Nucleic acid aptamers, often referred to as “chemical antibodies,” are versatile, specific, and easily modifiable functional nucleic acids. There is a growing focus on new methods for the selection and target validation of aptamers, with the aim of expanding their biomedical applications in molecular diagnostics and therapeutics, which is currently a research hotspot. This review is composed of eight sections. In the first section, we briefly introduce aptamers and review their development in molecular diagnostics and therapeutics. The “Advantages of aptamers in molecular diagnosis and therapeutics” section summarizes and discusses the advantages of aptamers in these fields. The “New methods for screening aptamers” section presents and discusses nucleic acid aptamer screening methods, including both classical and novel approaches. In the “New methods for target validation” section, we explore new methods for target validation, covering aptamer structure validation, target recognition validation, and aptamer–target interaction validation. The “New methods for molecular diagnostics” section summarizes and discusses recent applications of aptamers in molecular diagnostics, particularly focusing on new mechanisms and detection strategies as well as their applications in various diseases. The “New methods for molecular therapeutics” section summarizes and discusses recent applications of aptamers in molecular therapeutics, emphasizing new mechanisms and aptamer-based therapy strategies, along with their therapeutic applications in different diseases. The “Challenges and future perspectives of nucleic acid aptamers” section addresses the challenges and future perspectives of aptamers in disease diagnosis and treatment. Finally, the “Conclusion” section shares our views on the future directions of aptamers in clinical disease molecular diagnostics and therapeutics.
Aneuploidy is pervasive in cancers and contributes to chemoresistance; however, how aneuploidy-inducing stresses, such as infection and hypoxia, promote chemoresistance remains unclear. Here, we identify a prolyl hydroxylase domain protein 1 (PHD1)-E3 ubiquitin ligase TRIM21-cell division cycle protein 20 (CDC20) signaling axis that integrates infection- and PHD1-inhibitory signals to drive aneuploidy and chemoresistance. Analysis of clinical specimens revealed that HPV-positive cervical cancers exhibited reduced CDC20 expression and increased aneuploidy compared with HPV-negative tumors. Through proteomic screening, we found that CDC20 is targeted for degradation by TRIM21, which preferentially recognizes CDC20 when prolines 337 and 340 are non-hydroxylated. Hypoxia and α-ketoglutarate (α-KG) limitation impair the activity of the dioxygenase PHD1, thereby increasing the fraction of non-hydroxylated CDC20. In parallel, infection activates TRIM21. Thus, PHD1 inactivation and infection converge on CDC20 to reduce its abundance, leading to the accumulation of CDC20 substrates, including the separase inhibitor securin and the anti-apoptotic protein MCL1. Infection- and PHD1 inhibition-induced securin accumulation promotes aneuploidy, whereas MCL1 accumulation enhances chemoresistance. In cultured cancer cells and mouse xenograft models, stabilization of CDC20, either through TRIM21 inhibition or PHD1 activation, attenuates aneuploidy and restores chemosensitivity. Together, our study reveals a PHD1-TRIM21-CDC20 signaling axis that integrates hypoxic and infection-associated cues to regulate aneuploidy and chemoresistance, highlighting this pathway as a potential therapeutic target for overcoming chemoresistance.
Metabolic reprogramming forms the foundation of immune effector functions and the regulation of inflammation. As a pivotal node connecting the tricarboxylic acid cycle to immune signaling, the IRG1/ACOD1 and itaconate axes play a central role in coordinating inflammatory tone and redox balance. Itaconate, generated through the decarboxylation of cis aconitate, acts as an immunometabolic brake that engages multiple regulatory pathways to sustain the dynamic equilibrium between inflammation and tissue homeostasis. Across a broad spectrum of pathological conditions, including infectious diseases, metabolic disorders, ischemia‒reperfusion injury, neurodegenerative diseases, autoimmune disorders, and cancers, itaconate and its derivatives generally exert anti-inflammatory and cytoprotective effects. However, within specific microenvironments, these molecules may also be exploited by pathogens to evade immune clearance or promote immunosuppressive and protumorigenic responses. Future studies should further elucidate tissue- and lineage-specific functions, define bidirectional regulatory mechanisms, and optimize the pharmacokinetic properties of itaconate derivatives. With the advancement of multiomics integration, systems immunology, rational drug design, and engineered itaconate delivery technologies, the IRG1/ACOD1-itaconate axis and derivative-based therapeutic strategies are poised to emerge as key metabolic checkpoints and therapeutic targets in inflammatory-, metabolic-, immune-, and cancer-related diseases.
