Endoscopy is the mainstay of inflammatory bowel disease (IBD) evaluation and the pillar of colorectal cancer surveillance. Endoscopic equipment, both hardware and software, are advancing at an incredible pace. Virtual chromoendoscopy is now widely available, allowing the detection of subtle inflammatory changes, thus reducing the gap between endoscopic and histologic assessment. The progress in the field of artificial intelligence (AI) has been remarkable, and numerous applications are now in an advanced stage of development. Computer-aided diagnosis (CAD) systems are likely to reshape most of the evaluations that are now prerogative of human endoscopists. Furthermore, sophisticated tools such as endocytoscopy and probe-based confocal laser endomicroscopy (pCLE) are enhancing our assessment of inflammation and dysplasia. Finally, pCLE combined with molecular labeling could pave the way to a new paradigm of personalized medicine. This review aims to summarize the main changes that occurred in the field of IBD endoscopy and to explore the most promising novelties. Inflammatory bowel diseases (IBD) are a group of immune-mediated conditions of the gastrointestinal tract, associated with significant morbidity and reduction in quality of life. The two main types of IBD are Crohn’s disease (CD) and ulcerative colitis (UC). The role of endos in the management of IBD has gained importance over the past two decades. Coupled with histology, endoscopy is fundamental for diagnosis  and for In the follow up of IBD, symptoms are not reliable indicators of the underlying inflammatory state. Several studies show a mismatch between the clinical presentation and objective measures of inflammation, such as fecal calprotectin and endoscopic activity ,3. The pTechnological advances are reshaping the role of endoscopy and expanding it into other fields such as precision medicine. The aim of this review is to present the applications of endoscopy in the management of IBD and evaluate the latest advances and future developments .A literature review was independently performed by three authors  in the PubMed/MEDLINE databases up to December 2021 using the following search terms: “endoscopy”, “virtual chromoendoscopy”, “capsule endoscopy”, “artificial intelligence”, “machine learning”, “molecular endoscopy”, “molecular imaging” individually or in combination with “IBD”, “inflammatory bowel disease(s)”, “ulcerative colitis” or “Crohn’s disease”. Additionally, the abstracts presented at the ECCO congress, DDW and ESGE in 2021 were manually searched. The search focused on full-text papers published in English. Abstracts were selected when relevant. No publication date restrictions were imposed. Finally, articles were included in this review on the basis of their relevance, while additional publications were identified through their reference lists.The two main endoscopic scores validated for CD are the Crohn’s Disease Index of Severity (CDEIS) and the Simple Endoscopic Score for Crohn’s Disease (SES-CD)  21,22].,22.21,22In addition, as CD patients often undergo ileo-colonic resection, an ad hoc endoscopy score, Rutgeerts’ score, is used to predict the recurrence of disease  23]..23].Overall, the grading of CD activity is more challenging compared with UC for a number of reasons. The mucosal damage is usually discontinuous, hence the need to consider the location in the score, and transmural, whereas endoscopy is inherently limited to the mucosal surface. Moreover, different disease phenotypes exist: inflammatory, stenotic and fistulizing.Numerous scores have been proposed to assess UC endoscopic activity . The MayPatients with IBD have an increased risk of developing colorectal cancer (CRC). Consistently with the inflammation–dysplasia–cancer sequence, the risk is proportional to the duration and extension of disease . In partOver the past decade, the major changes in surveillance involved techniques rather than time intervals. Conventional endoscopic evaluation, including surveillance endoscopy, used to be performed in white-light (WL), meaning that the image is acquired after the mucosa is illuminated by a white light source. The picture is then digitally processed and presented on a screen . The resWhether to prefer dye or dye-less  chromoendoscopy, current evidence remains not conclusive. A recent meta-analysis of 11 randomized controlled trials  with a total of 1328 patients, comparing dye and virtual chromoendoscopy  did not find significant differences in detection of dysplasia . In lighFurthermore, the role of random biopsies has been questioned after the introduction of HD instruments, since dysplasia detected on random biopsies represent a small minority, when compared to dysplasia found in targeted biopsies . Scientip = 0.14). These results suggest that HD itself might overcome the need for chromoendoscopy. In addition, the trial confirmed the negligible diagnostic gain of quadratic random biopsies  [Finally, a multicenter multi operator randomized controlled trial (VIRTUOSO) compared the performance of HD VCE and HD WL for the detection of colonic neoplasia in 188 IBD patients at risk of cancer , not finding any significant difference between the two techniques  .Dye chromoendoscopy consists in the application of contrast dye to the mucosa via the endoscope channel . Dyes usFurther technical advancements led to the development of dye-less chromoendoscopy . This teVideo capsule (CE) can evaluate the whole gastrointestinal tract including the small bowels, beyond the reach of conventional endoscopy, with higher sensitivity for small findings compared to bowel ultrasound, entero-CT-scan (CTE) and entero-magnetic resonance (MRE) ,17 Figu. SuspectA prospective, blinded study of 93 suspected CD patients evaluated the diagnostic accuracy of CE, magnetic resonance enterography and CT enterography as compared to the gold standard, ileocolonoscopy . The senThe diagnosis of postsurgical CD recurrence is another application of CE in IBD. In a study by Beltran et al., CE was superior to colonoscopy for the detection of CD recurrence in the neoileum and the rest of the bowels, as well as better tolerated . FurtherThe ICCE consensus defines “capsule retention” as the capsule remaining in the gastrointestinal tract more than two weeks or when endoscopic, surgical or medical intervention is required to remove it . This evReviewing CE recordings is time-consuming and challenging. An experienced endoscopist needs to watch hours of videos searching for findings visible only in a few seconds, and hence easily missed. In fact, the main limitation of capsule endoscopy is the miss rate for solitary small bowel lesions (11%) . To speeMolecular imaging endoscopy aims to identify the presence of specific molecular targets in the gastrointestinal tract  Figure . This rep < 0.001). Furthermore, in patients with high mTNF expression, the response to treatment was sustained over a longer period of time and associated with mucosal healing at follow-up endoscopy.In a phase I trial, Atreya et al. predicted clinical response to anti-TNF therapy in CD through molecular imaging . With co4β7 integrin were applied topically, and then ex vivo confocal microscopy was used to detect them and estimate integrin expression. No response to vedolizumab was observed in the three patients that had no α4β7 integrin mucosal expression, while the other two patients did show clinical response to treatment. In a similar fashion, Iacucci et al. showed how ex vivo molecular imaging with CLE could predict response to anti-TNF therapy in 29 patients with CD and UC [In another pilot study by Rath et al., the response to vedolizumab in five CD patients with previous failure to anti-TNF was predicted in a similar way . FluoresD and UC . AltogetApplications of molecular imaging are not limited to the prediction of treatment response. Distinguishing CD and UC can be challenging, and as mentioned before, up to 10% of IBD cases do not reach a conclusive diagnosis . A studyFinally, molecular imaging may have a role also in the context of dysplasia and cancer detection. Recently, in a mouse model of colitis-associated cancer, Mitsunaga et al. studied the expression of gamma-glutamiltranspeptidase (GGT), an enzyme associated with malignancy: Through an enzymatically activatable probe (gGlu-HMRG) applied topically, after just 5 min, it was possible to detect fluorescent areas, which all proved to harbor dysplasia or cancer on subsequent pathological examination . When fuThe endocytoscope  is an optical microscope endoscope that, in addition to conventional WL and VCE, allows real-time ultra-magnified observation . Endocyt®) has been successfully developed to support the distinction of neoplastic from non-neoplastic polyps visualized in endocytoscopy [Endocytoscopy also holds promise in the field of colorectal lesions. Cellular-level magnification can help distinguish adenomas from malignant lesions and guide management . A CAD sytoscopy ,73. In tytoscopy . It is r®) showed a strong correlation with both endoscopic (MES r = 0.76 and UCEIS r = 0.74) as well as histologic scores (RHI r = 0.74) [®, a larger multicenter study (PROCEED) is ongoing.Artificial intelligence (AI) and computer-assisted diagnosis (CAD) systems are increasingly implemented in endoscopy, and IBD endoscopy is no exception. Over the last years, different groups have developed machine learning algorithms to assess disease activity in frames and videos of colonoscopies . In a pi = 0.74) . To valiTakenaka et al. used big data to develop an endoscopy AI for UC disease assessment. They trained and validated a deep neural network (DNN) algorithm using 40,758 colonoscopy images and 6885 biopsy results from 2012 UC patients. In each given frame, the algorithm predicted endoscopic remission with 93% sensitivity and 88% specificity compared to the endoscopist . The DNNAnother promising machine learning algorithm, recently presented by Byrne and colleagues, leveraged a large number of endoscopy frames (>33.000) to improve the efficiency and accuracy of the scoring process. This model predicted both MES and UCEIS, with a mean absolute error between machine and human of 0.30 and 0.72 for MES and UCEIS, respectively .The performance of computers does not fluctuate with tiredness or stress, thus improving human reliability and making up for endoscopist distraction. More importantly, AI results can be accurately reproduced. This represents a unique opportunity to standardize the assessment of disease activity, which suffers from high interobserver variability. For example, central reading in clinical trials could be simplified and expedited using the same machine learning algorithm .Finally, as CAD systems evolve to detect ever more subtle changes, the gap between endoscopy and histology narrows. Because endoscopy can assess larger areas of mucosa, in the future, enhanced endoscopy might reduce the need for biopsies. In this sense, AI models have also been successfully developed to interpret ultra-magnified imaging from endocytoscopy. In a recent study of 145 UC patients in clinical remission, Maeda and colleagues, accurately stratified the risk of relapse through an AI system developed on endocytoscopy images .In IBD, as interest shifts towards objective measures of disease activity, the importance of endoscopy grows. At the same time, numerous technological advances are reducing the gap with histology, increasingly considered the new reference standard. VCE, CLE and endocytoscopy converge towards a deeper characterization of the mucosa, paving the way to a more comprehensive endoscopic assessment. The clinical implication of a deeper disease characterization is becoming ever more relevant given the expansion of the therapeutic armamentarium. Until recently, the lack of medical options limited the change of treatment, thus confining stringent therapeutic targets, such as histologic remission, to mainly prognostic factors. At present, with a growing number of approved biologics and advanced small molecules for both CD and UC, physicians have more room for treatment adjustments and can afford a lower threshold for switching medications. Modern endoscopy will play a crucial role in guiding this process. Standardization is among the main challenges that lay ahead. Advanced endoscopy technologies have great potential though their application remain limited by high inter-observer variability. It is realistic that CAD systems will soon standardize complex evaluations such as grading inflammation. AI is also contributing to the simplification of complex image analysis such as endocytoscopy and pCLE, partially overcoming their long learning curve. More broadly, AI systems reliably supporting clinicians in the interpretation of findings could reduce the need for training, serving as supervision. Innovation will also affect dysplasia detection in IBD. Computer systems that detect adenomatous polyps are widely available, and it is reasonable that similar tools will soon be developed for IBD-associated dysplasia as well.Despite the advances, patient acceptance remains one of the main constraints of endoscopy. Video capsule is more tolerated than conventional endoscopy and could potentially be performed out of the hospital without medical supervision. AI software are dramatically reducing the time needed to review capsule videos while enhancing the detection of findings. Such advances might shift the balance in favor of a more common use of CE, particularly in settings of limited access to medical facilities, such as in the recent pandemic.Finally, pioneering studies on molecular endoscopy are paving the way to personalized medicine. As the number and cost of available medication increases, tools to predict response and guide choice of treatment are in great need. Assessing mucosal expression of biomarkers represents a promising approach not only to select treatment but more broadly to characterize tissue. In a similar way, molecular characterization has improved our understanding of cancer and its treatment.Innovations in AI for disease assessment and surveillance are expected to impact a large number of IBD patients in the next years, improving quality of care. We foresee a widespread adoption of CAD systems in capsule endoscopy reviewing, inflammation assessment and, soon, cancer surveillance. More sophisticated technologies such as endocytoscope and pCLE are likely to gain ground although, their adoption out of tertiary centers at present remains limited by high costs and need for special expertise. Finally, molecular imaging, while extremely promising from a theoretical perspective, needs more validation and cost effectiveness analysis before being proposed in clinical practice.New technologies also carry new challenges: first of all, costs. Medical care is already expensive, and access is often limited by budget constraints. Thus, new equipment could potentially widen the gap between resource-rich and poor settings. Secondly, machine learning algorithms are dependent on the population they are trained on, which tends to over-represent white Westerners to the detriment of other races and ethnicities. From a regulatory perspective, as interest in big data increases, so do privacy concerns. Finally, when computer algorithms will gain a more active role in medical decision making, legal implications will arise, requiring an update of the legislation.In conclusion, IBD endoscopy is evolving to encompass new aspects of disease assessment. The coming years are likely to witness a remarkable upgrade in the role of endoscopy in the care of UC and CD.


Drosophila model is used to investigate the effects of diet on physiology as well as the effects of genetic pathways, neural systems and environment on feeding behavior. We previously showed that Blue 1 works well as a dye tracer to track consumption of agar-based media in Drosophila in a method called Con-Ex. Here, we describe Orange 4 as a novel dye for use in Con-Ex studies that expands the utility of this method. Con-Ex experiments using Orange 4 detect the predicted effects of starvation, mating status, strain, and sex on feeding behavior in flies. Orange 4 is consumed and excreted into vials linearly with time in Con-Ex experiments, the number of replicates required to detect differences between groups when using Orange 4 is comparable to that for Blue 1, and excretion of the dye reflects the volume of consumed dye. In food preference studies using Orange 4 and Blue 1 as a dye pair, flies decreased their intake of food laced with the aversive tastants caffeine and NaCl as determined using Con-Ex or a more recently described modification called EX-Q. Our results indicate that Orange 4 is suitable for Con-Ex experiments, has comparable utility to Blue 1 in Con-Ex studies, and can be paired with Blue 1 to assess food preference via both Con-Ex and EX-Q.The  Drosophila (fruit fly) model has become a leading experimental platform for investigating genetic and environmental influences on feeding behavior as well as the impact of diet on many physiological and pathophysiological responses. For example, the fly model has been used to examine protein valuation1, compensatory feeding2, ethanol consumption3, effects of starvation4, the relationship between food intake and sleep6, effect of diet on mating7, the relationship between diet and insulin resistance8, nervous system control of behavior in response to hunger9, the effect of diet on subsequent food intake10, and the effect of diet on ethanol sedation11. Diet also affects rodent behaviors such as ethanol consumption12 as well as anxiety-like behavior and visual-spatial memory13. The fly model has the potential to greatly expand our understanding of the relationships between diet and disease, as well as the mechanisms that drive food consumption and food choice.Diet and dietary intake have long been associated with several chronic disease states including obesity, diabetes, heart disease, stroke and cancer. The 14. A related method, called EX-Q, also uses Blue 1 as a dye tracer for solid food consumption like Con-Ex, but provides dye-labeled media to flies in smaller containers15. The smaller containers of media used in EX-Q reduce the amount of dye excreted on  the surface of the food media, thereby increasing the fraction of consumed dye measured as excreted dye15. Radioactive tracers such as [α-32P]dCTP have also been used to label solid food media in flies, with consumption estimated by internal accumulation of the tracer in fly tissues16. A major advantage of the radioactive tracer methods is their sensitivity, whereas major advantages of the dye-based Con-Ex and EX-Q methods is their simplicity, low cost and ease of use.Given its popularity for studies on diet, several methods have been developed to assess consumption of agar-based food media in flies. We developed Con-Ex as a method using Blue 1 as a dye tracer for solid food medium in which excreted dye reflects the volume of food medium flies have consumed17. Understanding mechanisms that drive food choice could therefore ultimately lead to mitigation strategies for a wide range of disease-like states. In addition to its utility for investigating consumption of a single food type, the Drosophila model should be well suited for investigating food preference when presented with multiple food options. Accordingly, methods exist for assessing food preference when flies are presented with solid, agar-based media labeled with different dyes , solid media labeled with a single radioactive tracer20, solid media tracked by oligonucleotide ingestion21, or liquid media via CAFE3. These methods have demonstrated utility and their advantages include sensitivity21 as well as ease of measuring the tracers themselves20, but their implementation might be limited in some experimental settings due to the use of non-standard housing conditions or media19, inability to quantitate the volume of media consumed19, potential challenges with secure handling of radioactive flies20, difficulty in explicitly measuring consumption of different media simultaneously20, or use of sophisticated detection methods21. To the best of our knowledge, no dye-based methods have been described for assessing food preference in flies when they are provided with solid agar-based media under largely standard housing conditions.A key aspect of dietary intake is food preference or food choice. Food preference has major implications for human health and wellness including a role in obesity in adults and children, psychological health and even financial statusHere we report our studies on Orange 4 as an additional dye for use in Con-Ex experiments. Orange 4 can be used as an alternative to Blue 1 in Con-Ex or to confirm findings obtained with Blue 1 in Con-Ex. Additionally, Orange 4 can be paired with Blue 1 to monitor food preference in both the Con-Ex and EX-Q methods. Our findings establish Orange 4 as a dye suitable for Con-Ex and demonstrate the utility of Orange 4 and Blue 1 as a dye pair in the analysis of food preference in flies consuming solid media.Drosophila14. In Con-Ex studies, flies are provided with an agar-based food medium labeled with dye for prescribed amounts of time, the food medium with dye is consumed, excretion products containing dye accumulate inside the vials housing flies, and the amount of dye excreted is quantified by spectrophotometry14. Although it is possible to measure the amount of internal dye inside flies (INT) and the amount of dye deposited on the surface of the food medium (ExMedium) in Con-Ex studies, this approach is time-intensive and measuring ExVial  alone leads to comparable conclusions compared to measuring ExVial plus INT plus ExMedium14. Additionally, more dye is recovered as ExVial than as INT or ExMedium14. We therefore used ExVial as a measure of Con-Ex throughout the studies described here unless otherwise noted.We previously described Blue 1 as a dye suitable for use in Consumption-Excretion (Con-Ex) studies for monitoring intake of agar-based food media in We reasoned that identifying additional dyes for use in Con-Ex studies could provide several experimental advantages such as the ability to concurrently monitor consumption of different media in food preference studies. We therefore searched for additional dyes available from commercial vendors that were water soluble, non-hazardous, non-irritating upon skin contact, and had individual peak absorbances at wavelengths across the visible spectrum. We selected 29 dyes as candidates for use in Con-Ex studies (Table Two important properties of dyes suitable for use in Con-Ex studies are (i) they are readily detectable in ExVial samples and (ii) they do not substantially inhibit or increase consumption-excretion of food media at concentrations that allow their detection. We determined whether the 29 candidate dyes in Table The dyes Cochineal Red A  flies have greater ExVial than do GL flies in Con-Ex studies with Blue 1 as a tracerles Fig. a and malles Fig. b. In fem16. As expected, ExVial determined with Orange 4 was greater in mated females than in males in both the GL  was greater in GL females than males, and that ExVial measured with Yellow 6 and Yellow 10 was greater in males than in females  the volume of liquid medium consumed was measured by CAFE and the volume of medium excreted was determined as ExVial plus ExPlug (the volume excreted on the plug used to hold CAFE capillary tubes), (ii) flies were either fully fed or starved for 18 h prior to CAFE:Con-Ex experiments to provide a broader range of values for correlation analysis (see below), and (iii) Orange 4 is readily recovered and quantified from the plug ) raised the possibility of using Con-Ex to assess food preference in Drosophila. In principle, food preference could be determined by concurrently providing flies with 2 different media, each labeled with a different dye tracer, and then measuring the amounts of each dye in the same excretion samples. Peak absorbance of Orange 4 and Blue 1 occurred at distinct wavelengths  in separate vessels within each vial, allowed flies to consume-excrete media from both sources, and then measured the excreted Orange 4 and Blue 1 in the same samples from each vial. For both Con-Ex and EX-Q, flies consumed-excreted Orange 4 and Blue 1 determined as ExVial and did not exhibit a substantial preference  were indistinguishable when determined using Con-Ex and EX-Q  as Yang and co-workers in their initial development of EX-Qant Fig. a, b. FurX-Q Fig. a, b. OurDrosophila model has considerable utility for investigating molecular-genetic, neurobiological, dietary and environmental influences on feeding behavior and physiology. Flies are reared and housed on solid agar-based media in most studies, resulting in a need for methods to assess media consumption under ostensibly standard housing conditions. We previously showed that Blue 1 works well as a dye tracer to track consumption of agar-based media in Drosophila in Con-Ex studies14. We reasoned that identifying an additional dye tracer would expand the utility of Con-Ex by raising the possibility of (i) using an additional dye to confirm effects on feeding behavior determined with a single dye like Blue 1 and (ii) developing a dye that when paired with Blue 1 would be suitable for food preference studies. The studies reported here identify Orange 4 as a dye suitable for use in Con-Ex experiments and show that this dye has comparable utility to Blue 1.The 14 and this report), only Blue 1 and Orange 4 detect the anticipated effects of starvation, mating status, strain and sex and have other key features such as a lack of effect on consumption at workable concentrations as well as linear accumulation in excretion products over time. In the absence of formal validation of dyes in feeding assays, there is a risk of the tracer not being suitable for detecting meaningful differences between groups, the method lacking sufficient statistical power, unanticipated interactions between the dye and various food media, or the possibility that the dye itself could affect media consumption. Validation of dyes in Con-Ex and similar feeding methods, although time-consuming, is therefore critical for establishing methods suitable for measuring solid food intake in flies.Interestingly, of the 38 dyes we screened for use in Con-Ex14, flies unavoidably excrete dye on the food medium in Con-Ex studies14 and the fraction of the dye excreted on the medium is extremely challenging to directly quantify. Additionally, in studies using internal accumulation of [α-32P]dCTP, approximately 10% of the radioactive tracer is excreted and therefore not typically measured16. Although none of the tracers or tracer methods described to date formally quantify total consumption of agar-based media, methods using validated tracers like [α-32P]dCTP, Blue 1 and Orange 4 have excellent utility for estimating consumption and therefore assessing the effects of genetic and environmental manipulations on feeding behavior.In the studies we report here, the amount of Orange 4 we recovered in excretion products was 20–25% less than the amount of liquid Orange 4 consumed (measured via CAFE). Whether this loss of Orange 4 is due to breakdown of the dye in the fly digestive tract, incorporation of the dye into internal fly tissues, or some other phenomenon is currently unknown. Importantly, the 20–25% reduction in Orange 4 excreted is not a major limitation to its utility as a dye tracer when viewed within the context of limitations associated with other tracers and methods. Although we did not observe a difference between consumed liquid and excreted Blue 1 previously17. Blue 1 and Orange 4 can be measured independently in the same extracts. This makes it possible to provide flies with two kinds of media simultaneously, one labeled with Blue 1 and the other labeled with Orange 4, and then independently detect both dyes in the excretion products. As reported previously in studies with solid media19, we found that caffeine and NaCl were aversive tastants in food preference studies using Blue 1 and Orange 4 as a dye pair in Con-Ex experiments. We also found that caffeine and NaCl were aversive tastants in EX-Q studies with Blue 1 and Orange 4 as a dye pair, and that aversion to these two tastants was indistinguishable when measured with Con-Ex or EX-Q. Our studies indicate that Orange 4 and Blue 1 work well as a dye pair in food preference studies with aversive tastants in both Con-Ex and EX-Q, that Con-Ex and EX-Q provide comparable results in food preference experiments with aversive tastants, and raise the possibility that this dye pair might also work well in food preference studies with appetitive tastants or other media variations.One of the major motivations for identifying and validating an additional dye for Con-Ex was to explore the possibility of pairing it with Blue 1 to assess food preference. The collection of genetic tools and the detailed understanding of the central and peripheral nervous systems in flies make them extremely well suited for investigating the genetic and neurobiological mechanisms underlying food preference or choice, a phenomenon with major implications for human health and disease22, it would be informative to know the relative consumption of the two food sources and how that relative consumption affects life span. Additionally, providing flies with a high sugar diet leads to increased consumption10, making it important to understand whether consumption of a high sugar diet might alter subsequent food preference. It would also be informative to assess whether juvenile dietary manipulations Experiments were performed at Virginia Commonwealth University (Grotewiel laboratory) except those in Fig. Drosophila melanogaster) were reared to adulthood on a standard fly Grotewiel laboratory medium  at 25 °C/60% relative humidity under a 12:12 h light:dark cycle  as previously described14. GL, the standard control strain in the Grotewiel laboratory, was generated by backcrossing the w+ allele from Canton-S into a w1118 isogenic background strain contributed by Michael Ashburner . The GL stock is also reported as r[A]14 or w+26. Lausanne-S (LS) flies were from the BDSC (stock #4268).Flies .All comparisons were made between flies reared, housed and tested side-by-side to account for day to day variability in behavior. Flies were grown to adulthood on 2Y10S3C medium, collected under brief (i.e. < 5 min) COwww.formlabs.com). For EX-Q studies, agar-based media were poured into inverted 1 ml pipet tips. Media for Con-Ex and EX-Q studies were allowed to cool overnight at 4 °C and warmed to room temperature before the start of each experiment.Dyes, obtained from the suppliers in Table 14. Briefly, 15 three-five day-old adult flies were placed in each empty food vial, a feeder cap containing dye-labeled media was placed in the top of each vial, flies were allowed to consume-excrete media for the indicated amount of time , the feeder cap and the flies were discarded, the excretion products were collected in 3 ml of distilled water, absorbance values of the collected excretion products were obtained using a spectrophotometer (Pharmacia Biotech Ultraspec 2000) at the wavelengths indicated in the Figures and Table Con-Ex studies were performed as described14.When used, starvation was achieved by housing flies in vials with 1% agar as a water source and no other food components for 18 h. Flies were refed dye-labeled media for 4 h in Con-Ex experiments to capture the effects of starvation as described or for 8 h in coupled CAFE-excretion studies to produce vial to vial variance necessary for correlation analyses14. Briefly, flies  were fed 5% sucrose liquid medium labeled with Orange 4 for 8 h via capillary tubes (to determine CAFE), and then provided with 5% sucrose without dye for 16 h to excrete consumed Orange 4 . Recover of Orange 4 from foam plugs used to hold capillary tubes was assessed as described for Blue 114.Coupled CAFE-excretion studies were performed as described using capillary tubes 19, were added in increasing concentrations to either the media labeled with Orange 4 or Blue 1 in a counter-balanced design. Preference indexes were calculated for each vial as [volume of media with tastant – volume of media without tastant]/[volume of media with tastant + volume of media without tastant]. In principle, this preference index can vary from + 1 (indicating consumption of only media with tastant) to -1 (indicating consumption of only media without tastant).Con-Ex food preference studies were performed as described above for Con-Ex except that flies were allowed to consume two different media (one labeled with Blue 1 and the other labeled with Orange 4) from a divided feeder cap, and the amounts of Orange 4 and Blue 1 were determined in the same ExVial samples. NaCl and caffeine (Sigma), aversive tastants15 for food preference studies. Each empty feeder cap with two 9.4 mm holes bored through the top were retro-fitted with two 1 ml pipet tips containing food media. The larger ends of the tips were positioned toward the inside of the feeder caps (i.e. the fly side) with the rims of the pipet tips approximately 1 mm from the inside surfaces of the feeder cap. Flies  were added to an empty food vial, an EX-Q cap fitted with two pipet tips containing two different media (one labeled with Blue 1 and one labeled with Orange 4) was placed in the top of the vial, and the amount of Blue 1 and Orange 4 consumed-excreted was determined after 24 h of consumption as in Con-Ex studies except that dyes on the feeder cap as well as dyes on the inside of the vial was collected as previously described14.We adapted EX-Q methods from Yang and co-workers27. One- and two-way ANOVAs followed by Bonferroni’s multiple comparison tests (hereafter simply Bonferroni’s), non-linear regression , Pearson correlation, and one-sample t tests  were performed using Prism 9.0.1 . Whenever possible and unless otherwise noted, results are presented as individual replicates with the mean (as a bar) and error bars representing 95% confidence intervals of the means. Power calculations were performed using a tool available at https://www.stat.ubc.ca/~rollin/stats/ssize/n2.html.Of the 199 groups of results analyzed for this study, 90% (179 groups) did not have significantly non-Gaussian distributions and 10% (20 groups) had non-Gaussian distributions. Given that the vast majority of results did not have non-Gaussian distributions, we used parametric statistical analyses throughout this studywww.flystuff.com #8003) containing 25 ml of standard cornmeal-based growth medium. Following eclosion, adult flies were transferred into rearing bottles containing SY10% medium  and allowed to mate for two days, after which the sexes were separated under light CO2 anesthesia. Ten male or female flies were placed into individual vials (www.flystuff.com #8002) and allowed to age for 5–7 days before Con-Ex studies (performed as described above).Canton-S flies were used for Fig. Supplementary Information 1.Supplementary Information 2.


Wnt signaling is mainly transduced by β-catenin via regulation of the β-catenin destruction complex containing Axin, APC, and GSK3β. Transcription factor EB (TFEB) is a well-known master regulator of autophagy and lysosomal biogenesis processes. TFEB’s nuclear localization and transcriptional activity are also regulated by various upstream signals. In this study, we found that Wnt signaling induces the nuclear localization of TFEB and the expression of Wnt target genes is regulated by TFEB-β-catenin-TCF/LEF1 as well as β-catenin-TCF/LEF1 complexes. Our biochemical data revealed that TFEB is a part of the β-catenin destruction complex, and destabilization of the destruction complex by knockdown of either Axin or APC causes nuclear localization of TFEB. Interestingly, RNA-sequencing analysis revealed that about 27% of Wnt3a-induced genes were TFEB dependent. However, these “TFEB mediated Wnt target genes” were different from TFEB target genes involved in autophagy and lysosomal biogenesis processes. Mechanistically, we found that Tankyrase (TNKS) PARsylates TFEB with Wnt ON signaling, and the nuclear localized PARsylated TFEB forms a complex with β-catenin-TCF/LEF1 to induce the “TFEB mediated Wnt target genes”. Finally, we found that in various types of cancer, the levels of TFEB mediated Wnt target genes exhibit strong correlations with the level of Axin2, which represents the activity of Wnt signaling. Overall, our data suggest that Wnt signaling induces the expression of a subset of genes that are distinct from previously known genes regulated by the β-catenin-TCF/LEF1 complex or TFEB, by forming a transcription factor complex consisting of PARsylated TFEB and β-catenin-TCF/LEF1. The basic helix-loop helix leucine zipper transcription factor EB (TFEB) is a member of the microphthalmia-associated transcription factor (MITF) subfamily . TFEB reThe transcriptional activity of TFEB responds to environmental cues and is regulated through its post-translational modification such as phosphorylation . Under nWnt/β-catenin mediated gene expression is controlled by the transcription co-activator β-catenin. In the absence of Wnt, cytoplasmic β-catenin is located to the Axin complex, which includes Axin, APC (adenomatous polyposis coil), CK1 (casein kinase1) and GSK3. With β-catenin becoming phosphorylated by GSK3β and CK1α in the destruction complex, it is recognized and ubiquitinated by β-TrCP E3 ligase for subsequent proteasomal degradation. Binding of the Wnt ligand to Frizzled and the co-receptor LRP5/6 initiates the Wnt downstream signaling; this leads to relocation of the destruction complex to the plasma membrane via adapter DVL and breaking of the destruction cycle, followed by stabilization of cytoplasmic β-catenin. The accumulated β-catenin then enters the cell nucleus and interacts with TCF/LEF1, resulting in activation of various target genes , 10. GSKHere, we showed that TFEB, a master regulator of expression of genes related to lysosomal biogenesis, acts as a novel mediator of Wnt signaling cascade. We found that Wnt signaling induced PARsylation of TFEB and dissociation of TFEB from the destruction complex, followed by translocation of TFEB to the nucleus. Interestingly, nuclear TFEB induced by Wnt signaling did not induce lysosomal gene expression. RNA-sequencing analysis of cells with TFEB knockdown, which were then Wnt treated, revealed that about 27% of increased genes by Wnt treatment were TFEB dependent; we call these “TFEB-mediated Wnt target” genes. Interestingly, neither ectopic expression of TFEB nor nuclear TFEB induced by starvation induced expression of the TFEB-mediated Wnt target genes, which suggests that nuclear-localized TFEB induced by Wnt is different from the nuclear TFEB induced by starvation. We also found that TNKS1, a poly ADP-ribosylating enzyme, PARsylates TFEB upon Wnt treatment in turn induced nuclear localization TFEB and expression of TFEB-mediated Wnt target genes. The PARsylated TFEB was bound to TCF/LEF1-β-catenin, thus explaining the different profile of TFEB-mediated Wnt target genes from the profile of genes induced by TCF/LEF1-β-catenin or the starvation mediated TFEB.It is known that Wnt/β-catenin signaling represses GSK3β activity and inhibition of GSK3β induces nuclear localization of TFEB . Based oUsing live-cell imaging fluorescent microscopy, we found that treatment with Wnt3a-conditioned media (Wnt3a-CM) induced nuclear localization of TFEB-EGFP within 90 min Fig. ; for theTo further dissect the level of Wnt signaling pathway responsible for nuclear localization of TFEB, we utilized VSVG-LRP6ΔN  and FLAG-DVL1 constructs that can bypass the interaction between of Wnt ligand and receptor and directly activate the downstream pathway of the receptor complex , 15. OveAs subcellular localization of TFEB are strictly regulated by its phosphorylation of specific serine residue , we testWe mutated specific serine residues of TFEB, previously reported to modulate the localization of TFEB, and tested whether the nuclear localization of the mutated TFEB by Wnt treatment would be affected , 18. TheAs Wnt signaling mediated nuclear localization of TFEB was occurring independently of TFEB’s phosphorylation status and was regulated upstream of β-catenin  assays revealed that TFEB and Axin interacts at the overexpression condition as well as endogenous level Fig. . InteresTNKS binding motif (TBM: RXXPDG) [Previous reports have shown that binding partners or substrates of TNKS possess a consensus sequence known as the  RXXPDG) , 25. Mut RXXPDG) . Interes RXXPDG)   exhibited a much reduced interaction with TNKS1 and PARsylation levels compared to wild type TFEB Fig. . ImmunobTreatment of cells with Wnt3a-CM further increased PARsylation of TFEB Fig. . SurprisAfter reaching the conclusion described above, we next wondered how knockdown of APC or Axin1/2 alone, which may not induce PARsylation of TFEB, can induce nuclear localization of TFEB . The levels of TFEB mediated Wnt target genes  but not TFEB-mediated lysosomal gene (MCOLN1) exhibit strong correlations with the level of Axin2, which represents the activity of Wnt signaling   element containing genes, nuclear TFEB induced by Wnt signaling differentially recognized TFEB target genes independent of CLEAR. Since Wnt signaling induces PARsylation of TFEB, we speculated that PARsylation of TFEB contributes not only to its nuclear localization but also to its genomic occupancy. The latter might be achieved by PAR binding proteins (PAR readers), which would interact with PARsylated TFEB to recruit it to specific genomic locations as many of PAR readers are involved in transcription regulation . PARsylaThe relationship between the autophagy process and Wnt signaling is very complex. Several studies have shown that autophagy under starvation conditions reduces Wnt signaling by inducing degradation of Wnt pathway components, DVL  and β-caIn our model, we suggest that TFEB is a key regulator of various processes, sensitively responding to the surrounding environment of the cell, such as starvation or high Wnt levels, and transmits the appropriate signal. During starvation, TFEB moves to the nucleus and activates auto-lysosomal biogenesis to maintain cellular homeostasis, but in the Wnt ON condition, TFEB enters the nucleus and regulates homeostasis by inducing the expression of Wnt-TFEB target genes such as for mediating ion transport. We have therefore extended the role of TFEB as a master regulator of cellular homeostasis.Although much effort has been made globally in various laboratories to develop potent modulators of Wnt/β-catenin signaling, only a few such drug candidates have reached clinical trials . Therapi2 incubator. For the glucose-starvation experiment, we mixed no-glucose DMEM (Thermo Fisher Scientific) with 10% FBS and 1% antibiotics.HEK293T, HeLa, and TFEB-EGFP stable cells were cultured in Dulbecco’s modified Eagle’s Medium  supplemented with 10% FBS and 1% antibiotics at 37 °C in a humidified 5% COFor transient transfection, HEK293T cells were transfected with each plasmid by the calcium phosphate precipitation as described previously . For HeL3VO4, 1 mM PMSF, and 1 µg/ml leupeptin) for 30 min on ice and centrifuged at 13,000 x rpm for 30 min. Supernatants were collected for subsequent protein assays. For Western blotting, protein samples were separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to a PVDF membrane. The blocked transfer membrane was incubated overnight with primary antibodies at 4 °C. To detect the appropriate proteins, antibodies specific for anti-HA, Lamin B, TNKS1, EGFP , TFEB , Axin1 , β-actin, Flag, VSVG (Sigma), Poly(ADP-Ribose)Polymer (Abcam), Myc(Abm), β-catenin (BD Bioscience), active-β-catenin (Millipore), β-tubulin (Gene Tex) were used.For Western blotting, cells were suspended in lysis buffer  on ice for 20 min and centrifuged at 1500 × g, 4 °C for 5 min. Supernatants were collected in a new Eppendorf tube (for the cytosolic fraction). The pellets were washed with 150 μL nuclear washing buffer  and centrifuged at 13,000 rpm, 4 °C for 5 min. After removing these supernatants, the pellets were incubated in Buffer B  with homogenization using a syringe and being incubated on ice for 20 min, followed by centrifugation at 17,000 × g, 4 °C for 20 min. These supernatants were collected in a new Eppendorf tube (for the nuclear fraction). This method has been slightly modified from Sung et al, 2019.Harvested cells were lysed in Buffer A  was used.For immunoprecipitation, incubation of 800–1000 µg of lysates was performed with various antibodies including anti-GFP (Invitrogen), anti-TFEB  and IgG control (Stratagene), overnight at 4 °C and followed by incubation with Protein G (Millipore) or Protein A/G Plus agarose beads (Santa Cruz) for 2 hr. Before the bead incubations, the beads were blocked with 5% milk in lysis buffer. After incubation with the lysates, the beads were five-times washed with the lysis buffer; they were then pelleted and boiled in SDS sample buffer. These samples were then subjected to PAGE and Western blotting. For detection of PAR, harvested cells were lysed with RIPA buffer  on ice for 30 min and centrifuged at 13,000 rpm at 4 °C for 10 min. Supernatants were used for immunoprecipitation, where for nuclear lysates, 100 μg of nuclear proteins were incubated with anti-GFP (Invitrogen) antibody in lysis buffer, followed by bead capture and washing as described above.HEK293T cells were seeded in triplicate on 12-well plates and transfected with pSuperTop (0.5 μg), pGC1a (0.5 μg), or pRL-TK (0.05 μg) plasmids. The indicated plasmid was co-transfected into HEK293T or HeLa cells, and 24 h after transfection, the cells were lysed and luciferase activity was measured using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s instructions. Luciferase activity was measured by a GLOMAX 20/20 luminometer (Promega). Transfection efficiency was normalized to the internal control, thymidine kinase promoter-driven Renilla luciferase (pRL-TK).For establishment of the stable cell line, continuously expressing TFEB-EGFP, HeLa cells were transfected with TFEB-EGFP  by Turbofect transfection reagent (Thermo). On the following day, the transfected HeLa cells were cultured in the selection media containing 400 μg/ml of G418 for 4 days consecutively. The media was replaced every 4 days with an ever-increasing dose of G418 of up to 1,000 μg/ml for 16 days. After this selection period, stably green fluorescence positive colonies were isolated via flow cytometry (FACS). FACS ARIA2 (BD) was used for the sorting with the FACS data analyzed by FACSDiva version 6.1.3 software (BD).Cells were seeded in a 38-mm confocal dish  or on glass coverslips in 6-well plates. For the immunofluorescent staining, the cells were fixed for 20 min in 4% paraformaldehyde in PBS and then permeabilized with 0.1% Triton X-100 for 15 min at room temperature. Samples were washed 4 times with PBS. For pre-blocking, the cells were then incubated in 5% bovine serum albumin (BSA) in PBS for 1 h and then overnight incubated with primary antibodies. Primary antibodies were against TFEB (Bethyl Laboratories), β-catenin (BD Bioscience), active- β-catenin (Millipore) and Flag (Sigma). Samples were then washed with 1% BSA in PBS three times and incubated with the secondary antibody conjugated with Alexa488 or Alexa 546 for 1 h. Localization of the proteins was analyzed and imaged with confocal microscopy (see below for details). The Values for Nuclear TFEB were calculated using ImageJ software (Max intensity: 255).TM Reverse Transcriptase (Promega) with a random primer or using the ReverTra Ace® qPCR RT Kit (Toyobo). For quantitative real-time PCR, the experiment was performed as follows: quantitative real-time PCR was performed using THUNDERBIRD® SYBR® qPCR Mix (Toyobo) and according to the manufacturer’s instructions. All PCR products had a unique dissociation curve. Amplification was performed under the following conditions: 95 °C (10 min), followed by 40 cycles at 95 °C (30 s), then 58.5 °C (30 s). The threshold cycle (Ct) value for each gene was normalized to the Ct value for β-actin. Relative mRNA expression was calculated using the ΔΔCt method.Total RNA was isolated using the TRIzol reagent (Sigma) and according to the manufacturer’s instructions. cDNA was synthesized from total RNA using Improm-II5) in 0.3 ml of each conditioned media were seeded onto the upper chamber of cell culture inserts  and 0.7 ml of complete growth medium containing 10% FBS was added to the lower chamber. Following incubation for 24 h, non-migrated cells were removed from the upper chamber, and the migrated cells in the lower chamber were fixed with methanol, stained with 0.5% crystal violet and then photographed under a light microscope. For invasion assay, insert chambers were pre-coated with matrigel (BD Biosciences) diluted in serum-free medium. Remaining steps are the same as the protocol for transwell assay.Migration assays were performed according to the manufacturer’s instructions. For transwell assay, siRNA transfected cells were incubated with control CM or Wnt3a-CM for 24 hr. Then, cells were trypsinized and counted. Cells . For observing and capturing green fluorescence of Alexa 488, the sample was excited via an Argon 488 nm laser with 10% of output and 20% of laser power settings. For red fluorescence of Alexa 568, the sample was excited using a DSPP 561 nm laser with 5% laser power setting. Photomultiplier (PMT) detector /Hybrid Detector setup  was used for detection of emission with the scan speed of 400 Hz using 8 × line average with 1 Airy unit pinhole setting. Emission spectral detection ranges were 500–550 nm for green fluorescence and 580–650 nm for red fluorescence. Nuclear co-localization of β-catenin and TFEB respectively labeled for green and red fluorescence were analyzed using MetaMorph software . Intensity-based mean value of each fluorescent signal representing β-catenin and TFEB was automatically calculated with the equation of pixel sum intensity divided by a region of interest (ROI) area pixel. For molecular counting of β-catenin and TFEB areas, images were analyzed with IMARIS FL image analysis software V. 9.2 using the add spots and section mode. Estimated diameter of molecules for β-catenin and TFEB was determined in slice view as follows: the smallest XY dot in the images was ~250 nm and Z resolution of 600 nm. For live confocal microscope images, we used Zeiss AXIO Observer Z1. TFEB-EGFP stable cells were simultaneously imaged in a top-stage incubation system , which continuously provides 37 °C warmness and 5% COhttps://xena.ucsc.edu/). The correlation rates were analyzed using GraphPad Pearson correlation. For analyzing the Kaplan–Meier plot, survival data sets from TCGA breast cancer, GDC TCGA LUSC and TCGA bladder cancer database were downloaded from UCSC Xena browser (https://xena.ucsc.edu/).For analyzing the correlation of expression levels between Axin2 and TFEB-mediated Wnt target genes or TFEB-mediated lysosomal gene, gene expression data sets from TCGA breast cancer, GDC TCGA LUSC and TCGA bladder cancer database were downloaded from the UCSC Xena browser (Supplementaty Figure S1Supplementaty Figure S2Supplementaty Figure S3Supplementaty Figure S4Supplementaty Figure S5Supplementaty Figure S6Supplementaty Figure S7Supplementaty Figure S8Supplementary TablesSupplementary Figure and Table Legends


The function of P-M/epoxy composites as anti-corrosion coatings was explored using electrochemical and mechanical tests. Using electrochemical impedance spectroscopy (EIS), it has been noticed that the pore resistance and polarization resistance of the P-M/epoxy composites remain higher as compared to the neat epoxy. The P-M/epoxy composites have the greatest impact on the cathodic dis-bonded area and water absorption. