The characterization of the prepared nano-materials were performed using different analyses such as FTIR, XRD, SEM, EDS, TEM, and BET and the results proved the successful synthesize of UiO-66-NH2/CNTs nanocomposite. The corrosion protection performance of the coated panels was investigated by electrochemical impedance spectroscopy (EIS), salt spray, and contact angle measurement. The EIS results revealed that unmodified and UiO-66-NH2 containing coating in 3.5 wt.% NaCl electrolyte were failed after 45 days but the corrosion was negligible in UiO-66-NH2/CNTs coating due to high pore resistance values even after 45 days. Salt spray and contact angle measurements confirmed that UiO-66-NH2/CNTs containing coating acts as an efficient barrier against wet saline environment even at long exposure times. This is attributed to uniform dispersion in the epoxy matrix and formation of a uniform nanocomposite coating.In this study, a porous nanocontainer from UiO-66-NH Therefore, finding a suitable alternative for epoxy resin among organic coatings is challenging as these materials are not water-proof and totally perfect. Resultantly, in a corrosive environment, metals could not be preserved for long period. Formation of defects  in the structure of coatings is almost inevitable. The situation gets worse when the material is designed for outdoor application where it experiences harsh and uncontrolled conditions. Bypassing the coating material, the structural defects result in the corrosion of metal. So as to prevent such a kind of problem, a new generation of coating with the ability of anti-corrosion has been constructed. The protective ability could be improved by addition of nano/micro additives to the structure of epoxy-based coatings. Various investigations have dealt with the construction of high quality nanocomposites with excellent mechanical and thermal features5. A wide variety of nanofillers are investigated and chosen for enhancement of the coatings’ protectiveness. For example some of carbon-based nanofillers are: carbon nanotubes7, graphene and graphene oxide9, inorganic nanomaterials such as LDH10, fullerene11, and halloysite12 and clay13, etc.As a promising material for delaying ions and water molecules from reaching to metals’ surface, polymeric coatings act like a physical barrier. The epoxy resins could be considered as a group of distinguished thermosetting polymers with outstanding features of excellent resistance to moisture, remarkable resistance to solvents, excellent mechanical and thermal properties, and great adhesion properties to various surfaces of both metals and non-metals. Due to its excellent properties, this material has been utilized in a wide range of industries including aircraft construction, car industries, and petroleum industries; Nevertheless, suffering from some salient weaknesses such as poor crack deflection performance, and brittleness, it couldn’t be used in many applications19. Additionally, these porous materials have been contemplated as promising candidates for fabrication of anti-corrosion coatings with barrier capability. Furthermore, not only the resulted coatings will prevent corrosion properly, but also it has been observed that they are relatively impermeable. Unfortunately, rarely reports could be found covering the application of MOFs in the fabrication of anti-corrosion coatings or their application as a protective layer31. In this regard, there are some works that introduce bare MOFs imparted with corrosion resistance without the need of any post-synthetic modifications. Roy et al.32 synthesized a three dimensional supramolecular porous framework Zn(OPE-C18)0.2H2O (NMOF-1) with high water contact angles and corrosion resistance. In other study, Zhang et al.27 took an initiative to investigate the potential application of ZIF-8, one of the most widely studied hydrophobic and water stable MOFs, in the anticorrosion industry. Etaiw et al.23 obtained brown crystals of the MOF (AgCN)4(qox)2, and employed it as corrosion inhibitor for C-steel in 1 M HCl solution. Recently, Fouda et al.33 prepared silver based MOFs as corrosion inhibitors in acid environment and Kumaraguru et al.29 reported the preparation of nickel, copper, and cobalt MOFs employing the corresponding metal salts and trimesic acid as ligand and their anticorrosion properties.In recent decades, a group of porous materials with a 3D structure have been widely investigated and developed significantly. Since introduced as materials with nanopore structure, modifiable properties by replacing its ligands, and astonishingly large surface areas, metal–organic frameworks (MOFs) have attracted many attentions37, catalysis42, and photocatalysis47, could be mentioned. Therefore, the sophisticated composite materials could be fabricated using MOFs and their unique mechanical and thermal properties49 and anti-corrosion features51. Ma et al.52 added Sn-MOF@PANI and observed that the mechanical and thermal characteristics of the investigated epoxy composite coatings improved. Similarly, Zhang et al.53 showed that the epoxy coating’s resistance to fire was significantly intensified as a result of hybridization between MOFs and GO nanosheets.Although, MOFs are extremely porous , practical, and appropriate for many applications. The mechanical properties of the developed MOFs are yet to be understood in details. Having a plethora of outstanding features like low density, remarkable surface area, and modifiable porous structure, MOFs have been integrated into various fields. Among them, adsorption59. In recent investigations, zirconium-based MOFs have been paid attention due to their high elasticity module (G)/ shear modulus (more than 13 GPa) in comparison to the other MOFs. For example, it has been found that Zr-based MOFs generally have a shear modulus value about 5–10 times of other well-known MOFs like ZIF-8. Additionally, Zr-based MOFs have a mechanically, chemically, and thermally stable structures due to numerous organic–inorganic nodes between Zr atoms and 2-ATA. Having a perfect coordination between organic and inorganic parts, the zirconium-based MOFs benefit from outstanding mechanical features and a stable structure when experiencing a shear stress57. Sai et al.60 examined the effect of Zr6O4(OH)4 clusters and 1,4-benzodicarboxylic acid, as the metal node and organic ligands, respectively, on the thermal stability of the synthetized MOF in fire. Likewise, Guo et al.49 conducted research on covering CNTs with SiO2@UiO-66 particles and it was found that their resistance against flames improved significantly and the epoxy coating successfully acted like an organic–inorganic inhibitor against the corrosion issue. In order to protect metals against corrosion, scientists have synthesized new CNTs-based coatings known as the superhydrophobic coatings (SHCs)61. It is noteworthy to mention that the limited interactions between polymer chains and the CNTs used in the structure of epoxy and the low dispersion of CNTs in the matrix of epoxy coatings are challenging. Therefore, it is of utmost importance to control the CNTs entanglement and the uniform dispersion of them in the final coating. Surface modification of CNTs is a convenient way to reduce the interaction of nanotubes. A plethora of approaches just have focused on the enhancement of the oxygen/water/ion preventing properties of high-performance coatings which are filled with upgraded CNTs in the form of nanocomposite additive.In recent years, the thermal stability, high strength when exposed to shear forces, nanosize structure, and notable surface area of zirconium-based MOFs has caught the researcher’s imagination especially for some applications like separation of various components and drug delivery2 and CNTs can provide the anti-corrosion properties. Some UiO-66-NH2/CNTs composites have been synthesized for various applications such as fabricating transistors62, dye separation63, electrocatalytic sensing65, and photocatalytic CO2 reduction66. To the best of our knowledge, the report regarding decorating the UiO-66-NH2 with CNTs yet to be investigated. In the presented study, aiming for addressing the mentioned gap in the literature, authors have fabricated a UiO-66-NH2/CNTs nanocomposite for enabling the epoxy coating to act as an anti-corrosion coating. So as to accomplish these goals, a Zr-based MOF functionalized by amine group was employed. The properties of the developed MOF/CNTs were analyzed by various methods, such as FTIR, XRD, SEM, EDS, TEM and BET. Afterwards, electrochemical impedance spectroscopy (EIS) tests, salt spray and contact angle measurement were applied on CNTs, UiO-66-NH2, and UiO-66-NH2/CNTs incorporated epoxy composites to analyze their performance in their anti-corrosion characteristics.Considering the above concerns, composites based on the UiO-66-NH4, 98%) and 2-Aminoterephthalic acid  were used as metal and organic precursors of MOF, respectively bought from Sigma-Aldrich. Utilized solvents including methanol, N, N-dimethylformamide , nitric acid , sulfuric acid  and acetic acid  were provided by Merck company. Carbon nanotubes were purchased from PlasmaChem GmbH and used without further purification. Epoxy resin  was purchased from Saman Co. Furthermore, amido polyamide curing agent based on CRAYAMID 115 (Arkema Co.) was employed as curing agent. In this research work, mild steel (CK10) was used as substrate. The chemical composition (wt.%) of the steel was as follows: 0.1 wt.% C, 0.45 wt.% Mn, ma × 0.4 wt.% Si, and about 99 wt.% Fe. Steel panels were cut into the size of 30 × 20 × 1 mm and 80 × 50 × 1 mm. Then the surface was polished with SiC paper of graded grit sizes ranging from 120 to 1500 to achieve a mirror-shinning surface then ultrasonically cleaned in a mixture of acetone and ethanol for 10 min. Finally samples were washed with distilled water and then dried in air.Zirconium tetrachloride (ZrCl2-BDC (0.067 g) were separately dissolved in 15 mL of DMF solvent for 15 min using ultrasonic vibration. Next, the Zr4+ ions solution was mixed with the ligand solution containing 2 mL of acetic acid. Then, the final mixture was agitated for an hour, subsequently was moved into an autoclave (Teflon-lined stainless steel) and was heated at 120 °C in the oven during 24 h. Finally, the mixture was cooled down, centrifuged and washed several times with methanol and dried at 70 °C to obtain UiO-66-NH2 powder.Primarily, zirconium tetrachloride (0.095 g) and amino-functionalized ligand, NH2/CNTs nanocomposite, carbon nanotubes walls were primarily modified with negatively charged carboxyl groups (COOH−). This act was performed by dispersing CNTs powder (1 g) into the mixture of sulfuric acid and nitric acid (100 mL) solution using ultrasonic instrument followed by refluxing at 80 °C overnight. Then, the functionalized CNTs were filtered and washed with deionized water until pH of 6 was achieved. After that, the filter cake was dried for 24 h at 70 °C for the subsequent procedure. Similar to the mentioned method for synthesizing UiO-66-NH2 crystals, hybrid UiO-66-NH2/CNTs nanocomposite was prepared. Therefore, modified CNTs were dispersed into the DMF solution (15 mL) containing zirconium chloride and 2-Aminoterephthalic acid ligand agitated for an hour. Then, the final mixtures was transferred into stainless steel autoclave and heated at 120 °C for 24 h. Finally, the mixture was cooled down, centrifuged and washed several times with methanol and dried at 70 °C to obtain UiO-66-NH2/CNTs nanocomposite for further experimental tests. Fig. 2@CNTs hybrid nanocomposite.To prepare hybrid UiO-66-NHTo investigate the effect of synthesized composite material on the electrochemical behavior of epoxy coating, 0.5 wt.% of each materials were distributed in the epoxy resin and polyamide hardener mixture (at a ratio of 5/2 w/w) through an ultrasound-assisted procedure. The epoxy/polyamide mixture was diluted to reduce the viscosity. The epoxy coating with 0.5 wt.