Chronic kidney disease (CKD) has emerged as a critical public health challenge worldwide, and organ donor shortages underscore the urgent need for alternative therapeutic strategies. Advances in stem cell technologies have enabled the generation of kidney organoids, providing innovative platforms to model renal development, investigate disease mechanisms, support drug discovery, and explore applications in regenerative medicine. Yet, limitations such as immature tissue architecture, insufficient vascularisation, and unaddressed safety concerns still hinder their translation into regenerative medicine. In this review, we summarise the fundamentals of kidney development, current differentiation approaches, and the signalling and epigenetic mechanisms underlying organoid lineage specification. We further highlight the roles of bioengineering innovations and single-cell transcriptomics in establishing evaluation frameworks and enhancing structural complexity. We finally emphasise that existing optimisation frameworks, primarily focused on improving differentiation efficiency and enforcing relatively restricted lineage specification, may prove inadequate for bridging the gap to clinical translation. Instead, the most promising paradigm shift involves the convergence of bioengineering modulation and high-resolution functional assessment to facilitate the synchronised advancement of organoid complexity and physiological utility.

Plant roots dynamically respond to environmental changes and serve as an ideal system for studying cell development and gene regulation. Recent advances in imaging-based spatial transcriptomics have enabled high-resolution mapping of gene expression while preserving spatial context. However, existing sample preparation techniques remain inadequate for handling rigid plant tissues such as crop roots. Here, we present a detailed and practical protocol for preparing rigid plant tissue samples for imaging-based spatial transcriptomics. The workflow ensures effective tissue handling while maintaining RNA integrity and spatial organization. Within approximately eight days, samples can be processed and mounted onto commercial slides, making them ready for subsequent probe hybridization and imaging. This protocol also includes an integrated sample attachment test performed to assess slide quality. It has been optimized to produce consistent and reliable results across experiments. Overall, our method provides a robust solution for spatial transcriptomic analysis in rigid plant tissues, facilitating broader application of these technologies in plant research. Key features • Builds upon the method developed by Zhu et al. [1] and introduces an optimized sample preparation protocol for imaging-based spatial transcriptomics in rigid rice roots. • Ensures effective tissue fixation and sectioning, while preserving RNA integrity and spatial organization. • Includes an integrated sample attachment test to assess the adhesion of tissue sections to commercial slides. • Requires approximately 8 days to complete the sample preparation, with another 6 days for the attachment test.

Respiratory syncytial virus (RSV) bronchiolitis is the leading cause of hospitalization in infancy and exhibits pronounced age-dependent clinical heterogeneity. Fever becomes increasingly prevalent with age, yet whether febrile representation reflects a uniform inflammatory and immune phenotype across infancy remains unclear. In this prospective cohort of infants hospitalized with RSV bronchiolitis, we performed an integrated analysis of clinical features, pharyngeal microbiome composition, host transcriptomic profiles, and host-microbe interaction networks, with particular attention to age-related variation in fever-associated patterns. Clinically, fever prevalence exhibited a strong age-dependent increase across infancy. Correspondingly, canonical correspondence analysis identified age and fever as dominant gradients related to variation in both pharyngeal microbiome composition and host gene expression. Although no significant age-dependent correlations were observed at the global microbial and host transcriptomic levels in the fever-age interaction model, distinct patterns of microbial and host responses related to fever were observed across different age groups. Specifically, ranked gene set enrichment analysis indicated that febrile infants in early infancy showed relative attenuation of host defense-related programs, whereas older infants showed stronger enrichment of antiviral and inflammatory effector pathways, with more selective regulatory and signaling-associated patterns in late infancy. Integrated host-microbe network analysis further delineated a coherent developmental trajectory of fever-associated interaction architectures, evolving from densely interconnected regulatory networks in early infancy to modular, selectively coupled, host-centered configurations with advancing age. Together, febrile responses in RSV bronchiolitis should not be interpreted as a uniform biological phenotype across infancy and support age-aware interpretation of fever in pediatric RSV infection.

