Identification of a combined hypoxia and lactate metabolism prognostic signature in lung adenocarcinoma : Author.

BACKGROUND: In the tumor microenvironment (TME), a bidirectional relationship exists between hypoxia and lactate metabolism, with each component exerting a reciprocal influence on the other, forming an inextricable link. The aim of the present investigation was to develop a prognostic model by amalgamating genes associated with hypoxia and lactate metabolism. This model is intended to serve as a tool for predicting patient outcomes, including survival rates, the status of the immune microenvironment, and responsiveness to therapy in patients with lung adenocarcinoma (LUAD). METHODS: Transcriptomic sequencing data and patient clinical information specific to LUAD were obtained from comprehensive repositories of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). A compendium of genes implicated in hypoxia and lactate metabolism was assembled from an array of accessible datasets. Univariate and multivariate Cox regression analyses were employed. Additional investigative procedures, including tumor mutational load (TMB), microsatellite instability (MSI), functional enrichment assessments and the ESTIMATE, CIBERSORT, and TIDE algorithms, were used to evaluate drug sensitivity and predict the efficacy of immune-based therapies. RESULTS: A novel prognostic signature comprising five lactate and hypoxia-related genes (LHRGs), PKFP, SLC2A1, BCAN, CDKN3, and ANLN, was established. This model demonstrated that LUAD patients with elevated LHRG-related risk scores exhibited significantly reduced survival rates. Both univariate and multivariate Cox analyses confirmed that the risk score was a robust prognostic indicator of overall survival. Immunophenotyping revealed increased infiltration of memory CD4 + T cells, dendritic cells and NK cells in patients classified within the high-risk category compared to their low-risk counterparts. Higher probability of mutations in lung adenocarcinoma driver genes in high-risk groups, and the MSI was associated with the risk-score. Functional enrichment analyses indicated a predominance of cell cycle-related pathways in the high-risk group, whereas metabolic pathways were more prevalent in the low-risk group. Moreover, drug sensitivity analyses revealed increased sensitivity to a variety of drugs in the high-risk group, especially inhibitors of the PI3K-AKT, EGFR, and ELK pathways. CONCLUSIONS: This prognostic model integrates lactate metabolism and hypoxia parameters, offering predictive insights regarding survival, immune cell infiltration and functionality, as well as therapeutic responsiveness in LUAD patients. This model may facilitate personalized treatment strategies, tailoring interventions to the unique molecular profile of each patient's disease.

The Role of SCARA5 as a Potential Biomarker in Squamous Cell Carcinoma of the Lung.

Lung cancer is the leading cause of cancer-related deaths in the western world. Squamous cell carcinoma is one of the most common histological subtypes of this malignancy. For squamous cell carcinoma of the lung (LSCC), prognostic and predictive markers still are largely missing. In a previous study, we were able to show that the expression of THSD7A shows an association with unfavorable prognostic parameters in prostate cancer. There is also a link to a high expression of FAK. There is incidence that SCARA5 might be the downstream gene of THSD7A. Furthermore, there is evidence that SCARA5 interacts with FAK. We were interested in the role of SCARA5 as a potential biomarker in LSCC. Furthermore, we wanted to know whether SCARA5 expression is linked to THSD7A positivity and to the expression level of FAK. For this reason, we analyzed 101 LSCC tumors by immunohistochemistry. Tissue microarrays were utilized. No significant association was found between SCARA5 expression and overall survival or clinicopathological parameters. There was also no significant association between THSD7A positivity and SCARA5 expression level. Moreover, no significant association was found between FAK expression level and SCARA5 expression level. SCARA5 seems not to play a major role as a biomarker in squamous cell carcinoma of the lung.

Optimizing immunofluorescence with high-dynamic-range imaging to enhance PD-L1 expression evaluation for 3D pathology assessment from NSCLC tumor tissue.

