The detection of circulating tumor cells (CTCs) through liquid biopsy offers a non-invasive approach for accurately monitoring cancer dissemination and evaluating therapeutic efficiency. However, their rarity and heterogeneity limit conventional tumor antigen labelling-based methods in identifying and tracing CTCs. Here, we developed a novel metric, termed chromatin unwinding state (CUS), which leverages activated transcriptional regions related to cell-identity processes from single-cell transcriptomic data while overcoming technical variances. Using CUS features, we trained attention-based neural network models, panCTC, to in situ identify and lineage trace rare single CTCs directly from 5 mL of peripheral blood mononuclear cells scRNA-seq without enrichment. We benchmarked panCTC on various in silico-simulated, public, and in-house sequenced data, demonstrating its robustness across sample types and platforms. PanCTC could provide real-time scRNA-seq profiles of fresh CTCs, supporting early cancer detection and targeted anti-metastatic therapy.

Calcium is an essential inorganic element that acts as a crucial intracellular second messenger, regulating numerous cellular signaling pathways. Variations in calcium concentrations significantly influence a wide array of physiological and pathological processes. Nearly all human physiological activities are intricately linked to calcium signal regulation, with different cell types executing specific functions through distinct calcium signaling patterns. Dysregulation of calcium metabolism can lead to hypercalcemia or hypocalcemia, and hypercalcemia is often associated with various malignancies, where it facilitates cancer cell proliferation and metastasis, particularly in gastroesophageal and colorectal cancers. Conversely, studies indicate that dietary calcium and vitamin D supplements can reduce the risk of several cancers. In colorectal cancer, for instance, intracellular Ca2+ signaling can paradoxically promote both tumor initiation and progression, as well as induce tumor cell death. Similarly, in gastroesophageal carcinoma, specific calcium signals are critical for processes such as proliferation, migration, and invasion. This review elucidates the dual role of calcium signaling in gastrointestinal carcinomas, highlighting the paradox of its context-dependent, both promotive and protective, effects.

BackgroundHepatocellular carcinoma (HCC) has a poor prognosis, and identifying key driver genes and their molecular mechanisms is crucial for improving patient outcomes. While TOP2A is dysregulated in multiple cancers, its role and regulatory network in HCC remain incompletely understood.MethodsIntegrated bioinformatics analyses were performed using TCGA, ICGC, and GEO datasets. TOP2A expression and prognostic value were validated in 120 clinical samples via immunohistochemistry. Functional assays (including CCK-8, Transwell, and wound healing in vitro, as well as xenograft tumor models in vivo) were conducted to assess phenotypic effects. Mechanisms were explored using ChIP-PCR, WB, and RT-qPCR. Prognostic models were developed using multivariate regression and evaluated using calibration curves, ROC, and DCA.ResultsTOP2A was significantly upregulated in HCC tissues, with an AUC of 0.935 for diagnosis, and high expression correlated with advanced stage and poor survival (p<0.05). A prognostic model incorporating TOP2A expression, TNM stage, and tumor grade showed robust predictive accuracy for 1-, 3-, and 5-year survival (AUCs: 0.77, 0.85, 0.75). Knockdown of TOP2A suppressed proliferation, migration, and invasion. Mechanistically, E2F6 transcriptionally activated TOP2A by binding to its promoter, while TOP2A promoted EMT by upregulating DKK1 and activating β-catenin signaling. High TOP2A expression predicted poor response to TACE, sorafenib, and immunotherapy. The candidate targeted agent, A-443654, alone or combined with anti-PD-L1, potently suppressed tumor growth in vivo.ConclusionThis study delineates the E2F6-TOP2A-DKK1 axis as a key mechanism in HCC progression. TOP2A serves as a diagnostic and prognostic biomarker, and targeting this pathway offers a promising therapeutic strategy for HCC.

List of si RNA sequences, qRT-PCR primer sequences and antibody catalog numbers.