% of synthesized materials were applied on steel sheets by a film applicator. The coated samples were kept at 25 °C for 24 h and then cured at 80 °C for 1 h. The dry thickness of the coating was about 50 μm.2 adsorption–desorption isotherm at 77 K.The fingerprint and structural functional groups of the materials were determined using Fourier transmitter infrared spectroscopy . X-ray diffraction  was employed to distinguish crystal structure and the data was collected by Bruker D8 advance diffractometer utilizing Cu/Kα radiation source and voltage 40 kV, 40 mA, and 1.54056 Å. The sample was scanned between 5 and 65.95 degrees 2θ, in step sizes of 0.01° at a rate of 0.01° 2θ/second. The scanning electron microscopy (SEM)  and transmission electron microscopy (TEM)  were run to find out the crystal size and the surface morphology of all samples. The specific surface area of the synthesized MOF and nanocomposite were identified using Brunauer–Emmett–Teller (BET) measurements  by N2 exposed area), saturated calomel electrode (SCE) and a platinum electrode were employed as working electrode (WE), counter electrode and reference electrode, respectively. Impedance measurements were performed by an AC signal with the amplitude of 20 mV peak to peak at the open circuit potential in the range of 100 kHz to 10 mHz frequency. The coated samples were immersed in 3.5% NaCl solution and EIS measurements were performed over time. The Nova (version 1.6) and Z-View2 softwares were used to data measurements and fit the EIS data via the electrical equivalent circuits (EEC), respectively. All EIS experiments were repeated for three times under the same conditions to ensure that the results are reproducible.In order to study the electrochemical properties, a potentiostat/galvanostat  was used. Coated samples  on the different coating. Water droplets were carefully placed onto the surfaces of the samples and the shape of droplet was recorded by canon digital camera. The homemade apparatus has been calibrate with standard references before measurements. The contact angle was determined from the average of five measurements at various positions on the samples surface.2 crystals (50–400 nm) with a uniform and octahedral shape. The morphology of hybrid UiO-66-NH2/CNTs nanocomposite reveals a three-dimensional interlinked network  analysis to show the micro-cavities of the nanoparticles structure. Figure 2 particles, which confirms the octagonal structure with moderate aggregation. The TEM image of UiO-66-NH2/CNTs hybrid nanocomposite with two different magnifications, shown in Fig. 2 crystals on the outer wall of CNTs as clusters of grape. Also, the electron scattering pattern  analysis was employed to evaluate the surface morphology and structural characteristics of the synthesized nanoparticles. Figure ork Fig. c. It can2 and UiO-66-NH2/CNTs hybrid nanocomposite are shown in Fig. 2 spectrum illustrates three specific peaks  at 1400, 1580, and 1655 cm−1. The FTIR peak at around 1400 cm−1 attributed to symmetric vibration of carboxyl group. The carbonyl group vibration band appeared at 1655 cm−1 and the peak at 1580 cm−1 ascribed to aromatic C=C stretching vibration. In addition, two typical peaks located at 788 and 1260 cm−1 attribute to N–H and C–N stretching band and confirm the presence of –NH2 group in the UiO-66-NH2 structure. Furthermore, two characteristic peaks at 3518 and 3337 cm−1 are derived from the asymmetric and symmetric vibrational bands of the primary amine group, respectively67. Some peaks existing in the range of 400–700 cm−1 are belonged to in-plane and out-of-plane –COO groups’ vibrations69. The FTIR spectra of UiO-66-NH2/CNTs nanocomposite shows that the main bands are relatively remained with no variations in comparison with the pure UiO-66-NH2. However, some small changes can be observed at some peaks such as 574 cm−1, 1064 cm−1 (highlighted with orange color) and 1253 cm−1 (highlighted with green color), which is due to the interactions between CNTs and the MOF crystals (e.g. π–π interaction) and introducing functional groups brought with the acid-treated CNTs.The FTIR spectra of CNTs, UiO-66-NH70. The XRD pattern of the UiO-66-NH2 sample shows a great agreement with the patterns reported in literature , (200), and (600) planes, respectively65. According to the XRD pattern obtained for the UiO-66-NH2/CNTs nanocomposite, quite similar peaks to that of the UiO-66-NH2 can be seen with no significant displacement, confirming that the UiO-66-NH2 nanoparticles have been successfully loaded on the CNTs by employing the solvothermal preparation method.The phase structures of the prepared materials were further analyzed using XRD analysis for confirming the crystallinity. As shown in Fig. ure Fig. 68,71. Th2 adsorption–desorption isotherms of the nanomaterials were also investigated to measure the BET surface area, as shown in Fig. 2/g for CNTs, UiO-66-NH2, and UiO-66-NH2/CNTs, respectively  technique. Figure Nly Table . All samals Fig. . AdditioThe EIS technique was used to study the effectiveness of synthesized nanocomposite additive on the epoxy coating life-time applied on the steel plates. Four prepared samples See “” sectionZmax), the part of Bode phase plot that is in the maximum value (θ-90), number of time-constants (Ntc), and breakpoint frequency (fbr). These factors (except Ntc) are depicted in Fig. 2/epoxy samples after 45 days. The appearance of second time-constant could be a sign of double-layer formation in the organic–inorganic interaction face72. The double-layer creation is due to water uptake and consequent electrolyte accumulation in some areas of the metal-coating interface. So, the corrosion process initiated in the unmodified-epoxy and UiO-66-NH2/epoxy samples with longer exposures (45 days). However, the corrosion rate was not severe (See θ-90 description). Samples with one time-constant were in the immune region, although the corrosion rate could not be assumed to be zero. The resistance behavior observed in the bode-module plots of all samples showed that water penetrates to the metal surface through diffusion paths but the corrosion was negligible in CNTs/epoxy and composite samples due to high pore resistance values even after 45 days (See Zmax description). So, Ntc values and resistive-capacitance behavior of EIS plots confirmed that CNTs/epoxy and composite samples have relatively good corrosion resistance and two other samples were failed after 45 days.The Bode plots, as the most beneficial output of EIS technique, are reported in Fig. 2/CNTs/epoxy composite and UiO-66-NH2/epoxy samples showed the most Zmax values after 15 days. Also, the Zmax value of UiO-66-NH2/epoxy is higher than the unmodified/epoxy and CNTs/epoxy after 15 days. The observed better performance is due to the better barrier performance of UiO-66-NH2/epoxy. The UiO-66-NH2 are dispersed into the coating and reinforce it against electrolyte diffusion. However, as mentioned in the introduction section, the CNTs show improper dispersion in the epoxy coating and show weaker behavior at short exposure times. However, Zmax followed a considerable decreasing trend for UiO-66-NH2/epoxy and reduced below 6 MΩ.cm2 after 45 days. Thus, organic–inorganic composite based on UiO-66-NH2 and epoxy were not good candidates for corrosion protection of metals without any modification. The CNTs/epoxy exhibited more acceptable protective behavior related to the unmodified/epoxy and UiO-66-NH2/epoxy samples at longer exposures. Although, the best performance was recorded for composite sample which Zmax was about 1000 MΩ.cm2 even after 45 days73.As presented in Fig. θ-90 values of unmodified/epoxy and UiO-66-NH2/epoxy samples considerably decreased over time and reached close to zero after 45 days. Despite the remarkable reduction in the θ-90 values, the phase angle at high-frequency region still was near -90 degree, indicating that unmodified/epoxy and UiO-66-NH2/epoxy were not fully destructed as per mentioned in Ntc description part. The recorded results for CNTs/epoxy and composite samples were different and their ascending trend was not sharp over time. The θ-90 is a good criterion for estimating the activated interface area on the metal surface74. The higher θ-90 values, the lower active area. So, in accordance with the Ntc results, the most θ-90 values were observed for composite sample confirming the best performance against electrolyte penetration into the interfacial face. Finally, the fbr criteria was used to peruse the coating ability in contact with high saline solution over time. fbr is an efficient criteria to evaluate the electrochemical active area in the metal/electrolyte interface76. As observed in the Fig. fbr values were detected for composite sample at all exposure times. Also, all other samples showed positive log fbr values after 45 days and the worst results were obtained for unmodified/epoxy and UiO-66-NH2/epoxy. According to the obtained results, the better behavior of composite sample was proved based on all experimental criteria. If reinforcement constituent in a composite form a compatible interface with the matrix material and appropriately dispersed, the resulted compact composite coating turns to a good barrier against corrosive media.According to Fig. Q was used instead of ideal capacitor (C) in Fig. Rpore) and constant phase element parameters (Qc and nc) as well as double layer characteristics including charge transfer resistance (Rct) and constant phase element parameters (Qdl and ndl) were recorded versus exposure time to find out coating-electrolyte-metal interactions. The ideal coating (Cc) and double layer (Cdl) capacitance were calculated as per mentioned elsewhere78.In the second part of this section, all EIS plots were fitted with appropriate equivalent circuits (ECs) and the collected data were used to better understanding the anti-corrosion behavior of composite sample. The extracted results are reported in Table Cc values is detected in all samples versus immersion time. Water uptake is an inevitable phenomena in organic coating exposed to the aqueous electrolytes. So, the Cc is increased by water penetration into the composite coatings. Also, the lowest Cc values were recorded for composite sample confirming the minimum water uptake among all samples. So, although the Cc could be affected by coating swelling at prolonged exposure times, it could be concluded that the barrier action of epoxy coating was reinforced by UiO-66-NH2/CNTs nanocomposite additive even after relatively long exposures to high saline media79. However, further investigation was needed for finding the cause of lower water uptake and better corrosion protection in the composite sample. So, the hydrophobicity of the samples were studied by using of water contact angle measurement.As shown in Table 2 sample. In fact, the addition of UiO-66-NH2 nanoparticles to epoxy coating results in decreasing contact angle. It could be due to the hydrophilicity nature of UiO-66-NH280. On the other hand, the composite and UiO-66-NH2/epoxy samples show hydrophobic characteristics and a larger water contact angle81. So, it seems reasonable to accept that the hydrophobicity nature of composite effectively decreased the adsorption of water onto the surface; and composite and CNTs/epoxy coatings show good barrier properties in wet environments. However, the protection behavior of composite sample is considerably better than CNTs/epoxy, especially at longer exposures. Another factor that efficiently affects the barrier behavior of a composite coating is additive dispersion in the composite matrix. The addition of CNTs into the epoxy resin without any surface treatment is difficult due to the nanotubes aggregation83. The non-uniform dispersion of CNTs creates weak regions in the final cured epoxy system which are sensitive to wet conditions83. The water uptake and initiation of interfacial interactions are inevitable at prolonged exposures in the presence of these weak regions. On the other hand, UiO-66-NH2/CNTs additive is appropriately dispersed in the epoxy media and yield a uniform nanocomposite coating acts as efficient barrier against wet saline environment even at long exposures.According to Fig. 2/epoxy, CNTs/epoxy and composite samples. The visual performances of the samples are showed in Fig. 2/epoxy after 250 h. By increasing exposure time up to 500 h, corrosion process is intensified and consequent products are speared around the scratch on all samples. Also, UiO-66-NH2/epoxy is the only sample shows coating delamination after 500 h. However, blisters are not appeared in all samples. Comparing the intensity of damaged area on scribe region indicates that addition of UiO-66-NH2/CNTs nanocomposite to the epoxy coating caused significant decrease of the corrosion. The smaller area of corrosion damage and better visual performance of composite sample around the scratch could be attributed to the better barrier performance against salt spray. Creation of epoxy nanocomposite coating with uniform structure and less hydrophilic nature related to non-modified epoxy coating is the key.The salt spray test was carried out at different exposure times on the coating with artificial defect to reveal the inhibitive performance of UiO-66-NH2/CNTs nano-additive improve their dispersion into the epoxy matrix. So, the regions contained agglomerated additives (due to the CNTs entanglement) is not formed in the coating and the electrolyte diffusion is reduced, especially at shorter immersion times. This behavior is also observed in the UiO-66-NH2/epoxy after 15 days exposure due to the relatively good dispersion of UiO-66-NH2 additive. Another mechanism is based on the coating hydrophilicity. The water uptake and consequent corrosion of the substrate is proportional to the coating hydrophilic behavior. Despite acceptable anti-corrosion performance of the UiO-66-NH2/epoxy at short exposure times, its protectiveness shows considerable reduction after 30 days. However, other samples show lower reduction in Zmax value between 15 and 30 days. On the other hand, composite sample with the most contact angle provide the best protective behavior, even after 45 days. So, it can be concluded that lower hydrophilicity and more compactness are the two reasons for better corrosion protection action of composite coating in the presence of UiO-66-NH2/CNTs nano-additive.As presented in previous section, the protective behavior of prepared samples could be categorized into the two mechanisms. Addition of CNTs in the form of UiO-66-NH2/CNTs nanocomposite was synthesized and epoxy-based coating was performed using this nanocomposite additive. FTIR, XRD, SEM, EDS, TEM and BET characteristic analyses were performed to confirm the synthesizing of UiO-66-NH2/CNTs nanoparticles. The outcomes showed that the UiO-66-NH2/CNTs nanoparticles have been successfully synthesized. The most maximum impedance magnitude (Zmax) and the part of Bode phase plot that is in the maximum value (θ-90) and the lowest value of breakpoint frequency (fbr) for UiO-66-NH2/CNTs/epoxy coating after long exposures in 3.5% NaCl solution indicated better performance of composite coating. The contact angle measurements represented UiO-66-NH2/CNTs additive increase coating hydrophobicity related to the UiO-66-NH2/epoxy and CNTs/epoxy coatings. According to the salt spray exposure, the smaller area of corrosion damage and visual performance around the scribe region confirmed that the addition of UiO-66-NH2/CNTs nanocomposite to the epoxy coating could effectively enhance corrosion properties. So, it can be concluded that the main mechanisms for better corrosion protection behavior of the composite sample are the lower coating hydrophilic nature (leading to lower water uptake) and better additives uniformity resulting in better barrier performance.Fabrication of nanocomposite coating with uniform structure and hydrophobic nature related to non-modified epoxy coating is a key solution for designing an advanced protective organic coating. Accordingly, the UiO-66-NHSupplementary Information.