Assessing programmed death ligand 1 (PD-L1) expression through immunohistochemistry (IHC) is the golden standard in predicting immunotherapy response of non-small cell lung cancer (NSCLC). However, observation of heterogeneous PD-L1 distribution in tumor space is a challenge using IHC only. Meanwhile, immunofluorescence (IF) could support both planar and three-dimensional (3D) histological analyses by combining tissue optical clearing with confocal microscopy. We optimized clinical tissue preparation for the IF assay focusing on staining, imaging, and post-processing to achieve quality identical to traditional IHC assay. To overcome limited dynamic range of the fluorescence microscope's detection system, we incorporated a high dynamic range (HDR) algorithm to restore the post imaging IF expression pattern and further 3D IF images. Following HDR processing, a noticeable improvement in the accuracy of diagnosis (85.7%) was achieved using IF images by pathologists. Moreover, 3D IF images revealed a 25% change in tumor proportion score for PD-L1 expression at various depths within tumors. We have established an optimal and reproducible process for PD-L1 IF images in NSCLC, yielding high quality data comparable to traditional IHC assays. The ability to discern accurate spatial PD-L1 distribution through 3D pathology analysis could provide more precise evaluation and prediction for immunotherapy targeting advanced NSCLC.

Anticancer activity of Araguspongine C via inducing apoptosis, and inhibition of oxidative stress, inflammation, and EGFR-TK in human lung cancer cells: An in vitro and in vivo study.

The advanced non-small cell lung cancer (NSCLC) that harbors epidermal growth factor receptor (EGFR) mutations has put a selective pressure on the discovery and development of newer EGFR inhibitors. Therefore, the present study intends to explore the pharmacological effect of Araguspongine C (Aragus-C) as anticancer agent against lung cancer. The effect of Aragus-C was evaluated on the viability of the A549 and H1975 cells. Further biochemical assays were performed to elaborate the effect of Aragus-C, on the apoptosis, cell-cycle analysis, and mitochondrial membrane potential in A549 cells. Western blot analysis was also conducted to determine the expression of EGFR in A549 cells. Tumor xenograft mice model from A549 cells was established to further elaborate the pharmacological activity of Aragus-C. Results suggest that Aragus C showed significant inhibitory activity against A549 cells as compared to H1975 cells. It has been found that Aragus-C causes the induction of apoptosis and promotes cell-cycle arrest at the G2/M phase of A549 cells. It also showed a reduction in the overexpression of EGFR in A549 cells. In tumor xenograft mice model, it showed a significant reduction of tumor volume in a dose-dependent manner, with maximum inhibitory activity was reported by the 8 mg/kg treated group. It also showed significant anti-inflammatory and antioxidant activity by reducing the level of TNF-α, IL-1ß, IL-6, and MDA, with a simultaneous increase of superoxide dismutase and glutathione peroxidase. We have demonstrated the potent anti-lung cancer activity of Aragus-C, and it may be considered as a potential therapeutic choice for NSCLC treatment.

PRMT1 promotes Warburg effect by regulating the PKM2/PKM1 ratio in non-small cell lung cancer.

Abnormal epigenetic modifications are involved in the regulation of Warburg effect in tumor cells. Protein arginine methyltransferases (PRMTs) mediate arginine methylation and have critical functions in cellular responses. PRMTs are deregulated in a variety of cancers, but their precise roles in Warburg effect in cancer is largely unknown. Experiments from the current study showed that PRMT1 was highly expressed under conditions of glucose sufficiency. PRMT1 induced an increase in the PKM2/PKM1 ratio through upregulation of PTBP1, in turn, promoting aerobic glycolysis in non-small cell lung cancer (NSCLC). The PRMT1 level in p53-deficient and p53-mutated NSCLC remained relatively unchanged while the expression was reduced in p53 wild-type NSCLC under conditions of glucose insufficiency. Notably, p53 activation under glucose-deficient conditions could suppress USP7 and further accelerate the polyubiquitin-dependent degradation of PRMT1. Melatonin, a hormone that inhibits glucose intake, markedly suppressed cell proliferation of p53 wild-type NSCLC, while a combination of melatonin and the USP7 inhibitor P5091 enhanced the anticancer activity in p53-deficient NSCLC. Our collective findings support a role of PRMT1 in the regulation of Warburg effect in NSCLC. Moreover, combination treatment with melatonin and the USP7 inhibitor showed good efficacy, providing a rationale for the development of PRMT1-based therapy to improve p53-deficient NSCLC outcomes.

