Background: Although the contribution of matrix stiffness to aggravating the malignant features of HCC cells has been well documented, the effects of matrix stiffness on chemoradiotherapy resistance and its underlying mechanism remain largely elusive.
Methods: To delineate the role of matrix stiffness in HCC progression, we engineered novel in vivo animal models with defined liver stiffness and a complementary tunable hydrogel culture system. This integrated approach enabled a comprehensive investigation into how biomechanical cues modulate HCC cell proliferation and DNA repair both in vitro and in vivo.
Results: High stiffness stimulation noticeably enhanced cell proliferation and cell survival from DNA damage through changing the expression and distribution of metabolic enzyme PFKFB3. Specifically, high stiffness stimulation prominently suppressed PFKFB3 ubiquitination by downregulating E3 ubiquitin ligase NEDD4, and then increased the stability of PFKFB3 protein to enhance glycolysis, ultimately promoted HCC growth. Meanwhile, high matrix stiffness stimulation also effectively strengthened the DNA damage repair ability of irradiated HCC cells, and PFKFB3 nuclear translocation mediated in matrix stiffness-regulated DNA damage repair by interacting with Ku70.
Conclusions: Our results delineate a PFKFB3-mediated pathway that underpins how increased matrix stiffness potentiates HCC growth and compromises radiotherapy efficacy. These findings not only highlight the contribution of matrix stiffness to tumor growth and DNA damage repair in HCC, but also disclose a previously unidentified nonmetabolic function of PFKFB3.
Background: Gene expression-based molecular subtypes in glioblastoma from The Cancer Genome Atlas Network (TCGA-GBM) unraveled the pathological origins by identifying tumour cell driver genes. However, the causal inference between molecular subtype origins and their therapeutic efficacy remains obscure.
Methods: We integrated TCGA-GBM multi-omics (DNA, mRNA, and protein profiles) using correlation analysis to identify cis-regulation. We analyzed the exposure-mediated base substitution-level mutations and their potential triggers. Importantly, we performed Consensus Clustering based on the MSigDB database with Silhouette-correction to identify prognostically relevant pathway-based MSig subtypes. The tumour driver mutations (co-occurrence mutation pattern), aberrant pathways (tumour hallmarks), immune microenvironment (xCell), and pseudo-time analysis (dyno) were used to characterize the MSig subtype landscape. Furthermore, we evaluated potential drug sensitivities across MSig subtypes using the Genomics of Drug Sensitivity in Cancer database.
Results: We classified five MSig subtypes, characterized by neural-like, tumour-driving, low tumour evolution, immune-inflamed, and classical tumour features. We observed several key features in ‘tumour-driving’ GBM patients: (1) mutual exclusivity between prognostic factors TP53 and EGFR; and (2) IDH1 mutations co-occurring with TP53, which account for the protective role of IDH1 in TP53 mutant patients. The immune-inflamed GBM, characterized as a ‘hot’ tumour, exhibited upregulation of immune-related pathways, including PD-1 and IFN-γ signalling responses. DNA methylation landscape revealed 14 MGMT CpG-rich regions regulating expression. Evolutionary trajectories revealed progression from a primary tumour state (close to normal tissue) to two distinct endpoints (tumour-driving and immune-inflamed subtypes).
Conclusions: Our findings reveal interactions between tumour cells and their surrounding immune environment, classifying GBM into two newly identified subtypes: (1) the tumour-driving subtype is characterized by multiple oncogenic mutations, while (2) the immune-blockade subtype is marked by a high presence of immune cells. We highlight the importance of integrating multi-type data (somatic mutations, DNA methylation, and RNA transcripts, etc.) to decipher GBM biology and potential therapeutic implications.We report the interaction between tumor cells and environmental immune cells, classifying GBM into two main subtypes: 1) The tumor-driving subtype is characterized by multiple oncogenic mutations, while 2) the immune-blockage subtype is marked by a high presence of immune cells. We used integrated multidimensional analyses of somatic mutations, DNA methylation, and RNA transcripts to gain a deeper understanding of GBM biology and potential therapeutic implications.
Background: Valosin-containing protein (VCP) related disease, also known as multisystem proteinopathy 1 (MSP1), is an autosomal dominant disease caused by gain-of-function pathogenic variants of the VCP gene. The disease presents with variable combinations of inclusion body myopathy, early-onset Paget's disease of bone, frontotemporal dementia and may also overlap with familial amyotrophic lateral sclerosis. There is currently no treatment for this progressive disease associated with early demise resulting from proximal limb girdle and respiratory muscle weakness. We hypothesise that regulating VCP hyperactivity to normal levels can reduce the disease pathology.
Main topics covered: In this study, we assessed the effect of antisense oligonucleotides (ASOs) specifically targeting the human VCP gene in the patient (R155H) iPSC-derived skeletal muscle progenitor cells (SMPCs). ASOs were well tolerated up to a concentration of 5 µM and significantly reduced VCP protein expression in the SMPCs by 48% (95% CI [39–56]). We also treated the transgenic mouse model of VCP disease with the overexpressed humanised VCP severe A232E pathogenic gene variant (VCP A232E mice) with weekly subcutaneous ASO injections starting from 6 months of age for 3 months. In the skeletal muscle of transgenic mice, ASOs resulted in 30% (95% CI [27–32]) knockdown of VCP protein compared with control ASO. The ASO-mediated reduction of VCP expression in muscle tissue was associated with improvement in autophagy flux and reduction in TAR DNA binding protein 43 (TDP-43) expression, hallmarks of VCP related MSP1. In addition, ASO-treated VCP A232E mice showed improvements in functional tests of muscle strength, such as rotarod and inverted screen test compared with mice treated with control ASO.
Conclusions: These results suggest that targeting VCP could be beneficial in preventing the progression of the VCP myopathy and hold promise for the treatment of patients with VCP related MSP1.
Background: Early tumour vascular invasion contributes to cancer progression. Tip cells, a subset of tumour endothelial cells, significantly decline after anti-angiogenic therapy. However, their behaviour and the roles of their signature genes during early invasion are incompletely understood.
Methods: This study employed single-cell transcriptomic analysis and 10x Genomics Visium spatial transcriptomics on fresh lung tissues from patients with pulmonary nodules and from KrasG12D (K) and KrasG12DTgfbr2−/− (KT) mice. The role of plasma vesicle-associated protein (PLVAP), a tip cell marker, was further examined using survival databases, immunofluorescence, in vitro co-culture, cell migration, invasion assays and endothelial tube formation.
Results: Tip cell proportions were elevated in early-stage lung adenocarcinoma (LUAD) tissues and KT mice, with evidence suggesting they arise from capillaries type I. PLVAP expression was enriched in tumour endothelial cells, induced by TGFβ1, and negatively correlated with patient prognosis. Functionally, PLVAP promoted endothelial cell invasion, migration and angiogenesis, and regulated tumour cell invasiveness. Intercellular analysis revealed that some tip cells also expressed TGFβ1, which may act on adjacent tumour cells to enhance invasion during early tumour development.
Conclusion: Tip cells increased during early LUAD progression and likely evolved from capillaries type I. Their marker PLVAP was associated with poor prognosis and pro-invasive endothelial behaviour. Tumour-secreted TGFβ1 upregulated PLVAP in endothelial cells, promoting angiogenesis and tumour invasion. Additionally, tip-cell-derived TGFβ1 may further stimulate tumour aggressiveness, highlighting a reciprocal interaction that contributes to early tumour progression.
Acute myeloid leukaemia (AML) remains the most common type of leukaemia in adults. Despite advances in conventional therapies, high relapse rates persist, underscoring the need for novel approaches such as chimeric antigen receptor T (CAR-T) cell therapy. C-type lectin-like molecule-1 (CLL1)-targeted CAR-T emerges as a promising treatment for relapsed/refractory (R/R) AML. Although approximately 70% patients achieved remission, only a subset achieved minimal residual disease-negative remission, which still has much room for improvement. The main reasons for the failure of CLL1 CAR-T-cell therapy include: (1) persistence of CLL1-negative AML cells persist due to antigen escape; (2) downregulation of interleukin (IL)-12 and other cytokines by the immunosuppressive tumour microenvironment (TME), contributing to the exhaustion of both endogenous T cells and CLL1 CAR-T cells.
