The Wnt/β-catenin pathway is abnormally activated in most lung cancer tissues and considered to be an accelerator of carcinogenesis and lung cancer progression, which is closely related to increased morbidity rates, malignant progression, and treatment resistance. Although targeting the canonical Wnt/β-catenin pathway shows significant potential for lung cancer therapy, it still faces challenges owing to its complexity, tumor heterogeneity and wide physiological activity. Therefore, it is necessary to elucidate the role of the abnormal activation of the Wnt/β-catenin pathway in lung cancer progression. Moreover, Wnt inhibitors used in lung cancer clinical trials are expected to break existing therapeutic patterns, although their adverse effects limit the treatment window. This is the first study to summarize the research progress on various compounds, including natural products and derivatives, that target the canonical Wnt pathway in lung cancer to develop safer and more targeted drugs or alternatives. Various natural products have been found to inhibit Wnt/β-catenin in various ways, such as through upstream and downstream intervention pathways, and have shown encouraging preclinical anti-tumor efficacy. Their diversity and low toxicity make them a popular research topic, laying the foundation for further combination therapies and drug development. With an estimated 2 million new diagnoses and 176 million deaths worldwide, lung cancer (LC) is the most prevalent malignancy and a major cause of cancer-related deaths . Its proThe Wnt signal transduction cascade controls multiple embryonic and somatic processes, including cell fate classification, tissue repair, organ formation, and several pathological states . NotablyCharacteristic of adenocarcinomas, especially lung adenocarcinoma (LUAD), are abnormalities in the epigenetic control of the Wnt signaling system . CompareTargeted Wnt/β-catenin pathway therapy shows outstanding anti-LC efficacy, and a range of Wnt pathway inhibitors provide great potential for combination therapy in LC, according to preclinical studies conducted in recent years . UnfortuThe research prospects for plant-derived medicines are relatively broad owing to the variety of targets, good safety standards, and excellent synergism . Further2+ pathway. These three pathways form a network of mutual regulation that finely coordinates various biological activities. Among them, the canonical pathway is closely related to the occurrence and development of LC and the most widely studied pathway; it is also the subject of this review.Wnt ligands are cysteine-rich proteins whose family consists of 19 secreted glycoproteins. Wnt ligand-mediated signaling pathways have a variety of physiological functions that have been conserved throughout evolution . DependiThe β-catenin-dependent pathway consists of three major components: transduction of Wnt signaling in the membrane, modulation of β-catenin stability in the cytoplasm, and overactivation of Wnt target genes in the nucleus . Ligand-There are various abnormal Wnt activation modes in malignant tumors, including acquired mutations, decreased expression of pathway inhibitors, and abnormal expression of ligands and receptors . Most spAbnormally activated Wnt signalings frequently manifests in a variety of tumors that are widely involved in the occurrence and development of cancer. The Wnt signaling network controls a wide range of biological processes in LC . The undCancer stem cells (CSCs) are now considered the main “seeds” in tumorigenesis, development, metastasis and recurrence . Wnt sigWnt/β-catenin signaling is thought to be necessary for maintaining the CSC phenotype. In CSCS, β-catenin dependent Wnt signaling is extremely active and contributes to maintaining the expression of putative lung CSC markers such as cluster of differentiation 44 (CD44) and cluster of differentiation 133 (CD133) . CompareWnt signaling regulates complex biological behaviors that are beneficial for the survival and metastasis of lung CSCs. Through various target genes, Wnt pathway activation can aid CSCs in penetrating adjacent tissues and entering the bloodstream. During this time, tumor cells remain dormant, allowing them to survive and avoid immune monitoring through the adverse feedback of Wnt signaling. When CSCs reach appropriate sites, another burst of Wnt signaling induces successful CSC reinitiation and colonization of secondary organs . In addiControl of the cell cycle by Wnt signaling is responsible for the most mechanisms that promote proliferation during cancer . Wnt sigKIP), and dysregulation of β-catenin activity was shown to induce p53-dependent growth arrest in LC cells  . NuclearLC cells . In addiLC cells . SuppresLC cells .Tumor cell proliferation and malignant behavior require energy support  and CSCsLC is a highly vascularized tumor and microvessel density (MVD) is the primary characteristic of intratumoral angiogenesis . Wnt sigWnt signaling can also indirectly affect angiogenesis by regulating the tumor microenvironment. For instance, angiogenesis can be induced by tumor-associated macrophages (TAMs) in the tumor microenvironment, which is thought to be the “switch” that first causes tumor angiogenesis . TAMs reMetastasis and invasion of the primary tumor and systemic tumor dissemination are the most deleterious events in the development of cancer, which pose challenging therapeutic difficulties and are significant contributors to cancer-related mortality . ChangesCell-cell adhesion and matrix depletion are important steps in the progression of all cancers from localized malignancy to metastatic disease. Wnt/β-catenin and its downstream products may affect the actin cytoskeleton and destabilize adhesion molecule junctions . ActivatSystemic anti-LC treatments face great challenges, and drug resistance is primarily responsible for cancer patient mortality, whether directly or indirectly. The persistence of CSCs, enhanced multidrug resistance (MDR) and the downregulation of apoptosis are important causes of treatment failure . It has in vitro derived chemoresistant cell lines . Wnt signaling activation is an important mechanism of chemoresistance in recurrent LC. Activation of Wnt signaling by APC inhibition resulted in chemoresistance in chemosensitive human LC cell lines, and higher Wnt activity was observed ll lines . Overexpll lines . The levll lines . Frequenll lines . Upregulll lines . For exall lines . Experimll lines . Althougll lines .in vitro and in vivo effectiveness of EGFR inhibitors  gene and LC patients with activating mutations in the kinase domain of EGFR can be treated with tyrosine kinase inhibitors (TKIs) . Cross-rhibitors . Furtherhibitors . This imAberrant Wnt signaling can inhibit the recruitment of inflammatory anti-tumor T cells and may undermine cancer immunosurveillance, thereby negatively controlling innate immunity, fostering immunoevasion, and boosting resistance to a variety of immunotherapeutics, including immune checkpoint inhibitors (ICIs) . Wnt3a oTargeted Wnt therapy has become an important research direction in the development of cancer drugs; however, it also faces problems such as insufficient targeting and large toxic side effects. Therefore, the search for safer and broader drug sources has become the focus of Wnt-targeting research. Natural products have become valuable effective drug sources owing to their wide range of sources, high bioavailability, good safety, and low cost . In the Flavonoids, which are frequently found in human diets and have biological effects, including antioxidant, antibacterial, and anti-inflammatory properties, can lower the risk of disease and are thought to be natural tumor-preventive agents. The biological activity of flavonoids relies on the structural substitution of the C6-C3-C6 loop . The folEGCG, the main bioactive ingredient in green tea, is linked to a number of bioactivities, including, anti-cancer, antioxidant, anti-inflammatory and antibacterial activities . The inaGarcinia indica Choisy (kokum) fruiting bodies and having anti-inflammatory, antioxidant, acetyltransferase inhibitory, and anti-cancer effects, alters lung CSCs’ activity and the aggression that goes along with them. Garcinol interferes with the phosphorylation of LRP6, a shared Wnt and Stat3 receptor, and blocks the activation of the Wnt/β-catenin/Stat3 axis. In NSCLC-produced spheres, garcinol reduced the expression of Dvl2, Axin2, β-catenin, and CCND1, indicating that garcinol can control the Wnt/β-catenin signaling pathway. Garcinol therapy dramatically slowed the formation of tumors in a mouse model of H441 CSC, confirming the above findings in vivo. Garcinol was found to modulate the lung CSCs phenotype by modulating Wnt/β-catenin signaling and Stat3 inactivation, thus suggesting that garcinol might be a potential new anti-lung CSC medicinal substance  is a natural bioactive flavonoid present in a variety of fruits and vegetables, displaying a range of pharmacological properties, such as anti-invasive and anti-cancer actions . FisetinThe polymethoxylated flavonoid nobiletin  is specifically accumulated in citrus fruit peel, with many anti-cancer activities. Nobiletin was found to enhance regulators of Wnt/β-catenin signaling, such as Axin2 and WIF-1, which are related to the inhibition of miR-15-5p expression. Nobiletin significantly reduced the ability of NSCLC cells to form spheres and the expression of proteins linked to cancer stemness, namely, CD133 and ALDH1 . Study aTF isolated from Fructus viticis significantly reduced the mRNA expression levels of cyclooxygenase 2 (COX-2), Wnt, and β-catenin in A549 cells. Further research found that TF promoted the apoptosis of A549 cells by inhibiting the COX-2 mediated Wnt/β-catenin signaling pathway, increasing multiple pro-apoptotic genes expression such as apoptotic peptidase activating factor 1, and lowering expression of anti-apoptotic genes BCL2L2 and BCL2. The ability of A549 cells to proliferate was decreased by TF in a dose-dependent manner; however, this effect was reversed by activating Wnt signaling via COX-2. Additionally, animal studies revealed that TF therapy greatly slowed the growth of mouse tumors, with a tumor inhibition rate of 64.07%, and increased the survival time of the animals .Phenols in large numbers and diverse forms provide a variety of potential benefits to human health. Polyphenols have multiple conjugated double bonds and show strong antioxidant capacities. Therefore, when electrons at certain sites are removed by free radicals, the molecular structure is stabilized by conjugation, thereby protecting the cell . This reCurcuma longa L.), a commonly used traditional Chinese herbal remedy, is curcumin (diferuloylmethane), which has numerous pharmacological effects, including anti-tumor, anti-inflammatory, antioxidative, and neuroprotective properties  and other free radicals causing damage to antioxidant enzymes such as superoxide dismutase (SOD) . Previouoperties . Curcumioperties .Another study discovered that curcumin effectively reduces lung CSC activity by suppressing the Wnt/β-catenin and Sonic Hedgehog pathways to diminish tumor sphere formation, downregulate CSC marker expression, inhibit proliferation, and induce apoptosis. LiCl-triggered Wnt/β-catenin