The Role of Exhaled Breath Condensate in Chronic Inflammatory and Neoplastic Diseases of the Respiratory Tract.

Asthma and chronic obstructive pulmonary disease (COPD) are among the most common chronic respiratory diseases. Chronic inflammation of the airways leads to an increased production of inflammatory markers by the effector cells of the respiratory tract and lung tissue. These biomarkers allow the assessment of physiological and pathological processes and responses to therapeutic interventions. Lung cancer, which is characterized by high mortality, is one of the most frequently diagnosed cancers worldwide. Current screening methods and tissue biopsies have limitations that highlight the need for rapid diagnosis, patient differentiation, and effective management and monitoring. One promising non-invasive diagnostic method for respiratory diseases is the assessment of exhaled breath condensate (EBC). EBC contains a mixture of volatile and non-volatile biomarkers such as cytokines, leukotrienes, oxidative stress markers, and molecular biomarkers, providing significant information about inflammatory and neoplastic states in the lungs. This article summarizes the research on the application and development of EBC assessment in diagnosing and monitoring respiratory diseases, focusing on asthma, COPD, and lung cancer. The process of collecting condensate, potential issues, and selected groups of markers for detailed disease assessment in the future are discussed. Further research may contribute to the development of more precise and personalized diagnostic and treatment methods.

Effect of Flammulina velutipes polysaccharide on mitochondrial apoptosis in lung adenocarcinoma A549 cells.

FVP is a polysaccharide extracted from Flammulina velutipes with immunomodulatory, anti-tumor, and anti-oxidation activities. In this study, we obtained the crude polysaccharide FVP-C from the water extract of Flammulina velutipes, and its main component FVP-S1 was obtained after further purification. Upon structural identification, we found that FVP-C is a neutral polysaccharide, and FVP-S1 was an acidic golden mushroom polysaccharide, consisting of glucuronic acid, xylose, and glucose. Lung adenocarcinoma (A549) was treated with FVP-S1 and FVP-C, respectively, and we found that FVP-S1 and FVP-C inhibited the proliferation and migration ability of tumor cells, as well as changed the morphology of the tumor cells and caused chromosome sheteropythosis, among which FVP-S1 had the best inhibition effect. The results of flow cytometry experiments and mitochondrial membrane potential, RT-qPCR, and Western blot showed that FVP-S1 and FVP-C were able to decrease the mitochondrial membrane potential, increase the expression level of apoptotic proteins Casepase-3 and Casepase-9 proteins, and at the same time, increase the ratio of Bax and Bcl-2, which promoted apoptosis of tumor cells. In conclusion, these data indicated that FVP-S1 and FVP-C were able to induce apoptosis in A549 cells through the mitochondrial pathway, which played an important role in inhibiting tumor cells.

MRPL35 Induces Proliferation, Invasion, and Glutamine Metabolism in NSCLC Cells by Upregulating SLC7A5 Expression.

BACKGROUND: Mitochondrial ribosomal protein L35 (MRPL35) has been reported to contribute to the growth of non-small cell lung cancer (NSCLC) cells. However, the functions and mechanisms of MRPL35 on glutamine metabolism in NSCLC remain unclear. METHODS: The detection of mRNA and protein of MRPL35, ubiquitin-specific protease 39 (USP39), and solute carrier family 7 member 5 (SLC7A5) was conducted using qRT-PCR and western blotting. Cell proliferation, apoptosis, and invasion were evaluated using the MTT assay, EdU assay, flow cytometry, and transwell assay, respectively. Glutamine metabolism was analyzed by detecting glutamine consumption, α-ketoglutarate level, and glutamate production. Cellular ubiquitination analyzed the deubiquitination effect of USP39 on MRPL35. An animal experiment was conducted for in vivo analysis. RESULTS: MRPL35 was highly expressed in NSCLC tissues and cell lines, and high MRPL35 expression predicted poor outcome in NSCLC patients. In vitro analyses suggested that MRPL35 knockdown suppressed NSCLC cell proliferation, invasion, and glutamine metabolism. Moreover, MRPL35 silencing hindered tumor growth in vivo. Mechanistically, USP39 stabilized MRPL35 expression by deubiquitination and then promoted NSCLC cell proliferation, invasion, and glutamine metabolism. In addition, MRPL35 positively affected SLC7A5 expression in NSCLC cells in vitro and in vivo. Moreover, the anticancer effects of MRPL35 silencing could be rescued by SLC7A5 overexpression in NSCLC cells. CONCLUSION: MRPL35 expression was stabilized by USP39-induced deubiquitination in NSCLC cells, and knockdown of MRPL35 suppressed NSCLC cell proliferation, invasion, and glutamine metabolism in vitro and impeded tumor growth in vivo by upregulating SLC7A5, providing a promising therapeutic target for NSCLC.