INTRODUCTION:mRNA sequencing analysis suggested that ubiquinone oxidoreductase subunit A10 (NDUFA10) may function as a key downstream effector of circRAPGEF5 in lung adenocarcinoma (LUAD) progression; however, the underlying mechanism remains unclear. This study aimed to explore the expression and clinical significance of NDUFA10 in LUAD cells and its role in cell proliferation and metastasis. METHODS:Variations in NDUFA10 mRNA and protein expression between LUAD and adjacent normal tissues were assessed using the Cancer Genome Atlas Program (TCGA) and Tumor Immune Estimation Resource (TIMER) databases. These findings were subsequently validated in LUAD cell lines via Reverse Transcription Quantitative PCR (RT-qPCR). Further analyses included clinical correlation, Receiver Operating Characteristic (ROC), survival analysis, and Gene Set Enrichment Analysis (GSEA) of NDUFA10. The functional role of NDUFA10 was investigated in vitro at the cellular level. RESULTS:NDUFA10 mRNA expression was significantly upregulated in pan-cancer analyses and specifically in LUAD. Its expression levels correlated with clinical parameters, including metastasis (M stage) and patient gender. Elevated NDUFA10 expression demonstrated a negative correlation with overall survival (OS) and first progression (FP), and was associated with the best efficacy of anti-PD-1/PD-L1 antibody therapies. GSEA indicated significant enrichment of NDUFA10-related genes in pathways involving WNT and Notch signaling, as well as the TGFβ pathway regulating the epithelial-mesenchymal transition (EMT). In vitro, knockdown of NDUFA10 significantly suppressed LUAD cell proliferation, colony formation, migration, and invasion compared to control cells. DISCUSSION:This study focuses on the role of NDUFA10 in lung adenocarcinoma (LUAD). Through database screening and experimental verification, it was found that NDUFA10 is upregulated in LUAD. Its high expression is associated with advanced N stage (N2/N3), high pathological stage (III/IV), poor therapeutic effect and advanced age, suggesting its potential as a biomarker for poor diagnosis and prognosis. Further analysis indicates that NDUFA10 is associated with immune infiltration and involves pathways such as neuroactive ligand-receptor interaction and chemokine signaling. In vitro experiments have confirmed that knocking down NDUFA10 can inhibit the proliferation and metastasis of lung adenocarcinoma cells. This study provides a preliminary basis for the function and mechanism of NDUFA10 in LUAD. In the future, we will further explore how it affects tumor progression through the WNT/Notch/TGFβ pathway or ROS/ energy metabolism. CONCLUSION:Silencing NDUFA10 suppresses LUAD cell proliferation, migration, and invasion. The study suggested that circRAPGEF5 regulated NDUFA10 to facilitate the progression of LUAD.

Pazopanib, a first-line tyrosine kinase inhibitor for advanced renal cell carcinoma (RCC), faces significant clinical limitations due to acquired resistance. In this study, we reveal a novel mechanism underlying pazopanib resistance in RCC, driven by a senescence-like phenotype without cell-cycle arrest. Transcriptomic profiling identified ALYREF as a key upregulated m5C reader in pazopanib resistant cells. Mechanistically, ALYREF stabilizes CCNA1 mRNA via m5C modification, promoting Cyclin A1 expression. The Cyclin A1-CDK2 complex phosphorylates p21 at Thr-57, inducing its cytoplasmic translocation and abrogating its inhibitory effect on cell cycle progression, thereby facilitating G1-S transition. Our findings uncover a critical ALYREF-Cyclin A1-p21 axis in RCC and suggest that targeting this pathway may provide novel therapeutic strategies to overcome pazopanib resistance.

ABSTRACT Circulating tumor cells (CTCs) encounter multiple challenges within the blood microenvironment, including oxidative stress, flow shear forces, and immune surveillance, often leading to anoikis. Recently, a transitional state of CTC senescence has been identified, contributing to metastatic inefficiency. However, the molecular mechanisms linking senescent CTCs to disease relapse remain to be defined. By integrating a genetic model of cortactin knockdown-induced CTC senescence with single-cell multi-omic analyses, we revealed two distinct senescent CTC subpopulations marked by HES1 expression levels. These HES1 low and HES1 high subpopulations exhibited differential evolutionary trajectory dynamics and unique molecular and metabolic signatures, which were significantly correlated with adverse clinical outcome across several patient cohorts. HES1 low senescent CTCs displayed enhanced mitochondrial fitness, oxidative phosphorylation, and ROS-detoxifying capabilities, resulting in more efficient tumor regrowth with a pro-inflammatory and thrombotic phenotype when compared to the HES1 high group. Mechanistically, HES1 directly bound to the Sod1 promoter and repressed its expression, leading to redox imbalance and mitochondrial dysfunction that were linked to weakened tumor regrowth capacity. Both senescent CTC subpopulations were broadly resistant to cytotoxic and targeted therapies, yet they showed elevated dependency on anti-apoptosis programs that make them susceptible to dual blockade by SOD1 inhibitor and the anti-senolytic drug ABT737 in vivo . Finally, in a prospective cohort of on-treatment melanoma patients, HES1⁺ senescent CTCs were highly enriched in patients with progressive disease. Thus, the HES1-SOD1 antagonism shapes CTC senescence heterogeneity and contributes to differential tumor relapse, which can be therapeutically explored for eliminating residual metastatic disease. GRAPHIC ABSTRACT Trajectory analysis of senescent CTCs uncovers substantial heterogeneity, resolving two major subclones defined by HES1 expression (HES1⁺ and HES1⁻) with distinct molecular and metabolic programs. These subclones exhibit clinically prognostic correlation and linked to metastatic relapse. Mechanistically, HES1 and SOD1 are antagonistically regulated to control mitochondrial fitness and ROS homeostasis. HES1 represses the SOD1 expression, which elevates ROS and impairs OXPHOS capacity, thereby constraining tumor regrowth. Preclinical model showed the SOD1 inhibitor LCS-1 combined with the apoptosis inducer ABT737 synergistically suppresses senescent CTC-driven tumor relapse in vivo . Clinically, melanoma patients with progressive disease (PD) harbor significantly more senescent CTCs than non-PD patients, highlighting senescent CTCs as a noninvasive biomarker for therapeutic resistance and disease monitoring.