activation reversed the anti-tumor effects of curcumin . Curcumiin vivo, which increased the lifespan of mice and prevented the growth of LC. The addition of LiCl partially counteracts the inhibitory effect of RES on lung CSCs and reverses the inhibition of Wnt/β-catenin signaling by RES , which is obtained from a variety of plants, has tumor-suppressing effects on LC. RES successfully reduced IL-6 expression and the levels of the CSC-specific markers CD133, ALDH1A1, and Nanog in NSCLC cell lines. Moreover, RES dose-dependently decreased the expression of β-catenin, p-GSK-3β (Ser9), and Myc and inhibited Wnt/β-catenin signaling g by RES . Interesg by RES . These fThese contradictory results may be attributed to the different concentrations of RES. PD-L1 expression was induced after exposure to low doses of resveratrol in H1299 cells. However, at higher doses (>40 μM), RES treatment resulted in a gradual reduction of PD-L1, suggesting that RES can modulate PD-L1 expression in a dose-dependent manner . More imMorus alba L. and Garcinia mangostana L. are sources of the natural compound maclurin -methanone), a member of the benzophenone family ) is abundant in the essential oils of widely used Asian species, such as cloves. Eugenol, a naturally occurring β-catenin inhibitor, is an extremely important chemopreventive agent for LC. Eugenol can sequester β-catenin in the cytoplasm and regulate the phosphorylated form of β-catenin to promote its degradation, thus helping the cellular environment to resemble the normal state. Meticulous molecular analysis clearly revealed GSK-3β-mediated phosphorylation of β-catenin after eugenol treatment, leading to degradation of cytoplasmic β-catenin, which significantly limited its transfer to the nucleus. Eugenol activity reduced the expression of CSC markers regulated by β-catenin . Eugenol successfully slowed the activity of the small-molecule CK1 inhibitor D4476, perpetuating the mandatory phosphorylation of β-catenin by CK1α, thereby pushing it toward degradation and minimizing CSC expression. Eugenol treatment showed tumor-preventing effects and significantly prolonged the lifespan of mice with N-nitrosodiethylamine-induced LC .Natural alkaloids are regarded as valuable sources of anti-cancer medicines because they not only exhibit potent anti-tumor effects when taken alone but also when combined with other anti-tumor agents . Consequin vitro is the interaction between β-catenin and LEF1, based on a high-throughput sequencing assay  R. Br  R. Br . The maj.) R. Br .Nelumbo nucifera Gaertn.) leaves, has therapeutic effects such as anti-obesity, antioxidant, and anti-inflammatory properties (in vivo. NF effectively inhibited the proliferation of NSCLC cells pretreated with nicotine in vitro and exerted chemotherapeutic effects on tumor xenograft growth in vivo (Nuciferine (NF), an alkaloid originally isolated from lotus , the main bioactive component of tivities . β-asarotivities .Lycoris radiata (L’Her.), has many anti-cancer properties. After treatment with lycorine, the expression of GSK-3β was increased, the cellular level of β-catenin was inhibited, and the growth and metastasis of LC were significantly hindered. This is closely related to the inhibition of Wnt/β-catenin signal transduction by lycorine and the reversal of EMT in LC cells -nitidumalkaloid B is a novel alkaloid identified from Zanthoxylum nitidum (Roxb.) DC. and its antiproliferative activity is partly related to G2/M cell cycle arrest, induction of apoptosis and inhibition of the Wnt/β-catenin signaling pathway. It effectively suppressed the expression of β-catenin, Axin2, and p-GSK-3β but increased p-β-catenin expression in a concentration-dependent manner. Constitutive relationship analysis suggested that the C-6 substituent of benzophenanthridine alkaloids had a direct effect on its antiproliferative activity , an important active compound in  with TP . After t with TP . Triptol with TP . TP prom with TP .in vivo and in vitro. This property may be attained by influencing angiogenic mediators such MMP-2, survivin, and VEGF in Wnt target genes (Cucurbitacin B (CuB) is a natural plant triterpenoid that inhibits endothelial cell migration, invasion, and angiogenesis in NSCLC. CuB treatment inhibited the expression of Wnt3/Wnt3a, inactivating GSK-3 phosphorylation, and restricted β-catenin nuclear localization to influnce interaction with TCF-1 in NSCLC cells. Downregulation of the Wnt/β-catenin signaling axis conferred the ability of CuB to resist NSCLC stemness and metastasis. Additionally, CuB showed strong antiangiogenic properties both et genes .The sesquiterpene Curcumol, a bioactive substance purported to have anti-cancer effects, was isolated from rhizome of various Curcuma species. Curcumol treatment controlled the motility, migration, and proliferation of LC cells by altering the expression of Wnt signaling molecules. Curcumol inhibited the phosphorylation of LRP5/6, although Fzd8 was not altered, thereby greatly increasing the protein expression of downstream Axin, APC and GSK-3β. Thus, the β-catenin protein expression was dramatically reduced while its degree of phosphorylation was significantly higher .Sesquiterpenoids isolated from Curcuma species also contain furanedienone, which may be a potential adjuvant therapy for the treatment of LC. In A549 cells, furanodienone administration suppressed Wnt3 and β-catenin in a dose-dependent manner, thereby downregulating the Wnt signaling pathway. This was correlated with increased apoptosis and cell cycle arrest .Euphorbia lathyrism L., inhibited the expression of EGFR, Wnt3a, and Fzd-1 in NSCLC cells and triggered the activation of GSK-3β, which in turn induced β-catenin degradation. In EGFR-overexpressing and-depleted LC cells, EPBS regulated the constitutive activation of the Wnt/β-catenin pathway, and the nuclear translocation of β-catenin was considerably decreased upon EPBS administration, suggesting that EPBS may be implemented alongside EGFR-TKIs in drug-resistant LC (Euphorbiasteroid (EPBS), a tricyclic terpenoid extracted from stant LC .The antimalarial compounds artemisinin, dihydroartemisinin, and artesunate halt lung tumor progression by blocking the Wnt/β-catenin pathway. They suppressed Wnt5-a/b protein levels and efficiently lowered the expression of LRP6 and Dvl2 while increasing Axin2 expression. Ultimately, this results in downregulation of β-catenin and inhibition of LC proliferation and metastasis .20(S)-25-OCH3-PPD (PPD25), a dammarane-type triterpenoid saponin from the leaves of Panax notoginseng (Burk.) F. H., has been reported to have cytotoxic effects in several human cancer cell lines. PPD25 dose-dependently decreased β-catenin expression and inhibited β-catenin/TCF transcriptional activity in A549 cells. It inhibited the expression of Wnt pathway-associated proteins and effectively suppressed proliferation of LC cells, demonstrating the potential as a chemotherapeutic and/or chemopreventive agent for LC .Isothiocyanate members reduce the signature activation event of β-catenin nuclear localization in LC cell lines. Phenethylisothiocyanate was more effective in preventing the growth of a multicellular tumor spheroid model that mimicked micrometastases, whereas Benzyl isothiocyanate was better at preventing the Long-term colony formation dependent on CSC viability in Wnt-dependent squamous cells. Therefore, Phenethylisothiocyanate might work as chemotherapy to remove a large tumor and Benzyl isothiocyanate may be a safer alternative to target undifferentiated CSCs .in vitro and in vivo by decreasing viability and promoting apoptosis of LC cells, and its anti-tumor effects were mediated by inhibition of the Wnt signaling pathway  is a monomer of bufadienolides from the skin of toads. 19-HB reduced β-catenin as well as downstream target genes. It produced safe anti-tumor effects  pathway .Epibrassinolide is a steroid hormone in plants that acts via membrane receptors and GSK-3 pathway, resulting in stabilization of transcription factors. It significantly reduced β-catenin-dependent cytokinesis, metastasis, and anti-apoptotic gene expression in both drug-resistant (VPA17) and drug-sensitized (H69) small cell LC (SCLC) cells, but had no effect on normal lung epithelial cells .Penicillium vulpinum, exhibited antiproliferative, proapoptotic, and antimigration effects on human LUAD cells through inhibition of the Wnt pathway. It upregulated the expression of two endogenous inhibitors of Wnt pathway, Dkk and WIF-1, and downregulated the expression of CCND1  was synthesized using a computer-aided drug design method to reduce the expression and nuclear translocation of β-catenin. DUD significantly upregulated Axin expression and stabilized the β-catenin destruction complex to promote proteasome-mediated protein hydrolysis of phosphorylated β-catenin. DUD reversed EMT process and inhibited LC cell migration via Wnt/β-catenin and PI3K/Akt signaling . CompounGinkgo biloba exocarp extract (GBEE) contains a large number of components with bioactivities, such as antioxidant, antibacterial and anti-tumor. Ginkgo biloba seed exocarp polysaccharides and ginkgolic acids are be identified as the main components in GBEE contributing to the anti-tumor effect . GBEE siArtemisia vulgaris L. (AvL-EtOH) decreased the downstream activation of Wnt3a in A549 cells, thereby inhibiting tumor cell proliferation and promoting caspase cascade activation-induced apoptosis. The gas chromatography-mass spectrometry analysis of AvL-EtOH exhibited 48 peaks of various secondary metabolites such as terpenoids, flavonoids, carbohydrates, coumarins, amino acids, steroids and proteins  and Qiyusanlong (QYSL) decoction, significantly enhance the effect of Cisplatin on LC cells. Miao treatment inhibited the expression of Axin, β-catenin, and Myc, restricting the development of LC. Miao not only had similar effects as cisplatin on LC cells, but also induced an improvement in cisplatin resistance. The simultaneous use of Miao and cisplatin synergistically regulated cell proliferation and apoptosis and enhanced the inhibitory effect on LC cells . QYSL inThe Feiyiliu Mixture (FYLM) and Feiyanning Formula (FYN) are experienced Chinese medicinal formulas. FYLM reduced the expression of phosphorylated EGFR and Wnt pathway-related proteins, such as β-catenin and Myc. FYLM treatment delays the resistance of EGFR-mutant cell lines and EGFR-mutant tumor-bearing mice to osimertinib by inhibiting the Wnt/EGFR pathway . FYN in Additionally, almost every element of the Wnt signaling pathway, from the synthesis and secretion of Wnt ligands to the transcription of Wnt target genes, has the potential to become a molecular target for cancer therapy . NaturalAlthough natural products and their derivatives have achieved promising anti-tumor effects in basic research, they still face challenges in clinical applications, such as low solubility, limited oral bioavailability, poor metabolism and insufficient organ targeting. Because of these limitations, most the natural products have to apply to patients with a huge dosage, which may increase the risk of inducing side effects . Therefo+ CSCs (in vitro by reducing Wnt pathway-related gene expression (Nanotechnology helps reduce chaotic distribution and direct drug containment in cancer