Integrated network pharmacology and metabolomics to reveal the mechanism of Pinellia ternata inhibiting non-small cell lung cancer cells.

Lung cancer is a malignant tumor with highly heterogeneous characteristics. A classic Chinese medicine, Pinellia ternata (PT), was shown to exert therapeutic effects on lung cancer cells. However, its chemical and pharmacological profiles are not yet understood. In the present study, we aimed to reveal the mechanism of PT in treating lung cancer cells through metabolomics and network pharmacology. Metabolomic analysis of two strains of lung cancer cells treated with Pinellia ternata extracts (PTE) was used to identify differentially abundant metabolites, and the metabolic pathways associated with the DEGs were identified by MetaboAnalyst. Then, network pharmacology was applied to identify potential targets against PTE-induced lung cancer cells. The integrated network of metabolomics and network pharmacology was constructed based on Cytoscape. PTE obviously inhibited the proliferation, migration and invasion of A549 and NCI-H460 cells. The results of the cellular metabolomics analysis showed that 30 metabolites were differentially expressed in the lung cancer cells of the experimental and control groups. Through pathway enrichment analysis, 5 metabolites were found to be involved in purine metabolism, riboflavin metabolism and the pentose phosphate pathway, including D-ribose 5-phosphate, xanthosine, 5-amino-4-imidazolecarboxyamide, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Combined with network pharmacology, 11 bioactive compounds were found in PT, and networks of bioactive compound-target gene-metabolic enzyme-metabolite interactions were constructed. In conclusion, this study revealed the complicated mechanisms of PT against lung cancer. Our work provides a novel paradigm for identifying the potential mechanisms underlying the pharmacological effects of natural compounds.

Cigarette smoke promotes IL-6-dependent lung cancer migration and osteolytic bone metastasis.

Lung cancer stands as a major contributor to cancer-related fatalities globally, with cigarette smoke playing a pivotal role in its development and metastasis. Cigarette smoke is also recognized as a risk factor for bone loss disorders like osteoporosis. However, the association between cigarette smoke and another bone loss disorder, lung cancer osteolytic bone metastasis, remains largely uncertain. Our Gene Set Enrichment Analysis (GSEA) indicated that smokers among lung cancer patients exhibited higher expression levels of bone turnover gene sets. Both The Cancer Genome Atlas (TCGA) database and our clinic samples demonstrated elevated expression of the osteolytic factor IL-6 in ever-smokers with bone metastasis among lung cancer patients. Our cellular experiments revealed that benzo[α]pyrene (B[α]P) and cigarette smoke extract (CSE) promoted IL-6 production and cell migration in lung cancer. Activation of the PI3K, Akt, and NF-κB signaling pathways was involved in cigarette smoke-augmented IL-6-dependent migration. Additionally, cigarette smoke lung cancer-secreted IL-6 promoted osteoclast formation. Importantly, blocking IL-6 abolished cigarette smoke-facilitated lung cancer osteolytic bone metastasis in vivo. Our findings provide evidence that cigarette smoke is a risk factor for osteolytic bone metastasis. Thus, inhibiting IL-6 may be a valuable therapeutic strategy for managing osteolytic bone metastasis in lung cancer patients who smoke.

Analysis of Methylome of Different Forms of Basal Cell Hyperplasia and Squamous Cell Metaplasia of Bronchial Epithelium.

