Tertiary lymphoid structures (TLSs) are immune cell aggregates that emerge in nonlymphoid tissues during various disease states, including chronic inflammation, autoimmunity, and cancer. TLSs are structurally and functionally analogous to secondary lymphoid organs, and exhibit a maturation continuum (progressing from initial aggregation to mature structures with germinal centers). TLS formation is synergistically regulated by local chemokine networks (e.g. CXCL13, CCL19, and CCL21), lymphotoxin signaling axes, stromal cells, metabolic reprogramming, and the microbiome. This review comprehensively elucidates the biological foundations of TLSs, including their cellular composition, spatial architecture, and developmental dynamics of maturation. We explore the crucial roles of TLSs as favorable prognostic factors and predictors of the immunotherapy response in various solid tumors, including melanoma, breast cancer, lung cancer, hepatocellular carcinoma, and colorectal cancer. Additionally, we analyze their “pathogenic” role in causing tissue damage and disease progression in autoimmune disorders such as rheumatoid arthritis and Sjögren’s syndrome, as well as chronic inflammatory diseases such as COPD, IgA nephropathy, and atherosclerosis. In addition, we thoroughly examine TLS research methodologies, covering a wide range of approaches from conventional hematoxylin and eosin (H E) and immunohistochemical staining to advanced multiplex fluorescence staining, imaging mass spectrometry, and spatial transcriptomic techniques. We summarize multiple gene expression signatures (e.g. the 12-chemokine signature and TLS score) for TLS identification and quantification. Finally, we highlight multiple strategies for artificially inducing TLS formation, including cytokine delivery, immunotherapy, engineered scaffolds, microbiome modulation, and organoid technologies, designed to enhance antitumor immunity or reverse immunopathology. This review provides a comprehensive framework for understanding the complex functions of TLSs in human disease and explores their clinical translation potential as biomarkers and therapeutic targets.

The formation of the enteric nervous system (ENS) primarily involves the migration of enteric neural crest-derived cells (ENCCs) and the subsequent maturation of enteric neurons. The developmental dysfunction of ENCCs and enteric neurons can result in ENS disorders, such as hypoganglionosis (HG). Although neurite outgrowth is fundamental to neuronal maturation, the mechanisms by which neurite outgrowth influences neuronal maturation remain poorly defined. Here, we identified EMB as a critical regulator of enteric neuronal maturation. In EMB mutant patients, the expression of EMB is reduced in the enteric neurons of the HG-affected colon. In mice, knockdown of Emb exhibited HG-like features and defects. In vitro experiments, along with analyses using Smart-seq2 and immunoprecipitation-mass spectrometry, demonstrated that EMB is essential for autophagic flux and physically interacts with ATG7, recruiting it to the autophagosomal membrane to facilitate autophagosome formation, and then EMB/ATG7-mediated autophagy promotes neurite outgrowth. Our findings elucidate EMB-mediated autophagy as a pivotal pathway in regulating neurite outgrowth and promoting the maturation of enteric neurons, which provides a mechanistic basis for understanding ENS disorders.

Clear cell renal cell carcinoma (ccRCC) exhibits substantial molecular and clinical heterogeneity, contributing to variable disease progression and therapeutic outcomes. We previously identified a de-clear cell differentiation (DCCD) tumor state characterized by loss of canonical clear-cell features and aggressive clinical behavior. This study aimed to translate DCCD-associated biology into a prognostic framework and investigate its underlying molecular basis. Using TCGA-KIRC as the training cohort, we developed a five-gene prognostic signature comprising COL7A1, IGFN1, AJAP1, SMIM24, and ADGRV1, which consistently stratified patient survival across multiple public and institutional cohorts. Comparison with SSIGN, Leibovich, and ClearCode34-like models demonstrated that the DCCD signature provided complementary prognostic information. High-risk tumors exhibited enhanced proliferative and invasive programs, increased IL6-JAK-STAT3 pathway activity, and an immune-excluded microenvironment. Associations with treatment response were also observed in retrospective therapeutic cohorts. Integrated analyses of bulk and single-cell transcriptomics, spatial transcriptomics, tissue microarrays, CPTAC proteomics, and molecular interaction networks identified SMIM24 as an epithelial-associated factor linked to favorable outcomes and preserved metabolic programs. Functional experiments showed that SMIM24 overexpression suppressed migration, invasion, three-dimensional spheroid invasion, and clonogenic growth in ccRCC cells, accompanied by reduced JAK1 and STAT3 phosphorylation and decreased PD-L1 expression. Pharmacological activation of STAT3 with Colivelin partially restored PD-L1 expression in SMIM24-overexpressing cells. Collectively, these findings support the reproducibility of the DCCD-associated prognostic framework and identify SMIM24 as a candidate suppressor of ccRCC aggressiveness and PD-L1 expression, potentially acting in part through reduced STAT3 activation.

This study examined how 1-hydroxypyrene (1-OHP) affects bladder cancer (BCa) progression and tumor-associated immunosuppression. Public transcriptomic data and target-prediction databases were used to screen for targets related to 1-OHP and BCa. Machine learning was then used to narrow down the candidate genes. Molecular docking and single-cell RNA sequencing were also used to examine the selected targets and related pathways. The role of the main target was tested in vitro. CCK-8, colony formation, EdU, and Transwell assays were used to assess cell growth, migration, and invasion. Protein expression was examined by Western blotting. A tumor cell–CD8+ T-cell co-culture system was used to evaluate changes in CD8+ T-cell function. We identified 29 targets shared by 1-OHP and BCa. Machine learning further narrowed these targets to six genes, and macrophage migration inhibitory factor (MIF) was chosen for experimental validation. Treatment with 1-OHP increased BCa cell viability, proliferation, migration, and invasion. Silencing MIF partly reduced these effects. 1-OHP also increased MIF and CD74/CD44 expression and enhanced PI3K/AKT/mTOR signaling. These changes were reduced after MIF silencing. In the co-culture system, 1-OHP-treated BCa cells reduced CD8+ T-cell proliferation and function. Ki67, GZMB, and IFNG levels were decreased. MIF knockdown partly reversed these changes. Collectively, our findings show that 1-OHP promotes bladder cancer progression and tumor-associated immunosuppression, at least in part, through a MIF-dependent mechanism. By linking environmental exposure to activation of the CD74/CD44–PI3K/AKT/mTOR axis and impaired CD8+ T-cell function, our study provides new insight into the molecular basis of environmentally associated bladder carcinogenesis.

The global rise of drug-resistant Mycobacterium tuberculosis (Mtb) underscores an urgent need for antitubercular agents with novel targets and mechanisms of action. Among these, the de novo purine biosynthesis pathway is essential for Mtb growth and survival, making its constituent enzymes attractive targets for therapeutic intervention. Within this pathway, adenylosuccinate (ADS) synthetase (ADSS) Rv0357c catalyzes the first committed step in biosynthesis of adenosine monophosphate (AMP) by converting inosine monophosphate (IMP) to ADS through a GTP-dependent reaction with l-aspartate. Despite its importance, Mtb ADSS remains poorly characterized at the biochemical level. In this study, we report the expression, purification, and enzymatic characterization of recombinant Mtb ADSS. To overcome the challenge of the enzyme being predominantly expressed as inclusion bodies in Escherichia coli, we established both protein refolding and chaperone-assisted expression strategies to obtain soluble, catalytically active protein. Using complementary spectrophotometric, colorimetric, and fluorescence-based assays, we determined steady-state kinetic parameters and confirmed robust ADSS activity consistent with Michaelis-Menten behaviour. Furthermore, we developed scalable, nonradioactive assays compatible with high-throughput screening (HTS), enabling the quantitative monitoring of ADSS activity via GTP hydrolysis and phosphate release. As a proof of concept, the MESG assay successfully detected inhibition of Mtb ADSS by the previously reported ADSS inhibitor Aurodox, demonstrating its utility for inhibitor characterization and screening. Collectively, these results provide the first comprehensive biochemical framework for studying Mtb ADSS and establish a foundation for structure-guided inhibitor discovery targeting purine biosynthesis as a novel antitubercular strategy.