Although statistical significance was not reached (p = 0.15), cases expressing nuclear β-catenin showed a 5-year disease-free survival rate of 90.9% (SE 0.08) compared with 67.4% (SE 0.08) of cases expressing membranous β-catenin. Univariate Cox analysis revealed that membranous β-catenin expression was found to be associated with a relative risk of death of 33.9 (p = 0.04). The stage of disease (p = 0.0006), histology (p = 0.003), and grading (p = 0.008) were also significantly correlated with disease-free survival according to univariate Cox analyses. Determining β-catenin expression and localization patterns may predict survival in patients with endometrial cancer and, therefore, should be considered a potential prognostic marker of disease.Beta-catenin is involved in intercellular adhesion and participates in the Wnt signaling pathway. This study evaluated the expression pattern and prognostic value of β-catenin in a series of endometrial carcinoma patients. Immunohistochemical analyses were used to assess the expression and subcellular localization of β-catenin from tissue sections of 74 patients with endometrial carcinoma. No correlation was found between beta-catenin expression and clinicopathological parameters. Patients expressing nuclear β-catenin ( Endometrial cancer is the fourth most common cancer in women and the 15th most common cancer overall. Uterine cancer incidence rates continue to increase by about 1% per year, and it has been estimated that there are approximately 66,500 new cases of endometrial cancer with over 12,000 deaths reported annually in the United States ,4. For tβ catenin is a multifunctional protein, playing a central role in development and proliferation through the regulation of E-cadherin-mediated cell adhesion and Wnt/Wingless signaling pathways . In the The aim of this study was to investigate the expression patterns of the cellular adhesion marker β-catenin and its prognostic significance within a well-defined population-based series of endometrial carcinoma with complete follow-ups.http://publicationethics.org/, accessed on 31 October 2018), and the reporting of studies conducted using observational, routinely collected health data statements that are available using the Enhancing the Quality and Transparency of Health Research Network . The study was unadvertised, and no remuneration was offered to encourage patients to give consent for the collection and analysis of their data. Each patient enrolled in this study was informed about the aims and procedures and signed an informed consent to allow data collection for research purposes.This is a multicenter retrospective pilot study. The study was approved by the institutional review board of the Fondazione Policlinico Universitario “Agostino Gemelli”—IRCCS, a center promoting the study (CICOG-31-10-18/111). The design, analysis, interpretation of data, drafting, and revisions follow the Helsinki Declaration, the Committee on Publication Ethics guidelines , moderately (G2), and poorly (G3) differentiated using the World Health Organization (WHO) system. Clinical stages of disease were established according to the 2009 International Federation of Gynecology and Obstetrics’s (FIGO) system. Recurrence was considered as any documented relapse of the tumor either in the pelvis or systemically.Disease-free survival (dfs) was calculated from the date of surgery. Patients who had a residual tumor after surgery or had an early recurrence (within three months after surgery) were excluded from the disease-free analysis and considered alive with disease. Patients who died from a different cause than endometrial cancer were considered lost at follow-up; their survival time was concluded at the date of death.After surgical resection, each tumor specimen was immediately formalin-fixed and then paraffin-embedded for routine and immunohistochemical investigation. For immunohistochemistry staining, 3 μm-thick sections were taken on poly-l-Lysine coated slides. Briefly, the polyclonal antibody to β catenin (sc-1496)  was incubated overnight with tissue sections according to the kit’s instructions. The peptide was used to generate preabsorption to assess the staining specificity of β-catenin. The pattern of staining observed with the polyclonal antibody was confirmed on duplicate slides using a monoclonal antibody (sc-7963) . For the negative control, the primary antibody was substituted for PBS. There were two patterns of β-catenin expression described: membranous, if β-catenin was only expressed in the cell membrane; and nuclear, when β-catenin was localized in the nucleus, irrespective of the percentage of stained nuclei or simultaneous expression of β-catenin in the membrane and cytoplasm. As previously discussed, membranous β-catenin expression was estimated semi-quantitatively and subjectively  . At leasDNA was extracted from paraffin blocks containing a large area of epithelial tumor cells (>75%) expressing nuclear β-catenin using the QIAamp DNA FFPE tissues kit . The primers forward 5′-GCTGATTTGATGGAGTTGG-3′ and reverse 3′-CTCTTACCAGCTACTTGTTC-5′ were used to amplify DNA sequences of the third coding exon of the β1-catenin gene (MS) . A react2 and the Fisher’s exact test were used to analyze the distribution of β-catenin-positive specimens according to clinicopathological characteristics. Disease-free survival (dfs) and disease-related survival (drs) were calculated according to the Kaplan–Meier method and evaluated by the log-rank test. To evaluate the effect of each prognostic variable on dfs and drs, Univariate Cox analyses were used. Finally, a multivariate analysis  was used to estimate which possible risk factors yielded independent prognostic information. The reported p values are two-sided, and p values of less than 0.05 were considered to indicate statistical significance. Data analyses were carried out using SPSS Statistical Software, release 5.0.1 .The χEighty-one specimens were retrospectively collected from patients affected by endometrial carcinoma; however, clinicopathological parameters and follow-up were available in only 74 cases. Patients’ ages ranged from 46 to 84 years, with a median age of 64 years. Neither radiotherapy nor chemotherapy were received by patients before surgery.Adjuvant radiotherapy was administered to the whole pelvis (56 Gy) after surgery in patients with codified risk factors.Adjuvant chemotherapy with a standard regimen including carboplatin and paclitaxel every 21 days, for six cycles, was administered to those patients with a more advanced stage of the disease.After adjuvant treatments, patients were evaluated every three months for the first two years, every four months during the third and fourth years, and every six months thereafter.n = 12), simple and complex hyperplasia (n = 7), and atrophy (n = 3). Normal endometrium adjacent to the adenocarcinoma showed a high percentage of β-catenin staining in the examined specimens , whereas simple and complex hyperplasia showed decreased percentages of β-catenin-stained cells . In cystic atrophy, β-catenin staining also decreased . β-catenin immunostaining was mostly membranous, but weakly cytoplasmic staining was also observed. The expression of β-catenin in endometrial tissues was evaluated using immunohistochemical analysis. Lesions synchronous with invasive carcinomas were classified as normal human endometrium (p = 0.08). Post-operative therapy was also similar in tumors with a nuclear or membranous β-catenin expression pattern. As previously reported, only 14 of the 74 carcinomas with β-catenin expression in the membrane had preserved membrane expression (19%); the remaining 60 carcinomas had reduced membrane expression (81%). An investigation was performed on distribution of β-catenin according to a series of clinicopathological parameters  obtained from all 74 patients enrolled  was observed in only 13 (16%) endometrial carcinomas A,B; the enrolled . There wn = 13) with nuclear β catenin expression.No β-catenin gene mutations were observed in all endometrial cancers (p = 0.04) (p = 0.15), the 5-year disease-free survival (dfs) rate was 90.9% (SE 0.08) for cases expressing nuclear β-catenin compared with 67.4% (SE 0.08) of cases expressing membranous β-catenin (p = 0.04). Univariate Cox analyses also showed the stage of disease (p = 0.02) and grading (p = 0.01) to be significantly associated with disease-related survival (p = 0.0006), histology (p = 0.003), and grading (p = 0.008) were also significantly correlated with disease-free survival according to univariate Cox analyses . Progression and death from the disease were observed, respectively, in 13 and 12 patients. A more favorable prognosis was observed in nuclear β-catenin expression patterns than the membranous β-catenin expression pattern specimens; the 5-year disease-related survival (drs) rate was 100% for patients expressing nuclear β-catenin, compared with 73.8% (SE 0.08) of patients expressing membranous β-catenin ( = 0.04) A. Even i-catenin B. For bo-catenin . Membransurvival . The staanalyses .p = 0.02).In a multivariate analysis for disease-free survival, the stage of disease only retained an independent prognostic role with a hazard ratio of 5.89 . When β-catenin accumulates in the cytoplasm because it escaped the degradation mechanism, it translocates to the nucleus where the protein interacts with transcriptional factors Tcf/LEF, activating the expression of various genes such as cyclin D1 and c-myc ,22. β-caMutations of β-catenin have been reported in a large variety of human cancers such as colorectal cancer, gastric cancer, hepatocarcinoma , malignaCTNNB1 exon 3 mutations and thus might be considered in the endometrial cancer risk stratification [CTNNB1 mutations in our 13 cases. Since several other proteins, including S100P, are regulatory components of a novel ubiquitinylation complex involved in β-catenin degradation, further studies are in need with specific regard to trigger for β-catenin nuclear localization in endometrial cancer in the absence of CTNNB1 mutations [Several studies on β-catenin expression in endometrial carcinoma have been published ,28,29. Ification . Howeverfication , the exputations .A few studies have investigated the association between β-catenin expression and clinicopathological features of EC. A loss of β-catenin expression has been reported to be associated with advanced stages and lymph node involvement . As prevCTNNB1 and TP53 mutations were associated with worse recurrence-free survival at multivariate analyses in 342 patients with grade 1–2, stage I–II endometrioid endometrial cancer [A TCGA analysis focused on 271 endometrioid carcinomas of the endometrium revealed that the activation of the Wnt/β-catenin pathway in the tumor and the consequent nuclear β-catenin staining pattern had a lower overall survival of . Indeed,l cancer . Howeverl cancer ,17,34.p = 0.04). Although β-catenin nuclear expression has already been reported to identify a subset of patients with a favorable outcome in endometrioid ovarian carcinomas [In our study, nuclear β-catenin expression-pattern specimens showed a more favorable prognosis than membranous β-catenin expression pattern specimens; the 5-year disease-related survival (drs) rate was 100% for specimens expressing nuclear β-catenin, compared with 73.8% of specimens expressing membranous β-catenin . This could be because nuclear β-catenin staining is a feature of low-grade ECs, whereas membranous staining is more frequent in serous carcinomas and high-grade tumors [We also observed for the first time that membranous β-catenin expression is associated with a relative risk (RR) of dying of 33.9 (e tumors . With the increasing clinical availability of molecular tests, including molecular profiles along with the usual pathology and clinical data, the stratification of patients who may benefit from different therapies is becoming a realistic goal. Our current data suggest that β-catenin may have a different prognostic role in endometrial cancer based on its cellular localization. Prospective clinical trials on a broader number of cases are needed to confirm our hypothesis, with the ultimate goal of incorporating molecular information into routine endometrial cancer treatment algorithms.


Microneedles (MNs) as a novel transdermal drug delivery system have shown great potential for therapeutic and disease diagnosis applications by continually providing minimally invasive, portable, cost-effective, high bioavailability, and easy-to-use tools compared to traditional parenteral administrations. However, microneedle transdermal drug delivery is still in its infancy. Many research studies need further in-depth exploration, such as safety, structural characteristics, and drug loading performance evaluation. Finite element analysis (FEA) uses mathematical approximations to simulate real physical systems (geometry and load conditions). It can simplify complex engineering problems to guide the precise preparation and potential industrialization of microneedles, which has attracted extensive attention. This article introduces FEA research for microneedle transdermal drug delivery systems, focusing on microneedle design strategy, skin mechanics models, skin permeability, and the FEA research on drug delivery by MNs. Microneedles (MNs), as a non-invasive alternative to oral and I.V. (intravenous injections) administration, can be considered an invaluable route to bypass the stratum corneum (SC) barrier and avoid the gastrointestinal and hepatic first-pass metabolism . Thus, MThe past decade has seen the rapid development of finite element analysis (FEA) in many research fields, especially for medical purposes , which hIn recent years, the FEA has attracted considerable attention in the field of MNs. It is expected to be a prospective application in MN development, eliminating the need for many time-consuming and expensive experimental trials  and makiIn general, MNs can be made of a wide variety of materials, such as silicon, metal, titanium, glass, and polymers . Each maTo characterize the mechanical properties of materials, many studies used devices such as texture analyzers , micromeIn recent years, finite element analysis (FEA) has been used to test and verify the mechanic properties of polymer materials of MNs and complement the experimental findings, which attracted extensive attention. Eriketi Z. Loizidou et al.  performeAccording to the processing techniques, there are two types: in-plane and out-of-plane MNs . MNs canDrug molecules often enter the blood circulation through passive diffusion or combine with ion penetration to accelerate their diffusion efficiency. Transportation efficiency is mainly related to the size and depth of the channel . The desMany scholars have tried to improve the mechanical characteristics and drug-release performance of MNs by designing bio-inspired MNs. Inspired by the insertion mode of the mosquito, Aoyagi et al.  designedThe skin is mainly composed of the epidermis, dermis, and subcutaneous tissue. Since the structure and tissue composition of each layer are different , as showThe stiffness and strength of skin tissue are affected by many factors such as the external environment , differUnderstanding the maximum stress skin tissue can withstand is an essential precondition for MN design. In recent years, plenty of classic models have been used to describe the mechanical properties of the skin ,84,85, fPermeability is another important property of the skin. The penetration and absorption of the skin are mainly conducted through two pathways: the skin appendages and the stratum corneum. Skin appendages combined with hair follicles, sebaceous glands, and sweat glands account for only 0.1% of the skin area. Ions or macromolecules can be absorbed through this pathway . HoweverMathematical modeling of epidermal and dermal transport is essential for the optimization and development of products for transdermal delivery, especially for the optimization of microneedle transdermal drug delivery systems. Fortunately, FEA meets this requirement. For example, FEA can be used to decompose the complex physical model into disordered structures and heterogeneous media, and then vary the density of the mesh to simulate the diffusion properties of the skin . TransceIt can be concluded that the method of FEA based on a multi-layer geometry model is a promising strategy to predict the TDDS skin pharmacokinetics as well as to quantitatively describe the absorption and distribution behavior of the drug after transdermal administration. Nevertheless, many parameters required for advanced modeling are not readily available, which remains a considerable challenge. Improving the modeling parameters through experiments and establishing a more efficient model are all of great value for the in-depth study.After MNs penetrate the skin, their quality, safety, permeability, and biocompatibility are essential issues that must be clarified before the clinical application of MNs . Plenty Compared with traditional biomechanical methods, for instance, animal experiments, physical experiments, and in vitro experiments, the mathematical models and FEA methods constitute a powerful predictive tool to better understand the matrix degradation and drug release of the MNs. For solid MNs, the drug can be loaded into the microneedle matrix. In non-degradable ones, drugs with low MW will diffuse to the outer medium, while in degradable matrices, drugs with higher MW can be released as the polymeric matrix degrades . BenslimDespite the extensive research and advances in the field of transdermal drug delivery, there are still many technical issues related to microneedle drug delivery systems, including their mechanical strength, biocompatibility, dose limits, dose accuracy, and application methods . Pen-typAlong with biological simulations, the FEA can estimate biomechanical responses and the dynamic process of microneedle insertion. The FEA of simulated insertion and fracture forces can be used to understand the biomechanics of microneedles. The FEA plays an important role in MNs’ design by considering these categories. Compared with studies on real-life animal models, FEA as a computer-based design method can assist microneedle preparation techniques because they can be modified and altered to meet specific requirements and provided on an individual basis without ethical considerations, which can save a great deal of time , from thNevertheless, the model is not entirely realistic and may contain inaccuracies in segmentation, meshing, or biomechanical material variables . AlthougIn this paper, the applications of FEA in MN design strategy, skin mechanics characteristics, skin permeability, and drug delivery by MNs were reviewed, indicating that FEA could be useful in the study of microneedle transdermal drug delivery systems, as well as the design and mechanical properties testing of MNs. Before the preparation of MNs, the stress, penetration, and buckling of different types of MNs can be designed and predicted, which can avoid ineffective design. For the prepared MNs, the experimental results can be supplemented and verified using FEA simulation to better understand the properties of MNs. After administration of MNs, the drug dissolution, release characteristics, and skin pharmacokinetics can also be predicted. However, the following two main problems should be focused on and solved in the future: (1) How to obtain more realistic skin and MN parameters using the existing experimental method and (2) how to establish a comprehensive FEA method platform for microneedle delivery systems with the concept of “design MNs by computer simulation, calibrate the simulation parameters by experimental data, and support the experimental results by quantitative analysis.”We believe that the above problems will be solved through the deepening of research on transdermal drug delivery systems and the further improvement of microneedle preparation technology. In addition, an optimized simulation based on the experimental results can explain the other experimental data and ultimately guide the experiment design. It can be foreseen that FEA simulation will play an increasingly important role in the development of MNs TDDS, as depicted in


The tumor is an uncontrolled growth of tissue that can be localized (benign) or possesses the capability of metastasis . The conventional methods of tumor diagnosis, such as acupuncture, endoscopy, and histopathology, and treatment methods, such as injections, chemotherapy, surgery, and radiotherapy, are invasive, expensive, and pose severe safety and management issues for the patients. Microneedle technology is a recently developed approach for active transdermal drug delivery. It is minimally invasive, self-administrable, bypasses the first-pass effect, and effectively delivers chemotherapeutics and drugs at low doses, thus, overcoming the drawbacks of conventional delivery systems. This review provides an idea of the types, materials utilized in the fabrication, and techniques used for the preparation of microneedles (MNs), as well as their application in tumor diagnosis and treatment. Additionally, emphasis is given to the case studies related to MNs-assisted tumor therapy, such as photothermal therapy, gene therapy, photodynamic therapy, chemotherapy, immunotherapy, and various combination therapies. MNs also serve as a tool for diagnosis by the bio-sampling of blood and interstitial skin fluid, as well as biosensing various cancer biomarkers. The combined therapy and diagnostics provide theranostic MNs for enhanced and personalized tumor therapy. The limitations and prospects of MNs development are also discussed. As per the American Cancer Society, cancer is defined as a set of diseases categorized by unrestrained proliferation and propagation of abnormal cells that left untreated may lead to fatality. The uncontrolled growth of new tissue is termed neoplasia and results in tumor formation. A tumor can be localized (benign) or possesses the capability of metastasis  . As per The existing methods of chemotherapy  and diagnosis  pose severe safety and management issues for the patients and are also very expensive . The oraThe objective of this review is to give a broad overview of MNs-based tumor diagnosis, therapy, and recent advancements in tumor theranostics. The manuscript comprises the research outcomes of case studies on tumor therapy, including chemotherapy, photothermal therapy (PTT), photodynamic therapy (PDT), gene therapy, immunotherapy, and combination therapies, as well as discussion on tumor diagnosis, including MN-assisted sampling devices and biosensors. This review also highlights the upcoming MNs-based theranostic approach for a combined tumor diagnosis and therapy along with clinical status, and current perspectives for understanding the significance of MNs in cancer treatment.MNs technology is a form of active transdermal drug delivery and could be used as a substitute for conventional syringe injections. The direct injections of therapeutic agents into the tumor can cause leakage of the drugs to the surrounding healthy tissues, thereby decreasing safety and effectiveness. The basic mechanism of MNs-assisted drug delivery involves the disruption of skin layers by forming micron-sized pathways to directly deliver the drug to the epidermal or upper dermis region ; thus, tMNs, such as solid MNs, coated MNs, hollow MNs, dissolving MNs, and hydrogel-forming MNs, are explored for therapeutic delivery and the diagnosis of disease conditions. Each type has its own set of attributes: solid MNs are useful in generating pores in the stratum corneum as pre-treatment, but there is also medication-coated MNs, dissolvable MNs for sustained release, or hollow MNs filled with drug solution. All types are employed in cancer diagnosis and therapeutics . The difA variety of biomaterials, such as silicon, biodegradable and non-biodegradable polymers, metals, carbohydrates, and ceramics, are used to fabricate MNs for specific applications ,15. VariThe different types of MNs, along with their characteristics, advantages, and disadvantages, are summarized in To achieve effective topical tumor treatment, MNs are incorporated with many therapies such as chemotherapy, gene therapy, PTT, immunotherapy, and PDT . Figure MNs-assisted chemotherapy enables the topical, localized, and minimally invasive delivery of chemotherapeutic agents with reduced side effects . Hence, Cisplatin is a first-line chemotherapeutics agent; however, its application is rare because of toxicity in systemic circulation and severe side effects. For safe and effective cancer therapy, Lan et al. developed an MNs-mediated transdermal delivery of lipid-coated cisplatin nanoparticles (LCC-NPs). The pH-responsive tumor-targeting lipid NPs encapsulated cisplatin with a high loading rate of 80%, and this led to the enhancement of drug solubility and improvement in its in vitro antitumor potential. The model used for the in vivo study was a xenograft tumor animal model, which showed that MNs-mediated delivery had an apoptotic index of 58.6%, where it caused significant apoptosis and increased cytotoxicity in tumor cells, thereby considerably reducing tumor volume and weight . The in −2 h) [Microneedles are also used in the case of ocular melanoma; however, depending on the nature of the cornea, conventional polymeric microneedles will be relatively larger. Kim and coworkers fabricated miniaturized porous silicon (p-Si) bioresorbable MNs covalently associated with the drug at doses comparable to conventional polymeric MNs. These needles remain in the tissue and gradually degrade, enabling a sustained drug release. In in vivo studies on B16F10 murine melanoma cells using a doxycycline (DOX) drug incorporated with p-Si MNs, significant inhibition of melanoma was observed using DOX-loaded p-Si needles, whereas by syringe the same dose was administered and failed to inhibit the growth of the tumor. For ~24 h, more than 80% of the covalently linked DOX was released. This was significantly longer than the control specimens with the physically-bonded DOX (~8 h) and conventional polymeric MNs  . FurtherMNs-assisted PTT acts on the tumor cells by causing thermal ablation; this is due to hyperthermia generated from light energy strongly absorbed by photothermal agents such as nanomaterials of carbon, gold, silver, and nanocrystals of germanium. This method utilizes MNs with near infrared radiation (NIR) laser radiation  that kills tumor cells and triggers drug release from MNs .To obtain an efficient PTT through MNs, it is vital to optimize the mechanical strength to facilitate better penetration and quick dissolution after administration. Tian et al. incorporated PTT with dissolving microneedles (DMNs) fabricated from graphene and poly (acrylic acid) (PAA). They combined the property of mechanical strength from graphene and prompt drug delivery owing to good water solubility and the better biocompatibility of PAA. The prepared MNs showed sound photothermal effects under NIR and could also deliver the drug directly to tumor tissues and generate heat simultaneously .PTT with aggregation-induced emission luminogen (AIEgen) is effective for the superficial treatment of tumors, as AIEgens have better photothermal and photostability conversion proficiency; however, their systemic delivery is difficult due to solubility and biodistribution-related problems. To address this, Wei and colleagues developed a DMNs system incorporated with AIEgen (NIR950) for the treatment of malignant tumor melanoma in skin . The dru2C) and a dissolvable polyvinylpyrrolidone (PVP) MNs delivery system (PVP-Nb2C MNs). The nanosheets served as photothermal agents, and PVP acted as a dissolvable matrix system and provided higher strength for MNs penetration. This study was conducted on a BALB/c nude mouse with a 4T1 tumor model, where MNs, after insertion and subsequent dissolution, released the nanosheets in the tumor. This led to an increased temperature of about 70 °C and thermal ablation with the aid of NIR laser irradiation (1064 nm) at the second biological window. After two weeks, the tumor growth suppression was observed to be higher than the control group, and an improvement was seen in the survival rate of the mice [Another application of MNs-assisted PTT for superficial tumor treatment was made by Lin et al. by constructing two-dimensional (2D) MXene (nanosheets of Nbthe mice .MNs-assisted PDT involves the encapsulation of non-cytotoxic precursors—such as the mesotetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP) and Nile red (NR), and phthalocyanine or photosensitizers, such as 5-aminolevulinic acid, aluminum (III) phthalocyanine tetrasulfonate chloride (AlPcS4), and methyl aminolevulinate (MAL)—in MNs, which, when stimulated under laser irradiation with a specific wavelength, produce cytotoxic reactive oxygen species  that induce the death of a target cell .PDT with 5-Aminolevulinic acid (ALA) as a photosensitizer has been used to treat basal cell carcinoma, actinic keratoses, and squamous cell carcinoma. To prevent severe skin photosensitivity, topical local administration is more favorable; however, the transdermal penetration of ALA is restricted because of its zwitterionic character and hydrophilicity . To faci® AN- 139 with the hydrophobic dye NR-loaded PLGA NPs to facilitate transdermal penetration. Tissue penetration studies were conducted on excised porcine skin, which showed a high dye concentration (382.63 ng cm−3) in the tissue. This strategy enabled an effective topical administration of hydrophobic agents, hence, overcoming the drawbacks of existing therapies [One drawback of 5-ALA PDT is poor penetration in the skin and, therefore, the inability to treat deep skin neoplasias, such as nodular basal cell carcinoma. A better substitute is the delivery of NR (a model hydrophobic dye and a pre-formed photosensitizer) that enables the penetration of light into the skin when activated at longer wavelengths. Due to its lipophilic nature and high molecular weight, its topical administration is challenging . Donnellherapies .257–264: SIINFEKL)  . MesoporTo produce a strong immunogenic response in the target cells, Duong et al. devised a strategy of using an array of DMNs cocktails. It was prepared with a bioresorbable polypeptide matrix with nanopolyplex, comprising cationic amphiphilic conjugates with ovalbumin-expressing plasmid OVA (pOVA) and immunostimulant-polyinosinic: polycytidylic acid (poly(I:C)) synthesized in the one-pot synthesis method. The pOVA and poly(I:C) were effectively transferred into the intracellular compartments of macrophages and dendritic cells. This study, conducted on 6-week-old BALB/c female mice, showed a more robust antigen-specific antibody response than traditional vaccines. It demonstrated a remarkable enhancement in the reactivation of memory B cells and enhanced the lung clearance of cancer cells .A common problem with DMNs is that they are often fabricated with polymers which are water-soluble and unable to deliver drugs that have poor solubility in water. To solve such an issue, Park and coworkers devised amphiphilic block copolymer NPs that can incorporate poorly water-soluble drugs in MNs fabricated with water-soluble polymers. This system stimulated the immune system via tumor specific antigens and by the growth suppression of cancer cells using Pluronic F127 (F127) NPs loaded with R837 and coated with cancer cell membranes. The prepared NPs were then loaded into rapidly DMNs. These MNs inhibited the cancer cell growth during prophylactic vaccine therapy and immunotherapy. After immunization using MNs on day 21, in comparison to the control group, the tumor volume retarded eight-fold, which demonstrated the therapeutic effect on tumor growth suppression .MNs have also been amalgamated with controlled drug delivery technologies to augment the effectiveness of cancer vaccines. Kim et al. developed a biodegradable MNs patch to deliver a HA-antigenic peptide conjugate for prophylactic immunotherapy in cancer. The MNs were loaded with cytotoxic T-cell epitope peptide conjugated to HA to enable effective antigen delivery to the skin’s immune system. It exhibited an effective response for more than 24 h post administration and demonstrated statistically significant tumor growth inhibition in a B16 melanoma mice model .Gene therapy is performed by inserting the genetic material into the target cell to replace and rectify the faulty genes in the cells. MNs are also used as physical delivery and vector-based technologies  .The conventional methods for treating melanoma have limited therapeutic efficacy due to high toxicity, limited selectivity, and drug resistance. For melanoma, small interfering RNA (siRNA) therapy is gene therapy and has a relatively lower toxicity and high specificity. However, the nature of siRNA is hydrophilic and has a high molecular weight (~13 kDa) with a negative charge, which hinders the penetration of these agents through the stratum corneum . Pan et BRAF gene silencing is the leading therapy for melanoma, even though delivering macromolecular siRNA to the site of the tumor is challenging. Ruan and coworkers developed a siBraf delivery system based upon cell-penetrating peptide octaarginine (R8) nanocomplexes in combination with coated MNs, depending on the siRNA internalization by A375 cells. The study was conducted on a murine melanoma model induced in Balb/c-nude mice. There was ~31% downregulation of BRAF mRNA by R8/siBraf nanocomplexes in vivo, and significant downregulation of BRAF protein expression by coated MNs in vivo . This shMNs have a limited loading capacity, due to which pDNA transfer is challenging. Moreover, pDNA has a minimal transfection efficacy. Cole et al. combined RALA (a cell-penetrating cationic peptide) and pDNA to produce NPs and enhanced the loading capacity of MNs using the lyophilization technique. This new fabrication method enabled the loading of a seven-fold concentration of NP cargo, as well as57 μg of pDNA and 496 μg of RALA peptide per MN. The efficacy of this formulation was checked in a preclinical cervical cancer model, where it evoked a 1.7-fold higher antigen-specific humoral response than those elicited by intramuscular administration. This led to enhanced gene delivery and significant tumor suppression compared to injection delivery .−2 for 5 min). This system was in conjunction with chitosan’s pH sensitivity, providing a layer-dependent controlled drug release. This technique is used for cervical cancer therapy and exhibits a superior cytotoxic effect due to chemotherapy and the PTT combination [In cancer management, the combination of multiple treatment modalities becomes a safe and effective strategy . A combibination .In order to use Cu-PDA NPs for PTT and chemodynamic therapy synergistic treatment against cutaneous melanoma, the NPs had to first be prepared before being implanted into MNs. Cu-PDA NPs might be utilized to absorb the energy from NIR irradiation and transform it into heat. On the B16F10 tumor-bearing mouse, the obtained core-shell MNs showed excellent antitumor activities in vivo . The resChemotherapy and PDT combination have also been used to treat superficial tumors, wherein chemotherapeutic agent cisplatin and photosensitizer IR820 were incorporated into MNs for breast cancer therapy. Fu et al. reported this technique, wherein vinylpyrrolidone-vinyl acetate copolymer (PVPVA) was used as the base material for MNs and incorporated with cisplatin and IR820. It was observed that MNs-mediated cisplatin delivery (chemotherapy) and IR820 delivery (PDT) alone showed only a moderate ratio, i.e., 52.0% and 64.0%, of tumor inhibition, respectively, whereas an MNs-mediated combination delivery of cisplatin and IR820 showed a higher inhibition ratio of the tumor of 90.0%. The synergistic therapy-induced intracellular ROS generation, caspase III activation, and the death of the cell led to profound tumor growth suppression, reduced cell proliferation, and augmented apoptosis. Moreover, the synergistic therapy also showed lower toxicity and better biocompatibility .Transdermal delivery would be a better route for immunotherapy for the skin, as it consists of large amounts of resident antigen-presenting cells. In this case, MNs can facilitate efficient transdermal delivery in the tumor cells microenvironment. Lan et al. devised an MNs patch consisting of aPD-1 and cisplatin (CDDP) loaded into tumor-targeted lipid NPs for precise delivery to cancer tissues. An aPD-1/CDDP@NPs caused cell apoptosis of 24.5%, however only 2.79% cell apoptosis was caused by free CDDP. The cell apoptotic index was 61.4% in the aPD-1 MNs group and 73.2% in the aPD-1/CDDP@NP MNs group, considerably greater than 5.4% in the aPD-1 group. The amalgamation of immuno and chemotherapy has shown to be advantageous for cancer therapy .The p53 tumor suppressor gene therapy has been utilized for the treatment of cancer; however, it is insufficient as a monotherapy due to lack of a sustained treatment and lower therapeutic efficacy. An MNs-assisted combination of gene and PTT was developed to enhance treatment efficacy. An MNs patch fabricated with HA incorporated p53 DNA and IR820 as the photosensitizer. In vitro antitumor efficacy was studied in a BALB/c nude mice model with a subcutaneous tumor, using KB cells . The tumor volume was 832% that of the initial volume in the untreated group; the “IR820 MNs patch + NIR” group consisted of 248% that of the initial volume; and the “p53 DNA/ IR820 MNs patch without NIR” group consisted of 367% that of the initial volume. However, in the “p53 DNA/IR820 MNs patch + NIR” group, the tumor volume was only 40% of the initial volume and was considerably smaller, suggested that synergistic therapy had the highest efficacy in tumor therapy .Another MNs-assisted combination therapy, depicted in MNs for tumor diagnosis can be utilized in two ways: as biosensors that detect various biomarkers associated with a tumor, or as a sampling device for the collection of interstitial fluid, which can help in obtaining information for tumor diagnosis .Nitric oxide (NO) is one of the indicators used to detect cancer progression. The overexpression of the induced nitric oxide synthase (iNOS) enzyme is associated with the early stage of cancer that later promotes angiogenesis for cell growth. Various electrochemical sensors are available for the real-time detection of NO, but the detection of submicromolar concentrations is a challenge. A field-effect transistor (FET) that can detect the submicromolar concentrations of NO utilizes gallium arsenide (GaAS) semiconductor, which is cytotoxic and has a severe risk of carcinogenicity. To address this, D. Keum et al. developed an MNs sensor on the endomicroscope with polycaprolactone (PCL) MNs, and a poly  coating was developed. They were functionalized with hemin molecules for targeting action, as they have a high affinity to NO for the endomicroscopic imaging and biosensing of colon cancer. On testing the microsensor on a mouse-skin melanoma model, the NO levels observed in melanoma tissue were two times higher than in normal tissue. It provided a dual diagnostic system to identify polyp regions in the colon and detect increased NO production in the cancer-specific areas. This system was an effective tool for the rapid diagnosis of cancer. Another cancer biomarker is vascular endothelial growth factor (VEGF). Song et al. developed the first complementary metal-oxide-semiconductor (CMOS)-implemented capacitive biosensor that uses MNs . The factor VEGF on an electrochemical reaction with the peptide receptors on MNs altered the capacitance between two MNs. The biosensor measured both small and large variations in capacitance with a 15 fMrms resolution in the 0.1 to 1000 pM range, therefore, showing high selectivity of VEGF detection. Since the biosensor was MNs-based, the dimensions of the sensor system were considerably decreased in comparison to earlier works .Tyrosine kinase (TYR) is a polyphenol oxidase used for melanin synthesis, and its overexpression and deposition in skin cells cause skin melanoma; hence, it is a melanoma biomarker. TYR screening is, thus, essential for the early diagnosis of melanoma. The existing methods are electrochemical and involve biopsies and are, therefore, invasive. Ciui and coworkers developed a wearable bandage device incorporating MNs biosensors, enabling the non-invasive and rapid detection of melanoma. It consisted of immobilized catechol, which formed benzoquinone detected by amperometry upon reaction with TYR biomarker. The obtained signal from the current was proportional to the level of TYR present .Interstitial skin fluid (ISF) is a source for detecting various biomarkers for cancer diagnosis and progression. A swellable MNs patch made from methacrylated hyaluronic acid (MeHA) was developed and then crosslinked through UV irradiation. As MeHA had an excellent water affinity and structural integrity, no residues were left in the skin. MNs extracted 1.4 ± 0.3 mg (≈1.4 ± 0.3 µL) of ISF in 1 min and 2.3 ± 0.4 mg (≈2.3 ± 0.4 µL) of ISF in 10 min from an experiment conducted on mice. The extracted ISF was recovered by centrifugation; therefore, it was an easy-to-use and minimally invasive device . AnotherFor cancer diagnosis, a potential biomarker was reported to circulate—cell-free nucleic acids (cfNAs); among them, a class of 19–25 nucleotides in length—non-coding RNAs called microRNAs (miRNA)—had great potential as biomarkers for cancer, either individually or in combinations. MNs were developed for the sequence-specific sampling, isolation, and detection of nucleic acid biomarkers from ISF fabricated with an alginate–peptide nucleic acid (PNA) hybrid material. These MNs patches had the capacity of collecting samples of up to 6.5 μL of fluid in 2 min, hence, providing faster sampling kinetics and minimally invasive sample collection compared to reported methods that were either invasive (venous blood) or provided poor quality samples (fingerstick capillary blood) .Theranostic systems amalgamate therapeutics with diagnostics in a single system to enable the real-time monitoring of biomarkers for specific diseases and personalized therapy. This approach was reported by Yuan et al. for MNs-assisted deep brain imaging and in vivo laser ablation to diagnose and treat brain tumors. The ultracompact MNs were prepared by fusing 800 nm optical coherence tomography (OCT) imaging and laser ablation. This device provided histology-like imaging of the brain microstructure, which offered guidance of placement to the image of the ablation probe in the deep brain. In the study conducted on a patient-derived mouse model for the imaging of deep brain cancer, the device demonstrated its potential to detect in vivo lesions in the deep brain with a 1.23 mm imaging depth and 2.4 µm axial resolution in air; minimal invasiveness; quantitative tissue attenuation property (ability to differentiate brain cancer from non-cancer tissues); and high speed. The device was integrated with a 1448 nm continuous-wave laser at 350 mW of power, which performs the function of tissue ablation with an ablation depth of about 600 µm. This approach effectively integrated tissue imaging, tissue ablation, and characterization of the ablation process in vivo and in situ via real-time quantitative monitoring .2O3) microparticles, guiding them to the NPs inside tumor for activation. The effectiveness of this treatment was tested on the glioblastoma mouse tumor model, where a time-dependent alteration in the tumor model was assessed via T2-weighted MR imaging. It was observed that the negative control group showed a 520% change in mean tumor volume, and the therapy group showed a −12% change after two weeks. This affected the survival rate where the therapy group had a 42-day median survival time, which was longer than the negative control group by 24 days [Another theranostic approach that was reported by Lee et al. for brain tumor diagnosis and treatment via MNs involved the delivery of NPs and high-energy photons to the tumor site using MNs on a bioelectronics system . In this 24 days .Another interesting study by Gittard et al. highlighted quantum dots (QD) applications, which were nanostructured fluorescent semiconductor materials with a diameter of 2–10 nm as theranostic agents, as they had an ability to act as fluorophores and vehicles for drug delivery. The problem with these was that QDs  showed poor penetration, as they accumulated in the uppermost epidermal region. To facilitate the transdermal delivery of QD, hollow MNs devices were fabricated using a two-photon polymerization process from an acrylic polymer. Therefore, by using MNs, the QD solution was successfully delivered to the dermis and deep epidermis. This enabled the simultaneous multiphoton microscopic imaging of the QD and drug delivery, thus, providing theranostic treatment .The above-mentioned case studies are summarized in The majority of pre-clinical studies on the application of MNs in tumor therapy were found to be operational in multiple outlooks, but only a few have been successful in human subjects. The clinical trial currently recruiting participants to study the efficacy or safety of DOX MNs for basal cell carcinoma will be conducted in two parts. Part 1 will determine the factors that affect the rate and magnitude of the dissolution of MNs array, such as duration of exposure, age, anatomic location, and the cruciality of proper application technique. Part II will include a randomized study. The safety and efficacy of 50 µg, 100 µg, and 200 µg doses of DOX in a tip-loaded DMNs array will be evaluated via a biopsy of the treated subject . AnotherIn recent years, there has been great progress in the evolution of MNs as a transdermal drug delivery system . Table 4MNs are an emerging diagnostic and drug delivery system with unique features such as rapid drug delivery, painless administration, the prevention of injuries or infections due to hypodermic needle-based deliveries, bypass first-pass metabolism, localized therapy, enhanced bioavailability by transdermal administration, and a very compact and easy-to-use system with enhanced patient compliance . These aDespite all these successful applications and investigations, substantial challenges still need to be addressed before their use in real life. The short-term applications may not cause significant safety-related problems; however, long-term usage, especially in the case of cancer therapy, might be associated with concerns such as skin erythema—with a prompt recovery within 0.5–2 h —and in t


Rab8A has been reported as an oncogenic gene in breast and cervical cancer. However, the function and molecular mechanism of Rab8A in esophagus cancer has not been reported. β-catenin signaling pathway was determined by western blotting assay. M435-1279 was used to suppress the Wnt/β-catenin signaling pathway.  Rab8A expression was detected by qPCR and western blotting assays, small interference RNA (siRNA) was applied to reduce Rab8A expression, and the biological behaviors of esophagus cancer cells were estimated by cell counting kit-8, colony formation, and transwell and western blotting assays. The transcriptional factor of Rab8A was verified by dual-luciferase assay and chromatin immunoprecipitation assay. The protein expression of key genes in the Wnt/β-catenin signaling pathway were strengthened by PEA3 overexpression, which were reduced by si-Rab8A transfection. M435-1279 treatment significantly reduced the viability and colony formation of esophagus cancer cells.  A significant increase of Rab8A expression has been found in esophagus cancer cells. Knockdown of Rab8A suppressed the viability, colony formation, migration, and invasion abilities of esophagus cancer cells and induced apoptosis. PEA3 transcriptionally regulated Rab8A expression and promoted the viability, colony formation, migration, and invasion abilities of esophagus cancer cells and blocked apoptosis, which were diminished by si-Rab8A transfection. Additionally, the expression levels of key genes related to the Wnt/β-catenin pathway. The above results indicated that Rab8A may be considered as a promising biomarker for diagnosis and precision treatment in esophagus cancer. The data showed that Rab8A was transcriptionally regulated by PEA3 and promoted the malignant behaviors of esophagus cancer cells by activating the Wnt/ Esophagus cancer is still an integral cause of cancer-related death and has presented a drastic increase of greater than 6-fold in incidence rates worldwide . In ChinRab proteins belong to GTPases of the Ras superfamily, and functional damage of Rab8 proteins has been reported to be involved in tumorigenesis, such as Rab1, Rab2A, and Rab8 . Rab8, aThe concept of transcriptional control was put forward half a century ago in bacterial systems . Over thThis study was performed to detect the function of Rab8A in esophagus cancer. We estimated the effect of Rab8A depletion on cell proliferation, apoptosis, invasion, and migration. It is of great significance to estimate the molecular pathology to clarify the pathogenesis and disease progression of esophagus cancer patients.A total of 7-paired esophagus cancer specimens and adjacent normal control tissues were collected from esophagus cancer patients undergoing surgery at the First Affiliated Hospital of Jinzhou Medical University. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation and with the Helsinki Declaration of 1964 and later versions. Informed consent or substitute for it was obtained from all patients for being included in the study.2. siRab8A-1 (CGGAACTGGATTCGCAACATT) and siRab8A-2 (CGACTATGGAATCAAGTTCAT) were synthesized by Sangon  and transfected into TE-1 and KYSE150 cells to reduce Rab8A expression. si-PEA3 (GCTCCGATACTATTATGAGAA) and pcDNA3.1-PEA3 were purchased from Sangon and used for reducing or increasing PEA3 expression. M435-1279 was applied to inhibit the Wnt/β-catenin signaling pathway.Cells  were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and placed in a humidified incubator at 37°C in 5% COΔΔCt- method. Three independent experiments were carried out to determine Rab8A expression. GAPDH was regarded as the internal reference. The sequences of primers were listed as follows: Rab8A, forward: 5′-CTACGACATCACCAACGAGAAG-3′, Rab8A, reverse: 5′-CATCACACTTGTTCCCGAGTAT-3′; GAPDH, forward: 5′-GCACCGTCAAGGCTGAGAAC-3′, GAPDH, reverse: 5′-TGGTGAAGACGCCAGTGGA-3′.The whole RNA was extracted by using TRIzol kit  and reversely transcribed into cDNA by using the cDNA synthesis kit . SYBR Green qPCR mix  was applied to measure the relative expression of Rab8A in the ABI 7900 system. The expression of Rab8A was calculated by using the 2β-catenin , CyclinD1 , Tubulin , and GAPDH ) incubation. Subsequently, the PVDF membrane was blotted with the corresponding secondary antibody for 2 h. Last, the bands were visualized by an enhanced chemiluminescence detection kit. The relative protein expression was normalized to GAPDH or Tubulin.RIPA buffer was applied to extract the total protein from cell lysates, and protein concentrations were quantified by a BCA method. Next, the proteins were isolated by 12% SDS-PAGE gel, and then, the proteins were transferred from gels to a PVDF membrane. With sealing in 5% nonfat milk at 25°C for 1 h, the PVDF membrane was followed with the primary antibodies , Bcl-2 , BAK , caspase-3 , PEA3 , c-MYC , 3 cells/well) were implanted into 96-well plates and cultivated to about 80% confluence. After transfection with siRab8A, pcDNA3.1-PEA3, or pcDNA3.1-PEA3+siRab8A, cell counting kit-8 (CCK-8) reagent (10 μL) was put into per well and each well was cultured at 37°C incubator for 90 min. The optical density (OD) value of KYSE150 and TE-1 cells in per well was measured at 450 nm wavelength by a microplate reader.KYSE150 and TE-1 cells . After cultivation under standard condition for 2 weeks, the colonies were fixed and stained. Finally, cell colonies were counted and applied for comparisons of colony formation ability.μL) including 2 × 104 cells was added to the upper chamber of a 24-well transwell plate with 8 μm pore polycarbonate filters . In the lower chamber, 500 μL of DMEM including 10% FBS was added. After incubation for 24 h, the medium was removed from the upper chamber and cells in each group were erased by a cotton swab. The cells that had migrated to the lower chamber were fixed and stained. Migrating cells were captured under a microscope in five random fields of view. The steps of invasion assay were consistent with the migration assay, except that the upper chamber was precoated with Matrigel. Three independent experiments were performed.Cell suspension . Briefly, the treated cells were collected and lysed. Antibody against Rab8A and IgG was applied for immunoprecipitation. PCR amplification of the precipitated DNA was performed.https://genome.ucsc.edu/) and PROMO . To determine the activity of the Rab8A promoter in the presence of PEA3 overexpression, luciferase assay was performed. First, based on the predicted transcription factor binding sites, wild-type Rab8A (WT-Rab8A), mutant-1 Rab8A (Mut1-Rab8A), and Mut1-Rab8A were amplified and transfected into PGL3 vector. Subsequently, cells were cotransfected with PGL3-Rab8A promoter and PEA3 overexpression vector or the corresponding control vector using Lipofectamine 2000. A total of 48 h following transfection, the relative luciferase activity was measured as normalized to Renilla luciferase activity using the Dual-Luciferase® Reporter Assay System (Promega Corporation).The potential transcription factor binding sites between Rab8A and PEA3 were generated by online software UCSC (t-test (two groups) or one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test (three or more groups). P value less than 0.05 was deemed as a statistically significant difference.At least three independent experiments were carried out in each group. All experimental data were presented as mean ± standard deviation. The differences were processed by Student's First, an obvious increase of Rab8A was observed in esophagus cancer cells, including KYSE150, TE-1, and Eca109 cells Figures . To furtNext, the OD values, growth ability, apoptosis, migration, and invasion in KYSE150 and TE-1 cells were detected by CCK-8, colony formation, western blotting, and transwell assays, respectively. Results from the CCK-8 assay showed that the OD values in KYSE150 and TE-1 cells were obviously reduced after siRab8A-1 or siRab8A-2 transfection Figures . MoreoveTo further explore the molecular mechanism of Rab8A in esophagus cancer cells, the transcriptional factor of Rab8A was captured. PEA3 was discovered to have two binding sites in the promoter region of Rab8A by searching UCSC and PROMO website. Then, luciferase reporter vectors including WT or mutated PEA3 binding sequences (Mut1 and Mut2) were established and cotransfected with corresponding control or PEA3 overexpression (OE) vector into cells. The data showed that PEA3-OE treatment increased luciferase activity in the WT group whereas the luciferase activity in the Mut1 or Mut2 group has no obvious change . Data frTo further investigate the interaction between Rab8A and PEA3, con, PEA3-OE, or PEA3-OE+si-Rab8A was, respectively, transfected into KYSE150 and TE-1 cells. Data from CCK-8 and colony formation assays showed that upregulation of PEA3 elevated the growth ability of KYSE150 and TE-1 cells Figures . Howeverβ-catenin signaling pathway performs an important role for the initiation and development of esophagus cancer [β-catenin signaling pathway is overactivated, the patterns of target genes c-MYC and CyclinD1 related to cell proliferation and migration are increased, which promotes cell proliferation and migration, resulting in malignant transformation of cells [β-catenin, c-MYC, and CyclinD1 were significantly increased when PEA3 was overexpressed  was applied. Results from Figures β-catenin, c-MYC, and CyclinD1, which was more significant when siRab8A and M435-1279 were treated together. Additionally, functional experiments showed that the OD values and clone number of KYSE150 and TE-1 cells were obviously reduced after siRab8A or M435-1279 treatment, especially when siRab8A and M435-1279 were cotreated (Figures β-catenin signaling pathway.A growing number of evidence revealed that the Wnt/s cancer –13. Onceof cells . In our  Figures . Moreove Figures . To furt Figures . Based oThe tumor-promoting effect of Rab8A has been involved in various physiological activities and signaling pathways . In thisThe PEA3 subfamily, as a subgroup of the E26 transformation-specific family, includes ETV1, ETV4, and ETV5. Functionally, the PEA3 subfamily is related to motor coordination, axon guidance, metabolism, neuron development, hormonal regulation, fertility, and tumorigenesis . PEA3, aβ-catenin signaling pathway is a conserved signaling axis taking part in various physiological processes including proliferation, apoptosis, differentiation, invasion, migration, and tissue homeostasis [β-catenin signaling pathway promotes tumor stem cell renewal, cell proliferation, and differentiation, thus performing an important role in tumorigenesis and therapeutic response [β-catenin signaling pathway, aberrant regulation of β-catenin, which is the crucial component of the Wnt/β-catenin signaling pathway, resulted in early events in carcinogenesis [β-catenin signaling pathway [β-catenin, and CyclinD1, while siRab8A transfection diminished the effect of PEA3 overexpression and suppressed the protein levels of c-MYC, β-catenin, and CyclinD1. Additionally, the inhibitor of the Wnt/β-catenin signaling pathway was applied. We discovered that suppression of the Wnt/β-catenin signaling pathway could inhibit the growth ability of esophagus cancer cells.The Wnt/eostasis , 25. Abnresponse . In the ogenesis , 28. Addogenesis ; Rab8A aogenesis ; Rab8A h pathway . In our Several deficiencies in our study should be pointed out. First, our results were achieved only based on cell experiments; animal experiments will be needed to validate the results. Additionally, more molecular mechanism about Rab8A in esophagus cancer should be revealed.β-catenin signaling pathway was positively regulated by Rab8A/PEA3 in esophagus cancer cells. The above results indicated that Rab8A, transcriptionally modulated by PEA3, promoted the growth and metastasis abilities of esophagus cancer cells by activating Wnt/β-catenin signaling.Taken together, our results showed that knockdown of Rab8A could diminish the growth, invasion, and migration abilities as well as induce the apoptotic ability. Moreover, Rab8A was transcriptionally regulated by PEA3 and interacted with PEA3 to regulate the malignant behavior of esophagus cancer cells. The Wnt/


To investigate lymphoid enhancer factor 1 (LEF-1) protein expression in medulloblastomas (MBs) and its correlation with molecular grouping of MBs.Expressions of LEF-1 and β-catenin were detected by immunohistochemistry, and molecular grouping was performed based on the NanoString and sequencing techniques for 30 MBs.P < 0.0001). We also found focal nuclear β-catenin expression ( less than 1% of tumor cells) in 5 MBs. LEF-1 positivity was significantly correlated nuclear β-catenin expression (p < 0.001).By genetic defining, 3 MBs were WNT-activated, 11 were SHH-activated, 3 were in Group 3 and 13 in Group 4 respectively. Nuclear LEF-1 staining was found in 8 MBs using immunohistochemical method. Three out of 8 showed diffuse and strong nuclear LEF-1 staining which were proved to be WNT-activated genetically, while the other 5 MBs with focal staining were SHH-activated genetically. The expression of LEF-1 protein was significantly correlated with genetically defined WNT-activated MBs (Immunohistochemical staining of LEF-1 can be used as a supplement for β-catenin to diagnosis WNT-activated Medulloblastomas, when β-catenin is difficult to recognize for its cytoplasm/membrane staining background. Diffuse nuclear staining of LEF-1 indicates WNT-activated MB. Medulloblastoma (MB) is the most common malignant embryonal tumor of the central nervous system in childhood . It presAccording to the molecular characteristics of the fifth edition of the WHO Classification of Tumors of the Central Nervous System (CNS), published in 2021, medulloblastomas are defined into four groups: WNT-activated, SHH-activated and TP53-wildtype,SHH-activated and TP53-mutant and non-WNT/non-SHH . WNT-actCTNNB1gene mutation (more than 90%) is the main genetic variations of the WNT-activated MBs which results in the amino acid residues change at the phosphorylation site of β-catenin . TherefoA total of 30 formalin-fixed paraffin-embedded (FFPE) medulloblastomas samples were collected from the Department of Pathology, Qilu Hospital of Shandong University of China from 2018 to 2020. The H&E stained slides were reviewed independently by two pathologists blind to the clinical information to confirm the pathological diagnosis, and further defined the tumors to four histological subgroups as classic, desmoplastic/nodular, anaplastic/large cell or medulloblastoma with extensive nodularity (MBEN) according to the WHO 2016 classification of tumors of Central Nervous System. All protocols follow the ethical guidelines of the Helsinki Declaration and were approved by Shandong University Research Ethics Committee.Thirty formalin-fixed paraffin-embedded (FFPE) from selected MB tumor samples fixed in 10% neutral buffered formalin were retrieved for immunohistochemistry. Samples from case of desmoid-type fifibromatosis confirmed by CTNNB1 3 exon mutation and small lymphocytic lymphoma as positive control for β-catenin and LEF-1 respectively. After deparaffinization, sides were subjected to antigen retrieval by autoclave in 0.01 M EDTA buffer (pH 8.5) for 3 min 15 s, followed by incubation in 3% H2O2 for 10 min to quench endogenous peroxidase. Sections were incubated overnight at 4 °C with monoclonal antibody LEF-1  and β-catenin . Substitution of the primary antibody with PBS was served as a negative control. Slides were washed with PBS for 3 times and incubated with secondary antibody  at room temperature for 0.5 h. Staining was carried out by incubating the slides in 3, 3′-diaminobenzidine (DAB), followed by counterstained with hematoxylin, dehydrated in gradient ethanol and xylene. LEF-1 was localized in the nuclei of tumor cells. Its expression level was analyzed through assessing the percentage of stained tumor cells as follows: sections with no staining cells were regarded as negative, 1–50% positive cells as partial positive and 51–100% positive cells as diffuse positive. Any nuclear staining of β-catenin was defined as positive regardless of the percentage of stained tumor cells.Tumor DNA was purified from 5–10 pieces of 5 μm slides from FFPE blocks using QIAamp DNA FFPE Tissue Kit (Qiagen) following the manufacturer´s instruction. DNA was quantified with the Qubit Fluorometer V2.0 using the dsDNA Assay Kit (Thermo Scientific). DNA samples with a total amount greater than 100 ng were selected to perform whole exome sequencing.Whole genome DNA-sequencing libraries were constructed using the Rapid DNA Lib Prep Kit for Illuminafollowing the manufacturer’s guidelines. In brief, Genomic DNA was randomly fragmented to segments of 350 to 500 bp by Covaris sonication, end-repaired, A-tailed, adaptor ligated and PCR amplified followed by a quality control step using Agilent 4200 TapeStation System. Then, qualified samples were hybridized to KAPA HyperExome probes(Roche) to enrich human exome with KAPA HyperCapture Reagent kit (Roche). 150 base paired-end sequencing was performed on the Illumina Novaseq 6000.Sequenced Reads were aligned to the Human Genome Reference Consortium build 37 (GRCh37) using BWA-mem v0.7.16-r1188 with default settings. Then, SAMtools v1.9 and Picard tools v1.125 (Broad Institute) were used to sort the reads by coordinates and to mark the duplicates. Reads with a phred-scaled base quality below 20 were removed. The CNVkit v0.9.6 was used to estimate the status of copy number variation with default parameters. Varscan2 v2.4.1 was used to call SNVs and Indels. The filter procedures were performed as recommended and filter thresholds were set as default to identify high quality SNVs and indels. The vcf calls were then annotated using multiple publicly available tracks such as 1000 Genome variants, single nucleotide polymorphism database (dbSNP), COSMIC, Clinvar and other elements. The functional effect of the mutations was annotated using Annovar. Owing to the poor coverage of the TERT promoter region, TERT promoter mutation was tested by Sanger sequencing.Machine learning algorithm was utilized to build the MB classification model. Training sets derived from the combination of ICGC medulloblastoma cohort and own in-house data. Random Forest algorithm was used to build a probability model to calculate the probability of a given medulloblastoma sample belonging to one of 4 subtypes. Features used in this process included somatic mutation and copy number alteration (CNA). the number of chromosome level CNV. Besides, clinical features such as gender, age and histological defined subtype were also included in the classification model.Molecular subtyping based on the NanoString was performed for 5 MBs. Total RNA was purified from FFPE blocks using a RNeasy FFPE Kit (Qiagen) following the manufacturer´s instruction. RNA concentration was measured, and RNA integrity assessed using the Agilent 2100 bioanalyzer. Reporter Coreset and Capture ProbeSet were overnight hybridized to the total RNA in thermocycler (Applied Biosystem). Post Hybridization processing and data collection for gene expression was done using nCounter Sprint Profiler system, as per manufacturer’s protocol.P-value < 0.05 was considered statistically significant.Statistical analyses were performed using the SPSS for Windows version 19.0 . The relationships between LEF-1 expression and clinicopathological parameters were analyzed using Pearson Chi-square test and Fisher’s exact test. Two experienced pathologists confirmed the diagnosis of MB for all the enrolled cases with a group of the routine antibodies at least include Syn, CgA, NeuN, GFAP, INI-1, and Ki67 for possible differential diagnosis. Clinicopathologic characteristics of the MBs patients were summarized in Table Using the clone EP310 antibody, LEF-1 was detected in 8 of 30 26.7%) MBs .Detecting the protein expression using the clone UMAB15 antibody, All the MBs cases showed tumor cell membrane/cytoplasm expression of β-catenin. Only four MBs showed focal scattered nuclear staining of β-catenin with percentage of stained tumor cell less than 1% Fig. . Three oIndividualized treatment is the trend of therapeutic regime for patients with medulloblastoma. Previous studies have proved that the four molecular subgroups of medulloblastoma have significantly different characteristics regarding to clinical metastases and prognosis .DifferenWNT-activated medulloblastomas accounts for about 10% of all MBs. It mainly occurs in older children and adolescents with average age of 10 years old. Histologically, classic variants are most in this group. The 5-year overall survival rate is more than 90%. WNT-activated MBs frequently have deletions of chromosome 6, and more than 90% have CTNNB1 gene mutations. [Nuclear β-catenin immunohistochemical stain can be useful in the diagnosis of many tumors including desmoid-type fifibromatosis , hepatocAn early study showed two patterns of immunohistochemical expression of β-catenin in a series of 72 paediatric medulloblastomas, β-catenin extensive nuclear staining (> 50% of the tumor cells) and focal nuclear staining (< 10%). However in their study, only the extensive β-catenin nucleopositivity cases harboured CTNNB1 mutations . In our Another possible explanation might be the different clone of anti‐β-catenin antibody we use. But the antibody we used in our study showed excellence sensitivity in tumors of desmoid‐type fibromatosis and solid pseudopapillary neoplasm of the pancreas in our clinical pathology practice. Yamada’s research showed the sensitivity (ranging from 54 to 96%) and specificity (ranging from 62 to 98%) of nuclear β-catenin in the diagnosis of desmoid‐type fibromatosis were different among three anti‐β-catenin antibodies commonly used. Their work also showed comparable results with immunohistochemistry of β-catenin and LEF1 (sensitivity of 88% and specificity of 76%) . The facLymphoid enhancer-factor 1 (LEF-1), a transcription factor that binds DNA in a sequence-specific manner with β-catenin, but has not been fully studied in MBs. Activation of WNT / β-catenin signaling pathway results in reduced degradation of β-catenin, which leads to increased cytoplasmic β-catenin enter the nucleus, and combined with the nuclear transcription factor TCF/LEF-1 to form a transcription complex and regulate the transcription of downstream target genes, such as C-Myc and Cyclin D1 , 32. In In summary, this is the first report that LEF-1 immunohistochemical stain can be used as a more sensitive marker than β-catenin to predict WNT-activated MBs. Hopefully, these data can make contributions towards distinguishing future patients with WNT-activated MBs which need improved treatment scheme and reduce the treatment sequelae. Beyond clinical applicability, mechanistic questions about the functional role of LEF-1 expression needs further investigation in determining the outcome of MBs.