Squamous cell lung cancer (SCLC) occurs as a result of dysregenerative changes in the bronchial epithelium: basal cell hyperplasia (BCH), squamous cell metaplasia (SM), and dysplasia. We previously suggested that combinations of precancerous changes detected in the small bronchi of patients with SCLC may reflect various "scenarios" of the precancerous process: isolated BCH→stopping at the stage of hyperplasia, BCH+SM→progression of hyperplasia into metaplasia, SM+dysplasia→progression of metaplasia into dysplasia. In this study, DNA methylome of various forms of precancerous changes in the bronchial epithelium of SCLC patients was analyzed using the genome-wide bisulfite sequencing. In BCH combined with SM, in contrast to isolated BCH, differentially methylated regions were identified in genes of the pathogenetically significant MET signaling pathway (RNMT, HPN). Differentially methylated regions affecting genes involved in inflammation regulation (IL-23, IL-23R, IL12B, IL12RB1, and FIS1) were detected in SM combined with dysplasia in comparison with SM combined with BCH. The revealed changes in DNA methylation may underlie various "scenarios" of the precancerous process in the bronchial epithelium.

[Solasonine promotes apoptosis of non-small cell lung cancer cells by regulating the Bcl-2/Bax/caspase-3 pathway].

OBJECTIVE: To investigate the effect of solasonine, an active component of Solanum nigrum, on proliferation and apoptosis of non-small cell lung cancer PC9 cells. METHODS: PC9 cells were treated with 2, 5, 10, 15, 20, or 25 µmol/L solasonine, and the changes in cell proliferation were examined using CCK-8 assay. Tetramethyl rhodamine ethyl ester (TMRE) was used to detect the changes in mitochondrial membrane potential, and caspase-3/7 detection kit and GreenNucTM caspase-3/Annexin V-mCherry kit for live cell were used to analyze the changes in caspase-3 of the cells. Annexin V-FITC/PI double staining was employed to analyze the apoptosis rate of the cells. The effect of PTEN inhibitors on solasonine-induced cell apoptosis was examined by detecting apoptosis-related protein expressions using Western blotting. RESULTS: Solasonine treatment for 24, 48, and 72 h significantly lowered the viability of PC9 cells. The cells treated with solasonine for 24 h showed significantly decreased mitochondrial membrane potential and increased cell apoptosis with enhanced caspase-3/7 and caspase-3 activities and expression of cleaved caspase-3. Solasonine treatment significantly decreased phosphorylation levels of PI3K and Akt, increased the protein expressions of PTEN and Bax, and lowered the expression of Bcl-2 protein in the cells. CONCLUSION: Solasonine inhibits proliferation and induces apoptosis of PC9 cells by regulating the Bcl-2/Bax/caspase-3 pathway and its upstream proteins.

Myofibroblasts derived type V collagen promoting tissue mechanical stress and facilitating metastasis and therapy resistance of lung adenocarcinoma cells.

Lung cancer is a leading cause of cancer-related mortality globally, with a dismal 5-year survival rate, particularly for Lung Adenocarcinoma (LUAD). Mechanical changes within the tumor microenvironment, such as extracellular matrix (ECM) remodeling and fibroblast activity, play pivotal roles in cancer progression and metastasis. However, the specific impact of the basement membrane (BM) on the mechanical characteristics of LUAD remains unclear. This study aims to identify BM genes influencing internal mechanical stress in tumors, elucidating their effects on LUAD metastasis and therapy resistance, and exploring strategies to counteract these effects. Using Matrigel overlay and Transwell assays, we found that mechanical stress, mimicked by matrix application, augmented LUAD cell migration and invasion, correlating with ECM alterations and activation of the epithelial-mesenchymal transition (EMT) pathway. Employing machine learning, we developed the SVM_Score model based on relevant BM genes, which accurately predicted LUAD patient prognosis and EMT propensity across multiple datasets. Lower SVM_Scores were associated with worse survival outcomes, elevated cancer-related pathways, increased Tumor Mutation Burden, and higher internal mechanical stress in LUAD tissues. Notably, the SVM_Score was closely linked to COL5A1 expression in myofibroblasts, a key marker of mechanical stress. High COL5A1 expression from myofibroblasts promoted tumor invasiveness and EMT pathway activation in LUAD cells. Additionally, treatment with Sorafenib, which targets COL5A1 secretion, attenuated the tumor-promoting effects of myofibroblast-derived COL5A1, inhibiting LUAD cell proliferation, migration, and enhancing chemosensitivity. In conclusion, this study elucidates the complex interplay between mechanical stress, ECM alterations, and LUAD progression. The SVM_Score emerges as a robust prognostic tool reflecting tumor mechanical characteristics, while Sorafenib intervention targeting COL5A1 secretion presents a promising therapeutic strategy to mitigate LUAD aggressiveness. These findings deepen our understanding of the biomechanical aspects of LUAD and offer insights for future research and clinical applications.

Proteomics shows that brain metastases of lung adenocarcinoma overexpress ribosomal proteins in response to gamma knife radiosurgery.

Gamma knife radiosurgery (GKRS) is recommended as the first-line treatment for brain metastases of lung adenocarcinoma (LUAD) in many guidelines, but its specific mechanism is unclear. We aimed to study the changes in the proteome of brain metastases of LUAD in response to the hyperacute phase of GKRS and further explore the mechanism of differentially expressed proteins (DEPs). Cancer tissues were collected from a clinical trial for neoadjuvant stereotactic radiosurgery before surgical resection of large brain metastases (ChiCTR2000038995). Five brain metastasis tissues of LUAD were collected within 24 h after GKRS. Five brain metastasis tissues without radiotherapy were collected as control samples. Proteomics analysis showed that 163 proteins were upregulated and 25 proteins were downregulated. GO and KEGG enrichment analyses showed that the DEPs were closely related to ribosomes. Fifty-three of 70 ribosomal proteins were significantly overexpressed, while none of them were underexpressed. The risk score constructed from 7 upregulated ribosomal proteins (RPL4, RPS19, RPS16, RPLP0, RPS2, RPS26 and RPS25) was an independent risk factor for the survival time of LUAD patients. Overexpression of ribosomal proteins may represent a desperate response to lethal radiotherapy. We propose that targeted inhibition of these ribosomal proteins may enhance the efficacy of GKRS.

Automated PD-L1 status prediction in lung cancer with multi-modal PET/CT fusion.

Programmed death-ligand 1 (PD-L1) expressions play a crucial role in guiding therapeutic interventions such as the use of tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) in lung cancer. Conventional determination of PD-L1 status includes careful surgical or biopsied tumor specimens. These specimens are gathered through invasive procedures, representing a risk of difficulties and potential challenges in getting reliable and representative tissue samples. Using a single center cohort of 189 patients, our objective was to evaluate various fusion methods that used non-invasive computed tomography (CT) and 18 F-FDG positron emission tomography (PET) images as inputs to various deep learning models to automatically predict PD-L1 in non-small cell lung cancer (NSCLC). We compared three different architectures (ResNet, DenseNet, and EfficientNet) and considered different input data (CT only, PET only, PET/CT early fusion, PET/CT late fusion without as well as with partially and fully shared weights to determine the best model performance. Models were assessed utilizing areas under the receiver operating characteristic curves (AUCs) considering their 95% confidence intervals (CI). The fusion of PET and CT images as input yielded better performance for PD-L1 classification. The different data fusion schemes systematically outperformed their individual counterparts when used as input of the various deep models. Furthermore, early fusion consistently outperformed late fusion, probably as a result of its capacity to capture more complicated patterns by merging PET and CT derived content at a lower level. When we looked more closely at the effects of weight sharing in late fusion architectures, we discovered that while it might boost model stability, it did not always result in better results. This suggests that although weight sharing could be beneficial when modality parameters are similar, the anatomical and metabolic information provided by CT and PET scans are too dissimilar to consistently lead to improved PD-L1 status predictions.

Investigating the complex interplay between fibroblast activation protein α-positive cancer associated fibroblasts and the tumor microenvironment in the context of cancer immunotherapy.