Abstract Melanoma is the most lethal form of skin cancer, characterized by an increasing incidence and high mortality rates globally. However, the mechanisms underlying melanoma tumorigenesis and progression remain poorly understood. Analyses of public repositories have identified SERPINE2 as a key differentially expressed gene involved in the biological processes associated with melanoma. As a member of the serpin superfamily, SERPINE2 plays diverse roles in regulating proteolytic activity, inflammation, and tissue remodeling; however, its specific functions in melanoma warrant further investigation. This study aims to explore the potential mechanisms by which SERPINE2 influences tumorigenesis and progression in melanoma. In this study, we evaluated the expression and clinical relevance of SERPINE2 in melanoma by analyzing data from TCGA, GTEx, and TIMER2.0, and identified SERPINE2-associated biological processes through GO and KEGG enrichment analyses. Our findings revealed that SERPINE2 is significantly upregulated in melanoma tissues compared to normal skin tissues, and its expression correlates with cell growth, epithelial-mesenchymal transition, and immune response. Knockdown of SERPINE2 markedly inhibited cell viability, proliferation, and invasion of melanoma cells both in vitro and in vivo. RNA sequencing analysis of SERPINE2-knockdown cell lines demonstrated that silencing SERPINE2 induced cell cycle arrest in the G0/G1 phase and promoted apoptosis. To investigate the underlying mechanisms, we identified DDX3X as a binding partner of SERPINE2 through co-immunoprecipitation (CO-IP) and immunoprecipitation-mass spectrometry (IP-MS) analyses. We found that SERPINE2 attenuates the protein stability of DDX3X, promoting its degradation via the ubiquitin-proteasome system. Furthermore, silencing DDX3X resulted in the downregulation of nuclear protein expression of MITF and p21, suggesting that DDX3X downregulation may influence the transcription of downstream genes associated with cell cycle progression, thereby impacting melanoma growth. Additionally, downregulation of SERPINE2 enhanced CD8+ T cell infiltration, stimulated T-cell activation-related cytokines and chemokines, and improved the cytotoxic function of CD8+ T cells in vitro. Furthermore, in both the Lauss cohort and the IMvigor210 cohort, we observed that SERPINE2 expression correlated with the prognosis of melanoma patients undergoing immunotherapy; specifically, patients with low SERPINE2 expression exhibited better prognoses compared to those with high SERPINE2 expression. In summary, the expression level of SERPINE2 is a predictor of therapy response and prognosis in melanoma patients, suggesting its potential as a biomarker for melanoma immunotherapy. This work was supported by the National Natural Science Foundation of China (No. 82173336), and the MRI Project (G030410001). Citation Format: Qirui Liu, Xin Huang, Xiao Zhang, Xinyu Ye, Yi Lu, Jian Zhang. Serpine2 promotes melanoma progression through the modulation of the DDX3X/MITF/p21 signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1915.

The impact of SNX10 knockdown on phenotypes of WT parental cells.

Intratumoral heterogeneity supports cancer progression and metastasis while limiting therapeutic efficacy. Comprehensive biological characterization of heterogeneous subclones in breast cancer may hold promise in battling against this deadly disease. Using single-cell transcriptomic analysis and in situ profiling of primary tumors, metastases, and circulating tumor cells from multiple patients with breast cancer, we identified a unique EPCAM+ CD68+ TREM2+ tumor subpopulation, likely resulting from the fusion of tumor cells and lipid-associated macrophages (LAM). The presence of these tumor-LAM fusion cells in the blood or in distinct metastatic sites was significantly correlated with metastatic progression. Stable fusion clonal lines established in vitro exhibited substantially enhanced proliferation, tumor initiation, and metastasis formation in mice. Integrative molecular and functional analyses revealed a critical role for SNX10 in mediating tumor-LAM fusion. Mechanistically, SNX10 physically interacted with the phospholipid scramblase ANO6 and maintained its protein stability by suppressing proteasome-mediated degradation. Furthermore, the transcription factor CBX3 directly targeted SNX10 promoter and modulated its expression. Fusion cells accumulated abundant intracellular lipid droplets and were highly sensitive to simvastatin treatment in vitro and in vivo. Together, this study uncovered that CBX3-SNX10-ANO6 signaling facilitates generation of an aggressive tumor-LAM fusion cell subpopulation that promotes metastasis, revealing an alternative metastatic mechanism and exposing putative therapeutic vulnerabilities.Significance: Single-cell transcriptomic profiling combined with functional and clinical validation identifies fusion of tumor cells and lipid-associated macrophages mediated by the CBX3-SNX10-ANO6 axis as a potentially targetable mechanism driving cancer metastasis.

ANO6 depletion suppresses 3-D colony formation, proliferation, migration, and invasion of fusion clones.

Small cell lung cancer (SCLC) remains a highly lethal malignancy with limited therapeutic options. The purpose of this study was to investigate the central role of histone deacetylase 6 (HDAC6) in SCLC progression and its regulatory mechanisms to identify novel therapeutic strategies. Preclinical SCLC models were utilized alongside molecular, cellular, and immunological techniques to elucidate HDAC6's mechanistic functions. The deacetylation of S100A2 and its impact on downstream signaling were analyzed, compensatory responses to HDAC6 inhibition were assessed, and the efficacy of dual-target inhibition was evaluated. HDAC6 was found to deacetylate the calcium-binding protein S100A2 at lysine 27, thereby stabilizing TGF-β/SMAD signaling to promote epithelial-mesenchymal transition (EMT) and metastatic dissemination. Simultaneously, HDAC6 polarized macrophages toward tumor-promoting M2 phenotypes, fostering an immunosuppressive microenvironment. HDAC6 inhibition triggered compensatory CSF1R upregulation, revealing a resistance mechanism. Dual blockade of HDAC6 and CSF1R synergistically suppressed primary tumor growth and metastasis while reprogramming macrophages toward anti-tumor M1 states. SCLC patients with co-high expression of HDAC6 and CSF1R exhibited worse progression-free survival (PFS). This study defines the HDAC6-S100A2-TGF-β/SMAD and HDAC6-CSF1R-macrophage axes as actionable therapeutic vulnerabilities. The dual inhibition strategy provides a translational framework to overcome stromal and immune barriers in this recalcitrant cancer.

Related to Fig. 6d. Enrichment analysis for top important molecular features

Group-wise comparisons of dose–response curves and IC50 values between control and mTOR-knockdown cancer cell lines.

Lipid metabolism is among the most frequently dysregulated metabolic processes in human cancer, yet how cellular lipids, the end products of lipogenesis, and their composition are altered to support various aspects of cancer remains poorly understood. Here, we show that targeting SREBP-dependent lipogenesis via FGH10019, an orally available SREBP inhibitor, enhances docetaxel-induced cytotoxicity in human prostate cancer cells in vitro and in vivo. Mechanistically, suppression of lipid biosynthesis leads to a shift in cellular lipid composition toward polyunsaturated lipids, resulting in increased membrane permeability and intracellular docetaxel accumulation. Thus, our findings reveal a critical role of de novo lipogenesis in protecting cancer cells from chemotherapeutics and suggest that treatment with lipogenesis inhibitors could improve the efficacy of chemotherapy against human prostate cancer.

PCA of the WER-target expression profile in the TCGA cohort.

Related to Fig. 3d. The regulatory capacity of m6A machineries.

Related to Supplementary Fig. S3. Sample Information for the SRR Cohort.

Calibration and discrimination via the C-for-benefit method for m6A DE prediction.

The top 50 important determinants for the DE of m6A dysregulation in cancer.