搜索结果：Cell death discovery

NK cells are promising candidates for adoptive cell therapy; however, their proliferative capacity and functional persistence may be limited. Genetic modification with hTERT enhances their proliferative potential, while co-expression of the iCASP9 suicide gene provides a safety mechanism based on late-stage apoptosis induction by chemical dimerizer (CID). Whether hTERT overexpression interferes with iCasp9-mediated cell death remains unclear, and the non-canonical functions of telomerase in this context are poorly understood. This study served a dual purpose: to assess the efficacy of the iCasp9 "suicide switch" in NK cells, and to investigate a non-canonical role of telomerase in NK cell-mediated evasion from cell death. Here, we demonstrate that hTERT-modified NK cells exhibit significant resistance to CID-induced apoptosis, an effect independent of telomerase catalytic activity, as confirmed using a dominant-negative hTERT (DN-hTERT) mutant. Transcriptomic profiling revealed that both CID-resistant iCasp9-NK cells and hTERT-iCasp9-NK cells share common gene expression signatures: upregulation of cell cycle-associated genes and downregulation of splicing-related factors, including HNRNPH1 and SNRPD3, accompanied by shared patterns of alternative splicing. Among apoptosis-related transcripts, BIRC3, which encodes c-IAP-2, a direct inhibitor of caspase 9, was consistently elevated in both "resistant" and "survived" NK cells. However, shRNA-mediated knockdown of BIRC3 failed to restore sensitivity to CID, indicating that BIRC3 upregulation is not the unique determinant of resistance and suggesting involvement of additional compensatory pathways. Overall, our findings define specific transcriptional signatures associated with evasion of NK cells from iCasp9-mediated apoptosis, implying the contribution of cell cycle progression, enhanced anti-apoptotic signaling, and alterations in splicing regulation, and highlighting the complex role of non-canonical hTERT functions in these adaptations. In the rational design of next-generation gene-modified NK cell therapies with improved safety and persistence, the uncovered insights should be considered.

Inflammatory bowel disease (IBD) is characterized by a refractory, relapsing inflammatory state driven by a multifaceted and poorly understood interplay between host mucosal metabolic disturbances and immune microenvironment dysregulation. To uncover robust non-invasive diagnostic indicators, this study implemented an integrated analytical framework combining advanced machine learning feature selection, penalized logistic regression modeling, and leave-one-dataset-out cross-validation to construct a novel diagnostic signature derived from metabolic cell death-related genes (MCDRGs). Systematic multiple-testing correction and rigorous data harmonization were applied across all independent discovery and validation cohorts to control for potential batch effects. Through this approach, we successfully identified a core three-gene candidate panel comprising indoleamine 2,3-dioxygenase 1 (IDO1), lipocalin 2 (LCN2), and solute carrier family 6 member 14 (SLC6A14). Methodologically, we demonstrated that the initial near-perfect apparent discrimination within the discovery cohort was mathematically attributable to quasi-complete data separation rather than systemic model overfitting. This identified signature exhibited consistent cross-cohort validation and correlated tightly with the coordinated infiltration and functional states of multiple mucosal immune cell subsets. Furthermore, independent clinical assays substantiated the synchronized elevation of these markers during active clinical phases, while orthogonal single-cell RNA-sequencing confirmed their prominent, cell-type-specific enrichment within the myeloid, epithelial, and stromal compartments of inflamed intestinal mucosa. Collectively, the identified MCDRGs, IDO1, LCN2, and SLC6A14, link metabolic dysregulation, immune infiltration, and regulated cell death, offering insights into IBD pathophysiology and providing a transcriptomic candidate signature with exploratory diagnostic potential for IBD stratification.