Introduction: This study investigates the role of Fibroblast Activation Protein (FAP)-positive cancer-associated fibroblasts (FAP+CAF) in shaping the tumor immune microenvironment, focusing on its association with immune cell functionality and cytokine expression patterns. Methods: Utilizing immunohistochemistry, we observed elevated FAP+CAF density in metastatic versus primary renal cell carcinoma (RCC) tumors, with higher FAP+CAF correlating with increased T cell infiltration in RCC, a unique phenomenon illustrating the complex interplay between tumor progression, FAP+CAF density, and immune response. Results: Analysis of immune cell subsets in FAP+CAF-rich stromal areas further revealed significant correlations between FAP+ stroma and various T cell types, particularly in RCC and non-small cell lung cancer (NSCLC). This was complemented by transcriptomic analyses, expanding the range of stromal and immune cell subsets interrogated, as well as to additional tumor types. This enabled evaluating the association of these subsets with tumor infiltration, tumor vascularization and other components of the tumor microenvironment. Our comprehensive study also encompassed cytokine, angiogenesis, and inflammation gene signatures across different cancer types, revealing heterogeneous cellular composition, cytokine expressions and angiogenic profiles. Through cytokine pathway profiling, we explored the relationship between FAP+CAF density and immune cell states, uncovering potential immunosuppressive circuits that limit anti-tumor activity in tumor-resident immune cells. Conclusions: These findings underscore the complexity of tumor biology and the necessity for personalized therapeutic and patient enrichment approaches. The insights gathered from FAP+CAF prevalence, immune infiltration, and gene signatures provide valuable perspectives on tumor microenvironments, aiding in future research and clinical strategy development.

Hypoxia-enhanced YAP1-EIF4A3 interaction drives circ_0007386 circularization by competing with CRIM1 pre-mRNA linear splicing and promotes non-small cell lung cancer progression.

BACKGROUND: The progression of non-small cell lung cancer (NSCLC) is significantly influenced by circular RNAs (circRNAs), especially in tumor hypoxia microenvironment. However, the precise functions and underlying mechanisms of dysregulated circRNAs in NSCLC remain largely unexplored. METHODS: Differentially expressed circRNAs in NSCLC tissues were identified through high-throughput RNA sequencing. The characteristics of circ_0007386 were rigorously confirmed via Sanger sequencing, RNase R treatment and actinomycin D treatment. The effects of circ_0007386 on proliferation and apoptosis were investigated using CCK8, cloning formation assays, TUNEL staining, and flow cytometry assays in vitro. In vivo, xenograft tumor models were used to evaluate its impact on proliferation. Mechanistically, the regulatory relationships of circ_0007386, miR-383-5p and CIRBP were examined through dual luciferase reporter assays and rescue experiments. Additionally, we detected the binding of EIF4A3 to CRIM1 pre-mRNA by RNA immunoprecipitation and the interaction between YAP1 and EIF4A3 under hypoxic conditions by co-immunoprecipitation. RESULTS: Our investigation revealed a novel circRNA, designated as circ_0007386, that was upregulated in NSCLC tissues and cell lines. Circ_0007386 modulated proliferation and apoptosis in NSCLC both in vitro and in vivo. Functionally, circ_0007386 acted as a sponge for miR-383-5p, targeting CIRBP, which influenced NSCLC cell proliferation and apoptosis via the PI3K/AKT signaling pathway. Furthermore, under hypoxic conditions, the interaction between YAP1 and EIF4A3 was enhanced, leading to the displacement of EIF4A4 from binding to CRIM1 pre-mRNA. This facilitated the back-splicing of CRIM1 pre-mRNA, increasing the formation of circ_0007386. The circ_0007386/miR-383-5p/CIRBP axis was significantly associated with the clinical features and prognosis of NSCLC patients. CONCLUSIONS: Circ_0007386, regulated by YAP1-EIF4A3 interaction under hypoxia conditions, plays an oncogenic role in NSCLC progression via the miR-383-5p/CIRBP axis.

A Spatial Omnibus Test (SPOT) for Spatial Proteomic Data.