We synthesise a combination of CAR-engineered dendritic cells (CAR-DCs) and CLL1 CAR-T cells to overcome current limitations. CAR-DCs enhance antigen cross-presentation to activate endogenous T cells against antigen-negative clones, secrete immunostimulatory cytokines (e.g., IL-12) to sustain CAR-T activity, and remodel the TME. Key challenges involve optimising CAR designs (e.g., incorporating Fms-like tyrosine kinase 3 ligand [FLT-3L] or CD40 signalling domains), mitigating toxicity and establishing clinical administration protocols.
In this review, a focused discussion was provided on the specific challenges limiting CLL1-targeted CAR-T-cell therapy in R/R AML, namely antigen escape and the TME, and a novel combination strategy of CAR-DCs with CLL1 CAR-T cells was proposed as a promising approach to mitigate these barriers. Here, the rationale, current research advances, and future perspectives of this synergistic strategy were critically examined.
Precision medicine has evolved through distinct phases, from the origins of the Human Genome Project to mutation-based targeted therapies. This editorial posits that “stereological cell biomedicine” could be a new approach promoting the development of the next generation of precision medicine. This emerging discipline transitions the focus from genomic data to the multi-dimensional and spatiotemporal complexity of single cells. Driven by advances in Stereo single-cell multi-omics (Stereo Cell-seq), spatial transcriptomics (Stereo-seq), and single-cell surfaceomics (sc-surfaceome), this approach aims to capture the stereologically dynamic interactions between organelles within a cell and between cells in the tissue. We argue that understanding the spatiotemporal location of molecules, particularly protein interactions at organelle interfaces and on the cell surface, is as critical as their abundance for defining cellular function in health and disease. Integrating these high-resolution measurements with artificial intelligence and computational modelling will bridge the gap between advanced omics and pathology. Initiatives such as the newly established European Stereo Cell Center (ESCC) signal a global shift towards this new paradigm, which promises to unlock novel diagnostic biomarkers and therapeutic targets for truly multi-factorial and dynamic precision medicine.
Background: Nasopharyngeal carcinoma (NPC) patients who develop distant metastasis have significantly reduced survival rates. Therefore, understanding of metastasis and identifying high-risk patients are important, and a robust predictive model for accurately assessing the distant-metastasis risk before treatment is needed for personalised treatment.
Methods: NPC patients diagnosed at four Hong Kong public hospitals and at Sun Yat-Sen University Cancer Center in Guangzhou were selected. Patients were divided into two training cohorts (n = 77 and 30, respectively) and one testing cohort (n = 70). Two independent NPC cohorts collected from Sun Yat-Sen University Cancer Center (n = 88), and a randomised phase III trial (NPC-0501) in Hong Kong (n = 81) were used for external validation of the model-based risk prediction.
Results: Our RNA-based risk score could stratify the patient groups into high and low risk of metastasis and disease progression in two independent external validation cohorts. In predicting NPC 3-year distant metastasis, the score significantly improved the area under the curve from 84.8% to 90.4% when combined with the known prognostic clinical parameters. This RNA-based risk score was highly associated with dysregulated functions of B cells and T helper 17 cells and reduced plasma B cells and tertiary lymphoid structure (TLS) formation. The analysis of biopsy samples revealed a significant enrichment of the TLS in non-metastatic NPC patients.
Conclusions: This study improved the accuracy of NPC metastasis prediction and highlight the potential association of TLS against metastatic NPC, encouraging future studies to understand how TLS interacts with NPC to prevent distant metastasis. Furthermore, the multi-cohort Pareto-optimisation-based feature selection approach offers a practical method to explicitly avoid model overfitting and achieve a more robust model.
Novelty and Impact: In this multicentre study, we established a new and robust predictive model for NPC distant metastasis using markers selected by a Pareto optimisation approach designed for multi-cohort data. When combined with clinical parameters, our RNA-based risk score significantly improved the area under the curve to 90.4%. This study revealed that reduced B-cell immunity, and TLS formation, may be associated with NPC metastasis, providing insights for the future studies in NPC metastasis.
Background: Advanced maternal age (AMA) increases pregnancy risk. However, uterine-specific mechanisms independent of oocyte and embryo quality remain poorly defined. This study aimed to characterise the decidual microenvironment in women with AMA to identify key pathological changes and regulatory pathways.
Methods and results: Through integrated histology, organoid modelling, and high-resolution scRNA-seq of first-trimester decidua from women of AMA and controlled reproductive age, we uncovered a pathologically remodelled decidual microenvironment characterised by aberrant cellular states and pathological differentiation pathways, leading to a pro-fibrotic state and accompanied by immune cell dysfunction, and disrupted intercellular communication in the AMA decidua. Central to this pathology was hyperactivated TGF-β signalling, driving fibroblast-to-myofibroblast transition and extracellular matrix overproduction, thereby fuelling fibrosis. Aberrant TGF-β further impaired decidual stromal cell (DSC) differentiation, leading to the failure of the essential mesenchymal-to-epithelial transition. We identified PRR15 as a novel DSC-specific regulator that is markedly suppressed in AMA. PRR15 deficiency unleashed hyperactive TGF-β/SMAD signalling, directly causing decidualisation failure, enhanced fibrosis, and aborted DSC differentiation. Epithelial–mesenchymal transition and immune cell reprogramming towards pro-fibrotic transcriptional signatures further amplify the fibrotic pathology.
Conclusion: This study established the aged decidual microenvironment, orchestrated by dysregulated TGF-β signalling and PRR15 loss, as a critical independent determinant of reproductive failure in AMA. Thus, it unveils novel diagnostic and therapeutic targets and strategies.
Background: Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal haematopoietic stem cell disorder. Immune escape is crucial in PNH, and our previous studies revealed that natural killer (NK) cells potential participate in the immune escape of PNH. This study aimed to investigate the subtypes and functional changes of NK cells in PNH patients.
Methods: We analysed CD59+ and CD59− bone marrow mononuclear cells using single-cell RNA sequencing (scRNA-seq). The results were validated through flow cytometry and co-culture experiments.
Results: We classified NK cells into seven subtypes by scRNA-seq, and found significant differences in the distribution of subtypes in CD59+ and CD59− NK cell of PNH patients. Compared with controls, the proportion of active and adaptive NK cells was higher in CD59+ NK cells. Conversely, the proportion of CD56bright NK cells and terminal NK cells was elevated in CD59− NK cells. Additionally, the proportion of mature NK cells decreased in both the CD59+ and CD59− groups. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis revealed impaired function of CD59− NK cells, whereas CD59+ NK cells showed minimal change. Furthermore, similar results were verified by flow cytometry and co-culture in vivo and in vitro. And the proportion of NK cells was closely related to the proportion of CD8+ T cells and the clinical indicators of disease.
Conclusions: The quantity and function of NK cells in PNH patients are insufficient, in which CD59− NK cells have functional defects, whereas CD59+ NK cells were mainly activated and potential involved in immune escape by regulation of T cells.
Background: Colorectal cancer (CRC) remains a major global health concern, partly due to resistance to therapy and the lack of new effective treatments for advanced disease. The combination of 5-Fluorouracil (5FU, a thymidylate synthase inhibitor) and irinotecan (a topoisomerase 1 inhibitor) is widely used in first-line and subsequent treatments. This study aimed to identify novel therapeutic targets to enhance combinatorial therapy, improving treatment efficacy and durability of response.
Methods: We performed a loss-of-function screen using HT29 CRC cell line and a retroviral library containing 7296 shRNAs targeting 912 chromatin genes. Cells were then treated with 5FU and SN38 (the active metabolite of irinotecan) or left untreated for 4 weeks. Genes enriched in resistant clones were identified through next-generation sequencing. Amongst candidate genes, PARG was selected for functional validation.
Results: CRISPR/Cas9-mediated knockout (HT29 PARG-KO) resulted in increased global poly(ADP-ribosyl)ation after 5FU and SN38 treatment. PARG depletion led to reduced cell viability and increased apoptosis, particularly after 5FU exposure. Pharmacological PARG inhibition (PDD00017273) synergised with 5FU and SN38 across three CRC models (HT29, DLD1, HT115). In vivo, HT29 PARG-KO xenografts were more sensitive to 5FU. Immunohistochemical analysis of 170 CRC patient tumours revealed that positive PARG expression correlated with poor response to 5FU + Irinotecan, increased liver metastases, and worse long-term survival.
Conclusions: Our findings highlight PARG as a promising therapeutic target for CRC, where its inhibition enhances the efficacy of standard chemotherapy.
The complex pathogenesis of Alzheimer's disease (AD), combined with the presence of the blood‒brain barrier (BBB), severely limits the effectiveness of conventional therapeutic approaches. Engineered exosomes—nanoscale extracellular vesicles of natural origin—have emerged as a promising platform for innovative AD therapy due to their excellent biocompatibility, low immunogenicity and intrinsic ability to cross the BBB. This review provides a systematic overview of the synthetic and structural biological characteristics of exosomes, with a focus on their functionalisation through physical, chemical and genetic modifications. These approaches enable the targeted loading of therapeutic cargo and the conjugation of brain-targeting peptides, thereby facilitating precise delivery to specific brain regions and offering a multi-target therapeutic strategy for AD. We further examine the potential of engineered exosomes in modulating core AD pathological pathways, including amyloid-beta deposition, tau hyperphosphorylation, neuroinflammation and synaptic dysfunction, and highlight their utility as an integrated delivery system for the co-delivery of multiple therapeutic agents to achieve synergistic therapeutic effects. Finally, key challenges in clinical translation are addressed, such as scalable production, standardised drug loading protocols and comprehensive assessment of safety and immunogenicity. Unlike previous reviews that primarily focus on general engineering techniques, this article emphasises a rational design strategy tailored for multi-target synergistic therapy and presents a comprehensive roadmap from basic research to clinical application, thereby providing both theoretical insights and practical guidance for the development of next-generation AD treatments.
Background: Oncofetal reprogramming—the reactivation of fetal-like gene programmes in malignant cells—has been implicated in the progression and stemness of hepatocellular carcinoma (HCC), yet its protein landscape and connection to tumour stemness remain incompletely defined.
Methods: We integrated multi-omics datasets to derive an oncofetal reprogramming-based prognostic signature (oncoScore) and validated it across multiple independent HCC cohorts. Candidate oncofetal proteins were validated in murine fetal liver and HCC tissue microarrays. Functional characterization of dual-specificity phosphatase 9 (DUSP9) was performed using gain- and loss-of-function studies, including sphere and colony formation, proliferation, migration and invasion assays, sorafenib resistance testing, and limiting-dilution tumourigenicity assays. Mechanistic studies employed Oil Red O staining, co-immunoprecipitation, chromatin immunoprecipitation, pharmacologic inhibition and genetic rescue experiments.
Results: The oncoScore demonstrated robust prognostic value across multiple independent HCC cohorts. DUSP9 emerged as a key regulator of stemness, promoting self-renewal and aggressive phenotypes, enhancing sphere and colony formation, proliferation, migration, invasion, sorafenib resistance, and tumourigenicity. Mechanistically, DUSP9 drives lipid metabolism by upregulating stearoyl-CoA desaturase (SCD) through the ERK1/2peroxisome proliferator-activated receptor gamma (PPARG) signalling axis.
Conclusion: Our results establish the oncoScore as a reliable prognostic marker for HCC and identify a DUSP-9ERK1/2-PPARG-SCD pathway that links lipid metabolism to stemness. Targeting the oncofetal protein DUSP9 may offer a therapeutic avenue for aggressive, stemness-driven HCC.
Background: Emerging evidence suggests that ferroptosis resistance may drives colorectal cancer (CRC) pathogenesis and limits therapeutic efficacy. A previous study has reported that lactate can enhance ferroptosis in CRC cells. The objective of this study was to elucidate how lactate regulates ferroptosis in CRC and to identify potential therapeutic targets.
Methods: Cellular viability and proliferative capacities were determined via cell counting kit-8 (CCK-8) and colony formation. Ferroptosis-related and inflammatory markers, including malondialdehyde (MDA), Fe2+, glutathione (GSH), IL-1β, IL-12 and IL-10, were quantified by commercial kits. Protein and RNA interactions were investigated using co-immunoprecipitation (Co-IP), RNA pull-down, dual-luciferases reporter and RNA immunoprecipitation （RIP) assays. Flow cytometry analysed M1 and M2 macrophage populations. Chromatin immunoprecipitation followed by quantitative polymerase chain reaction (ChIP–qPCR) examined histone H3 lysine 18 lactylation (H3K18la) and EP300 binding at the insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2)promoter. Methylated RNA Immunoprecipitation-qPCR (MeRIP–qPCR) measured the m6A modification of nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA. Transmission electron microscopy determined mitochondrial morphology. C11-BODIPY immunofluorescence staining analysed lipid peroxidation.
Results: Our findings revealed that lactate up-regulates IGF2BP2 through H3K18la-mediated transcriptional activation in both CRC cells and tumour-associated macrophages. Elevated IGF2BP2 directly bound to and stabilised Nrf2 mRNA, resulting in increased Nrf2 levels and enhanced resistance to ferroptosis in CRC cells. Notably, this lactate–IGF2BP2–Nrf2 axis also promoted M2 macrophage polarisation, fostering an immunosuppressive tumour microenvironment (TME). In xenograft models, lactate-driven Nrf2 up-regulation accelerated CRC tumour growth and metastasis. Conversely, pharmacological inhibition of ferroptosis resistance with dichloroacetate (DCA) or depletion of IGF2BP2 significantly reduced tumour burden.
Conclusion: Our study identified a novel lactate–IGF2BP2–Nrf2 signalling pathway that drives ferroptosis resistance and immune evasion in CRC.
Background: Autism spectrum disorder (ASD) is increasingly recognized as a neurodevelopmental condition with systemic immunological involvement, yet the underlying immune mechanisms remain incompletely defined.
Aims: To delineate the peripheral immune landscape in ASD using integrated multi-omics profiling and to determine how immune and immunometabolic alterations relate to clinical severity.
Materials & Methods: Circulating immune cells from individuals with ASD were profiled using multicolor flow cytometry, single-cell RNA sequencing, and bulk RNA sequencing. Plasma proteomic and metabolomic analyses were performed to identify immune-related and metabolic biomarkers. Immune features were evaluated for associations with clinical severity measures.
Results: Multi-omics profiling revealed marked immune dysregulation in ASD, with significant shifts in immune cell subsets and inflammatory signatures that correlated with clinical severity. T cell abnormalities included reduced frequencies and a skewed Th1/Th2 balance, consistent with a chronic inflammatory milieu. Natural killer (NK) cells showed increased activation but impaired cytotoxic capacity, accompanied by expansion of an atypical NK subset. Myeloid-derived suppressor cells (MDSCs) and hyperinflammatory CD56+ monocytes were elevated. Transcriptomic analyses corroborated broad immune activation, prominently implicating interferon-driven and antiviral signaling pathways. Plasma metabolomics and proteomics further indicated disruptions in purine metabolism and oxidative phosphorylation, alongside increased inflammatory markers, which were significantly associated with symptom severity.
Discussion: These findings support a systemic immunometabolic framework in ASD characterized by concurrent immune activation and altered myeloid/NK cell states, providing mechanistic context for peripheral biomarkers linked to clinical phenotype.
Conclusion: Integrated multi-omics profiling identifies robust peripheral immune and metabolic disturbances in ASD. The dysregulated immune subsets, activated immune pathways, and plasma biomarker signatures highlight potential avenues for biomarker-driven stratification and immune-targeted therapeutic development in ASD.
Background: As a critical component of the tumour microenvironment, cancer-associated fibroblasts (CAFs) actively drive the malignant advancement of non-small-cell lung cancer (NSCLC); however, their underlying mechanisms continue to be poorly characterized. This work examined the role of CAFs-derived exosomal miR-3126-5p in the glycolysis of NSCLC cells.
Methods: Glycolysis was evaluated by lactate production, glucose uptake, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Cell proliferation and cycle were evaluated by CCK-8, EdU staining, and flow cytometry. Src homology 2B adaptor protein 1 (SH2B1) and insulin receptor substrate 1 (IRS1) protein interaction was tested by Co-IP and GST pull-down assay. ChIP, dual-luciferase reporter assay, and EMSA determined the binding of kruppel-like factor 13 (KLF13) to the SH2B1 promoter. Dual-luciferase reporter assay was applied to assess miR-3126-5p binding to KLF13 3′-UTR. In vivo growth of NSCLC was determined in the mouse xenograft and Lewis lung carcinoma models.
Results: CAFs-derived exosomal miR-3126-5p was highly expressed in NSCLC tissues, and its elevated plasma level was significantly associated with poor prognosis of NSCLC patients. CAFs-derived exosomal miR-3126-5p facilitated glycolysis to accelerate the malignant progression of NSCLC cells. KLF13 exhibited reduced expression in NSCLC, while its overexpression suppressed NSCLC growth via repressing glycolysis. Exosomal miR-3126-5p targeted KLF13 3′-UTR to inhibit its expression in NSCLC cells. KLF13 transcriptionally inhibited SH2B1 expression to abolish the interaction between SH2B1 and IRS1 proteins, thus repressing PI3K/AKT pathway-mediated glycolysis. KLF13 knockdown counteracted the anti-cancer action of exosomal miR-3126-5p inhibition.
Conclusion: CAFs-derived exosomal miR-3126-5p accelerated NSCLC progression via inhibiting KLF13 expression, which transcriptionally activated SH2B1 to promote its interaction with IRS1, thereby promoting PI3K/AKT pathway-mediated glycolysis. Our findings position CAFs-secreted exosomal miR-3126-5p as a novel therapeutic intervention with potential in NSCLC management.
Background: The worst pattern of invasion (WPOI) is a critical histological prognostic indicator in oral squamous cell carcinoma (OSCC), yet the underlying mechanisms driving high WPOI remain poorly understood. While cancer-associated fibroblasts (CAFs) and their secreted factor serglycin (SRGN) are implicated in tumour progression, the regulation of SRGN secretion within the hypoxic tumour microenvironment is unknown.
Methods: We performed single-cell RNA sequencing (scRNA-seq) on 6 OSCC samples (3 each of WPOI 1–3 and 4–5) to identify subgroups of CAFs and their characteristic gene expression profiles. Using Western blot, qRT-PCR, and immunofluorescence, we investigated hypoxia-induced SRGN secretion pathways. Complementary CRISPR-Cas9 knockout, Co-IP assays, and xenograft models elucidated SRGN's role in ECM remodelling.
Results: ScRNA-seq revealed significant enrichment of CAFs, particularly an SRGN-expressing myCAF subpopulation, in high-WPOI (4–5) OSCC tissues. Under hypoxia, CAFs switched SRGN secretion from the conventional ER-Golgi pathway to an unconventional secretory autophagy pathway, dependent on autophagosome formation but independent of lysosomal degradation. Secreted SRGN directly interacted with matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) in the extracellular matrix (ECM), enhancing ECM remodelling and OSCC invasion and migration. In vivo, either genetic ablation of SRGN in CAFs or pharmacological inhibition of autophagy significantly suppressed tumour growth, inhibited collagen I degradation, and restored E-cadherin expression.
Conclusion: Our study identifies a novel mechanism whereby hypoxia induces CAFs to secrete SRGN via secretory autophagy. This SRGN-MMP2/9 axis drives ECM remodelling and promotes OSCC invasion, which histologically manifests as high WPOI. Targeting secretory autophagy or SRGN represents a promising therapeutic strategy for aggressive OSCC.