Epithelial ovarian cancer (EOC) is a highly lethal disease characterized by a high rate of platinum (PT)-resistant recurrence. Dysregulation of transcriptional and alternative splicing (AS) has been suggested as a possible mechanism of PT resistance involving transcriptional cyclin-dependent kinases (CDKs). We report that pharmacological CDK12/CDK13 inhibition (CDK12i) synergistically enhances PT-induced cytotoxicity in a large number of PT-sensitive and PT-resistant EOC models. CDK12i effectively delayed the emergence of PT-resistant recurrences in vitro and in vivo. Acquired resistance to CDK12i enhanced PT sensitivity, revealing a therapeutically exploitable vulnerability to PT. CDK12i strongly reshapes transcriptional elongation and RNA processing, impacting multiple categories of AS, with marked enrichment of transcripts involved in the DNA damage repair (DDR) pathway. We proved that CDK12 interacts with the splicing master regulator SFPQ/p54nrb complex, driving its distribution in transcriptional condensates. The p54nrb subunit of the complex is required for the CDK12-SFPQ interaction and to ensure splicing reprogramming and CDK12i-induced cytotoxicity in all the EOC models tested. The use of CT7439, a novel clinical-grade CDK12i, which is now in phase I/II clinical trials, confirmed the therapeutic potential of CDK12i, either as monotherapy or in combination with PT. These findings emphasize that the therapeutic potential of CDK12i extends beyond DDR regulation, encompassing a critical role in RNA processing and AS. This dual impact affects EOC survival and may provide a novel, clinically relevant therapeutic strategy for hard-to-treat PT-resistant patients for whom effective treatment options remain limited.
T cell exhaustion and T cell senescence constitute distinct yet partially overlapping differentiation states that collectively constrain T cell functionality. T cell exhaustion arises under conditions of chronic antigen exposure and is characterised by a progressive, hierarchical loss of effector capacity, sustained expression of inhibitory receptors, and extensive transcriptional, epigenetic and metabolic reprogramming. By contrast, T cell senescence represents a more stable and terminal state, driven by replicative history, age-associated decline or stress-induced damage, and is defined by durable cell cycle arrest, altered differentiation, metabolic remodelling and acquisition of a pro-inflammatory secretory phenotype. In the context of cancer, dysfunctional T cells contribute to tumour progression, while also representing a major barrier to the success of T cell-based immune therapies, which strongly rely on the fitness, persistence and functional plasticity of T cells. Although substantial efforts have focused on overcoming exhaustion and optimising T cell manufacturing, senescence remains comparatively underexplored and presents unique therapeutic challenges due to its relative resistance to functional reprogramming. This review provides a comprehensive overview of T cell replenishment in homeostasis, followed by the molecular hallmarks and signalling pathways of T cell senescence and exhaustion. We discuss the current landscape of T cell-based immune therapies, including immune checkpoint blockade, T cell engagers and adoptive cell therapies, and explain how T cell dysfunction impacts their therapeutic outcomes. Finally, we highlight emerging strategies to prevent or overcome T cell dysfunction in adoptive cell therapy products.
The global prevalence of metabolic diseases, notably obesity, diabetes mellitus, and thyroid disorders, has risen dramatically in recent decades, posing a significant threat to public health. While conventional pharmacological interventions remain the cornerstone of management, they face considerable limitations, including adverse effects and the development of drug resistance, underscoring the urgent need for alternative therapeutic strategies. Intriguingly, immune checkpoint molecules, originally recognized for their transformative role in cancer immunotherapy, are now gaining attention as pivotal regulators of metabolic homeostasis. Growing research demonstrates that immune checkpoint pathways, particularly the PD-1/PD-L1 axis, play dual roles in immune regulation and metabolic modulation by orchestrating inflammatory responses and energy metabolism. For instance, in obesity, PD-1/PD-L1 signaling promotes the polarization of adipose tissue macrophages from a proinflammatory M1 phenotype toward an anti-inflammatory M2 state, thereby mitigating chronic low-grade inflammation and associated metabolic dysfunction. These findings highlight the potential of immune checkpoint modulation as a novel therapeutic strategy for metabolic disorders. However, despite these advances, the role of immune checkpoints in metabolic diseases remains underexplored, with limited comprehensive reviews on the subject. This review comprehensively elucidates the pathophysiological mechanisms and translational potential of immune checkpoint regulation in obesity, diabetes, and thyroid disease while proposing novel directions for clinical intervention.
miRNAs are important metabolic regulators and are altered at both the cellular and secreted levels in diseases, including type 2 diabetes (T2D). However, to what extent these alterations are in response to factors in the in vivo milieu or are cell-intrinsic remains unclear. Here we used a disease-in-a-dish model in which iPSCs from T2D patients and controls were differentiated into myoblasts (iMyos), and their cellular and secreted miRNAs were profiled. We found that iMyos from T2D donors exhibit cell-intrinsic alterations in miRNA expression and secretion in small extracellular vesicles (sEVs)/exosomes. Integrating miRNA-predicted targets with transcriptomic and proteomic data revealed that miRNAs altered in T2D iMyos were associated with coordinated changes in their predicted targets, but with a much greater impact on protein than on mRNA levels. This effect was validated by miRNA overexpression in control iMyos. The upregulated miRNAs targeted pathways related to aerobic respiration, membrane trafficking, and RNA metabolism. Even more marked changes were observed in sEV-associated miRNAs secreted by T2D iMyos, indicative of T2D-associated effects on miRNA sorting and release. Target genes of secreted miRNAs altered in T2D iMyos were enriched in metabolic pathways including insulin signaling and mitochondrial metabolism. Consistent with this, sEVs derived from control iMyos increased glucose uptake and mitochondrial function in recipient human white adipocytes, whereas sEVs from T2D iMyos did not. Thus, in T2D, muscle exhibits cell-intrinsic alterations in expression and secretion of miRNAs, which function as epigenetic regulators of protein expression locally, as well as potentially in distal tissues.
As a key regulator of the immune system, regulatory T cells (Treg cells) suppress excessive immune responses and maintain self-tolerance through various immunosuppressive mechanisms, both contact-dependent and contact-independent. They have demonstrated significant therapeutic potential in immune-related diseases and some non-immune diseases. In this paper, we systematically review the induction strategies of Treg cells, covering various aspects including antigenic stimulation, cytokine modulation, metabolic pathways, epigenetic regulation, drug induction, and engineered technology. Although each strategy has its own advantages, no single approach can comprehensively address the complex issues surrounding Treg cell stability, target specificity, and large-scale production. Therefore, we propose that integrating multiple induction strategies during the Treg cell induction process is crucial for designing Treg cell therapies with superior functionality and broader applications, thereby overcoming the current limitations of Treg cell therapies. In addition, we introduce their therapeutic applications in immune diseases (such as autoimmune disease, organ transplantation, allergic asthma, and cancer) and non-immune diseases (such as insulin resistance, tissue repair, fibrotic diseases, and osteoporosis), and recent advancements. We also discuss current research bottlenecks of Treg cell-based therapeutic strategies and provide an outlook on their future development, aiming to deepen the understanding of Treg cells and promote the advancement of Treg cell-based cellular therapeutic strategies.
The intratumoral microbiota is increasingly recognized as an active component of the tumor microenvironment, yet whether it directly drives tumor mutagenesis remains unclear. Here, integrated multi-omics analysis of human non-small cell lung cancer (NSCLC) identifies Mycobacterium abscessus as a microbial determinant of APOBEC3A-associated mutagenesis. Mechanistically, the bacterial effector nucleoside diphosphate kinase (NDK) directly targets the host transcription factor IRF3 and installs a non-canonical 1-phosphohistidine modification at H263, thereby amplifying type I interferon signaling and sustaining APOBEC3A expression. This inter-kingdom phosphotransfer event links intratumoral microbial colonization to an endogenous mutational process that promotes genomic diversification. Genetic inactivation of NDK, or pharmacologic elimination using an engineered NDK-PROTAC, suppresses APOBEC3A activation and attenuates microbe driven mutagenesis. Together, these findings establish a direct microbial effector mechanism that promotes APOBEC3A-associated mutagenesis and provide a therapeutic framework to intercept microbiome driven mutagenesis in NSCLC.
The pulmonary system is a vital interface between the body and the external environment, making it highly vulnerable to environmental, infectious, and genetic insults. Precision nanomedicine offers a promising strategy to overcome the limitations of conventional gene and drug therapies, including safety concerns associated with viral vectors, instability of therapeutic agents, suboptimal cellular internalization, and a critical lack of tissue- and cell-specific targeting. Nanoparticle-based delivery platforms address these challenges by enhancing therapeutic stability and bioavailability, enabling controlled release, facilitating cellular uptake and endosomal escape, and achieving targeted delivery to specific lung compartments. While recent literature often focuses on specific nanoparticle types or isolated pathologies, this work provides a comprehensive overview of the current state of respiratory nanomedicine, bridging fundamental nanoparticle bioengineering with a wide range of pulmonary pathologies and the obstacles to clinical translation. We discuss the key physicochemical properties of nanoparticles for pulmonary biomedical applications, along with advanced design strategies for targeted delivery. Given the unique architecture and physiology of the lung, we compare the advantages and limitations of pulmonary versus systemic administration routes, emphasizing context-specific delivery strategies. Nanoparticle design and therapeutic applications are explored across a broad spectrum of diseases, including pulmonary fibrosis, chronic obstructive pulmonary disease, infections, pulmonary vascular disease, cystic fibrosis, asthma, lung cancers, and neonatal pulmonary disorders. Finally, we evaluate the current status of clinical trials, highlighting translational challenges such as biological barriers, long-term safety, and manufacturing. Future perspectives and interdisciplinary strategies are proposed to advance the clinical translation of nanocarriers for respiratory diseases.
Bevacizumab plus chemotherapy is the standard first-line therapy for metastatic colorectal cancer (mCRC). To date, no phase 3 trial has compared first-line oral multitargeted TKI versus bevacizumab plus chemotherapy in RAS/BRAF wild-type mCRC. The open-label, noninferiority, randomized, phase 3 trial (ANCHOR; NCT04854668; CTR20210940) evaluated first-line anlotinib versus bevacizumab plus oxaliplatin and capecitabine (CapeOX) in this setting. Patients were centrally randomized (1:1) to receive 4-8 cycles of CapeOX in combination with either anlotinib (12 mg once daily on days 1-14) or bevacizumab (7.5 mg/kg on day 1) every 3 weeks, followed by maintenance therapy with anlotinib or bevacizumab plus capecitabine until unacceptable toxicity or disease progression. The primary endpoint was progression-free survival (PFS) assessed by an independent review committee in the intention-to-treat population. The hazard ratio (HR) for the noninferiority margin was 1.09. Between May 25, 2021, and August 30, 2023, 373 patients were assigned to the anlotinib group and 375 to the bevacizumab group. As of February 2, 2025, the median follow-up was 25.1 months (95% confidence interval [CI] 23.8-26.3). The median PFS was 11.0 months (95% CI 9.8-11.2) in the anlotinib group versus 11.0 months (9.7-11.2) in the bevacizumab group (stratified HR, 1.00; 95% CI 0.84-1.18; p = 0.87). The incidences of grade ≥3 treatment-related adverse events were 64.9% and 44.8%, respectively. Compared with bevacizumab plus CapeOX, anlotinib plus CapeOX showed similar antitumor activity but failed to reach the prespecified noninferiority margin for PFS and was associated with increased manageable toxicity.
The PI3K/AKT signaling pathway serves as a pivotal regulatory hub that governs essential cellular processes, including growth, proliferation, metabolism, survival, and migration. In the context of tumorigenesis and cancer development, aberrant activation of this pathway drives malignant transformation, enhances invasive and metastatic potential, and confers resistance to therapeutic interventions, ultimately resulting in tumor progression and poorer clinical outcomes. This study comprehensively investigates PI3K/AKT signaling, elucidating its involvement across diverse human systemic tumors and analyzing the interplay between this pathway and other signaling cascades implicated in tumorigenesis. Emphasis is placed on mechanistic insights into the role of the PI3K/AKT pathway in tumor initiation and progression across various systems, alongside an overview of therapeutic strategies targeting PI3K/AKT signaling. Notably, advances in research on the PI3K/AKT pathway have identified a wide array of potential therapeutic targets, facilitating significant progress in related treatment modalities. Given the extensive involvement of PI3K/AKT signaling in diverse biological processes, optimizing the therapeutic efficacy of PI3K/AKT-targeted interventions while minimizing associated adverse effects remains a critical challenge, necessitating innovative approaches for substantial therapeutic breakthroughs. Therefore, a systematic and nuanced understanding of the pathway’s role in tumor biology is essential for advancing foundational research and informing future clinical developments in this field.