MOTIVATION: Spatial proteomics can reveal the spatial organization of immune cells in the tumor immune microenvironment. Relating measures of spatial clustering, such as Ripley's K or Besag's L, to patient outcomes may offer important clinical insights. However, these measures require pre-specifying a radius in which to quantify clustering, yet no consensus exists on the optimal radius which may be context-specific. RESULTS: We propose a SPatial Omnibus Test (SPOT) which conducts this analysis across a range of candidate radii. At each radius, SPOT evaluates the association between the spatial summary and outcome, adjusting for confounders. SPOT then aggregates results across radii using the Cauchy combination test, yielding an omnibus P-value characterizing the overall degree of association. Using simulations, we verify that the type I error rate is controlled and show SPOT can be more powerful than alternatives. We also apply SPOT to ovarian and lung cancer studies. AVAILABILITY AND IMPLEMENTATION: An R package and tutorial are provided at https://github.com/sarahsamorodnitsky/SPOT.

Leucine rich α2 glycoprotein 1 derived from malignant pleural mesothelioma cells facilitates macrophage M2 phenotypes.

Background: Macrophages constitute the main part of infiltrating immune cells in Malignant pleural mesothelioma (MPM) and abnormally high ratios of M2 macrophages are present in both pleural effusion and tissue samples of MPM patients. Whether MPM cells affect formation of M2 macrophages is poorly understood. In this study, we focused on identification of MPM-cells-derived soluble factors with M2-promoting effects. Methods: Media of malignant pleural mesothelioma cells were collected and soluble factors affecting macrophages were analyzed by mass spectrometry. TGF-ß receptor inhibitor SB431542 was used as the entry point to explore the downstream mechanism of action by qRT-PCR, WB and immunofluorescence. Results: The serum-free culture media collected from the human MPM cells Meso1 and Meso2 significantly enhanced expression of the M2 signature molecules including IL-10, TGF-ß and CD206 in the human macrophages THP-1, while the culture medium of the human MPM cells H2452 did not show such M2-promoting effects. Analysis of proteins by mass spectrometry and ELISA suggested that Leucine rich α2 glycoprotein 1(LRG1) was a potential candidate. LRG1 time- and dose-dependently increased expression of the M2 signature molecules, confirming its M2-promoting effects. Furthermore, LRG1's M2-promoting effects were reduced by the TGF-ß receptor inhibitor SB431542, and LRG1 increased phosphorylation of Smad2, indicating that M2-promoting effects of LRG1 were via the TGF-ß receptor/Smad2 signaling pathway. Conclusions: Our results provide a potential M2-promoting new member, LRG1, which contributes to the immune escape of MPM via the TGF-ß receptor/Smad2 signaling pathway.

PARP4 interacts with hnRNPM to regulate splicing during lung cancer progression.

BACKGROUND: The identification of cancer driver genes from sequencing data has been crucial in deepening our understanding of tumor biology and expanding targeted therapy options. However, apart from the most commonly altered genes, the mechanisms underlying the contribution of other mutations to cancer acquisition remain understudied. Leveraging on our whole-exome sequencing of the largest Asian lung adenocarcinoma (LUAD) cohort (n = 302), we now functionally assess the mechanistic role of a novel driver, PARP4. METHODS: In vitro and in vivo tumorigenicity assays were used to study the functional effects of PARP4 loss and mutation in multiple lung cancer cell lines. Interactomics analysis by quantitative mass spectrometry was conducted to identify PARP4's interaction partners. Transcriptomic data from cell lines and patient tumors were used to investigate splicing alterations. RESULTS: PARP4 depletion or mutation (I1039T) promotes the tumorigenicity of KRAS- or EGFR-driven lung cancer cells. Disruption of the vault complex, with which PARP4 is commonly associated, did not alter tumorigenicity, indicating that PARP4's tumor suppressive activity is mediated independently. The splicing regulator hnRNPM is a potentially novel PARP4 interaction partner, the loss of which likewise promotes tumor formation. hnRNPM loss results in splicing perturbations, with a propensity for dysregulated intronic splicing that was similarly observed in PARP4 knockdown cells and in LUAD cohort patients with PARP4 copy number loss. CONCLUSIONS: PARP4 is a novel modulator of lung adenocarcinoma, where its tumor suppressive activity is mediated not through the vault complex-unlike conventionally thought, but in association with its novel interaction partner hnRNPM, thus suggesting a role for splicing dysregulation in LUAD tumorigenesis.