Dysregulation of cholesterol homeostasis is closely associated with the development of various cancers, but the underlying mechanisms remain unclear. This study found that the circadian clock gene Period 2 (PER2) is downregulated in oral squamous cell carcinoma (OSCC), promoting intracellular cholesterol synthesis and cell proliferation. Conversely, PER2 overexpression inhibits cell proliferation by suppressing intracellular cholesterol synthesis. Mechanistically, PER2 binds to Myosin Heavy Chain 9 (MYH9) and recruits Tripartite Motif Containing-21 (TRIM21) to promote ubiquitin-dependent degradation of MYH9, thereby inhibiting the cholesterol synthesis rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), reducing intracellular cholesterol synthesis, and suppressing OSCC proliferation. In OSCC xenograft models, overexpression of PER2 or treatment with the HMGCR inhibitor simvastatin significantly inhibited OSCC growth, with the combination showing markedly enhanced anti-OSCC efficacy compared to either approach alone. To explore the circadian regulatory role of PER2, overexpression or silencing experiments in OSCC demonstrated that PER2 modulates the circadian rhythms of HMGCR expression, intracellular cholesterol, and cell proliferation. Further investigations in subcutaneous OSCC mouse models revealed sex-specific circadian rhythms in PER2 and HMGCR expression, intracellular cholesterol, cell proliferation, and tumor growth in OSCC cells. Specifically, the acrophases of these indicators in OSCC of male mice were delayed relative to females, and the mesors of HMGCR expression, intracellular cholesterol, and cell proliferation were significantly higher than in females. The anticancer efficacy of simvastatin against OSCC in mice varied up to 2.5-fold across six different time points within 24 h, with broader time windows for optimal therapeutic efficacy and adverse effects in female mice than in males. This study identified a novel mechanism by which PER2 reprograms intracellular cholesterol, demonstrating that PER2 upregulation combined with simvastatin represents a potential novel strategy to improve the prognosis of OSCC patients, and clarifies that the anti-OSCC effect of simvastatin exhibits time-dependent and sex differences.

MLKL pseudokinase is a critical executioner of necroptotic cell death. MLKL drives necroptosis by forming pores in the cell membrane. A growing body of data indicates that, in addition to this well-established role, MLKL can promote cell survival in certain contexts. Moreover, pharmacological or genetic MLKL inhibition was shown to suppress in vivo growth of several tumor types. It was found that MLKL protects cancer cells from various cell death-inducing stimuli by promoting autophagy or preserving the mitochondrial function of the cells. It was proposed that both of these MLKL effects prevent parthanatos, a cell death type mediated by hyperactivation of PARP1 and subsequent PARP1-dependent chromosomal DNA degradation. In addition, MLKL was found to protect tumor cells from the death receptor-induced demise and trigger the secretion of the growth-promoting cytokines by the cells. Notably, MLKL-deficient mice are healthy, while pharmacological MLKL inhibitors are not significantly toxic to mice. Hence, targeting MLKL in vivo to block MLKL-dependent cancer cell survival is feasible. The mechanisms of the pro-survival MLKL effects is the subject of this review.