cells through the movement of DDSs to a greater extent, increasing local utilization, maintaining a higher bioactivity and improving metabolic stability . The imm+ CSCs . Encapsu+ CSCs . Co-delipression . Liposompression .etc., have also shown non-negligible development potential, and they not only improve bioavailability in vivo, but also enhance the synergistic delivery efficiency of chemotherapeutic agents (Nanoparticles can be modified to allow delivery of drugs by inhalation, which offers a promising non-invasive therapeutic option for the treatment of LC, such as a nanocomposite system loaded with RES for nebulization, demonstrating higher anti-cancer selectivity index . Nanocryc agents . Quercetc agents . Nano-dec agents . HoweverCurrent Wnt studies in LC have focused on LUAD-dominated NSCLC, and markers suggesting efficacy remain inadequately studied, although there have been reports on Wnt ligand-dependent tumor markers . In the Targeted toxicity of existing Wnt inhibitor therapeutic-grade molecules is a major limitation hindering their clinical development . CompareAlthough the limited number of studies and the incomplete study of Wnt inhibition modalities may have influenced us to draw definite conclusions, we found from the relevant studies that different types of compounds seem to play their own roles in interfering with the Wnt pathway in LC. Polyphenolic compounds, such as curcumin, have an excellent ability to inhibit oxidative stress-mediated upregulation of the abnormal Wnt pathway, an ability not possessed by other compounds. Half of the studied alkaloids and their derivatives could promote the stabilization of β-catenin destructive complex by up-regulating Axin, GSK-3β or downregulating its inactive phosphorylation. Terpenoids appear to act upstream of the Wnt pathway to inhibit it, and most terpenoids and derivatives, such as triptolide, inhibit the Wnt pathway in LC through the upregulation of Wnt antagonists and the downregulation of Wnt ligands. Natural small-molecule products have greatly increased the diversity and potential for drug development, with diverse mechanisms of action, thus allowing them to act on multiple targets of the Wnt pathway in LC and present partial inhibition of some of the more difficult targets, such as c-Myc, which may reduce the risk of future drug resistance.In the context of the limited clinical studies on existing Wnt inhibitors, our study provides pharmaceutical companies and researchers with safer and more diverse options for the future development of inhibitors targeting the key Wnt pathway in LC, thereby enriching the clinical treatment options for LC.The complexity of the molecular interactions of the Wnt/β-catenin pathway and its involvement in different physiological functions make drug discovery challenging . The intIn addition, the direct tumor-killing effect of Wnt inhibitors alone is limited. We are particularly interested in the impact of Wnt in mediating clinically relevant therapies for LC, such as chemotherapy, radiotherapy, EGFR-TKI, immunotherapy, and even antiangiogenic therapy. Therefore, Wnt combination therapy is a good strategy for overcoming drug resistance in LC, and should be an overriding direction for future research. Many naturally occurring small molecules used as Wnt inhibitors have good effects when used in combination with chemotherapy and targeted drugs, with the advantage of inhibiting proliferation and overcoming drug resistance, but the effectiveness and safety of clinical treatment still need to be further determined . The the


One system involves the fueled activation of a reactionmodule leading to the temporal formation and depletion of the bioreactorconjugate operating the nickase-guided transient biocatalytic cascades.The second system demonstrates the fueled activation of a reactionmodule yielding a bioreactor conjugate operating the exonuclease III-dictatedtransient operation of the two biocatalytic cascades. The temporaloperations of the bioreactor circuits are accompanied by kinetic modelsand computational simulations enabling us to predict the dynamic behaviorof the systems subjected to different auxiliary conditions.Glucose oxidase-loadedZIF-90 metal–organic frameworknanoparticlesconjugated to hemin-G-quadruplexes act as functional bioreactor hybridsoperating transient dissipative biocatalytic cascaded transformationsconsisting of the glucose-driven H Several applications of transient nucleic acid-basedcircuits were demonstrated, including the temporal aggregation/deaggregationof metal nanoparticles or semiconductor quantum dots and control overtheir optical properties of the systems,43 the temporal formation and separation of DNA nanostructures, suchas microtubules or wires,44 and the conjugationof the DNA circuits to enzymes, driving transient biocatalytic cascades.45Emulating native transient dissipativecircuits by synthetic networks attracts substantial research effortswithin the rapidly developing area of systems chemistry.48 such as metal,52 metal oxide,58 metal–organic framework nanoparticles,63 or carbon-based nanoparticles,66 “nanozymes”,attracts substantial research efforts in catalysis and materials science.Also, integration of biocatalysts and catalytic nanoparticles intonanoparticle frameworks generated hybrid composites acting as bioreactorsystems driving catalytic cascades.67 Tetheringof aptamer strands to nanozymes led to aptananozymes revealing thebinding and catalytic functions mimicking native enzymes68 and to cell-targeting catalytic nanoparticlesfor therapeutic applications.69 Moreover,catalytic nanoparticles conjugated to catalytic nucleic acids, DNAzymes,acted as hybrids operating as bioreactor systems driving catalyticor biocatalytic cascades.70 Diverse applicationsof nucleic acid-modified nanozymes were reported for sensing,73 imaging,75 and therapeutic uses.78 Particularly, the loading of metal–organicframework nanoparticles with nanoparticle catalysts,79 biocatalyst,80 or drugs81 and capping the loaded nanocarriers with stimuli-responsivenucleic acid gates82 for controlled releaseof the loads were demonstrated.Development of catalytic nanoparticles,70Accordingly, the conjugationof transiently operating nucleic acidgates to nanozymes could introduce dynamic dimensions to nucleic acid/nanozymehybrids. Here, we report on the assembly of supramolecular bioreactorsystems consisting of nucleic acid-modified glucose oxidase (GOx)-loadedZIF-90 metal–organic framework nanoparticles (NMOFs) and hemin-G-quadruplexconstituents demonstrating transient-peroxidase like activities inthe presence of the nickase, Nt.BbvCI, or exonuclease III, Exo III,as control units. The ZIF-90 nanoparticles were selected due to theease to integrate GOx into the ZIF framework and due to the surfacecarboxaldehyde functionalities of ZIF-90 that allow the covalent conjugationof the nucleic acids to the scaffold.1)+(2)/(3) supramolecular GOx-loaded ZIF-90 NMOFshybrid as the functional conjugate for the nickase-activated, transientbioreactor system. Glucose oxidase (GOx)-loaded ZIF-90 NMOFs wereprepared according to the literature70 byreacting imidazole-2-carboxaldehyde with Zn2+-ion, in thepresence of GOx, 1, (1), thatprovide the anchoring sites for the assembly of the GOx-loaded ZIF-90NMOFs/G-quadruplex conjugate. Reacting the (1)-functionalizedNMOFs with the strand T1, (3), and the G-richstrand B1, (2), in the presence of K+-ion yielded the GOx-loaded ZIF-90 NMOFs/G-quadruplex hybrid, andthe subsequent treatment of the assembly with hemin yielded the GOx-loadedZIF-90 NMOFs/hemin-G-quadruplex bioreactor hybrid system. 1)+(2)/(3)-modified GOx-loadedZIF-90 NMOFs. Identical dodecahedral crystalline NMOFs are observed,indicating that the functionalization of the NMOFs with the biomaterialsdoes not affect the crystalline structure of the particles. This isfurther supported by identical XRD patterns of the NMOFs in the differentstates of modification of the NMOFs (1) on the NMOFs and of the G-quadruplexes on the NMOFs both correspondedto 10 nmol/mg . Confocal microscopy imaging experiments, 1)+(2)/(3) supramolecular GOx-loaded ZIF-90 NMOFs/G-quadruplex conjugate.The loading of the hybrid with FITC (fluorescein isothiocyanate isomerI)-modified GOx demonstrated green fluorescent (λ = 420 nm)particles, panel I, consistent with the GOx-loaded NMOFs. Subjectingthe particles to Zn(II) protoporphyrin IX, Zn(II)PPIX, resulted inthe characteristic red fluorescence, λ = 590 nm, of Zn(II)PPIXbound to G-quadruplexes,83 panel II. Thebright-field image of the particles is depicted in panel III, andthe overlay of the images is displayed in panel IV, revealing a yellowfluorescence of the combined constituents associated with the hybridNMOFs. The successful construction of (1)+(2)/(3) supramolecular GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplexbioreactor was further characterized by the efficient biocatalyticcascades of glucose-driven H2O2-channeled oxidationof Amplex-Red or generation of chemiluminescence, Figure S4. +(2)/(3)supramolecular GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplex bioreactor;see Figure S4B, panels I and II, curvec.)he NMOFs 1C. The l1)+(2)/(3) GOx-loadedZIF-90 NMOFs/hemin-G-quadruplex bioreactor system. The initial reactionmodule consists of the A1, (1)-functionalizedGOx-loaded ZIF-90 NMOFs, the hemin-G-quadruplex, B1, (2) constituent, the duplex composed of L1/T1, (4)/(3), and the nicking enzymeNt.BbvCI. Subjecting the reaction module to the fuel strand L1′, (5) results in the displacement ofthe duplex L1/T1, (4)/(3) to yield the duplex L1/L1′, (4)/(5) and free T1, (3). The released T1, (3) self-assembles the(1)+(2)/(3) GOx-loaded ZIF-90NMOFs/hemin-G-quadruplex bioreactor hybrid system that drives twodifferent biocatalytic cascades. One biocatalytic cascade, panel I,involves the aerobic oxidation of glucose, yielding gluconic acidand H2O2 and the subsequent hemin-G-quadruplex-catalyzedoxidation of Amplex-Red by H2O2 to form thefluorescent resorufin. The second biocatalytic cascade, driven bythe GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplex bioreactor, is displayedin 2O2 and the subsequent hemin-G-quadruplex-catalyzedoxidation of luminol by the H2O2 to generatechemiluminescence, λ = 425 nm.84 Theresulting duplex L1/L1′, (4)/(5), generated upon the fueled operation of the reactionmodule and the formation of the hybrid NMOFs bioreactor, is engineeredto include in the sequence of L1′, (5) the nicking site to be cleaved by Nt.BbvCI. Cleavage of the L1′, (5) leads to fragmented “waste”products that are separated from the duplex, leading to the separationof L1, (4). The separated strand L1, (4) displaces T1, (3) fromthe (1)+(2)/(3) hybrid bioreactorstructure, resulting in the separation of two bioreactor constituents,the blockage of the two biocatalytic cascades displayed in panelsI and II, and the regeneration of the parent inactive reaction module.That is, the L1′, (5) triggered activationof the reaction module leads to the temporal activation of the twobiocatalytic cascades that reveal a guided transient operation anddepletion mechanism to regenerate the parent inactive state.1′-triggeredoperation of the reaction module (L1′ = 3 μMand Nt.BbvCI = 0.046 μM). The rates of resorufin formation increasefor a time interval of 2 h and then decrease for a depletion timeinterval for 10 h leading to regeneration of the parent system. Usinga calibration curve relating the fluorescence intensities of resorufinto the concentrations of (1)+(2)/(3) supramolecular structure , the transient concentrations of (1)+(2)/(3) supramolecular bioreactor,corresponding to the transient catalytic formation of resorufin, werecalculated, and these are displayed in 1′triggering the reaction module and by the concentration of the nickingenzyme Nt.BbvCI. Qualitatively, increasing the concentration of thefuel strand L1′ is anticipated to enhance and enrichthe content of the transiently formed resorufin whereas elevatingthe content of the Nt.BbvCI is expected to decrease the peak contentof the catalytically generated resorufin and to shorten the depletiontime-interval of the temporal process. The efficiency of the GOx-loadedZIF-90/hemin-G-quadruplex biocatalytic cascade is controlled by theloading of GOx in the NMOFs and the resulting H2O2 generated by the GOx-catalyzed aerobic oxidation of glucose. Theresults are displayed in Figure S5 andaccompanied discussion. As the loading of GOx increases, the biocatalyticcascade is enhanced, yet the background signal of the separated GOx-loadedZIF-90/hemin-G-quadruplex constituents is intensified.Panels I and II of Figure S8. Thismodel was adopted to simulate the experimental results (dotted transientpoints a) in Table S1 . This set of rate-constants has avalue provided that it can predict the transient behavior of the supramolecularbioreactor at other auxiliary conditions. Accordingly, the transientbehavior of the formation and depletion of (1)+(2)/(3) supramolecular bioreactor generating fluorescentresorufin was predicted at different auxiliary concentrations of L1′ +(2)/(3) supramolecular GOx-loadedNMOFs/hemin-G-quadruplex catalytic bioreactor system that undergoestransient depletion by the nickase-induced separation of the bioreactorconjugate, leading to the recovery of the original reaction module.The transient concentration changes of (1)+(2)/(3) supramolecular bioreactor generating chemiluminescenceare displayed in 1′ that activates the reaction module resultsin lower temporally generated yields of the bioreactor conjugate,leading to lower chemiluminescence intensities, Similarly, 2, (6) , andthe hemin-G-quadruplex B2, (7). The enzymeExo III is also included in the reaction module. Subjecting the reactionmodule to the fuel strand T2, (8), resultsin the assembly of the (6)+(7)/(8) supramolecular bioreactor complex consisting of the (8)-bridged GOx-loaded ZIF-90 NMOFs and hemin-G-quadruplex constituents.The supramolecular complex operates two different biocatalytic cascades.One biocatalytic cascade, panel I, involves the aerobic GOx-catalyzedoxidation of glucose to yield gluconic acid and H2O2 and the subsequent hemin-G-quadruplex-catalyzed oxidationof Amplex-Red to resorufin. The second biocatalytic cascade drivenby the (6)+(7)/(8) supramolecularGOx-loaded ZIF-90 NMOFs/hemin-G-quadruplex bioreactor involves theaerobic GOx-loaded ZIF-90 NMOFs-catalyzed oxidation of glucose togluconic acid and H2O2 and the subsequent hemin-G-quadruplexcatalyzed oxidation of luminol by H2O2 and thegeneration of chemiluminescence, panel II. The Exo III included inthe system selectively degrades the 3′-ended fuel strand T2, leading to the separation of the supramolecular complexregenerating the rest of the reaction module and producing base fragmentsT2 as waste products. The separation of the supramolecularcomplex by Exo III leads to the temporal activation of the biocatalyticcascades shown in panels I and II and to the recovery of the parentreaction module. That is, the T2-triggered activation ofthe reaction module leads to the temporal and transient formationof the (6)+(7)/(8) supramolecularcomplex consisting of the GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplexthat guides the temporal biocatalytic cascades shown in An alternative transient GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplexreaction module leading to the fuel-triggered and transient operationof the bioreactor catalyzing the biocatalytic oxidation of Amplex-Redto resorufin or the biocatalytic generation of chemiluminescence isdepicted in 2 that forms the duplex with B2 is being digestedby Exo III. We find that it is essential to tether to the G-quadruplexB2 a single strand 3′-terminated tether; otherwise,the G-quadruplex is also degraded by Exo III.It should be noted that in order to operate theExo III driventransient generation of the bioreactor, the supramolecular GOx-loadedZIF-90 NMOFs/hemin-G-quadruplex complex had to be engineered to retainan intact, Exo III-resistant structure, to the extent that only theT2 and withdrawing at time interval samples driving the glucose-initiatedH2O2-mediated oxidation of Amplex Red to resorufin.The withdrawn samples reveal a temporal initial increase in the oxidationrates of Amplex-Red for a time interval of ∼2 h, and afterward,the withdrawn sample shows a continuous temporal decline in the bioreactorcatalyzed oxidation rates, which are fully blocked after 8 h, reachingthe parent background rates of Amplex-Red oxidation to resorufin bythe parent reaction module. Readdition of the fuel strand T2 to the system reactivated the transient operation of the reactionmodule, following the oxidation of Amplex-Red to form resorufin. Theseresults are consistent with the T2-fueled/Exo III-guidedoperation of the reaction module shown in 2-triggered, transient formation of (6)+(7)/(8) supramolecular bioreactor generating fluorescentresorufin is controlled by the concentrations of T2, 2 increases, the peak ratesof temporal resorufin formation are higher, and as the concentrationof Exo III increases, the peak rates of catalyzed formation of resorufinare lower and the depletion of the catalytic rates generating resorufinis enhanced. Figure S11 formulates thekinetic model corresponding to the transient operation of the T2/Exo III driven (6)+(7)/(8) supramolecular GOx-loaded NMOFs/hemin-G-quadruplex bioreactor system. 6)+(7)/(8) supramolecular structure generating resorufin by the T2/Exo III-driven bioreactor system, overlapping the experimenttransient curve a, using the kinetic models. The derived rate constantsfollowing the kinetic model are summarized in Table S2. Using this set of rate constants, the predictedcurves corresponding to temporal transient concentrations of the catalyticbioreactor system at different concentrations of T2 andExo III were computed, curves b′ and c′, 2/Exo III-driven activation of the GOx-loaded ZIF-90 NMOFs/hemin-G-quadruplexbioreactor, 2/Exo III-activated reaction module. The triggered reaction modulereveals intensified chemiluminescence spectra for a time intervalof 2 h and, subsequently, temporal depletion and blockage of the chemiluminescenceafter 8 h. 6)+(7)/(8) supramolecular structuregenerating chemiluminescence upon the T2/Exo III-triggeredactivation of the bioreactor system in the presence of 3 μMT2 and 0.4 μM Exo III. 6)+(7)/(8) supramolecular bioreactor leading to chemiluminescenceupon activation of the reaction module at different concentrationsof T2 and Exo III. As the concentration of T2 increases, the temporal peak chemiluminescence intensity is higher,and as the concentration of Exo III is elevated, the peak chemiluminescenceintensity is lower and the dissipative depletion of the bioreactordriven process is enhanced.Panels I and IIof 69 Alternatively, the triggered temporal unlocking of the NMOFs couldbe used for the dose-controlled release of drugs, e.g., insulin fromthe NMOFs carriers.85 Moreover, the presentstudy introduced the fueled strand displacement principle and coupledenzyme driven transformation as control motives of the dynamic process.Other triggering stimuli for temporal operation of the reaction modulessuch as miRNA/RNase,86 redox-triggeredaptamer-ligand complexes,87 or light,38 may be envisaged.The study introduced nucleic acid-modified/GOx-loadedZIF-90 conjugatesas functional assemblies guiding transient biocatalytic cascades.Such frameworks could find important nanomedical applications fordose-controlled, temporal release of therapeutic agents. For example,the GOx-stimulated activation of the hemin-G-quadruplex peroxidase-mimickingDNAzyme yields reactive oxygen species (ROS) that might act for temporalchemodynamic treatment of cancer cells.


Long noncoding RNAs (lncRNAs) are involved in glioma initiation and progression. Glioma stem cells (GSCs) are essential for tumor initiation, maintenance, and therapeutic resistance. However, the biological functions and underlying mechanisms of lncRNAs in GSCs remain poorly understood. Here, we identified that LINC00839 was overexpressed in GSCs. A high level of LINC00839 was associated with GBM progression and radiation resistance. METTL3-mediated m6A modification on LINC00839 enhanced its expression in a YTHDF2-dependent manner. Mechanistically, LINC00839 functioned as a scaffold promoting c-Src-mediated phosphorylation of β-catenin, thereby inducing Wnt/β-catenin activation. Combinational use of celecoxib, an inhibitor of Wnt/β-catenin signaling, greatly sensitized GSCs to radiation. Taken together, our results showed that LINC00839, modified by METTL3-mediated m6A, exerts tumor progression and radiation resistance by activating Wnt/β-catenin signaling. Glioblastoma (GBM) is the most aggressive and prevalent primary brain tumor in adults . DespiteLong noncoding RNAs (lncRNAs) are a class of RNAs consisting of more than 200 nucleotides (nt) and posing a negative ability of encoding proteins . LncRNAsIn this study, we identified a novel lncRNA, LINC00839 is involved in stemness maintenance and radiotherapy resistance. Mechanistically, METTL3-YTHDF2 mediated m6A modification elevated LINC00839 levels; LINC00839 functions as a scaffold for c-Src phosphorylates β-catenin at Y654, promoting Wnt signaling activation; β-catenin inhibitor treatment sensitizes brain tumor to radiotherapy.A total of 30 primary GBM specimens and 20 recurrent GBM specimens were obtained from the First Affiliated Hospital of Nanjing Medical University. The use of clinical samples was approved by the medical ethics committee of the First Affiliated Hospital of Nanjing Medical University. The written informed consent have been collected from all patients. Detailed patient information is presented in Supplementary Table Two GSCs (MES28 and GSC2907) and two neural stem cell NSC (HNP1 and NESA), gifted from Professor Xiuxing Wang, were maintained in Neurobasal media (Invitrogen) supplemented with B27 (Invitrogen), EGF, and bFGF . We synthesized full-length complementary cDNAs of human LINC00839 and cloned into the expression vector pcDNA3.1 (Invitrogen). LINC00839 ASO and control ASO were synthesized by Ribobio. Lipofectamine 3000 reagent (Life Technologies) was used for vector, LINC00839, control ASO, and LINC00839 ASO transfection as suggested by the manufacturer’s protocol. After 48 h of transfection, the cells were collected for analysis.TM Fast SYBR Green qPCR Mix (Monad) and an LC96 Real-Time PCR Detection System (Roche) were used for qRT-PCR analysis. The primers used are listed in Supplementary Table Total RNA was extracted from GBM specimens and GSCs using Trizol reagent (Invitrogen) as previously described . The purTotal protein was extracted from GBM specimens and GSCs using RIPA cell lysis buffer (Invitrogen). The FractionPREP Cell Fractionation kit (BioVision) was used for subcellular fractions extraction as previously described . Preparehttp://bioinf.wehi.edu.au/software/elda) software.In vitro limiting dilution was used to measure the Neurosphere formation ability of GSCs as previously described . BrieflyThe co-localization of LINC00839 and YTHDF2 or β-catenin was detected by FISH and IF double staining. Alexa Fluor 555-labeled LINC00839 FISH probe was synthesized by RiboBio. YTHDF2  and β-catenin  antibodies were used for IF. Images were collected using the fluorescence microscope (Olympus FV1000).GSCs were radiated at 0, 2, 4, 6, 8, or 10 Gy and seeded into 6-well plates at 500 cells per well in 4 ml. Two weeks later, the formed colonies were fixed with 4% formaldehyde and stained with 0.5% crystal violet. Colony numbers were counted. And the ratio of the plating efficiency of the radiated GSCs to that of control cells was calculated.TM Magnetic RNA-Protein Pull-Down Kit (Thermo) following the manufacturer’s instructions. Magnetic streptavidin-coated beads were used to extract LINC00839-associated proteins for western blotting assays.LINC00839 RNA was transcribed using Ribo™ RNA max-T7 Transcription Kit (RIBOBIO) and Pierce RNA 3’ End desthiobiotinylation Kit (Thermo) was used to label LINC00839. RNA pull-down assays were performed using PierceTM Kit (Millipore) according to the manufacturer’s instructions. Briefly, cell lysis was incubated with indicated antibodies-coated magnet beads at 4 °C for 24 h. The protein-associated RNAs were eluted and purified for further qRT-PCR analysis.RIP assays were performed using the Magna RIPTM m6A Kit (Millipore) according to the manufacturer’s instructions. Briefly, protein A/G magnetic beads were coated with anti-m6A antibody at 4 °C for 24 h. GSCs RNA was fragmented to 94 °C for 5 min, and incubated with coated beads at 4 °C for 2 h mixture with RNase inhibitor. m6A RNA was eluted and purified for further qRT-PCR analysis.MeRIP assays were performed using the Magna MeRIPGSCs were lysed using the Pierce™ IP Lysis Buffer (Thermo). Protein A/G magnetic beads were coated with β-catenin antibody, and incubated with cell lysis at 4 °C for 24 h. Proteins were eluted for further western blotting assays.In vitro kinase assay was performed as previously described . Active −1 celecoxib daily. On day 28, after injection, mice were euthanized. And mice brains were harvested, fixed in 4% formaldehyde, embedded in paraffin, and cut into 4 μm sections. Hematoxylin-eosin (HE) staining was performed on sections for histological analysis. Parallel survival experiments were performed to observe the mice until the development of neurological signs.The animal studies were approved by the Institutional Review Board of Nanjing Medical University . All mice were age and sex-matched and then randomized into the different groups. The investigators were not blinded to group allocation during experiments. Briefly, MES28 cells were intracranially injected into athymic nude mice  as previously described . Mice reP < 0.05 was considered statistically significant.The statistical analyses were performed using GraphPad Prism. The two-tailed unpaired Student’s t-test or one-way ANOVA with Dunnett’s posttest, and log-rank test were employed. All data represent the mean ± standard deviation (SD) of three independent experiments unless specifically indicated. To identify the potential oncogenic lncRNAs in GSCs contributing to the malignant phenotype of GSCs, HNP1, and MES28 were collected for RNA-seq. Heatmaps showed a clear distinction between HNP1 and MES28 Fig. . Top fivLINC00839 is located in 10q11.21  modification has been considered as the most prevalent internal RNA modification in tumor cells . PreviouNext, we tend to explore the post-transcriptional regulation of METTL3 on LINC00839. METTL3 was depleted or overexpressed in GSCs  assays and found that LINC00839 was markedly enriched using anti-YTHDF2 antibodies, which was undermined when YTHDF2 silenced Fig. . Next, wTaken together, YTHDF2 is essential for LINC00839 stability and expression in GSCs.To explore the molecular mechanism underlying the oncogenic activity of LINC00839 in GSCs radiation resistance, we performed LC-MS to identify the proteins associated with LINC01138 in GSCs. The results showed that sense but not antisense LINC00839, was specifically associated with β-catenin Fig. . We obseTaken together, our results indicate that LINC00839 physically interacts with β-catenin to promote GSC maintenance and radiation resistance.Next, we tend to characterize the molecular consequences of the interaction of LINC00839 and β-catenin. Firstly, we found that neither of LINC00839 overexpression nor knockdown had significant effects on mRNA and protein levels of β-catenin  are essential for nuclear localization . AccidenA co-immunoprecipitation assay demonstrated that LINC00839 knockdown inhibited the phosphorylation of c-Src on β-catenin Y654 instead of Y333, both residues have been identified as phosphorylation sites of c-Src on β-catenin  Fig. 6E6E. METTLTaken together, these results suggested that LINC00839 promotes GSC maintenance and radiation resistance through c-Src-mediated β-catenin phosphorylation.Given that LINC00839 confers GSCs radiotherapy resistance by activating Wnt/β-catenin signaling, we tested whether β-catenin inhibitor could reverse the function of LINC00839. Celecoxib was chosen for further study on account of its ability to transport across the blood–brain-barrier (BBB) and inhibit Wnt signaling activation . In addiWe next tested the preclinical effects of celecoxib on sensitizing radiation therapy in vivo Fig. . The resGlioblastoma is one of the most refractory solid tumors because of its high heterogeneity . Recent Recently, the biological significance of m6A modifications has drawn much attention for its vigoroso regulation on mRNAs and noncoding RNAs . m6A mod“Readers” can recognize and bind m6A sites leading to the degradation or stabilization of target RNAs. Using RNA pull-down assays, we found LINC00839 could directly interact with YTHDF2. YTHDF2 initiates the decay and deadenylation of m6A mRNAs by recruiting the CCR4-NOT deadenylase complex . YTHDF2 Further mechanism research found that LINC00839 knockdown significantly suppressed Wnt/β-catenin signaling activation. Wnt/β-catenin signaling, one of the most evolutionarily conserved signaling, has been uncovered to play crucial roles in tumors . In glioCelecoxib and other Wnt/β-catenin signaling inhibitors have been approved by FDA as effective anti-tumor drugs . CelecoxIn conclusion, we elucidated the functions of LINC00839 in GSCs, and proposed the molecular mechanism model in which METTL3/YTHDF2 maintains LINC00839 expression in an m6A-dependent way. Stabilized LINC00839 functions as a scaffold guiding the binding and phosphorylation of c-Src on β-catenin, thereby promoting Wnt signaling activation. Combinational use of Wnt/β-catenin signaling inhibitor, celecoxib, sensitized brain tumors to radiation therapy in animal studies .supplementary tablessupplementary figure legendsFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8


High expression of low-density lipoprotein receptor-related protein 6 (LRP6), a key component of the Wnt/β-catenin signaling pathway, is reported to be associated with malignant potential in some solid tumors including breast cancer and hepatocellular carcinoma. Few reports, however, have examined its function and clinical significance in colorectal cancers (CRC) demonstrating constitutive activation of Wnt signaling. Here, we compared the expression level and function of LRP6 in CRC with that of esophageal squamous cell carcinoma (ESCC) bearing few Wnt/β-catenin pathway mutations. On immunohistochemical staining, high LRP6 expression was noted in three of 68 cases (4.4%), and high β-catenin in 38 of 67 cases (56.7%) of CRC. High LRP6 expression was found in 21 of 82 cases (25.6%), and high β-catenin expression in 29 of 73 cases (39.7%) of ESCC. In our in vitro studies, LRP6 knockdown hardly changed Wnt signaling activity in CRC cell lines with mutations in Wnt signaling downstream genes. In contrast, in ESCC cell lines without Wnt signaling-related mutations, LRP6 knockdown significantly decreased Wnt signaling activity. LRP6 function may depend on constitutive activation of Wnt signaling. In epithelial tissue, the processes of cell proliferation and differentiation are coordinately regulated ,2. One oIn the presence of Wnt, Wnt ligands bind to the seven-pass transmembrane receptor Frizzled and the low-density lipoprotein receptor-related protein (LRP) 6, inducing LRP6 phosphorylation. The active form of LRP6 then binds by phosphorylation to AXIN, suppressing degradation of β-catenin and releasing it into the cytoplasm . β-catenCTNNB1) has been observed in liver cancer [APC, a key tumor suppressor gene, have been detected in most CRCs [The abnormal activation of Wnt/β-catenin pathway by gene mutation is involved in the pathogenesis of various diseases, especially human cancers . For exar cancer , endometr cancer , and colr cancer . Mutatioost CRCs , as wellost CRCs .LRP6 is an indispensable transmembrane receptor for Wnt and is overexpressed in several solid tumors, including colorectal , liver , and breIncreased LRP6 phosphorylation involved in nuclear accumulation of β-catenin also has been observed in CRC, with relevance for tumor malignancy and staging , along wAlthough the aberration of LRP6 is observed in many types of cancers with various gene mutation frequencies in the Wnt/β-catenin pathway, there are no reports showing whether LRP6 could behave differently in cancers based on Wnt/β-catenin pathway genetic abnormalities. For this reason, the purpose of this study was to elucidate the role of LRP6 in CRC and esophageal squamous carcinoma (ESCC), which shows a low frequency of gene mutations in the Wnt/β-catenin pathway , and no APC and wild-type β-catenin; HCT116, with a Ser45 deletion in one β-catenin allele and one wild-type allele, wild-type APC) and ESCC cell lines  were purchased from the American Type Culture Collection . These cell lines were authenticated by morphologic inspection, short tandem repeat profiling, and mycoplasma testing by the ATCC. The authors also performed the mycoplasma testing on a regular basis. Cell lines were maintained in Dulbecco’s modified Eagle’s medium and Roswell Park Memorial Institute 1640 with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin in humid 5% CO2 at 37 °C.Human CRC cell lines  between 2006 and 2010. As positive controls, we used samples from three cases of breast cancer. Written informed consent was obtained from all patients, in accordance with guidelines approved by the Institutional Research Board. This study was conducted under the supervision of the Ethics Board of Osaka University Hospital (No. 15144).Small interfering (si)RNA against LRP6 (siRNA-LRP6) and negative control siRNA (NC-siRNA) were purchased from GeneDesign Inc. . Transfection was performed at a final concentration of 30 nM with Lipofectamine 2000 , based on the manufacturer’s protocol.Formalin-fixed, paraffin-embedded tissues were prepared and sectioned into 4 μm slices. Sections were deparaffinized with xylene and then rehydrated in graded alcohols. Immunostaining was performed using antibodies and the VECTASTAIN Elite ABC Kit , according to the manufacturer’s protocol. Anti-LRP6 rabbit polyclonal antibody (PA5-13145) was obtained from Thermo Fisher Scientific, and anti-β-catenin mouse monoclonal antibody (610153) was obtained from BD Transduction Laboratories . The stain intensity was graded as 0 to 2 , and the percentage of positive cells was scored as 1 to 3 . The two scores were multiplied together to determine a staining score, with possible scores of 0, 1, 2, 3, 4, or 6. Immunostaining for the LRP6 protein was performed in 68 sets of normal colon tissues and cancer tissues and 82 sets of normal esophageal tissues and cancer tissues. The breast cancer tissue served as a positive control for the primary antibody . A seriaThe protein samples were subjected to SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Cell lysates and the antibodies against LRP6 (C47E20) and β-actin (ACTB) were obtained from Cell Signaling Technology, and the antibody against β-catenin (610153) from BD Transduction Laboratories. We collected protein lysate after processing by siRNA at 48 and 72 h. After incubation with secondary antibodies, signals were detected with the Pierce Western Blotting Substrate (Thermo Fisher Scientific).Total RNA from cell lines was isolated using TRIzol reagent (Thermo Fisher Scientific) following the manufacturer’s protocol. RNA concentration and purity were assessed with a Nano Drop One spectrophotometer W1F1 (Thermo Fisher Scientific). Total RNA was reverse transcribed using the High-Capacity RNA-to-cDNA kit (Thermo Fisher Scientific). To measure LRP6 expression level, a real-time quantitative polymerase chain reaction (qRT-PCR) was performed using a LightCycler 2.0 Instrument  with LightCycler TaqMan Master (F Hoffman-La Roche AG). The product numbers of the TaqMan Gene Expression assay were as follows: ACTB ID Hs01060665_g1 and LRP6 ID Hs00999795_m1. Relative expression was quantified using the ΔΔct method.Cells were seeded onto 96-well plates , and transfection of siRNA was performed using Lipofectamine 2000 (Thermo Fisher Scientific). At 24 h after siRNA transfection, cells were transfected with 100 ng/well of the TOPFLASH luciferase reporter vector  using Lipofectamine 2000 and treated with 100 ng/mL human recombinant Wnt3a . After 24 h, cells were collected with the reporter lysis buffer for luciferase activity measurement using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized against Renilla luciferase activity for each transfected well. The transfection efficiency was evaluated with 1 ng/well of the Renilla luciferase reporter vector . We incubated four kinds of cultured cells under unstimulated conditions, collected the lysate, and measured firefly luciferase activity (TOPFLASH), as well as Renilla luciferase activity (Renilla) as endogenous control.t-test for continuous variables and the chi-squared test for noncontinuous data. Survival curves were generated using the Kaplan–Meier method and assessed using the log-rank test. A value of p < 0.05 was considered statistically significant.Data were expressed as means ± standard deviations. Statistical analysis was performed using JMP Pro 14 software . Statistical differences were analyzed with the Student’s In normal mucosa of colon tissues, LRP6-positive cells were generally localized at the bottom of the colonic epithelium A, arrow,In contrast to the results for LRP6 staining, many cases showed strong β-catenin expression (strong: 2) at a high incidence (90–100%), mainly in the cytoplasm A–D. As aIn normal squamous epithelium of esophageal tissues, LRP6 expression was found mainly in the parabasal layer A. In ESCWith the introduction of LRP6-siRNA into HCT116 and TE-8, LRP6 mRNA expression decreased compared with expression in the control group receiving NC-siRNA A. LikewiAPC, we found no response to stimulation with Wnt3a, along with unchanged Wnt/β-catenin signaling under LRP6 suppression , and introduction of LRP6-siRNA significantly dampened this increase (p < 0.01 or 0.05)  G,H.p = 0.035, To explore the relationship of LRP6 expression and β-catenin protein with clinicopathological correlation and prognosis in CRC and ESCC, we divided the cases with clinical follow-up data by high LRP6 expression (staining score >3) and low LRP6 expression (staining score < 2). Similarly, we performed analyses with two other groups, one with high β-catenin expression (staining score > 4) and one with low β-catenin expression (staining score < 3). The clinicopathological status of 58 CRC patients and 68 ESCC patients, which was stratified by LRP6 and β-catenin expression level, are shown in APC (67%), CTNNB1 (6%), and AXIN2 (5%), are often observed [CTNNB1 (25%) and AXIN1 (8%) genes [In CRC, gene mutations in proteins downstream of the Wnt/β-catenin signaling pathway, such as observed . Liver c%) genes . On the  [CTNNB1 % and AXI%) genes  cases ra%) genes , it is nAPC mutation. One possible explanation is that the signal was activated in DLD-1 because of an APC mutation affecting the downstream signaling in the Wnt/β-catenin pathway, so that the upstream factor LRP6 did not significantly affect this activity. To confirm whether LRP6 knockdown only affects the Wnt/β-catenin pathway when it is not constitutively active, an experiment with overexpression of APC protein in DLD-1 to reduce Wnt/-catenin pathway activation may be helpful. Because APC is a huge protein, the center third of APC (cAPC), which contains both β-catenin and AXIN binding domain and is sufficient to promote degradation of β-catenin protein [CTNNB1, responded to stimulation by Wnt3a but not to LRP6 knockdown. The wild-type allele in HCT116 provided susceptibility to Wnt3a, but the mutant SER45 allele likely caused accumulation of β-catenin. These results suggest that LRP6 is not important for the activation of Wnt/β-catenin signaling in CRC, which harbors genetic abnormalities in this pathway. There are several controversial studies showing whether LRP6 expression contributes to Wnt/β-catenin signal activation in CRC with APC and CTNNB1 mutations. Raisch et al. demonstrated that LRP6 did not affect tumorigenesis in Min/+APC mice, and they also showed that LRP6 knockdown did not affect the colony formation activity and cell growth in CRC cells with APC or CTNNB1 mutations, suggesting that LRP6 is dispensable for tumorigenesis induced by an aberrant Wnt/β-catenin pathway [APC mutations did not alter the activity of the Wnt/β-catenin pathway [APC mutations, but not in CRC cells with CTNNB1 mutations [We performed an LRP6 knockdown in CRC and ESCC cells to investigate how LRP6 expression affects Wnt/β-catenin signaling. In the luciferase reporter assay using the TOPFLASH plasmid to evaluate Wnt/β-catenin signaling, we found almost no change under Wnt3a stimulation or LRP6 knockdown in DLD-1 cells carrying a homozygous  protein , may be  pathway . Chen et pathway . On the  pathway . Saito-Dutations , and Cabutations . Our resAPC and CTNNB1 show no enhanced signal activity downstream of the Wnt/β-catenin pathway. Therefore, they responded to Wnt3a stimulation and showed a decrease in this response under LRP6-siRNA exposure, suggesting that LRP6 is important for regulating the Wnt/β-catenin pathway in ESCC. Our results support previous findings that LRP6 is associated with cell migration, invasion, and epithelial-to-mesenchymal transition in ESCC cell lines [Unlike DLD-1 and HCT116 cells, the TE-1 and TE-8 ESCC cell lines without mutations in ll lines .Taken together, our results suggest that the function of LRP6 may differ depending on genetic abnormalities in the Wnt/β-catenin pathway .LRP6 is a membrane protein associated with the Wnt/β-catenin signaling pathway  and an iAPC or CTNNB1 mutations.In cancer, many CRC samples demonstrate a high positivity rate for β-catenin and strong staining intensity, which is consistent with previous reports. Although the staining intensity for LRP6 was not as strong in many CRC samples, the positivity rate was high. This result supports the idea that LRP6 may not be important for the activation of the Wnt/β-catenin pathway in CRC with genetic abnormalities such as In contrast, for β-catenin staining scores in ESCC samples that were lower than in CRC samples and related to LRP6, the positivity rate did not differ significantly compared with CRC samples, but the number of ESCC samples with strong intensity tended to increase. This result suggests that increased expression of LRP6 is associated with the activation of the Wnt/β-catenin pathway in ESCC.These results are consistent with our in vitro data and further support the idea that LRP6 functions differentially in cancers depending on the presence or absence of genetic abnormalities in the Wnt/β-catenin pathway.APC or CTNNB1 mutations. Therefore, further studies will be needed to reveal the underlying mechanism.By analyzing the relevance of LRP6 expression to prognosis and clinicopathological parameters, we found a significant correlation between high LRP expression and lymphatic invasion in CRC, suggesting that LRP6 is associated with the invasive activity of CRCs. This is consistent with the studies that show LRP6 is associated with metastasis and poor prognosis in multiple types of cancers such as breast cancer , liver cAlthough high LRP expression was significantly correlated with lymphatic invasion in CRC, Kaplan–Meier curves showed no significant difference in OS or RFS between groups with high vs. low LRP6 expression in CRC. This discrepancy may be because we analyzed only small number of samples for survival analysis in this study. Therefore, further investigation using a higher sample number will be required.Small molecule inhibitor, monoclonal antibody and modified peptides, which target the Wnt/β-catenin pathway have been developed and have proceeded to clinical trial . The tarOur results suggest that LRP6 inhibition and other Wnt/β-catenin inhibitors may be effective for ESCC in case of LRP6 overexpression and upregulated Wnt/β-catenin pathway. Meanwhile, LRP6 inhibition may not be effective against CRC with genetic abnormalities in the Wnt/β-catenin pathway.However, it is reported that LRP6 affects not only the Wnt/β-catenin pathway, but also other pathways such as the noncanonical Wnt pathway , Wnt-depIf LRP6 overexpression is associated with the malignant phenotype of CRC independent of the Wnt/β-catenin pathway, it is possible that the combined inhibition of LRP6 with an intracellular component or transcription cofactors of the Wnt/β-catenin pathway may have additive or synergistic effects. Further studies will be needed to investigate this possibility.APC or CTNNB1 mutations [Nucleic acid medicine, including small interference RNA (siRNA) and microRNA (miRNA), is considered a next-generation cancer therapy . We prevutations . We deveutations . TherefoOur findings indicate that LRP6 could behave differently in different cancers based on genetic abnormalities related to the Wnt/β-catenin pathway. Further studies are needed to elucidate the function of LRP6 in cancer and characterize its potential as a therapeutic target.


Although bioderived flame retardants are environmentally sustainable and less toxic, their impact on the thermal stability and flammability of polymers remains poorly understood. In this study, we assessed the influence of mycelium on the thermal stability and flame spread characteristics of epoxy through thermogravimetric analysis, Fourier transform infrared spectroscopy, the UL94 flammability test, and scanning electron microscopy. We observed a decrease in the maximum mass loss rate temperature when mycelium was incorporated into epoxy, indicating an earlier onset of thermal degradation. The inclusion of mycelium increased char yields above 418 °C due to mycelium’s inherent char-forming ability. However, mycelium did not alter the thermal degradation pathway of epoxy. Furthermore, according to the UL94 test results, the incorporation of mycelium reduced the flame spread rate compared to that of neat epoxy. These findings contribute to our understanding of the interaction between bioderived flame retardants and polymers paving the way for the development of more sustainable fireproofing materials. Formaldehyde is a toxic gas that irritates the respiratory system and can also cause various other health effects5. Phenols also irritate the respiratory system and have toxic effects on human and animal health6. VOCs, which may include hydrocarbons, can cause dizziness and they are potentially carcinogenic8. Incomplete combustion of epoxy can produce highly toxic CO, leading to asphyxiation when in elevated concentrations. Additionally, the thermal decomposition of epoxy can release acrid smoke and irritating fumes from the thermal decomposition of a thermal degradation by-product, 4,4ʹ-methylenediphenol10. Heavy smoke and irritating fumes hinder evacuation in fire situations causing death or serious injury.When polymer matrix composites are exposed to high temperatures during a fire, they ignite, thermally decompose and burn. The thermal decomposition of polymer matrix composites releases toxic volatile compounds that can impair visibility, and cause death by asphyxiation. For example, the thermal decomposition of epoxy produces hazardous volatiles such as formaldehyde, phenols, volatile organic compounds (VOCs), carbon monoxide (CO), carbon dioxide  and chlorinated paraffin. When subjected to high temperatures, halogenated flame retardants release free radicals that interrupt the flaming process by combining with by-product radicals which would otherwise propagate the fire. However, despite their effectiveness in reducing the flame spread and smoke generation, halogenated flame retardants have been phased out due to their potential environmental persistence and toxicity, as well as their contribution to ozone depletion17.To improve fire resistance and reduce the volume of toxic gases and smoke released during the thermal decomposition of polymer composites, flame retardants are typically added to the polymer matrix19 as well as inorganic salts such as aluminium hydroxide, magnesium hydroxide, layered double hydroxide, and hydroxy double salts22. Phosphorous-based flame retardants act through a combination of mechanisms, including char formation, dilution of volatile components, and flame quenching. While the environmental persistence and toxicity of phosphorous flame retardants may be substantially lower compared to halogenated compounds, higher loading levels are required which may adversely impact the mechanical properties of the host polymer matrix24. Nitrogen-based flame retardants release inert gases during decomposition, diluting oxygen, and combustible volatiles, and promoting the formation of a protective char. While effective in reducing smoke and toxic gas emissions, these compounds have limitations in achieving high flame retardancy levels and they are also sensitive to moisture. Inorganic flame retardants release water vapour when heated, thereby having a cooling effect while also diluting combustible gases27. These compounds contribute to char formation through the formation of metal oxides within the condensed phase. Inorganic flame retardants are less toxic and highly thermally stable, but higher loading levels may be required, impacting the mechanical properties and processability of modified polymers.Halogenated flame retardants have now been replaced by phosphorus- and nitrogen-based compounds such as phosphates, phosphonates, phosphazenes, melamine, melamine polyphosphate, and melamine cyanurate29, compared to fossil fuel-derived or synthetic inorganic flame retardants. Biodegradable flame retardants can be broken down by natural processes, reducing their long-term environmental impact. Moreover, the by-products generated from the thermal decomposition of bioderived materials, such as water and CO2 have reduced toxicity. Despite holding promise as more sustainable alternatives to traditional flame retardants, further research is required to improve our understanding of the flame retardation mechanisms inherent in bioderived materials.Recently, the focus has shifted to a new class of flame retardants derived from renewable biomass sources, such as plants or agricultural waste. Bioderived materials with potential flame retardation effects such as mycelium are more sustainable and environmentally friendly, with versatile multifunctional properties31. Previous research conducted by our team30 revealed that alkaline-based deacetylation of mycelium can further increase char yields at high temperatures. Based on these findings, we developed mycelium-containing epoxy composites to enhance our understanding of the interaction between unmodified and deacetylated mycelium and epoxy resin, specifically the effects of mycelium derivatives on the thermal degradation behaviour of epoxy. We aimed to test the hypothesis that high char-forming mycelium (either unmodified or deacetylated) can improve the thermal stability of epoxy. To this end, we evaluated the performance of mycelium-containing epoxy composites in which the ratio of mycelium-to-epoxy was varied. This research will pave way for the integration of bioderived flame retardants into composite polymer matrices or flame-retardant coatings.In this paper, we employed thermogravimetric analysis (TGA), Fourier Transform infrared (FTIR) spectrometric analysis, and scanning electron microscopy (SEM) techniques to explore the interaction with and influence of mycelium on the thermal degradation of epoxy. Mycelium was selected as a representative bioderived flame retardant due to its proven ability to produce elevated char yield at temperatures above 325 °CThe outcomes of our research hold significant implications for fire sciences and fire engineering research communities. Firstly, this research holds the promise of providing insights into the influence of bioderived materials such as mycelium on the thermal decomposition of polymers, which can pave the way for sustainable fire safety solutions, improved fire performance, and reduced toxicity. Secondly, by reducing the reliance on chemically synthesized flame retardants, our research will contribute to a greener and more environmentally friendly approach to fire safety. Finally, our findings can help design mycelium-containing polymer coatings that can thermally protect flammable composites and minimise the release of toxic by-products during thermal decomposition. This new information has the potential to enhance flame retardancy, reduce smoke generation, and increase fire resistance, ultimately leading to the development of fire safe solutions for flammable materials.Ganoderma australe, for this study was based on its exceptional growth performance31 and relatively high chitin content in its cell wall32. A subculture of Ganoderma australe, provided by RMIT University’s Fungal Culture Collection , grown on malt extract agar was used as an inoculum for mycelium cultivation. The liquid feed solution, blackstrap molasses, was sourced from E&A Salce , while reagent-grade sodium hydroxide (NaOH) was obtained from Sigma Aldrich . An epoxy resin, diglycidyl ether of bisphenol A (DGEBA) (West System 105), and the corresponding slow-hardener (West System 206) were supplied by ATL Composites under the Gougeon Brothers Inc, (United States) licence.The selection of the non-pathogenic mycelium fungus, 30. Control and deacetylated mycelium sheets were denatured at 60 °C for 3 h and kept in hermetic plastic bags to prevent moisture absorption during storage. Mycelium sheets were conditioned at 60 °C for 3 h before TGA experiments.Molasses feed solution 10 wt%) was prepared by diluting blackstrap molasses using deionized water. The feed solution was sterilised at 121 °C for 30 min to prevent bacterial contamination during mycelium incubation. Mycelium inoculum (6 mm diameter) was transferred to the dilute molasses solution (100 mL) in a sterilised polypropylene container. The culture was incubated in an environmental chamber under controlled temperature (~ 25 °C) and white light for 17 days. Mycelium sheets were harvested by peeling off the brownish gel at the feed/mycelium interface. Mycelium sheets were washed with water to remove molasses residue. Some mycelium sheets were deacetylated by submersion in 2 M NaOH at 40 °C for 8 h. Unmodified (control) and deacetylated mycelium samples are referred as the control and 2 M/40 °C/8 h, respectively. Deacetylation conditions, biomass yields, and the calculated degrees of deacetylation are given in Table 0 wt% wasFlame retardants can be integrated into flammable composite laminates as thin interleaving layers, or micro-particulate additives to the polymer resins. To understand how the introduction of mycelium can influence the thermal degradation behaviour of an epoxy polymer matrix or coating, mycelium-containing epoxy composites were fabricated. Two layers of wet mycelium sheets (unmodified or deacetylated) were stacked and consolidated under elevated temperature (60 °C) and applied compression pressure (0.25 MPa) for at least 30 min to ensure complete drying. After cooling the two-ply mycelium composite to ambient temperatures, an epoxy/hardener slurry was uniformly applied on either side of the mycelium composite ensuring uniform resin spread across the whole surface. Mycelium-containing epoxy composites were cured at room temperature for 24 h and then post-cured at 60 °C for 8 h in a conventional oven.Two sets of mycelium-containing epoxy composites were prepared. The first set of composites was used to investigate the effect of the mycelium-to-epoxy ratio on the thermal degradation behaviour of neat epoxy. Composites having mycelium-to-epoxy ratios of 1:2 (M1:E2), 1:1 (M1:E1) and 2:1 (M2:E1) were prepared using unmodified (control) mycelium. The same manufacturing methodology was followed in the preparation of the second set of composites in which the ratio of mycelium (control or deacetylated) to epoxy was fixed at 1:2. Composites in the second set are identified as epoxy/control and epoxy/2 M/40 °C/8 h. The post-cured mycelium-containing epoxy composites were shredded into small flakes and stored in hermetic plastic bags to prevent moisture absorption prior to TGA analysis. The composites were further dried at 60 °C for 3 h in a conventional oven just before conducting TGA experiments. The incubated and denatured mycelium, and the epoxy/mycelium composites are shown in Fig. Mycelium-only laminate specimens UL94 tests were manufactured by compression loading (0.25 MPa) wet mycelium sheets into 2 mm thick panels at 60 °C for at least 3 h. The consolidated mycelium-only laminates were cut into 125 × 13 × 2 mm specimens for UL-94 tests. Separately, mycelium-containing epoxy composite laminates in which the mycelium-to-epoxy ratio was 1:2, were manufactured for the UL-94 test. Epoxy-coated mycelium sheets were stacked to achieve a thickness of approximately 2.0 ± 0.2 mm. Separately, the neat epoxy sample was prepared by pouring the epoxy resin/hardener slurry into a flat-bottom rectangular container achieving a thickness of approximately 2 mm. The neat epoxy and mycelium-containing epoxy laminates were left to cure at room temperature for 24 h and subsequently post-cured at 60 °C for 8 h. The post-cured panels were cut into 125 × 13 × 2 mm specimens using a diamond saw. For the horizontal UL-94 tests, the post-cured specimens were marked at 25 and 100 mm from the free end where the flame was introduced.2 gas flowing at 20 mL/min. Some TGA experiments were stopped at 600 °C so that the primary residual char could be recovered for microstructural and FTIR analysis. An evolved gas transfer line connecting the TGA to the FTIR was maintained at 300 °C and continuously flushed with N2 gas (flow rate of 50 mL/min) to prevent condensation of evolved volatiles. Evolved gases were analysed between 4000 and 600/cm wavenumbers. A minimum of three repeat experiments were performed for each sample.Changes to the thermal degradation behaviour epoxy in the presence of mycelium were investigated via thermogravimetric analysis using the Perkin Elmer STA 6000 TGA instrument interfaced with the Perkin Elmer Frontier FTIR Spectrometer for real-time chemical analysis of evolved gases. Flakes of test samples weighing ~ 12 mg were loaded into an alumina crucible which was then transferred into the TGA furnace. Initially, the furnace was set at 35 °C for 10 min to allow for sample equilibration. The furnace temperature was then increased at a rate of 30 °C/min until 850 °C under a continuous stream of N–3 mbar. SEM images were collected using a spot size of 5 and accelerating voltages ranging between 15 and 30 kV depending on specimen thickness.The FEI Quanta 200 scanning electron microscope was used for microstructural and morphological characterisation of both pristine mycelium and residual char recovered at 600 °C during TGA experiments. SEM specimens were attached to an electrically conductive carbon tape, which was then affixed to a sample holder. An iridium coating ~ 5 nm in thickness was applied onto the specimen using the Leica ACE 600 sputter coater, which was operated at 8 × 10−1 between 4000 and 600 cm−1. Absorption peaks were assigned using the built-in Perkin Elmer Spectrum 10.5.2 software. At least three repeat experiments were conducted for each sample.The functional groups and chemical interaction between mycelium and epoxy were investigated using the Perkin Elmer (Spectra 100) FTIR Spectrometer equipped with an attenuated total reflection (ATR) lens at room temperature. Biochemical analysis was performed on mycelium-containing epoxy as well as the residual char recovered at 600 °C during TGA experiments. For each FTIR-ATR experiment, an average of 32 scans were recorded at a resolution of 4 cm33. During the UL-94 vertical test, the flame was applied for 10 ± 5 s and then removed. The flameout time was recorded as t1 in seconds. Once the specimen flamed out, the flame was immediately applied for the second time for about 10 s. After the flame was removed for the second time, the after-flame and afterglow times were respectively measured as t21 and t3, in seconds. Furthermore, the UL-94 horizontal flame test was performed to evaluate the flammability of neat epoxy, unmodified and deacetylated (2 M/40 °C/8 h) mycelium-containing epoxy composites, following ASTM D635-22 guidelines34. For the horizontal test, the flame was applied only once to each specimen, with a flame-impingement time of 30 ± 1 s. The time it took for the flame front to travel the 75 mm distance between the 25- and 100-mm reference markings on the test specimen was recorded. This data was then used to calculate the burning rate in mm/min. Five specimens were evaluated for each configuration.The flammability of unmodified and deacetylated (2 M/40 °C/8 h) mycelium was assessed via the UL-94 vertical flame test, in accordance with ASTM D3801-20 guidelinesThe thermal degradation profile of neat epoxy consisted of two distinct mass loss events, as shown in Fig. 30, we reported that deacetylation of mycelium converted chitin into chitosan, which promoted char formation at high temperatures. However, we also observed that deacetylation reduced biomass yields. The reduction in biomass yields was attributed to deproteinisation, which involves the breaking of chemical bonds between chitin and proteins present in the cell wall of mycelium35. Consequently, deacetylated mycelium exhibited increased brittleness compared to unmodified mycelium36. Since a detailed discussion on this topic was presented in our previous work30, we provide a brief overview of the microstructural and morphological changes caused by deacetylation. Scanning electron microscopy images of unmodified (control) mycelium revealed a 3D hyphae filamentous network as shown in Fig. In our previous study30. Key findings regarding the impact of deacetylation on the thermal degradation properties of mycelium, which provide the necessary background for this study, are briefly presented. Mass loss (TGA) and derivatised mass loss (dTG) profiles revealing five thermal degradation steps for both the control and deacetylated mycelium are shown in Fig. Changes in the chemical, microstructural, and morphological features resulting from deacetylation can affect the thermal degradation behaviour of mycelium. The correlation between the degree of deacetylation and the thermal degradation behaviour of mycelium was extensively discussed in our previous study37. The second step, recorded between 130 and 235 °C, was associated with the loss of physically and chemically absorbed water molecules. The third step, occurring between 235 and 465 °C, was considered the primary thermal degradation step and attributed to the thermal decomposition of the fungal cell wall, which is composed of chitin, amino acids, carbohydrates (including polysaccharides), and glucan. The degradation of mycelium and its derivatives during this step involved deacetylation, depolymerisation, or cleavage of glycosidic linkages in the chitin segments of mycelium39. The fourth step, between 465 and 600 °C, formed primary char while the fifth and final step, occurring between 600 and 810 °C, involved the conversion of primary into secondary char. The char yields increased with the increase in the degree of deacetylation due to the superior thermal stability of chitosan compared to chitin. Additionally, the presence of low molecular weight OH-terminated units resulting from deacetylation promoted the formation of dense and consolidated char40.The thermal degradation behaviour of mycelium and its derivatives was characterised by several distinct mass loss steps. The first step, occurring between 35 and 130 °C, involved moisture desorption and the degradation and volatilisation of low molecular weight (< 20 kDa) hydrophobin fragments present on the surface of myceliumFigure TGA experiments were performed to examine the effect of unmodified mycelium on the thermal degradation behaviour of epoxy. The TGA and dTG profiles are shown in Fig. The dTG analysis of neat epoxy Fig.  showed tTmax) in mycelium-containing epoxy to a slightly lower value was caused by the early onset of mass loss in unmodified mycelium as revealed in Fig. The experimental TGA mass loss data for the M1:E1 ratio was compared with theoretically estimated data using an analytical approach based on fractional mass. The theoretical mass loss-temperature data for M1:E1 was calculated by summing the remaining mass fraction, entclass1pt{minima2 (~ 2349 and 669 cm−1)41, glyoxylic acid 42, ethylene oxide 43, H2O (4000–3400 cm−1 and 1940–1277 cm−1)44, as well as a mixture of formaldehyde 45, and 4,4'-methylenediphenol 46. H2O is formed from a combination of hydroxy and hydrogen radicals, while CO2 is generated from the oxidative cleavage of the epoxy groups47. Similar evolved products were identified at higher temperatures of 515 and 755 °C, albeit with an increased relative proportion for CO2 and a reduced proportion for formaldehyde and 4,4′-methylenediphenol. Additionally, the presence of CO at ~ 2114 cm−141 was observed at the higher temperature of 755 °C, indicating incomplete oxidation of the thermal degradation products under an inert atmosphere.The analysis of evolved gases during the thermal degradation of TGA samples can provide valuable insights into potential changes in the degradation mechanisms of materials. In the case of neat epoxy, gases evolved at temperatures of 418, 515, and 755 °C were analysed using Fourier Transform Infrared (FTIR) spectroscopy, and the corresponding spectra are shown in Fig. −1 were identified as the aldehyde carbonyl group produced during the thermal decomposition of mycelium48. Furthermore, the sharp peaks at approximately 1591 and 1512 cm−1 are characteristic of aromatic heterocyclic compounds formed during the thermal degradation of mycelium48. At 418 °C, the volatiles released from mycelium-containing epoxy had similar FTIR spectra to neat epoxy. Similar patterns were observed at 515 and 755 °C, where the gaseous thermal decomposition products of mycelium-containing epoxy composites exhibited the same characteristic features as those reported for neat epoxy. These findings provide insights into the volatiles released during the thermal degradation of mycelium-containing epoxy and more important they suggest subtle changes to the thermal degradation pathway of epoxy due to the presence of mycelium especially when considering evolved gases.Figure To investigate the relationship between char morphology and the thermal degradation behaviour of unmodified mycelium and mycelium-containing epoxy, char material recovered at 600 °C were analysed using the scanning electron microscopy. Figure −1, which was attributed to C–H bending or aromatic C=C bonds49. Remarkably, this absorption peak persisted in the residual char recovered at 850 °C (spectra not shown here), suggesting its association with aromatic C=C bonds. Additionally, another distinct and moderately strong absorption peak, centred around 875 cm−1, was observed in the FTIR spectra of char recovered at 600 °C for both unmodified mycelium and mycelium-containing epoxy composites. This absorption band was assigned to C–H bending of the furanose ring51. Furanose, is a product of ring-chain tautomerism of pyranose at high temperatures and is likely present in the recovered char. Furthermore, a weak yet sharp peak at approximately 710 cm−1 appeared in both the char derived from unmodified mycelium and mycelium-containing epoxy, which was attributed to aromatic ring C–H bending49. The similarity in FTIR spectroscopic features between unmodified mycelium and its mycelium-containing epoxy counterparts suggests the absence of chemical interactions between mycelium and epoxy, as well as their thermal degradation products.Figure In essence, the presence of unmodified mycelium did not substantially affect the thermal degradation behaviour of neat epoxy, as indicated by the analysis of evolved gases and FTIR characterisation of the recovered char. The thermally degradation of epoxy preceded the formation of primary char from the thermal decomposition of mycelium. By the time mycelium formed the primary char, the epoxy resin component had already been depleted. These findings suggest that the by-products of the thermal decomposition of mycelium at temperatures below 418 °C have very little impact on the primary pyrolysis mechanism of epoxy. While mycelium may not alter the thermal degradation pathways of epoxy, its ability to promote char formation when incorporated into polymer matrices can be harnessed to create fireproofing coatings for flammable composites. When exposed to high temperatures, mycelium-containing epoxy coatings will rapidly generate a consolidated char layer on the surface of a flammable substrate. The physical char layer then serves to reduce heat transfer to the flammable substrate and prevent the escape of pyrolysis products into the combustion zone thereby limiting combustion fuel.30. The increased char formation was attributed to the presence of more thermally stable chitosan in deacetylated mycelium. Furthermore, deacetylation produced hydroxyl-group terminated low molecular weight polysaccharides which have been shown to promote char formation48. In this study, we investigated whether the enhanced char formation in deacetylated mycelium would have different effects on the thermal degradation behaviour of epoxy compared to unmodified mycelium. A deacetylated mycelium-containing epoxy composite (epoxy/2 M/40 °C/8 h) with a mycelium-to-epoxy ratio of 1:2 ratio was prepared. Typically, fire retardant additives are integrated into polymers at loading fractions lower than 40 wt% to preclude processing issues and mechanical property degradation of the resultant composite23.In our previous studies, we reported that deacetylated mycelium generated higher char yields than its unmodified counterpartFigure 52. However, if the mycelium/epoxy formulations were to be used for thermal surface protection, it would be critical to evaluate their flammability or flame spread characteristics.Interestingly, the ATR-FTIR analysis of the char recovered from the epoxy/control and epoxy/2 M/40 °C/8 h composites revealed identical spectroscopic features as shown in Fig. UL-94 flammability tests were performed to assess the fire safety characteristics of mycelium and mycelium-containing epoxy composites. Both vertical and horizontal configurations of UL-94 flammability tests were conducted to measure the rate of fire propagation and identify whether the material promotes or inhibits flame spread. Parameters such as the time of flame persistence, dripping behaviour, and burning rate were measured.Under the vertical test mode, the V-rating of unmodified, deacetylated mycelium, and mycelium-containing epoxy composites are presented in Table t1 ≥ 30 s). Although the presence of mycelium did not lead to a V-rating under vertical UL94 conditions, it did alter the burning behaviour of neat epoxy. While epoxy dripped and ignited the cotton batting during combustion, composites containing both unmodified and deacetylated mycelium did not exhibit dripping. Furthermore, the difference in burning rate between epoxy and mycelium-containing epoxy composites was noticeable, as reflected in Fig. Figure  °C/8 h samples were 9% and 29% respectively slower than the neat epoxy. That is, the flame spread in mycelium-containing epoxy composites was supressed suggesting improved fire safety ratings in comparison to the neat polymer. The superior fire safety ranking of mycelium-containing epoxy composites was achieved despite a relatively high weight fraction of the highly flammable epoxy polymer. Thus, if mycelium were to be used as fireproofing material with minimal polymer binder content, it would be expected to perform even better. In summary, this research has demonstrated the feasibility of developing mycelium-based fireproofing materials with improved fire safety for flammable substrates including flammable polymer matrix composites.The experimental results obtained from UL-94 horizontal test are reported in Table The inclusion of mycelium in epoxy resulted in a shift in the maximum mass loss rate temperature from 418 °C for the neat epoxy to a range of 405–411 °C for the mycelium-containing epoxy composites. This reduction in temperature was attributed to the early onset of thermal degradation caused by the presence of mycelium.No apparent synergistic effects were observed due to the presence of mycelium in epoxy, apart from the shift in the peak temperature (515 to 535 °C) for the conversion of primary into secondary char.Primary and secondary char formation by mycelium occurred after the epoxy resin had already thermally degraded, suggesting minimal effects of mycelium-produced char on the thermal degradation behaviour of epoxy.The char yield obtained from mycelium-containing epoxy composites was relatively higher than that of the neat epoxy, particularly at temperatures above 418 °C. This increase in char was attributed to the intrinsic char-forming ability of mycelium due the presence of hydroxyl-terminated polysaccharides and the superior thermal stability of deacetylated mycelium.Improved char formation in mycelium-containing epoxy composites paves the way for the development of polymer coating formulations for thermal protection of flammable composite substrates.Unmodified and deacetylated mycelium achieved the V-1 and V-0 UL94 ratings suggesting superior resistance to flame spread. Further, when mixed with epoxy resin, mycelium-containing epoxy composites had a reduced flame spread rate to neat epoxy suggesting improved fire safety ratings.This study investigated the influence of mycelium, both unmodified and deacetylated, on the thermal degradation behaviour and char-forming characteristics of epoxy matrix composite formulations. The key findings can be summarised as follows:The integration of mycelium into epoxy does not alter the thermal degradation pathways of the polymer matrix. However, improved char formation in mycelium-containing epoxy composites suggests the potential for developing sacrificial surface coatings for fireproofing flammable substrates. Upon exposure to extreme temperatures, mycelium-containing polymer coatings would thermally decompose to form a residual char layer that then acts as a protective barrier for the underlying substrate. Moreover, mycelium-containing epoxy composites had reduced flame spread rates compared to the neat epoxy polymer. These findings contribute to our understanding of the interaction between bio-derived mycelium and epoxy resin, guiding the development of sustainable and environmentally friendly fire-retardant solutions. In conclusion, this study expands our knowledge of the interaction between mycelium and epoxy resin, aiding in the design and development of sustainable, non-toxic, and biodegradable fire safety solutions.