Activation of retinoic acid-inducible gene-I-like receptors (RLRs) is important for type I interferon (IFN-I) production and antiviral innate immunity initiation. However, the epigenetic mechanisms that regulate RLR signaling remain poorly understood and require further investigation. Here, we demonstrate that Fizzy-related protein 1 (FZR1), which is essential for mitotic exit and G1/S transition, potentiates antiviral innate immune responses against RNA viruses. Mechanistically, vesicular stomatitis virus infection increases N6-methyladenosine (m6A) modification of FZR1 mRNA, which enhances FZR1 translation and elevates intracellular FZR1 protein levels. Upregulated FZR1 attenuates mitochondrial antiviral-signaling protein (MAVS) binding to 6-Phosphofructo-2-Kinase/Fructose-2, 6-Biphosphatase 3, a glycolytic rate-limiting enzyme, thereby promoting MAVS aggregation. Furthermore, FZR1 facilitates tumor necrosis factor receptor-associated factor 3/6 (TRAF3/6) autoubiquitination independently of the anaphase-promoting complex/cyclosome, subsequently activating interferon regulatory factor 3 and P65 of nuclear factor κB to drive the production of IFN-I and proinflammatory cytokines. Consequently, FZR1 deficiency impairs antiviral responses and increases viral titer in vitro and in vivo. Pharmacological inhibition of FZR1 significantly attenuates MAVS activation and TRAF3/6 ubiquitination, thereby abolishing FZR1-mediated antiviral immunity both in vitro and in vivo. Collectively, these findings reveal a molecular mechanism by which m6A modification of FZR1 activates the MAVS-TRAF3/6 signaling axis to potentiate IFN-I-dependent antiviral innate immunity.

Renal cancer is a common malignant tumor in the urinary system. Current research has found that immune escape in kidney cancer can prevent immune system attacks through specific mechanisms, making it difficult for the immune system to effectively kill cancer cells, and promote the progression and metastasis of kidney cancer. Therefore, despite the continuous updating of immunotherapy methods for kidney cancer, the high recurrence rate, high drug resistance, and severe side effects of kidney cancer patients are still difficult to solve. This review systematically summarizes the latest mechanisms of immune escape in the renal cancer immune microenvironment, including abnormal expression of histocompatibility complex (MHC), secretion of immunosuppressive factors, programmed death ligand-1 with abnormal expression, recruiting immunosuppressive cells, and VHL gene deletion. This article also summarizes new treatment strategies proposed for these immune escape mechanisms. We hope this will help future researchers further explore the immune escape mechanism of renal cell carcinoma and propose new immunotherapy strategies.

CD8+ T lymphocytes are critical for rejecting cancer cells but often lose functionality in the immunosuppressive tumor microenvironment. Understanding this process could improve cancer immunotherapy. The enzyme GGPS1, which produces geranylgeranyl pyrophosphate (GGPP), has an unclear impact on CD8+ T cell cytotoxic function and tumor immune escape in clear cell renal cell carcinoma (ccRCC). To investigate how GGPS1 deficiency in T cells affects tumor progression, we established Renca tumor models in mice with T-cell-specific deletion of GGPS1. A combination of approaches—including real-time PCR, western blotting, flow cytometry, in vitro cytotoxicity assays, Seahorse metabolic analysis, and measurements of glucose uptake and lactic acid secretion—was employed to dissect the role of GGPS1 in CD8⁺ T cell cytotoxicity and glycolysis. In parallel, adoptive transfer studies were conducted to evaluate the antitumor potential of CD8⁺ T cells engineered to overexpress GGPS1. CD8⁺ T cells isolated from ccRCC patients who failed to respond to PD-1 blockade exhibited markedly lower GGPS1 expression. In mice, T-cell-specific deletion of GGPS1 accelerated tumor growth, accompanied by diminished infiltration of cytotoxic CD8⁺ T cells and elevated expression of exhaustion markers—including PD-1, TIM-3, LAG-3, and TOX—on tumor-infiltrating CD8⁺ T cells. Silencing GGPS1 impaired cytokine production and antitumor activity, whereas restoring GGPS1 expression reinforced both functions. Notably, exogenous GGPP fully rescued the defects in cytotoxicity and glycolysis induced by GGPS1 knockdown. Mechanistically, GGPS1 promoted MEK/ERK signaling, and the MEK inhibitor trametinib abolished GGPP-driven enhancement of CD8⁺ T cell cytotoxicity and glycolysis. Finally, elevating GGPS1 levels in CD8⁺ T cells boosted the efficacy of both adoptive immunotherapy and immune checkpoint blockade in ccRCC models. These findings highlight the essential function of GGPS1 in regulating glycolysis and cytotoxicity in CD8+ T cells, offering potential therapeutic strategies for clinical tumor immunotherapy in ccRCC.