Bone marrow suppression is a common side effect of radiotherapy and a major cause of mortality following exposure to medium or high doses of total body irradiation (TBI). In this study, naked mole rats (NMRs) have been demonstrated to exhibit significantly greater tolerance to high-dose TBI than mice. Surviving NMRs maintain red blood cell (RBC) counts and restore the bone marrow microenvironment. A comprehensive single-cell RNA sequencing (scRNA-seq) analysis of NMR bone marrow cells in response identified two TBI-resistant subpopulations of HSPCs (C19 and C24) in NMRs, and C24 increased post-TBI. These subpopulations exhibit unique regulatory mechanisms related to oxidative stress damage, cell cycle regulation, DNA repair, and HSPC proliferation and differentiation. These mechanisms contribute to the enhanced survival of NMRs under high-dose TBI conditions. These findings suggest that NMRs serve as a valuable model for seeking radioprotection mechanisms and potential therapeutic and protective strategies, particularly in the context of long-term or high-dose irradiation therapies.

Breast cancer incidence is rising globally, presenting challenges such as treatment side effects and drug resistance. Bufalin is a bufadienolides compound with potential anti-cancer effects. This study shows that bufalin inhibits malignant proliferation of MDA-MB-231 and MCF-7 cells and protects mice against breast cancer. Of note, GTF3C4 was identified as the target protein by Limited Proteolysis-Mass Spectrometry. GTF3C4 is overexpressed in breast cancer and associated with poor prognosis. RNA sequencing analysis reveals that the PI3K/AKT signaling pathway is a key contributor. Using cell thermal shift assays, drug affinity response target stability assays, and surface plasmon resonance, it was verified that bufalin can specifically bind to GTF3C4. Bufalin reduces GTF3C4 protein levels in vivo and in vitro, effectively inhibiting breast cancer progression by suppressing the PI3K/AKT signaling pathway. After the knockdown of GTF3C4, the PI3K/AKT signaling pathway is also suppressed, thereby inhibiting the proliferation of breast cancer cells and promoting apoptosis. Single-cell RNA sequencing results indicated that bufalin reduces the proportions of macrophages, neutrophils, and monocytes, and affects the strength of receptor-ligand signals between cells. Collectively, this study demonstrates that bufalin targets GTF3C4 to inhibit the PI3K/AKT pathway and remodels the tumor microenvironment, thereby hindering the malignant progression of breast cancer.

Innate immune activation is a major driver of unmodified in vitro-transcribed (IVT) mRNA degradation; however, how modified IVT mRNAs are degraded, and the related regulation mechanisms, remain poorly understood. Through a focused screen of viral- and host-derived immune suppressors, we identify 13 factors that enhance mRNA performance, with SOCS1 and the coronaviral membrane protein (M) emerging as the most potent. Multi-omics analyses reveal that pseudouridine-modified IVT mRNA undergoes rapid deadenylation and predominant 3'-5' decay, followed by bidirectional degradation, closely resembling endogenous mRNA decay kinetics, and is extensively associated with canonical mRNA decay machineries. Mechanistically, IVT mRNA activates IFN-β signaling, which promotes processing body (P-body) formation and XRN1-mediated 5'-3' degradation. Suppression of IFN signaling by SOCS1 or M markedly enhances mRNA expression across diverse cell types, organoid systems, and murine disease models. Together, these findings define a type I interferon-P-body-XRN1 axis that constrains modified IVT mRNA stability and provides a framework for enhancing mRNA therapeutics.

