Circulating tumor cell (CTC) detection in hepatocellular carcinoma (HCC) is limited not only by the rarity of CTCs but also by a heavy reliance on cell surface markers such as EpCAM, which are variably expressed or lost during tumor progression. Detecting intracellular markers, such as cytokeratin offers an important complementary and comprehensive strategy but remains technically limited in flow cytometry due to the need for fixation and permeabilization, which often lead to cell loss and surface epitope damage. In this study, we systematically evaluated the feasibility of using fixed samples for flow cytometry, using HepG2 cells, PBMCs, and CTCs from patients with HCC. Our results demonstrate that fixation enabled intracellular staining without compromising cell surface marker detection, even after short-term storage at 4 °C and long-term storage at -80 °C. Fixed samples, particularly fixed unfrozen, exhibited comparable staining performance to fresh samples with only a 7-10% reduction in cell recovery. Clinical validation in HCC patients confirmed successful CTC detection, and tumor-specific CTNNB1 mutations were identified in CTC-derived DNA but not in matched plasma cfDNA. These findings support fixed CTC sample workflows as a reliable and practical approach for CTC analysis in HCC.

Hepatocellular carcinomas (HCC) with multinodular morphology were typically polyclonal and exhibited extensive heterogeneity. We explored the multiple-layer heterogeneity of HCC utilizing single-cell multiomics sequencing together with multiregional sampling. We found that Confluent Multi-Nodular samples exhibit more heterogeneous immune landscapes characterized by increased transcriptome heterogeneity and more complex immune-related interactions compared to Single Nodular (SN) samples. We identified differential DNA methylation patterns among distinct tumor foci in a subset of liver cancer patients. Global DNA hypomethylation in cancer cells predominantly occurs in partially methylated domains. We predicted and validated two genes, GADD45A and SNHG6, as potential drivers of DNA hypomethylation. Lastly, we demonstrated that DNA methylation distance serves as a more robust metric than gene expression distance for reconstructing tumor evolutionary trajectories.

To identify genetic markers that can predict the prognosis of bladder carcinoma (BC) patients using single-cell RNA sequencing (scRNA-seq). The RNA-seq data for BC were obtained from the GSA-Human database (HRA000212). Quality control and normalization were performed using the Seurat package, followed by identification of highly variable genes. Cell clustering and annotation were based on these genes. Functional enrichment analyses (GO and KEGG) were conducted, along with cell-cell communication and trajectory analysis. Prognostic modeling was performed using LASSO-Cox regression, with Receiver Operating Characteristic (ROC) analysis for risk group evaluation. Gene expression validation was carried out via RT-qPCR and western blotting in normal (SV-HUC-1) and BC cell lines (T24, J82, EJ, UM-UC-3, 5637, RT112). A total of 473 upregulated genes and 106 downregulated genes were identified in BC samples. These differentially expressed genes (DEGs) were significantly enriched in apoptosis-related signaling pathways and IL-17 signaling pathway. Cell-cell communication analysis suggested that the CXCL2/MIF-CXCR2 signaling pathway may mediate interactions between epithelial cells and fibroblasts. By integrating bulk RNA sequencing data, we identified 49 genes that are associated with the prognosis of BC. Using the LASSO-Cox regression model, 17 prognostic genes were selected. Stratification of patients into high- and low-risk groups based on these genes allowed for effective survival prediction, supported by the ROC curve analysis. Univariate and multivariate Cox regression analyses further confirmed that the risk score was an independent predictor of overall survival. Furthermore, IGFBP5, KRT14 and SERPINF1 were found to be linked to poor survival outcomes in BC, with their expression levels notably elevated in BC cell lines compared to a normal bladder cell line. This study identified potential prognostic marker genes for BC, offering valuable insights into the prediction of patient survival outcomes.

Immune checkpoint blockade (ICB) offers an in vivo approach to activate CD8+ tumor-infiltrating lymphocytes (CD8+TILs) in cases of advanced non-small cell lung cancer (NSCLC). A large fraction of NSCLC patients is unresponsive to ICBs and relapse due to the development of dysfunctional CD8+TILs with impaired cytotoxicity. Therefore, an improved understanding of regulator(s) that favor the development of cytotoxic Teff cells over dysfunctional CD8+TILs is required for the success of ICB therapy in NSCLC patients. Here, our metaVIPER-based scRNA-seq analysis of deep CD8+ cell scRNA-seq data from 14 treatment-naïve NSCLC patients revealed that the master regulon ZEB2 may drive CD8+ differentiation along the cytotoxic effector trajectory in NSCLC tumors. In vitro, ZEB2 acts downstream of T-bet to stimulate lung tumor-reactive Teff cell differentiation. This T-bet/ZEB2 axis displays immunotherapeutic effects on KP.SIY lung tumors independent of ICB therapy and mediates the therapeutic effects of murine serum albumin-fused IL-2 + IL-12 combination immunotherapy (IL2-MSA + IL12-MSA) in mice. IL2-MSA + IL12-MSA operates through a parallel STAT4/FOXO1-mediated mechanism that promotes CD8+TIL T-bet/ZEB2 expression and lung tumor-reactive Teff cell differentiation. In conclusion, immunotherapeutic regimens that support ZEB2 activity in CD8+ cells may show promise in NSCLC patients.