Clear cell renal cell carcinoma (ccRCC), the most common and lethal subtype of renal cell carcinoma, exhibits marked intratumoral heterogeneity and complicates clinical management. Although long noncoding RNAs (lncRNAs) regulate diverse cellular processes, their landscape and biomarker potential in ccRCC remain poorly defined. Here we performed single-nucleus and bulk transcriptomic, proteomic, and metabolomic analyses on a cohort of 100 ccRCC patients. The expression pattern of lncRNAs were described based on metacells. Malignant cells displayed broader but lower lncRNA expression, likely reflecting copy number alterations, whereas low-abundance lncRNAs in normal epithelial cells showed individual variability. Multi-omics integration was used to establish a preliminary lncRNA functional inference pipeline, identifying lncRNAs involved in metabolic and immune processes and validating their roles through functional and in vivo experiments. Candidate biomarkers lncRNAs were identified to build diagnostic (DMRlnc) and prognostic models (PMRlnc), which were validated in TCGA, CheckMate, and IMmotion151 cohorts. DMRlnc achieved high diagnostic accuracy in both discovery and TCGA-KIRC cohorts (AUC 0.98 and 0.93). PMRlnc stratified patients into distinct risk groups with significant differences (p < 0.0001) across TCGA-KIRC and IMmotion151 cohorts. PMRlnc further indicated that low-risk patients may benefit more from nivolumab, while high-risk patients might respond better to atezolizumab plus bevacizumab.

Bladder cancer, particularly muscle-invasive disease, has a high metastatic potential and limited treatment options, highlighting the need for new therapeutic targets. CD276 (B7-H3), a type I transmembrane protein of the B7 family, is traditionally considered an immunomodulatory ligand with an unidentified receptor. Here, we investigated the role of CD276 in bladder cancer and sought to identify its binding partner. Using a high-throughput human proteome microarray, we identified the secreted lectin galectin-1 (LGALS1) as a high-affinity binding partner of CD276, suggesting that CD276 may function as a receptor. Mechanistically, we uncovered a novel LGALS1-CD276 axis in endothelial cells, where CD276 serves as a functional receptor for tumor cell-derived LGALS1, with their interaction mediated by N-linked glycosylation at the N433 site within the D4 domain of CD276. This interaction activates the MAP4-dependent PI3K/AKT signaling pathway, thereby promoting angiogenesis and bladder cancer progression. Disruption of the LGALS1-CD276 interaction or inhibition of the downstream MAP4/PI3K/AKT pathway markedly suppressed endothelial proliferation, migration, and tube formation. In subcutaneous and orthotopic bladder cancer mouse models, anti-CD276 monoclonal antibody treatment significantly inhibited tumor angiogenesis, delayed tumor growth, and extended survival. Consistently, Cd276-/- and/or Lgals1-/- mouse models confirmed that the tumor-promoting effect of LGALS1 depends on CD276 expression. Clinically, elevated CD276 and LGALS1 expression was associated with poor prognosis and positively correlated with angiogenesis. Together, our findings identify CD276 as a functional cell-surface receptor and demonstrate that LGALS1 binding to CD276 promotes tumor angiogenesis through MAP4-mediated activation of the PI3K/AKT signaling pathway. These findings establish a new mechanistic foundation for bladder cancer therapy and highlight the potential of targeting the LGALS1-CD276 axis for anti-angiogenic treatment.

The basic clinical information of the human specimens analyzed in the manuscript

Supplementary data related to Figure 3

BACKGROUND:The enteric nervous system (ENS), which arises from enteric neural crest cells (ENCCs), plays important roles in many aspects of gastrointestinal tract function, including motility, secretions, blood flow and hormone release. Defects in ENS development could lead to a broad range of disorders, including Hirschsprung's disease (HSCR), which is characterized by missing nerve cells in the distal segment of the colon. Here, we identify EMB as an evolutionarily conserved regulator of ENS development. METHODS:We first examined EMB expression in human and mouse intestines using scRNA-seq data and immunofluorescence staining. To investigate its role in ENS development, we constructed Emb-knockout zebrafish and mouse models. To explore the underlying mechanisms, we focused on ENCCs and analyzed their proliferation and migration using migration assays in explant guts and organoid cultures. Finally, we assessed rare EMB variants in a cohort of HSCR patients. RESULTS:In zebrafish, loss of emb leads to a decrease number of enteric neurons and impaired intestinal transit ability. In mice, knockout of Emb causes HSCR-like phenotypes and defects. In vitro experiments, including explant mouse gut and organoid cultures, show that EMB is required for both the proliferation and migration of ENCCs. Mechanistically, EMB binds to and recruits the phosphatase complex PP2A to the cellular membrane to facilitate the activation of PI3K-AKT pathway, thereby promoting ENCCs development. Indeed, application of PI3K or AKT agonists partially restores the ENS developmental defects in zebrafish emb mutants. Furthermore, rare variants of EMB may potentially contribute to the pathology of HSCR in humans. CONCLUSIONS:EMB is required for ENS development by regulating the proliferation and migration of the ENCCs. Mechanistically, EMB recruits PP2A to the cell membrane, reducing cytoplasmic dephosphorylation activity and promoting the activation of the PI3K signaling pathway.

Immunogenic cell death (ICD) is a type of cell death sparking adaptive immune responses that can reshape the tumor microenvironment. Exploring key ICD-related genes in bladder cancer (BLCA) could enhance personalized treatment. The Cancer Genome Atlas (TCGA) BLCA patients were divided into two ICD subtypes: ICD-high and ICD-low. High ICD expression linked to increased immune cell infiltration and longer survival, but with potentially suppressed immune function. The high ICD group responded better to PD1-targeted therapy. A risk-scoring model with four ICD-related genes (CALR, IL1R1, IFNB1, IFNG) was validated across TCGA, GEO datasets, and tissue samples, showing higher risk score correlated with weaker anti-tumor immune function, more tumor-promoting elements, lower immunotherapy response rates, and shorter patient survival. This study connects ICD-related genes to BLCA prognosis and immune infiltration, offering a vital tool for personalized treatment guidance.

The levels of MLYCD expression and Ran K141 malonylation correlates with the clinical prognosis of PCa patients

AML is a complex disease caused by multiple molecular mechanisms. As an important regulatory molecule, the role of circRNA in AML is not fully understood. By performing high-throughput sequencing on clinical samples, we systematically identified the differences in circRNA expression and distribution between AML and healthy donor samples. One circular RNA, circAFF2, was found to be significantly upregulated in AML patients. Functional studies showed that knockdown of circAFF2 could significantly inhibit the proliferation of AML cells and promote their apoptosis. Overexpression of circAFF2 can have opposite effects. In vivo experiments showed that transplantation of AML cells with circAFF2 knockdown slowed the proliferation and infiltration and prolonged the survival time of mice compared to controls. Further studies showed that circAFF2 can promote the degradation of PML mRNA by binding to the 3’UTR of PML mRNA, thereby affecting the proliferation and apoptosis of AML cells. In conclusion, our work demonstrates that circAFF2 can bind to PML mRNA to regulate AML cell function, providing new insights into the mechanism of AML development and potential targets for clinical diagnosis and treatment.

Background and objective:Olaparib is one of the earliest approved treatment options for metastatic castration-resistant prostate cancer (mCRPC). This systematic review and network meta-analysis aimed to determine the optimal olaparib strategy for treating mCRPC. Methods:The Cochrane, Embase, PubMed, and Web of Science databases were searched comprehensively using "mCRPC" and "olaparib" as keywords. Study quality was appraised with the National Institutes of Health tools. Data were analyzed in R version 4.4.1. The primary endpoints included progression-free (PFS) and overall (OS) survival. The secondary endpoints included adverse events and severe adverse events (grade ≥3). Effect sizes were reported as hazard ratios (HRs) and risk ratios, with 95% credibility intervals (CrIs). Key findings and limitations:Nine studies from seven clinical trials involving 2355 patients were identified. For homologous recombination repair-mutated mCRPC, combination therapies did not demonstrate significant benefits compared with olaparib alone. However, for BRCA-mutated mCRPC, olaparib combined with abiraterone improved PFS (HR = 0.61, 95% CrI = 0.41-0.91) and OS (HR = 0.41, 95% CrI = 0.21-0.80) significantly. These significant advantages of olaparib combined with abiraterone were also observed in patients from different prostate-specific antigen subgroups. Conclusions and clinical implications:These findings suggest that olaparib combined with abiraterone offers substantial benefits in BRCA mutated type (BRCAmt) mCRPC patients. For those with BRCA wild type homologous recombination repair-mutated mCRPC, olaparib monotherapy is effective. Patient summary:We reviewed the published studies comparing different treatment options using the drug olaparib (alone or combined with other therapies) for advanced prostate cancer that has spread and no longer responds to standard hormone therapy (metastatic castration-resistant prostate cancer). We found evidence that the effectiveness of olaparib depends significantly on specific genetic features of the cancer. For patients whose cancer has changes in the BRCA genes, the combination of olaparib and the drug abiraterone was more effective in delaying cancer growth and improving survival than olaparib alone. For patients with changes in other related DNA repair genes (but not BRCA), olaparib alone was an effective treatment. This information may assist doctors and patients in choosing the most suitable treatment based on the cancer's genetic characteristics.

Background Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of metastatic renal cell carcinoma (mRCC), but response rates remain heterogeneous, and reliable predictive biomarkers are lacking. Recent studies suggest that androgen receptor (AR) signaling plays a role in regulating CD8+ T-cell function, implying that 5α-reductase inhibitors (5-ARIs), which lower androgen activity, could enhance antitumor immunity and improve clinical outcomes in patients receiving immunotherapy. This study retrospectively investigates the impact of a history of 5-ARI use (≥12 months) on the efficacy of ICIs in mRCC.Methods We conducted a multicenter retrospective cohort study of 185 patients with mRCC who received ICIs. Patients were stratified based on their history of 5-ARI use. Baseline characteristics included age, body mass index, International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) risk group, programmed death-ligand 1 (PD-L1) expression levels, tumor stage, and metastasis sites. The primary endpoints were progression-free survival (PFS) and overall survival (OS), analyzed using Cox proportional hazards models. Secondary endpoints included objective response rate (ORR) and disease control rate (DCR). Key immunological insights were gained through single-cell RNA sequencing analysis of tumor samples.Results Patients with a history of 5-ARI use demonstrated improved ORR (59.8% vs 39.8%, p=0.0075) and DCR (87.0% vs 78.7%, p=0.1747) compared with those without. The median PFS and OS were significantly longer in the 5-ARI group, with HRs of 0.64 (95% CI: 0.47 to 0.86, p=0.0085) for PFS and 0.65 (95% CI: 0.47 to 0.90, p=0.0271) for OS. Subgroup analysis further indicated enhanced ICI efficacy with 5-ARI use across age, IMDC risk scores, and PD-L1 expression levels. Single-cell RNA sequencing analysis revealed that 5-ARI treated patients exhibited a reduced presence of regulatory T cells and CD8 T-cell exhaustion (CD8 Tex), and lower programmed cell death protein-1 expression in CD8 Tex cells, suggesting an immunologically favorable modification of the tumor.Conclusion A history of 5-ARI use is associated with improved responses to ICI therapy in mRCC, potentially through AR-related modulation of CD8+ T-cell activity and favorable alterations in the immune microenvironment. These findings support further investigation into androgen-targeted approaches as adjunctive strategies in immunotherapy for RCC.

Ran K141 malonylation promoted AR nuclear localization, related to Figure 5

Neuroblastoma (NB), the most common extracranial solid tumor of childhood, originates from developing sympathetic nervous system. While cuproptosis has emerged as a critical regulator in oncobiology, its mechanistic involvement in NB remains poorly characterized. RNA-seq data of NB patients was analyzed using limma and ClusterProfiler. CRlncRNAs were identified through Pearson correlation between lncRNAs and CRGs, with co-expression networks visualized via ggalluvial. Prognostic signature was constructed through Lasso-penetrated Cox regression and validated via EFS analysis, C-index, and ROC curves. Somatic mutations and TMB were profiled using maftools. Immune infiltration landscapes were deciphered by CIBERSORT, while single-cell analysis integrated AddModuleScore, AUCell, and inferCNV to map CNV-driven transcriptional heterogeneity. Functional validation via siRNA knockdown confirmed the oncogenic role of CRGs. The risk model: Risk Score = (-1.637×DIRC3-AS1) + (0.6758×FOXN3-AS1) + (0.3032×LINC00682) + (-0.6812×RASSF8-AS1) was constructed. Stratification by median risk score revealed significantly prolonged EFS and OS. No significant TMB difference was observed between subgroups. scRNA-seq analysis highlighted malignant cell dominance with marked CNVs in high-risk patients. Functional validation confirmed CRlncRNAs’ roles in modulating proliferation and migration. Our findings establish a novel prognostic framework for NB that enhances risk stratification accuracy and provides actionable biomarkers to guide precision clinical management.