4092 Background: The SCALE-1 Phase Ib trial demonstrated that short-course neoadjuvant radiotherapy combined with toripalimab and chemotherapy, followed by esophagectomy, exhibited manageable toxicity and promising efficacy in patients (pts) with resectable locally advanced ESCC (RLaESCC). This Phase II trial aims to further valuate efficacy and safety of this novel short-course regimen in a larger cohort. Methods: RLaESCC pts with clinical stages cT3-4aN0M0/cT1-4aN+M0 received neoadjuvant paclitaxel (135 mg/m 2 ), carboplatin (AUC=5), and toripalimab (240 mg) every 3 weeks for two cycles. Short-course neoadjuvant radiotherapy (30 Gy/12f; 5 days per week) was administered between the two doses of neoadjuvant immune-chemotherapy. Compared to SCALE-1, high-risk nodal regions were selectively included in the irradiation volume. Weekly weight changes were used to assess preoperative waiting time. Esophagectomies were scheduled 8-10 weeks after completing neoadjuvant treatment. The primary endpoint was pathological complete response (pCR) rate, with secondary endpoints included disease free survival (DFS), overall survival (OS) and safety. Results: From April 28, 2024 to February 26, 2025, 63 pts were enrolled (intention-to-treat population, ITT), with stage distribution:I (3.2%), II (14.3%), III (63.5%) and IVA (19%). Following the exclusion of 3 pts who refused surgery, pathological assessment of the surgical specimens revealed that 56.7% (34/60) pts achieved pCR (ypT0N0 or ypTisN0) and 91.7% (55/60) achieved MPR. The median number of resected lymph nodes was 19 (range: 6–45). At a median follow-up of 23.9 months (cutoff date: Jan 19, 2026), median OS and DFS were not reached. In the ITT population, the 2-year OS and DFS rates were 90.4% (95% Cl: 82.4-98.4%) and 81.5% (95% Cl: 70.5-92.5%), respectively. Postoperative complications occurred in 45% (27) pts, with 18.3%(11) experiencing Grade ≥3 events. The most frequent Grade 3 and above complications were anastomotic leakage (n=4) (with or without pleural effusion or hemothorax) and airway mucus obstruction (n=4). Moreover, one case of postoperative delirium was observed (Grade IV), and one patient died of septic shock secondary to an anastomotic leak. All 63 pts completed neoadjuvant radiotherapy. However, 2 pts discontinued the second dose of toripalimab, and 16 pts required dose reductions during the second cycle of neoadjuvant chemotherapy. The most frequent grade 3/4 adverse events were neutropenia (n=27, 42.9%) and leukopenia (n=21, 33.3%). Notably, no grade 3 esophagitis or pneumonitis occurred. Conclusions: The SCALE regimen demonstrated potent antitumor activity, achieving 56.7% pCR and 91.7% MPR. Favorable 2-year OS and DFS further highlight its potential to improve long-term outcomes for RLaESCC pts. Clinical trial information: NCT05424432 .

Usher syndrome can cause loss of vision, hearing, and balance. There are four clinical subtypes, USH1-4, which are associated with mutations in genes important for structure, function, and survival of photoreceptor cells in the retina and sensory hair cells in the inner ear. Genetic mutations in the USH2A gene, which encodes usherin protein, are the most common cause of Usher syndrome worldwide, with c.2299delG (p.Glu767Serfs∗21) being the most frequent pathogenic variant. An investigational antisense oligonucleotide (ASO) for USH2A c.2299delG, QR-421a, designed to bypass the mutation, has already shown promise in phase 1/2 clinical trials. While recently developed chemistry provides longer ASO half-lives, repeated injection of ASOs may be required to provide long-term efficacy. To overcome this limitation, we screened novel USH2A exon 13 skippers and 20 AAV capsids with the goal of developing a vectorized ASO exon skipping strategy. Optimized vectors and skippers were evaluated in inner ear and retinal organoids derived from human stem cell lines bearing the USH2A c.2299delG mutation. The data revealed enhanced skipping of the pathogenic exon, offering an alternative strategy for treatment of USH2A patients using a single local injection which may prevent progression of vision and hearing loss.