Pancreatic cancer (PC) is highly lethal because of its immunosuppressive tumor microenvironment and resistance to immunotherapy. This study explored the role of NAT10-mediated N4-acetylcytidine (ac4C) RNA modification in pancreatic cancer progression and immune evasion. NAT10 (N-acetyltransferase 10) is overexpressed in pancreatic cancer tissues and correlates with poor prognosis. Mechanistically, NAT10 stabilizes ETS2 mRNA through ac4C acetylation, forming a positive feedback loop that upregulates NAT10 and PD-L1, thereby suppressing CD8 + T cell infiltration and promoting immune evasion. In addition, NAT10 stabilizes KRT8 mRNA via ac4C acetylation, which drives cancer cell proliferation and metastasis. Single-cell RNA sequencing analysis revealed enhanced interactions between pancreatic cancer epithelial cells with high NAT10 and KRT8 expression, and T cells, thereby providing new insights into the immune microenvironment. In vivo, NAT10 knockdown significantly inhibited tumor growth, enhanced CD8 + T cell infiltration, and reduced lung metastasis. Notably, combination therapy with an NAT10 inhibitor and anti-PD-L1 antibody demonstrated superior antitumor efficacy compared to monotherapy. In conclusion, NAT10 promotes pancreatic cancer progression and immune evasion by regulating the ETS2-PD-L1 axis and stabilizing KRT8 mRNA, highlighting its potential as a therapeutic target for overcoming immunotherapy resistance.

Cholangiocarcinoma (CHOL) is a highly fatal malignancy originating from the bile ducts, with most patients diagnosed at an advanced stage, limiting treatment options. This study explores the role of Muscleblind-like 3 (MBNL3), a conserved RNA-binding protein, in CHOL progression, particularly its involvement in metastasis and immune microenvironment regulation. Using data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO), we investigated MBNL3 expression and its correlation with patient prognosis. High MBNL3 expression was associated with significantly better overall survival (OS), progression-free survival (PFS), disease-specific survival (DSS), and disease-free interval (DFI) in CHOL patients. Functional analysis revealed that MBNL3 modulates epithelial-mesenchymal transition (EMT) and interacts with critical pathways, including focal adhesion, collagen signaling, and TNF signaling. Additionally, MBNL3 influences immune cell infiltration, with high expression linked to increased macrophage and Th17 cell presence, suggesting potential roles in immune response modulation. Experimental validation confirmed that silencing MBNL3 promotes EMT, enhances tumor cell migration and invasion, and increases metastasis in vivo. Furthermore, genetic analyses highlighted the impact of MBNL3 mutations and methylation on CHOL progression, identifying potential targets for immunotherapy. This is the first study to investigate MBNL3's role in CHOL, positioning it as a prognostic biomarker and a promising therapeutic target for improving outcomes in CHOL patients. Further research is needed to elucidate the molecular mechanisms underlying MBNL3's regulation of tumor metastasis and the immune microenvironment.

Gastric cancer (GC) metastasis remains a major cause of poor prognosis, yet its molecular drivers are poorly understood. Here, we integrated single-cell RNA sequencing (scRNA-seq) of primary tumors and matched metastatic lymph nodes from six GC patients to identify a metastatic epithelial subpopulation characterized by EGR4 overexpression. Kaplan-Meier analysis revealed that high EGR4 expression correlated with reduced survival in GC patients. Mechanistically, chromatin immunoprecipitation sequencing (ChIP-seq) and luciferase assays demonstrated that EGR4 directly bound to the GDF15 promoter, driving its transcriptional activation. Functional studies showed that EGR4 promoted migration and metastasis via GDF15-mediated ErbB3/ErbB1 hetero-dimerization, which activated PI3K/AKT and MAPK/ERK pathways. Furthermore, CellChat analysis identified robust interactions between EGR4+ GC cells and cancer-associated fibroblasts (CAFs), particularly extracellular matrix (ECM)-remodeling eCAFs. Secreted GDF15 induced CAF activation through TGF-β receptor signaling, creating a pro-metastatic niche. Collectively, our study establishes the EGR4/GDF15 axis as a critical driver of GC metastasis, offering possible therapeutic targets for intervention.