Activation of quiescent or extremely slow-cycling breast cancer stem-like cells (qsBCSCs) contributes to tumor progression, yet the regulatory mechanisms involved in triple-negative breast cancer (TNBC) remain elusive. We developed a dual-reporter system combining NANOG-EGFP and H2B-mCherry pulse-chase labeling to separately identify qsBCSCs and active BCSCs (aBCSCs) in vitro and in vivo. aBCSCs display significant enrichment of glycolysis and lactylation signatures, a finding corroborated by single-cell RNA sequencing (scRNA-seq) of TNBC patient samples. Glycolytic enzyme Enolase 1 (ENO1) expression is positively correlated with elevated histone H4 lysine 12 lactylation (H4K12la) in aBCSCs. Functional studies revealed a positive feedback circuit in which ENO1-increased lactate promotes H4K12la, which in turn activates ENO1 transcription. The ENO1-lactate-H4K12la axis enhances proliferating cell nuclear antigen (PCNA) transcription to activate qsBCSCs. Suppression of ENO1 or H4K12la prevents PCNA upregulation and qsBCSC activation. Importantly, PCNA knockdown alone blocks the activation of qsBCSCs induced by exogenous sodium L-lactate (NALA). In orthotopic breast cancer mouse models, both genetic depletion of ENO1 and pharmacological inhibition of lactate production attenuated tumor growth by blocking qsBCSC activation. Clinically, ENO1 expression was strongly correlated with PCNA expression, with high expression predicting poor OS, RFS, and DMFS. Our results establish the ENO1-H4K12la-PCNA axis as a key metabolic‒epigenetic driver of qsBCSC activation and a therapeutic target in TNBC.
Triple-negative breast cancer (TNBC) is an aggressive and immunogenic subtype lacking targeted therapies. While tumor-derived exosomes are known to modulate immune function, their direct impact on human T cell plasticity and antigen specificity remains poorly defined. Here, we conducted a comprehensive single-cell multiomic analysis of primary human T cells exposed to exosomes derived from 17 genomically diverse TNBC cell lines and 35 patient samples. Integrating single-cell RNA-seq, V(D)J sequencing, non-coding RNA profiling, bulk and single-cell cytokine analyses, we uncovered conserved and subtype-specific immunomodulatory programs induced by TNBC exosomes. Exosome-treated T cells displayed skewing toward regulatory and dysfunctional phenotypes, including Th17-like, Treg, and PD-1⁺/PD-L1⁺ Tfh cells. Functional profiling revealed suppression of early activation markers and cytokine responses, alongside selective preservation of cytotoxic features in γδ T and NKT subsets. Transcriptomic and miRNA network analyses demonstrated widespread downregulation of immune effector genes (e.g., HBEGF and TNFSF9) mediated by exosome-delivered regulatory miRNAs (has-miR-98-5p). Notably, exosome-stimulated T cells displayed distinct clonotypic expansions, characterized by the emergence of five tumor-specific γδ TCR clonotypes and 30 unique αβ TCR CDR3 sequences that were absent in mock-treated controls, underscoring the role of exosomes in shaping TCR repertoire dynamics.
The retinoblastoma (RB1) gene is a critical tumor suppressor that regulates cell cycle progression and genomic stability. Although RB1 alterations have been reported in hepatocellular carcinoma (HCC), the biological and clinical consequences of biallelic RB1 inactivation (RB1-Bi) remain poorly defined. We performed a comprehensive allele-specific genomic analysis of HCC patients from the TCGA-LIHC (n = 355) and in-house AMC (n = 206) cohorts, collectively comprising the AMC-TCGA discovery cohort. In this combined cohort, RB1-Bi was identified in 14.6% of tumors, was enriched in poorly differentiated HCCs and was independently associated with significantly reduced overall survival (adjusted hazard ratio 3.32, 95% CI 1.93-5.72, p < 0.001). Additionally, a deep learning-based histopathology model using hematoxylin and eosin-stained slides (i.e., FR-MIL model) accurately predicted RB1-Bi status (F1 score 84.39% [95% CI, ±0.02]), making it readily identifiable in routine clinical practice. The prevalence and prognostic impact of RB1-Bi, as well as FR-MIL model performance, were consistent across independent validation cohorts, including advanced-stage tumors and external institutions. High-throughput drug screening in isogenic HCC models revealed that RB1-Bi HCC cells were particularly sensitive to inhibitors targeting mitotic regulators (e.g., AURKA, PLK1, KSP) and DNA damage response pathways (e.g., PARP inhibitors). Synthetic lethal interactions between RB1-Bi and these compounds were demonstrated in vitro and in vivo, and combination treatment with mitotic and PARP inhibitors had synergistic effects with acceptable tolerability. We conclude that RB1-Bi represents a clinically actionable biomarker that identifies a high-risk HCC subtype with specific therapeutic vulnerabilities, offering new opportunities for precision medicine.