Gastric cancer (GC) is a common malignant tumor with high mortality, recurrence, and metastasis rates. Compound Kushen injection (CKI) combination chemotherapy has been clinically used for the treatment of GC in China for many years, but its underlying mechanisms of action remain unclear. Recent reports have highlighted the important role of the competing endogenous RNA (ceRNA) mechanism of noncoding RNA (ncRNA) and messenger RNA (mRNA) formation in GC and other tumors. This study aimed to investigate the effects of CKI on GC from the ceRNA perspective. We confirmed the inhibitory effect of CKI on GC in mouse models and cell lines. By examining the GC cell lines sensitive to CKI treatment, we developed the CNScore method to analyze the ceRNA network, revealing that the CKI-GC ceRNA network promotes GC proliferation and metastasis through the PTPRG-AS1/hsa-miR-421/KITLG axis. Finally, we constructed GC cell models with PTPRG-AS1 overexpression or knockdown and GC liver metastasis models and found that PTPRG-AS1 can sponge hsa-miR-421, releasing KITLG and promoting GC proliferation and metastasis through the KITLG/KIT pathway. Taken together, CKI can suppress these malignant phenotypes by regulating the PTPRG-AS1/hsa-miR-421/KITLG axis.

Colorectal cancer is one of the most common malignant tumors worldwide, significantly impacting human health. Cantharidin (CTD), an active compound derived from the Spanish fly, exhibits antitumor properties. Its derivative, norcantharidin (NCTD), is synthesized by removing methyl groups from positions 1 and 2 of cantharidin. NCTD has demonstrated lower toxicity while maintaining similar antitumor effects compared to CTD. However, the mechanism by which NCTD exerts its effects against colorectal cancer remains unclear. Here, we conducted a comprehensive analysis of the effects of NCTD on colorectal cancer both in vitro and in vivo. Whole-transcriptome sequencing and bioinformatics tools were employed to identify potential key targets of NCTD in the treatment of colorectal cancer. Additionally, we designed folate-receptor-targeting NCTD liposomes (FA-NCTD) and assessed their anticancer efficacy in vivo. NCTD effectively inhibited cell viability, clonal formation, and migration in HCT116 and HT-29 cell lines. NCTD also induced apoptosis, influenced the cell cycle, altered mitochondrial membrane potential, and increased reactive oxygen species levels. The whole-transcriptome sequencing and bioinformatics analysis identified TRAF5 as a key target for NCTD’s action against colorectal cancer. Furthermore, NCTD was found to regulate the TRAF5/NF-κB signaling pathway in both HCT116 and HT-29 cells. The FA-NCTD liposomes demonstrated effective tumor targeting and significantly inhibited tumor growth in vivo. This result showed that NCTD effectively suppresses the malignant proliferation of colon cancer cells by modulating the TRAF5/NF-κB signaling pathway and inducing programmed apoptosis, thereby offering a novel strategy for colorectal cancer treatment. The prepared FA-NCTD liposomes provide a promising approach for achieving the precise targeting and controlled release of NCTD.

Objective: To explore the key molecules regulated by norcantharidin (NCTD) in colon cancer treatment. Methods: We used cell counting kit-8 and 5-ethnyl-2′-deoxyuridine/Hoechst staining assays to study the effects of NCTD on cell proliferation in colon cancer. Annexin V-fluorescein isothiocyanate/propidium iodide staining was used to evaluate apoptosis, whereas Transwell assays were conducted to evaluate migration and invasion. We performed RNA sequencing to analyze the changes in gene expression after treatment. Differential analysis was performed using differential expression sequencing 2 (Deseq2) in R. Cytoscape was used to construct a competing endogenous RNA (ceRNA) network and Gene Expression Omnibus (GEO) datasets were used to validate sine oculis homeobox homolog 4 (SIX4) expression in colon cancer tissues. Furthermore, the prognostic potential of SIX4 was evaluated using receiver-operating characteristic curves. We conducted an immune infiltration analysis to explore the SIX4 relationship with the immune microenvironment in colon cancer. Finally, SIX4 expression, pan-cancer prognosis, tumor mutation burden (TMB) correlations, microsatellite instability (MSI), and mismatch repair (MMR) were analyzed. Results: NCTD inhibited colon cancer cell proliferation (P < .0001), induced apoptosis (P = .0007), and suppressed the migration and invasion of colon cancer cells. The H19/miR-193b-3p/SIX4 axis was identified as the key ceRNA network involved in the anticancer activity of NCTD. SIX4 is highly expressed in colon cancer tissues, shortening patient survival and affecting immune infiltration. A pan-cancer analysis showed that SIX4 overexpression affects the survival of various cancers. Finally, we correlated SIX4 expression with TMB, MSI, and MMR expression. Conclusion: NCTD inhibits the malignant behaviour of colon cancer cells. SIX4 is abnormally expressed in multiple tumor types, significantly affecting the overall survival of patients with cancer, and is a core regulatory target of NCTD in the treatment of colon cancer.