Long non-coding RNAs (lncRNAs) and RNA N⁶-methyladenosine (m6A) have been linked to leukemia drug resistance. However, whether and how lncRNAs and m6A coordinately regulate resistance remain elusive. Here, we show that many differentially expressed lncRNAs enrich m6A, and more lncRNAs tend to have higher m6A content in CML cells resistant to tyrosine kinase inhibitors (TKIs). We demonstrate the broad clinical relevance of our findings, showing that upregulation of top-ranked lncRNAs (e.g., SENCR, PROX1-AS1, LINC00892) in TKI-resistant cell lines occurs in CML patients at the diagnostic stage, blast crisis phase, or not responding to TKIs compared to the chronic phase or TKI responders, respectively. Higher lncRNAs predict drug resistance and shorter survival duration. The knockdown of SENCR, PROX1-AS1, or LINC00892 restores TKI sensitivity. Mechanistically, upregulation of PROX1-AS1, SENCR, and LINC00892 results from FTO-dependent m6A hypomethylation that stabilizes lncRNA transcripts and empowers resistant cell growth through overexpression of PI3K signaling mediators (e.g., ITGA2, F2R, COL6A1). Treatment with PI3K inhibitor alpelisib eradicates resistant cells in vitro and in vivo, with prolonged survival of leukemic mice through downregulation of F2R, ITGA2, and COL6A1. Thus, the lncRNA-m6A-PI3K cascade represents a new non-genetic predictor for drug resistance and poorer prognosis in cancer, and a pan-cancer mechanism underlying TKI resistance.

SET domain-containing 7 (SETD7, also known as KMT7 or SET7/9), a histone lysine methyltransferase (HKMT) responsible for catalyzing histone H3 lysine 4 monomethylation (H3K4me1), has emerged as a key regulator in multiple cancers. However, the biological functions and epigenetic regulatory mechanisms of SETD7 in esophageal squamous cell carcinoma (ESCC) remain unclear. Our study found that SETD7 expression is significantly upregulated in ESCC tissues and positively correlates with clinical staging. Functional analyses revealed that SETD7 promotes ESCC cell proliferation and migration in vitro, while accelerating tumor growth in vivo. Additionally, SETD7 knockdown increased ESCC cell sensitivity to ferroptosis induction, indicating its dual functionality in tumorigenesis and ferroptosis resistance. Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing analysis systematically mapped H3K4me1 modifications in ESCC cells, identifying ALDH1A3 (aldehyde dehydrogenase 1 family member A3) as a key downstream target. Mechanistically, SETD7-mediated H3K4me1 deposition at the ALDH1A3 promoter drives transcriptional activation, increasing the level of reduced coenzyme Q10 (CoQ10H₂) and inhibiting lipid peroxidation. This study reveals a novel epigenetic-metabolic axis (SETD7-H3K4me1-ALDH1A3/NADH/CoQ10H₂) that regulates ESCC progression and ferroptosis sensitivity, which highlights the clinical translational value of SETD7 in ESCC prognosis assessment and therapeutic development. Schematic diagram illustrating the mechanism by which SETD7 accelerates ESCC progression through enhancing ferroptosis resistance. Created with BioRender.

Stress granule (SG) regulators affect tumor progression in patients with gastric cancer (GC). This study aimed to explore the effects of SG-related genes on the prognosis, immune characteristics, and drug sensitivity of patients with GC.

To enhance the effectiveness of irradiation (IR) and optimize PD-1/PD-L1 blockade therapy for hepatocellular carcinoma (HCC), we conducted in vivo CRISPR-Cas9 metabolic sublibrary screening, pinpointing UDP-glucose dehydrogenase (UGDH) as a key target. Post-IR, protein kinase R (PKR) translocates to the nucleus. There, elevated UGDH produces UDP-GlcUA, which binds PKR's dsRNA-binding domain (dsRBD). This binding triggers PKR dimerization, autophosphorylation (T451), and activation. Activated PKR then phosphorylates TOP2A at S1467, inducing its liquid-liquid phase separation (LLPS) and enhancing topoisomerase activity, and finally protects tumor cells from IR-induced DNA damage. Disrupting the UDP-GlcUA/PKR complex reduces TOP2A-S1467 phosphorylation, lowers topoisomerase activity, increases cGAS-STING signaling, and improves anti-PD-L1 immunotherapy efficacy. Clinical HCC sample analysis confirmed the relevance of UDP-GlcUA and the phosphorylation of PKR and TOP2A in response to radiation. Critically, blood UDP-GlcUA may serve as a biomarker for PKR/TOP2A axis activation, guiding patient suitability for anti-PD-L1 immunotherapy after IR and enabling personalized treatment strategies.

Cancer metastasis causes the majority of cancer-related deaths. During metastasis, invasive tumor cells enter the bloodstream and become circulating tumor cells. These cells eventually populate the secondary organs. In addition to genetic mutations, epigenetic changes (such as DNA methylation, histone modifications, and noncoding RNA-associated changes) are important for regulating gene expression and preserving chromatin integrity in tumor cells. Dysregulation of these epigenetic patterns is critical for carcinogenesis, tumor growth, and metastatic spread. We presented an overview of CTC biology, including essential mechanisms such as epithelial-mesenchymal transition (EMT) and immune evasion. We discussed how epigenetic reprogramming of CTCs also occurs post-transcriptionally via non coding RNAs, The subject then moves to how epigenetic changes in CTCs affect these processes throughout migration, survival in circulation, and metastatic seeding. The following sections discuss the clinical implications of addressing CTC epigenetics, proposing CTC epigenetic changes as prospective biomarkers for early cancer detection, prognosis, and targeted therapy intervention to improve patient outcomes.

Cervical cancer is a malignant gynecological tumor, and cancer cell metastasis remains poorly controlled despite surgery, radiotherapy, and chemotherapy. Traditional Chinese medicine, with advantages of multi-target effects and low toxicity, has emerged as an important therapeutic approach. In this study, we screened Amygdalin-related targets and cervical cancer differentially expressed genes using databases (SwissTargetPrediction, TCGA). Key modules were identified via WGCNA, and core targets (CA9 and HK2) were determined through PPI network analysis and the MCODE algorithm. Single-cell RNA sequencing further localized Amygdalin-affected cell populations. Molecular docking and molecular dynamics simulations verified the binding affinity of Amygdalin to CA9 and HK2, which were further confirmed by enzymatic activity assays. These key targets were upregulated in cervical cancer tissues, significantly correlated with patient survival, and exhibited good diagnostic value (ROC curve AUC > 0.9). Cell experiments have shown that Amygdalin inhibits the proliferation of cervical cancer HeLa and SiHa cells in a dose- and time-dependent manner, and induces cell apoptosis. Amygdalin-treated HeLa cells were arrested at the G1 phase, while Amygdalin-treated SiHa cells were arrested at the G2 phase. Finally, RNA-seq transcriptomics analysis elucidated the pathway regulation mechanisms. Collectively, this study systematically confirms that Amygdalin exerts significant anti-cervical cancer effects by targeting CA9 and HK2 to regulate multiple pathways, providing an experimental and theoretical basis for its development as a candidate drug for cervical cancer treatment.

Understanding therapy resistance requires deconvolving heterogeneous cell populations and tracking clonal trajectories. While CRISPR-based cellular barcoding is powerful for lineage tracing, many platforms suffer from low efficiency and limited compatibility with single-cell transcriptomics. We developed Oligo-CALL (Oligonucleotide-inducible CRISPR transcriptional activator-Assisted Lineage Labeling), an advanced barcoding system enabling precise lineage tracing, live clone isolation, and seamless integration with single-cell RNA sequencing. Applied to lung cancer cells treated with a KRASG12C inhibitor, Oligo-CALL identified clones consistently enriched posttreatment, supporting a model of predestined resistance. Oligo-CALL achieved >95% efficiency in linking lineage identity to transcriptomes, uncovering diverse clone-specific pathways with underlying resistance. Paired analysis of barcode-matched clones from naïve and resistant populations revealed transient and fixed resistance phenotypes. Notably, DNA repair pathways are recurrently altered in resistant clones, and inhibition of poly(adenosine 5'-diphosphate-ribose) polymerase synergizes with KRAS G12C inhibition to overcome resistance. Together, Oligo-CALL provides a versatile platform for dissecting lineage evolution and molecular dynamics of targeted therapy resistance.