Disulfidptosis and cuproptosis are recently identified forms of regulated cell death whose component genes may harbor prognostic information in oral squamous cell carcinoma (OSCC). However, no validated multi-gene prognostic signature derived from these pathways has been established. Differentially expressed genes from two GEO discovery cohorts (GSE30784 and GSE37991) were intersected with 28 disulfidptosis- and cuproptosis-related genes. A LASSO-Cox model was developed in a TCGA oral HNSC training cohort (n&#x2009;=&#x2009;268) and validated in GSE41613 (n&#x2009;=&#x2009;97), GSE65858 (n&#x2009;=&#x2009;270), and GSE42743 (n&#x2009;=&#x2009;55). An extended clinical-genomic model incorporating age, AJCC stage, histological grade, alcohol use, radiation, and chemotherapy was evaluated in a complete-case subcohort (n&#x2009;=&#x2009;212). Single-cell RNA-seq data (GSE103322; 5,902 cells) were analyzed to characterize hub gene expression across cell types. Hub gene expression was validated by qRT-PCR and ELISA in OSCC cell lines. A four-gene signature (CDKN2A, FLNA, GLS, TLN1) was identified. The risk score was an independent prognostic factor (HR&#x2009;=&#x2009;1.36 per SD, 95% CI: 1.08-1.72, P&#x2009;=&#x2009;0.009) after adjustment for six clinical covariates. The extended combined model achieved a C-index of 0.669 and a 5-year AUC of 0.805, outperforming the extended clinical model (C-index 0.650; 5-year AUC 0.722) and gene-only model (C-index 0.563; 5-year AUC 0.684). The signature achieved significance in GSE41613 (P&#x2009;=&#x2009;0.046, HR&#x2009;=&#x2009;1.81) and GSE65858 (P&#x2009;=&#x2009;0.0013, HR&#x2009;=&#x2009;1.99), with a concordant trend in GSE42743 (P&#x2009;=&#x2009;0.079, HR&#x2009;=&#x2009;2.10). Single-cell analysis showed that CDKN2A was preferentially expressed in malignant cells, while FLNA and TLN1 were enriched in fibroblasts, consistent with ECM-mediated tumor-stromal crosstalk. CellChat revealed that high-activity malignant cells exhibited enhanced LAMININ, COLLAGEN, and THBS signaling. qRT-PCR and ELISA confirmed upregulation of all four hub genes in CAL-27 and SCC-9 cells (P&#x2009;<&#x2009;0.01). A four-gene prognostic signature derived from disulfidptosis- and cuproptosis-related genes provides independent prognostic value in OSCC and improves stratification when combined with standard clinical variables.

The Glucocorticoid-Induced Leucine Zipper (GILZ) is a key mediator of the anti-inflammatory effects of glucocorticoids, primarily within the immune system. Recent evidence has implicated GILZ as a secretive protein in goblet cells, with reduced expression linked to active Inflammatory Bowel Disease (IBD), suggesting a role in intestinal cell homeostasis. In this context, GILZ has been found also in enteroendocrine cells (EEC), but its role remained undefined. This study aimed at identifying GILZ-expressing EEC subtypes, dissecting how the secretive function is affected by inflammation in human ulcerative colitis (UC) and exploring its role in these cells. GILZ was predominantly expressed in glucagon-like-peptide-1 (GLP-1)-secreting L-cells, across all stages of EEC differentiation. GILZ was also expressed in serotonin (5HT)-producing enterochromaffin cells (EC), even though at low levels. Such an expression profile was supported by analysis of publicly available single-cell RNA sequencing datasets, identifying both EEC progenitors and mature subsets. Interestingly, confocal immunofluorescence localized GILZ to cytoplasmic granules partially co-staining with GLP-1-containing vesicles. Histological analysis of mucosal colonic biopsies revealed a global reduction in EEC during active UC as compared to healthy individuals and quiescent UC. Specifically, GILZ-expressing L-cells were significantly reduced in active UC and only partially restored in quiescent disease. In contrast, 5HT-producing EC cells were still reduced during active UC but fully recovered in quiescent disease. In vitro, NCI-H716 L-cell line-secreted products reduced IL-8 secretion in Caco2 epithelial cells, indicating an anti-inflammatory effect. This activity was attenuated following GILZ silencing. Interestingly, treatment with a recombinant TAT-GILZ protein directly diminished IL-8 in Caco2 cells. Collectively, our findings identify GILZ as a novel secretory product of L-cells with potential anti-inflammatory properties. Restoring GILZ secretion may represent a promising therapeutic strategy to mitigate chronic intestinal inflammation in UC.

