeted therapies and immunotherapies, the clinical benefit remains limited. Therefore, there is an urgent need to further investigate the molecular mechanisms underlying lung cancer. The aim of this study was to investigate the expression and function of NPM3 in the tumor microenvironment of lung adenocarcinoma (LUAD).MethodsWe utilized bioinformatics tools and databases, including UALCAN, GEPIA2, HPA, and Sangerbox, to analyze NPM3 expression in LUAD samples and its association with prognosis and mutational landscape. NPM3 expression in various cell types was assessed at the single cell level using the TISCH database. We also used algorithms such as TIMER and EPIC to explore the crosstalk between NPM3 expression and immune features. KEGG enrichment analysis was performed to identify potential signaling pathways of NPM3. Finally, we employed siRNA knockdown strategy to investigate the effect of NPM3 on LUAD cell proliferation and migration in vitro.ResultsNPM3 was significantly upregulated in LUAD tissues and was strongly associated with poor prognosis and TP53 gene mutations. Single-cell sequencing analysis revealed that NPM3 was expressed in immune cells (dendritic cells and monocytes/macrophages) in the tumor microenvironment. Moreover, NPM3 expression was negatively associated with immune B cell and CD4 T cell infiltration, as well as with several immune-related genes (including CCL22, CXCR2, CX3CR1, CCR6, HLA-DOA, HLA-DQA2). KEGG enrichment analysis indicated that NPM3 expression was associated with cell cycle, CAMs, and NSCLC pathway genes. Finally, in vitro experiments showed that NPM3 knockdown inhibited LUAD cell proliferation and migration in NCI-H1299 and SPC-A1 cells, and suppressed the expression of CCNA2 and MAD2L1.ConclusionElevated NPM3 expression predicts poor clinical outcome and an immunosuppressive microenvironment in LUAD tissues. NPM3 promotes LUAD progression by promoting cell proliferation and migration, and targeting NPM3 may represent a novel therapeutic strategy for LUAD.Graphical

Abstract PR07: Oncogenic Kras signaling shapes the tumor microenvironment in lung adenocarcinoma

Abstract Despite advancements in targeted therapy and immunotherapy, lung cancer remains the leading cause of cancer-related deaths in both men and women worldwide, with an estimated 2.2 million deaths each year. At 85%, non-small cell lung cancer (NSCLC) is by far the most common subtype, comprising adenocarcinomas and lung squamous cell carcinoma. Mutations in Kirsten rat sarcoma viral oncogene homolog (KRAS), epidermal growth factor receptor (EGFR) and anaplastic lymphoma receptor tyrosine kinase (ALK) genes are common, with the worst overall survival in KRAS mutant adenocarcinoma patients. About 40% of lung tumors with KRAS mutations also harbor mutations in tumor protein p53 gene (TP53), a tumor suppressor gene. KrasG12C and KrasG12D are both prevalent mutations in lung cancer. Sotorasib, a KrasG12C inhibitor, was recently approved for treatment of NSCLC. However, only 37% of patients have an objective response to Sotorasib, with a median duration of just 11 months. The KrasG12D inhibitor MRTX1133 is entering clinical trials; elucidating the mechanism of action for potential resistance is imperative to design combination treatment approaches. While cell-intrinsic mechanisms of resistance in epithelial oncogenic KRASG12D (Kras*) are well studied, our group and others have shown that KRASG12D also extrinsically regulates the tumor microenvironment (TME) in pancreatic cancer. Whether and how this oncogene regulates the TME of lung cancer is less understood. To study the crosstalk between epithelial lung cells expressing oncogenic KRAS and the surrounding TME, we have established a triple transgenic murine model of Kras*-driven NSCLC, wherein both lung epithelial-specific expression of Kras* and expression of mutant TP53R172H (Tp53*) can be controlled in an inducible and reversible manner. Our preliminary data show that expression of Kras* drives tumor growth, while Kras* depletion results in regression, even in the presence of Tp53*. Here we interrogate how Kras* modulates the TME in lung adenocarcinomas. Harnessing scRNA sequencing, immunohistochemistry, and multiple biochemical approaches, we will provide insight into changes in stromal and immune contexture upon Kras* depletion in mice. Citation Format: Rachael K. Baliira, Emily Lasse Opsahl, Elyse McLintock, Katelyn L. Donahue, Ivana Barravacchia, Marina Pasca di Magliano, Stefanie Galbán. Oncogenic Kras signaling shapes the tumor microenvironment in lung adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR07.

Investigating the interactions between regulatory T cells and cancer-associated fibroblasts and their role in lung cancer progression

Abstract The tumor microenvironment (TME) is a highly complex multicellular system and has been shown to play a fundamental role in dictating tumorigenesis. High expression of regulatory surface molecules, cytokines, and chemokines in fibroblast niches within lung tumor stroma, paired with their colocalization with tumor infiltrating lymphocytes (TILs), suggests that cancer-associated fibroblast (CAF) niches may alter the functional response of TILs and influence the anti-tumor immune response. Here, utilizing a transcriptomic approach, we have uncovered a distinct CAF population, which we term “iCAFs,” that is enriched for immunomodulatory gene signatures, T cell ligands, and chemoattractants. This population expresses a unique set of cell surface molecules that permit their identification and isolation from lung tumor tissue. Given the demonstrated importance of Tregs in suppressing anti-tumor immunity, we chose to evaluate how Tregs may be interacting with iCAFs within the TME of lung adenocarcinoma. We uncovered significant colocalization patterns of Foxp3 +Tregs with iCAF—a pattern which was found to be unique for Tregs when compared to conventional CD4 +Foxp3 −T cells. Moreover, the Treg to conventional CD4 +T cell ratio is significantly decreased in tumor tissue relative to normal adjacent tissue and healthy controls. Our analyses demonstrate that Tregs preferentially associate with iCAF populations within the tumor, leading us to hypothesize that interactions between Tregs and iCAFs may, in turn, influence Treg trafficking, phenotype and function within the TME. T32AI106711

Abstract LB104: Preclinical evaluation of the role of Galectin-1 in tumor immune evasion associated mechanisms using a highly selective Galectin-1 small molecule inhibitor

Abstract Galectin-1 (Gal-1) is a β-galactoside-binding lectin that is highly expressed within the tumor microenvironment (TME) of some aggressive cancers and whose high expression correlates strongly with poor survival. Following a data mining exercise of external publication data and additional clinical biomarker datasets, we found strong evidence concluding the potential role of Gal-1 in the establishment of a fibrotic tumor stroma and mechanisms associated with tumor invasion, metastasis and angiogenesis. However, there was limited data regarding the role of Gal-1 in immune escape by tumors to make any strong conclusions/pathway reconstruction around this specific TME mechanism of action (MOA). To further strengthen the potential role of Gal-1 in tumor immune evasion we completed a series of preclinical in vivo and in vitro activities, using a highly selective Gal-1 small molecule inhibitor developed at Galecto Biotech. To examine the role of Gal-1 inhibition in vivo, we investigated the effects on tumor volume of the highly selective Gal-1 inhibitor, in a syngeneic mouse model of lung adenocarcinoma. RNA profiling of vehicle and compound treated tumors was also implemented following completion of the in-life phase. Oral administration of the Gal-1 inhibitor reduced mouse lung adenocarcinoma growth and increased the immune gene expression within the tumors, showing a strong association to that of exacerbated NK cell activity. No additional cytotoxic T cell gene changes were uncovered in the Gal-1 inhibitor treated tumors. We further evaluated the overall T cell compartment, relevant to the clinical TME setting, by stimulating human peripheral blood mononuclear cells (hPBMC’s) incubated with and without the Gal-1 inhibitor to observe the potential blockade of any known immune checkpoint inhibitor (ICI) resistance mechanisms. Interestingly, we found a variety of immune suppressive proteins to be inhibited, including IL-17, when hPBMC’s were incubated with the Gal-1 inhibitor. In summary, the data presented suggests highly selective Gal-1 inhibition could provide an effective, monotherapy (or be used in combination with ICI's) to boost immune infiltration and/or activation in the TME of lung adenocarcinoma and potentially other aggressive cancers. Citation Format: Ian Holyer, Rob Slack, Kristoffer Petersen, Fredrik Zetterberg, Alison Mackinnon. Preclinical evaluation of the role of Galectin-1 in tumor immune evasion associated mechanisms using a highly selective Galectin-1 small molecule inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB104.