BackgroundEvidence for the benefits of resveratrol (Res) in the treatment of asthma is progressively accumulating. However, the full spectrum of its molecular targets and the precise mechanisms remain incompletely characterized.MethodTargets of Res were obtained from Swiss Target Prediction, TCMCP, and DrugBank. Targets of asthma were obtained from DisGeNET, Therapeutic Target Database, GeneCards, and DrugBank. Intersecting target genes were identified by using jvenn. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomics (KEGG) enrichment analyses were performed using the R package clusterProfiler in R version 4.4.0. Protein–protein interaction networks were constructed using Cytoscape 3.9.1 software. Molecular docking validation of the binding capacity between Res and targets was performed using AutoDock Vina and visualized in PyMOL version 3.0.4. ELISA and Western blotting were used to verify the reliability of Res effects on the top five targets in both house dust mite (HDM)-induced asthma mouse model and BEAS-2B cell model.ResultsAfter the intersection of the 236 Res targets and the 2,382 asthma targets, 120 targets for Res against asthma were obtained. The top five therapeutic targets based on weighted degree score were TNF, IL6, STAT3, TP53, and IL1B. GO enrichment analysis identified 2,595 significant terms, associated with 2,402 biological processes, followed by 153 molecular functions and 40 cellular components. KEGG enrichment analysis identified 107 relevant pathways, including “apoptosis,” “TNF signaling pathway,” and “MAPK signaling pathway.” Molecular docking showed that Res had a strong binding affinity toward the top five targets with binding energies less than −5.8 kcal/mol. Res treatment normalized the dysregulated expression of TNF-α, IL-6, STAT3, p53, and IL-1β both in vitro and in vivo.ConclusionRes may target TNF-α, IL-6, STAT3, p53, and IL-1β to act as a therapeutic agent for asthma. These findings reveal the potential therapeutic targets for Res against asthma and provide theoretical bases for the clinical application of Res.

BACKGROUND:With extended gefitinib treatment, the therapeutic effect in some non-small cell lung cancer (NSCLC) patients declined with the development of drug resistance. Aidi injection (ADI) is utilized in various cancers as a traditional Chinese medicine prescription. This study explores the molecular mechanism by which ADI, when combined with gefitinib, attenuates gefitinib resistance in PC9GR NSCLC cells. METHODS:In vitro and in vivo pharmacological experiments were conducted in PC9GR cells and NSG mice with PC9GR cell-derived tumors, respectively. The molecular mechanism of ADI was further studied using whole-transcriptome sequencing technology. Bioinformatics and molecular biology methods were employed to validate the critical targets of ADI. RESULTS:Firstly, ADI treatment alone and combined with gefitinib significantly inhibited the proliferation, migration, and invasion of PC9GR cells. Then, whole-transcriptome sequencing and bioinformatics analysis revealed that PLAT is a key target for the increased efficacy of ADI combined with gefitinib. Additionally, ADI downregulates the expression of PLAT, TNC, ITGB3, p-AKT, p-PI3K, and p-FAK. ADI inhibits the migration and invasion of PC9GR cells by regulating the PLAT/FAK/AKT pathway. CONCLUSIONS:Aidi injection inhibits the migration and invasion of gefitinib-resistant lung adenocarcinoma cells by regulating the PLAT/FAK/AKT pathway. This study provides essential evidence for elucidating the mechanism of ADI in synergistic therapy for lung cancer.