Although iron accumulates in brain regions impacted by neurodegenerative diseases such as Alzheimer's and Parkinson's, how chronic elevated iron levels contribute to neuronal dysfunction remains unclear. Here, we show that sustained iron overload, but not acute exposure, leads to a state of ferroptotic stress where nerve cells remain viable but become hypersensitive to oxidative injury. Retinoic acid-differentiated SH-SY5Y neuronal cells were exposed to acute (6-8&#x2009;h) or chronic (9 days) iron loading to model transient versus prolonged age-related iron stress. While acute iron exposure produced minimal biochemical changes and did not sensitize cells to oxidative or ferroptotic challenges, chronic iron exposure induced ferritin upregulation, mitochondrial superoxide accumulation, suppression of GPX4 expression, elevated lipid peroxidation and loss of cellular glutathione (GSH). In addition, chronic but not acute GSH depletion by buthionine sulfoximine (BSO) recapitulated the iron-induced phenotype. Cells under chronic ferroptotic stress exhibited increased sensitivity not only to the ferroptosis inducer RSL-3 but also to hydrogen peroxide. Ferrostatin-1 significantly mitigated these effects suggesting that lipid peroxidation drives this state. Together, these findings demonstrate that, in contrast with acute exposure, chronic disruption of iron homeostasis with consequent GSH depletion remodels cellular redox homeostasis over time, inducing a state we term chronoferroptosis: a persistent ferroptotic adaptation characterized by coordinated alterations in iron-handling and antioxidant defense proteins that may represent early vulnerability to neurodegenerative pathology. Thus, these studies highlight the importance of sustained stress paradigms for modeling the progressive nature of neurodegenerative diseases.Graphical abstract illustrating how prolonged iron or BSO exposure drives a persistent ferroptotic stress state in RA differentiated SH-SY5Y cells. These cells were subjected to either acute exposure for 6-8&#x2009;h or chronic exposure for 9 days. Acute iron or BSO treatment caused minimal biochemical changes and did not increase vulnerability to oxidative or ferroptotic challenges. In contrast, chronic exposure stimulated ferritin production, mitochondrial superoxide buildup, GSH depletion, reductions in GPX4 expression and increased lipid peroxidation without altering cell viability. However, these chronically stressed neuronal cells became markedly more sensitive to secondary stressors such as RSL-3 or hydrogen peroxide resulting in decreases in cell viability, whereas viability was not altered in acutely treated cells.

Machine learning (ML) has the potential to revolutionize antibody design and selection, but its success depends on access to well-curated datasets of antibody-antigen interactions. We developed a synthetic Fab yeast display library optimized for seamless integration with ML processes, focusing on sequence diversity within the complementary determining region heavy chain CDRH3 loop. The library incorporates key sequence features derived from human B cell repertoires captured in a compact antigen recognition module (ARM) format. Built with the VH1-69 heavy chain and four light chains, the library was evaluated against ten human and murine cell surface antigens, including programmed cell death ligand 1 (PD-L1), T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domains (TIGIT), and roundabout guidance receptor 1 (ROBO1). This approach yielded hundreds of antibodies with robust biophysical properties, some of which were validated by flow cytometry and immunohistochemistry. Furthermore, ML analysis identified additional antibodies for ROBO2 and PD-L2 from the aggregate sequencing data. The publicly available dataset establishes an ML-compatible framework designed to accelerate and streamline antibody discovery and development. A record of this paper's transparent peer review process is included in the supplemental information.

Glioblastoma (GBM) is shielded by both the blood-brain barrier (BBB) and an immunosuppressive tumor microenvironment. Here, we develop a chimeric biohybrid nanovesicle (BEV-RVG29-PTX) that integrates viral tropism, bacterial vesiculation, and chemotherapeutic cytotoxicity into a single genetically programmable platform. Genetic fusion of rabies virus glycoprotein 29 (RVG29) to the AIDA1 autotransporter translocator domain enables robust, autonomous surface expression on bacterial extracellular vesicles (BEVs) without the need for chemical conjugation. The BEV-RVG29-PTX drives receptor-dependent BBB transcytosis and achieves efficient glioma accumulation. Encapsulated paclitaxel (PTX), otherwise restricted by BBB impermeability, is effectively delivered to intracranial tumors and induces reactive oxygen species-driven immunogenic cell death. Bone marrow-derived dendritic cells immune-activation experiments further confirmed an approximately 2-fold increase in CD80/CD86 activation. Synergizing with the pathogen-mimetic characteristics of BEVs, these signals also elicit an approximately 2-fold increase in intratumoral CD8&#x207a; T-cell infiltration, overcome immune exclusion, and achieve durable tumor control with extended survival in orthotopic GBM models. Accordingly, this virus-bacteria-drug biohybrid strategy enables targeted brain delivery while simultaneously amplifying antitumor immunity, offering a promising and translatable approach for GBM treatment.

Cuproptosis is a recently identified form of copper-dependent cell death that depends on ferredoxin 1 (FDX1)-mediated protein lipoylation. Here, we reveal that CD8+ T cell-mediated antitumor immunity enhances tumor cell susceptibility to cuproptosis, leading to a more potent tumor-suppressive effect of cuproptosis inducers in immunocompetent hosts compared with immunodeficient ones. Mechanistically, cuproptotic tumor cells act as a form of immunogenic cell death, releasing damage-associated molecular patterns that activate dendritic cells and enhance antitumor immunity. Reciprocally, CD8+ T cell-derived interferon (IFN)-&#x3b3; enhances FDX1 transcription in tumor cells by activating the signal transducer and activator of transcription 1 (STAT1)-IFN regulatory factor-1 (IRF1) signaling axis, resulting in heightened tumor cell sensitivity to cuproptosis. Consequently, combining a cuproptosis inducer with anti-programmed cell death ligand 1 (PD-L1) therapy amplifies tumoral cuproptosis and demonstrates efficacy in overcoming PD-L1 therapy resistance across multiple preclinical models. Our findings unveil a previously unrecognized connection between antitumor immunity and cuproptosis and highlight a potential therapeutic approach to counteract tumor immunotherapy resistance by targeting this unique cell death pathway.

Immune checkpoint inhibitor (ICI) therapy improves survival in head and neck squamous cell carcinoma (HNSCC), yet only some patients benefit, highlighting the need for early identification of responders and functional evaluation of tumor-immune interactions. We developed an autologous coculture platform combining patient-derived organoids (PDOs) with peripheral blood mononuclear cells (PBMCs) to assess immune responses and correlate preclinical findings with clinical outcomes. PDOs from HNSCC specimens were expanded and cocultured with PBMCs, followed by treatment with ICIs targeting programmed cell death protein 1 (PD-1), lymphocyte-activation gene 3 (LAG-3), or their combination. PBMCs were enriched for CD4+ and CD8+ T cells (&#x223c;90%) and CD16+CD56+ natural killer cells, with baseline exhaustion (LAG-3+, CTLA-4+). T cell-mediated tumor killing occurred in 44% of cases and was accompanied by Th1/Th17 cytokine signatures. ICI treatment modestly shifted T-cell phenotypes, reducing exhaustion (PD-1) and enriching cytolytic subsets (CD4+IFN-&#x3b3;+, CD8+TOX+). Interleukin (IL)-17A emerged as a relevant cytokine, correlating with FasL secretion and suggesting activation of the Fas/FasL axis. High circulating IL-17A levels in treatment-na&#xef;ve HNSCC patients were associated with improved survival, whereas other cytokines showed no prognostic value. In a noninterventional observational trial, increases in IL-17A positively correlated with favorable ICI responses, indicating its potential as a biomarker. PDO viability decreased in response to ICIs in a patient-specific manner, consistent with cytokine signatures and clinical outcomes. Collectively, this autologous PDO-PBMC coculture platform, combined with longitudinal cytokine profiling, captures patient-specific tumor-immune interactions and interpatient heterogeneity, providing a framework for real-time response prediction, biomarker discovery, and personalized immunotherapy in HNSCC.

Despite effective antiretroviral therapy, HIV persists in the central nervous system (CNS) and may contribute to neuroinflammation and cognitive impairment. How viral persistence, immune responses, and regional CNS T-cell architecture relate to cognitive functioning remains unclear. We performed a cross-sectional, multi-compartmental immune-genomic study in 12 people with HIV on long-term viral suppression enrolled in the Last Gift rapid autopsy program. Quantitative HIV reservoir measures (total-episomal DNA, unspliced-multiply spliced RNA) and paired &#x3b1;&#x3b2; T-cell receptor repertoire (TCRR) sequencing were performed in peripheral blood mononuclear cells and five CNS regions: hippocampus, frontal motor cortex, basal ganglia, occipital cortex, and spinal cord. Cognitive performance was assessed within one year of death. Tissue-resolved associations between cognition and HIV reservoir, TCRR architecture (richness, diversity, clonality), and pathogen-specific T-cell clonotypes (HIV, CMV, EBV, and riboflavin derivatives) were evaluated using participant-clustered multivariable models. False discovery rate was applied. HIV DNA and RNA were detectable across all tissues but were not associated with cognitive performance or TCRR metrics. Peripheral TCRR architecture was unrelated to cognition, whereas higher TCRR richness and diversity in the hippocampus and spinal cord were associated with worse verbal, motor, and attention/working memory scores. Higher TCRR richness in the spinal cord was also associated with better recall. T-cell receptor clonotype frequency distributions differed across CNS regions, consistent with regional immune compartmentalization. Epitope-inference analyses revealed pathogen-dependent associations: higher number of HIV-specific T-cell clonotypes in the basal ganglia was associated with better global and attention/working memory scores, whereas riboflavin derivative-specific clonotypes in frontal motor cortex were associated with better motor performance. CMV-specific clonotypes showed nominal associations with worse learning and memory. CNS-localized T-cell receptor architecture and antigenic imprinting related more closely to neurocognitive variability than quantitative measures of HIV persistence under viral suppression, highlighting regional specialization of T-cell responses as a potential correlate of brain health.

Aberrant phosphatidylinositol 3-kinase (PI3K) activation drives many cancers, but PI3K inhibitors like Pictilisib often induce cytostasis rather than cytotoxicity, limiting their therapeutic potential. Here we demonstrate that PI3K inhibition combined with nutrient stress triggers methuosis, a non-apoptotic form of programmed cell death characterized by dysregulated macropinosomes. This response occurs selectively in PI3K-aberrant cancer cells that maintain macropinocytic uptake despite PI3K inhibition. Methuosis-associated vacuoles originate from macropinosomes that retain endosomal markers but fail to undergo lysosomal fusion. Active macropinocytic uptake is essential for methuosis, as demonstrated by suppression with EIPA and Bafilomycin A1, whereas the AKT inhibitor MK2206 has no effect, establishing that direct PI3K inhibition, rather than AKT signaling, is required. Mechanistically, PI3K blockade prevents conversion of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) to phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) causing PI(4,5)P2 to accumulate on internalizing macropinosomal membranes. This aberrant PI(4,5)P2 enrichment impairs ion channel function across multiple channel families, disrupting intracellular osmotic balance. Ion dysregulation triggers aquaporin-1-mediated water influx, driving catastrophic vacuolar expansion and cell death. Although Pictilisib activates pro-survival autophagy, this fails to prevent methuosis-mediated cytotoxicity. In xenograft models, dietary restriction synergizes with Pictilisib to suppress tumor growth, correlating with pronounced intratumoral vacuolization. These findings reveal that combining PI3K inhibition with nutrient restriction converts cytostatic responses into methuosis-driven cytotoxicity via PI(4,5)P2-dependent macropinocytic dysregulation, providing a rational pharmacologic-dietary strategy to enhance PI3K-targeted cancer efficacy.

Accurate assessment of cell viability is fundamental in biomedical research, with applications ranging from cancer biology to drug discovery. Traditional assays based on metabolic activity or membrane integrity are cost-effective but limited to endpoint measurements, often overlooking the dynamic nature of cell survival and death. Here, we present a workflow that combines live cell imaging with automated image analysis to provide continuous, unbiased quantification of cell viability. Using a fluorescent marker of membrane integrity and nuclear staining, time-lapse microscopy captures cell fate dynamics under diverse experimental conditions. Automated segmentation and classification, implemented through the open-source DIPlib library, enable reproducible distinction between viable and non-viable cells while minimizing observer bias. The method is scalable, adaptable to different imaging platforms and suitable for high-throughput applications.

Lung cancer is a leading cause of cancer-related mortality, with metastasis significantly reducing patient survival. Interleukin 11 (IL11), a member of the IL-6 cytokine family, has been associated with cancer progression, yet its role in non-small cell lung cancer (NSCLC) metastasis remains unclear. This study analyzed public datasets and demonstrated that IL11 is upregulated in NSCLC and correlates with lymphatic metastasis and poor prognosis. Functional assays revealed that IL11 enhances lung cancer cell migration through upregulation of matrix metalloproteinase 12 (MMP12). Mechanistically, IL11 acts via the IL11 receptor subunit alpha (IL11RA)/ IL6 cytokine family signal transducer (IL6ST) complex to activate the PI3K/Akt/NF-&#x3ba;B signaling pathway, which in turn drives MMP12 expression and promotes metastatic behavior. Notably, the clinically approved selective estrogen receptor modulator bazedoxifene effectively inhibited IL11-induced signaling, reduced MMP12 levels, and suppressed cancer cell migration in vitro. In an orthotopic lung cancer mouse model, IL11 knockdown significantly reduced tumor growth and intrapulmonary spread, accompanied by decreased IL11, MMP12, and phosphorylated NF-&#x3ba;B p65 levels in lung tissues. These findings uncover a novel IL11-driven pathway contributing to metastatic behavior in NSCLC and identify the IL11/IL11RA/IL6ST axis as a potential therapeutic target.Schematic representation of bazedoxifene-mediated suppression of IL11-induced pro-metastatic signaling in lung cancer. Non-small cell lung cancer (NSCLC) cells secrete IL11, which binds to the IL11RA/IL6ST receptor complex and activates the PI3K/Akt signaling pathway. This activation enhances NF-&#x3ba;B transcriptional activity, leading to upregulated MMP12 expression and promoting lung cancer cell migration. Notably, bazedoxifene effectively blocks IL11-induced PI3K/Akt activation, thereby suppressing NF-&#x3ba;B signaling and reducing MMP12 expression, which ultimately attenuates NSCLC metastasis.

Non-small cell lung cancer (NSCLC) patients with oncogenic driver mutations such as EGFR, ALK or ROS1 (mutant-type [MT]) exhibit poor responses to PD-1/PD-L1 immune checkpoint inhibitors (ICIs) compared to wild-type (WT) patients. The mechanisms underlying this limited response to ICIs in MT patients remain unclear. This study aimed to identify key immune biomarkers and elucidate immune cell dynamics in peripheral blood contributing to ICI resistance in MT-NSCLC. A total of 262 NSCLC patients who received PD-1/PD-L1 inhibitor monotherapy between February 2018 and July 2024 were included. Of these, 43 patients were assigned to the discovery cohort, where immune profiling was performed on peripheral blood mononuclear cells using Cytometry by Time-of-Flight (CyTOF) at baseline and cycle 2, day 1 (C2D1). The findings were validated in an independent cohort (n=57) using flow cytometry. Baseline CXCR3+ CD127+ effector CD8+ T cells were significantly elevated in WT compared to MT patients (P&#xa0;=&#xa0;0.0120) and were a robust predictor of favorable response (AUC&#xa0;=&#xa0;0.745). Patients with CXCR3+ CD127+ CD8+ T cell frequencies above 3.54% exhibited superior progression-free survival (PFS, P&#xa0;=&#xa0;0.0005) and overall survival (OS, P&#xa0;=&#xa0;0.0012). In contrast, MT patients demonstrated a distinct reduction in CD27+ PD-1&#x207b; effector memory CD4+ T cells during treatment, correlating with poor outcomes (AUC&#xa0;=&#xa0;0.716). This reduction was associated with diminished conventional dendritic cell (cDC) abundance, suggesting impaired T-cell differentiation and function in MT patients. These findings were consistently validated using flow cytometry in an independent cohort. Distinct immune cell profiles highlight elevated baseline CXCR3+ CD127+ effector CD8+ T cells predicting favorable outcomes and impaired cDC-CD4+ T cell dynamics as critical contributors to anti-PD-1/PD-L1 resistance in MT-NSCLC.

High-content screening (HCS) is a powerful approach for rapidly and efficiently assessing the harmfulness of numerous compounds across a wide range of cultured cell types. We recently developed a fully automated, miniaturized HCS wet-plus-dry pipeline (MITOMATICS) which leverages mitochondrial morphology as a sensitive and dynamic biomarker of cellular health or damage. Mitochondria are indeed not only vital for energy production and homeostasis, but also serve as critical gatekeepers of apoptotic cell death. MITOMATICS incorporates a proprietary software tool (MitoRadar) designed in-house to perform fast, comprehensive and cost-effective analysis of mitochondrial morphology in live cells. Together, the pipeline and its associated big data analytics software provide a valuable framework for early detection of acute mitotoxic effects of chemicals agents or physical stressors. To illustrate this, we present here a complete protocol for quantifying the impact of the pesticide chlorpyrifos-methyl on mitochondrial morphology of human lung epithelial BEAS-2B cells. Our results show that chlorpyrifos-methyl, even as a single compound, induces profound disruptions in mitochondrial subcellular structure. Beyond this case study, MitoRadar opens up promising avenues for investigating mitotoxicity across diverse cell types and environmental exposures, paving the way for a new generation of cellular diagnostics that could be of interest to the cell death community.

BECLIN1 is a central regulator of autophagy and endocytic trafficking essential for epithelial homoeostasis. While complete intestinal epithelial loss of BECLIN1 causes fatal enteritis originating in the small intestine, the consequences of its partial loss in the gut remain unclear. Given that BECLIN1 expression can vary in human disease, we investigated whether reduced BECLIN1 is sufficient to impair gut barrier function. Heterozygous Becn1 deletion (Becn1IEC+/-) in the mouse intestinal epithelium caused subtle but significant defects. These included shortened small intestines and altered epithelial architecture, despite preservation of basal autophagy, implicating trafficking-related functions. Supporting this conclusion, Becn1IEC+/- small intestinal epithelial cells showed modest increases in RAB5+ve vesicles, redistribution of E-CADHERIN and F-actin along lateral membranes and altered apico-basal cell morphology. Given the absence of overt small intestinal epithelial disruption or inflammation, as seen with complete loss of BECLIN1, we next addressed whether BECLIN1 insufficiency manifests a phenotype under stress or in other gut regions. Indeed, in the colon, Becn1IEC+/- mice exhibited reduced colonic crypt length, baseline goblet cell loss and reduced mucin production, particularly in mature goblet cells, indicating vulnerability of the mucus barrier. When challenged with dextran sulfate sodium (DSS), Becn1IEC+/- mice exhibited greater weight loss, higher disease activity, more severe histological colitis, and disproportionate loss of neutral mucins, with inflammation confined to the mucosa. Together, these findings show that BECLIN1 insufficiency does not trigger spontaneous inflammation but destabilises epithelial organisation and barrier defence, thereby sensitising the gut to inflammatory challenge and further positioning BECLIN1 as a threshold-dependent determinant of intestinal resilience.

Inhibition of the Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) represents a promising therapeutic strategy for cancer. However, resistance to SHP2 inhibition, mediated by multiple mechanisms, has limited the clinical efficacy of SHP2 inhibitor monotherapy. Herein, we identified that nicotinamide phosphoribosyltransferase (NAMPT) inhibition could potentially overcome resistance to SHP2 inhibition in tumor cells. Compound A4 was identified as the most potent dual inhibitor targeting SHP2 and NAMPT, exhibiting high inhibitory activity against both SHP2 and NAMPT. A4 effectively inhibited proliferation in SHP099-insensitive tumor cell lines and reversed programmed cell death ligand 1 (PD-L1)-mediated immunosuppression. Furthermore, A4 displayed significant in vivo antitumor efficacy in an MDA-MB-231 mouse model and strongly promoted in vivo antitumor immunity in a 4T1 mouse model. Our results identified A4 as a promising dual SHP2 and NAMPT inhibitor, providing a novel therapeutic strategy for overcoming resistance to allosteric SHP2 inhibition.