The LRIG gene family consists of LRIG1-3. While LRIG2 has been described as a tumor promoter, LRIG1 and LRIG3 have been identified as tumor suppressors in previous literature. Because of these contrasting roles, the expression of LRIG1-3 was examined across different grades of glioma, between primary and secondary glioblastoma and with focus on chemotherapy treatment. Human tumor tissue samples were extracted during neurosurgery and grouped among the WHO classification valid at the time of surgery. Quantitative western blot analysis, qPCR and immunofluorescence staining were performed. LRIG1 was less expressed in glioma compared to peritumoral tissue with additional decrease with ascending tumors grade. Further, secondary glioblastoma expressed more LRIG1 protein than primary. On mRNA level, the same was seen for LRIG2, were low grade glioma expressed significantly more LRIG2 than high grade glioma. And on protein level, secondary glioblastoma showed higher expression than primary. LRIG3 mRNA expression, in contrast, was significantly higher in grade II gliomas compared to surrounding control tissue, whereas chemotherapy did not significantly affect expression levels in glioblastoma. Our results reinforce suggestions that LRIG1-3 could function as diagnostic markers and therapeutic targets in the treatment of gliomas.

Colorectal Cancer (CRC) is the third most prevalent malignancy, leading to significant morbidity and mortality globally. Epidemiological studies suggest that chronological age and diet are among the major contributing factors correlated with the incidence of CRC. Our study aimed to provide insights into the association between age, diet, and gut microbiome in CRC using molecular techniques including RNA sequencing, cytokine analysis, and metagenomic analysis. We used syngeneic MC38 mice model divided into two age groups (old and young) and three diet groups (standard chow, calorie-restricted and high-fat). The major findings of this study are that age and diet impact intratumoral gene signaling (nuclear and mitochondrial), and hub genes we identified are associated with prognosis in CRC. Fecal microbiome analysis showed that old microbiomes have higher alpha diversity compared to young mice. Our results demonstrate that interactions between host (age) and external (diet) factors regulate tumor growth mediated by cytokines, mitochondrial derived proteins, and the gut microbiome. Collectively, our findings advance current understanding of the mechanisms by which aging, diet and gut microbiota impact CRC onset and progression though further investigation is warranted.

Glioblastoma multiforme (GBM) is a complex and aggressive central nervous system (CNS) tumor that has a poor prognosis, and restricted therapeutic options are available despite the increasing research conducted. Moreover, the cells in our body package microRNAs, ubiquitous modulators of numerous biological processes, into exosomes for cell-to-cell signaling. Indeed, exosomal miRNAs contribute to several aspects of glioma, such as development, occurrence, metastasis, and immune evasion. Additionally, exosomal miRNAs play a key role in cellular functions and glioma pathogenesis by regulating numerous pathways, including the Wnt/β-catenin, PTEN/PI3K/Akt, EGFR/MAPK, notch signaling, and NF-κB. Notably, exosomal miRNAs are recognized to have promising potential in clinical applications; in fact, exosomal miRNAs are emerging biomarkers for glioma diagnosis and prognosis and are additionally considered as putative therapeutic candidates by inhibiting tumor progression, occurrence, and metastasis. This review presents the current knowledge regarding clinical potential and application of exosomal miRNAs in glioma, as well as the miRNA-mediated regulatory network underlying glioma immunopathogenesis.

Pleomorphic adenoma (PA) is the most common benign salivary gland tumor, yet its histologic diversity can resemble malignant neoplasms, posing diagnostic challenges. Molecular characterization, including fusion gene analysis, can aid in distinguishing these cases.

Gains of chromosome 12p11.21, encoding for the cancer-specific lncRNA LISRR, correlate with poor survival across different cancers. In melanoma, LISRR is upregulated in immunotherapy-resistant patients to contribute to the generation of drug-tolerant cells by activating an immune-suppressive translational program, affecting the synthesis of PD-L1 and of the glycocalyx. Accordingly, downregulation of LISRR initiates robust immune responses and resensitizes to immunotherapy ex vivo and in vivo. The use of glycans to evade immunity exhibits shared characteristics with the testis, where defects in the glycocalyx cause infertility. Mechanistically, we showed that LISRR affects the ribosome core composition and recruits deleted in azoospermia-associated protein 1 to polysomes to prime the integrated stress response. Our study reveals the contribution of lncRNAs to the generation of cancer-specific ribosomes and identifies an RNA-based strategy to overcome resistance to immune checkpoint blockade.

Glioblastoma multiforme (GBM), the most invasive primary malignant tumor of the central nervous system, is characterized by an extremely poor prognosis and a high recurrence rate. Its significant molecular heterogeneity challenges precise diagnosis and treatment. Recently, with the rapid development of molecular pathology, the combination of histological and molecular typing has become the mainstream method for GBM diagnosis. Here, we review the impact of classic molecular markers on patient prognosis in GBM, as well as the different values of traditional and novel molecular markers in prognosis assessment. We initially discuss the correlation between molecular markers and recurrence, as well as the research progress of molecular markers in emerging technological fields. Moreover, we propose the challenges currently faced by molecular markers in glioblastoma and discuss future research directions in this field.