Vesicular glutamate transporter 3 (VGLUT3) is prominently expressed in the inner hair cells of the cochlea, playing a vital role in auditory signal transmission to the brain. Previous studies have shown that Vglut3 gene knockout in mice causes severe sensorineural hearing loss without affecting hair cell integrity. However, the cochlear structure of the aged Vglut3KO remains inadequately explored. In this study, we analyzed the cochlear structure of aged Vglut3KO mice, revealing significant degeneration of inner hair cells, synapses, and stereocilia. To explore the potential of gene therapy to restore cochlear structure, we employed AAV8 vectors to express Vglut3 in the cochleae of 5-week-old Vglut3KO mice. Twenty-seven weeks post-injection, we conducted a series of experiments to evaluate the efficacy of our gene therapy approach. Auditory brainstem response (ABR) testing demonstrated restoration of auditory function following gene therapy. Immunohistochemical staining and scanning electron microscopy (SEM) analysis revealed substantial recovery of inner hair cells and stereocilia post-injection. Our findings provide important insights into the development of novel therapeutic strategies for age-related hearing loss.

e16119 Background: The standard of care for unresectable locally advanced esophageal squamous cell carcinoma (ESCC) is definitive chemoradiation (dCRT). However, long-term prognosis for patients(pts) receiving dCRT remains unfavorable. Our previous study adopting neoadjuvant short course chemo-radiotherapy in combination with ICIs (ICRT) showed promising locally control efficacy in locally advanced ESCC. Here we report the safety and antitumor activity of short course ICRT as conversion therapy for pts with unresectable ESCC. Methods: Pts with unresectable primary tumor site (invades adjacent organs such as trachea, bronchus, and aorta) and/or with unresectable regional lymph nodes or metastatic sites limited to non-regional lymph nodes (supraclavicular or abdominal lymph nodes outside the 3-field lymphadenectomy scope) ESCC were eligible for inclusion. Tislelizumab (200mg) was given with paclitaxel (135 mg/m2) and carboplatin (AUC = 5) on D1, D22. Short course radiotherapy (30Gy in 12 fractions on 5 days per week) was administered following the completion of immunochemotherapy. Target volumes were delineated after deliberations among radiation oncologists and surgeons. Lymph nodes outside the extended two-field lymph node dissection area were boosted to a radiation dose of 45Gy in 18 fractions. A third dose of tislelizumab and following esophagectomy were given at 3 and over 8 weeks after the completion of conversion radiotherapy, respectively. Target total enrollment was 30 pts. The primary endpoint was treatment safety. The secondary outcomes including conversion esophagectomy rate, pathological responses and patient survival. Results: From May 27th, 2022 to Jan 20, 2025, 30 pts were included in the trial. The median age was 63 years. 12 (40%) had supraclavicular lymph node metastasis; 13 (43.3%) with borderline resectable T4a or unresctable T4b disease; 16 (53.3%) had stage IVa while 14 (46.7%) had stage IVb disease. Common grade 3 and above AEs included leukopenia (n = 13), neutropenia (n = 13), heart injury (n = 2) and thrombocytopenia (n = 2). Among the 30 pts , 19 underwent R0 resection; 3 were still waiting for surgery. Reasons for not undergoing surgery were disease progression (n = 4), ICIs related grade 5 and 4 heart injury (n = 2), patients’ choice (n = 1) and tuberculosis reactivation (n = 1). Six pts with Grade III surgical complications were observed during the perioperative period. No postoperative death occurred. 9/19 (47.4%) complete pathological responses (ypT0N0) were observed. With a median follow-up time of 16.8 months (range: 12.5–21.1), 12-months progression free survival for pts who underwent surgery were 60.1% (95% CI: 34.8%,86.3%). Conclusions: Conversion short course chemo-radiotherapy in combination with ICIs followed by surgery was a promising treatment strategy for unresectable ESCC. Clinical trial information: NCT05394415 .

The inner ear has a pivotal role in auditory and vestibular perception. Despite the vast number of individuals worldwide affected by hearing loss and balance disorders, therapeutic options have been largely limited to technological aids. The recent advent of gene therapies for genetic hearing loss in human patients underscores the urgency of developing scalable platforms to investigate a broader spectrum of inner ear disorders. Although animal models are powerful for assessing auditory and vestibular dysfunction, in vitro human inner ear models have shown promise in disease modeling and as platforms for studying developmental biology. Several studies have demonstrated that stem cells can be guided to differentiate into otic progenitor cells by mimicking environmental cues present during normal fetal inner ear development. Here we present a step-by-step approach to creating inner ear organoids (IEOs), which is an extension of our previous method for skin organoid generation, with which it shares foundational methodology and reagents. We used these organoids to elucidate the subtle signaling cues that govern their developmental trajectories. Generating sensory hair cells takes about 40 d, and cultures can be maintained for up to 150 d to allow further development. Moreover, we outline methods for assessing late-stage organoids, including whole-mount imaging of cleared IEOs, vibratome sectioning of live and fixed IEOs and other endpoint analyses, to study inner ear biology. IEOs are ideal for investigating human inner ear development, studying the mechanisms of inner ear disorders and developing therapeutic strategies. This protocol requires proficiency in basic stem cell culture techniques. This protocol details the generation and characterization of vestibular inner ear organoids from human pluripotent stem cells. This is an extension of a previous protocol for skin organoid generation, with which it shares foundational methodology and reagents.

Purpose: Since TNM staging has limitations for predicting post-operative outcomes and relapse, more effective prediction tools need to be researched and developed. Lymphovascular invasion, LVI, as a histopathological feature, has been widely shown to have a correlation with poor prognosis and early recurrence of lung adenocarcinoma (LUAD). However, LVI assessment is limited by subjective bias, and therefore its efficacy in practical clinical application needs further clarification. The aim of this study was to formulate a new signature based on LVI-related genes to predict prognosis and recurrence in patients with lung adenocarcinoma.Methods: Clinicopathological information, gene sequencing data and whole slide images (WSIs) of LUAD patients were downloaded from the Cancer Genome Atlas (TCGA) databases. LVI statue were evaluated by professional pathologists, and then the differentially expressed genes (LVI DEGs) associated with LVI were screened. The least absolute shrinkage and selection operator (LASSO) and Step Cox regression models were used to construct LVI-associated risk scores (LVRS), including PAQR4, ARGHEF6, CKS1B, CFTR and SEC14L4. The validity of the LVRS score was evaluated on multiple external datasets and our JSSZL cohort dataset. Using LVRS scores and clinical information, nomogram were constructed for use by clinicians. In addition, we further explored the relationship between LVRS score and clin-icopathological features, immune infiltration, tumor mutational load, and immunotherapy response, and confirmed the expression of key genes in LVRS score in lung adenocarcinoma tissues using qRT-PCR and immunohistochemistry (IHC) techniques.Results: Based on the LVRS, patients could be classified into high-LVRS and low-LVRS groups. It was found that OS and PFS were significantly worse in the high-LVRS group than in the low-LVRS group (p < 0.001). By ROC curve analysis, it could be found that the nomogram combining LVRS and clinical information could accurately predict the prognosis of LUAD patients with the area under the curve of 1,3,5-year survival rate could reach 0.754, 0.741 and 0.735. The results of univariate and multivariate analysis showed that LVRS was an independent prognostic factor. At the same time, there were significant differences in the mutation profiles and immune microenvironment between the high-LVRS and low-LVRS groups, with the high-LVRS group having a significantly higher mutation rate than the low-LVRS group and exhibiting immunological "cold" features. By the experimental results, higher expression levels of PAQR4 and CKS1B were found in LUAD tissues, while lower expression levels of ARGHEF6, CFTR and SEC14L4 were observed.Conclusions: The LVRS established in this study serves as a valid tool to predict the prognosis and recurrence status of lung adenocarcinoma patients and has a predictive effect on the response to postoperative treatment. The establishment of LVRS may offer some theoretical support to clinical treatment strategies for patients with lung adenocarcinoma following surgical intervention.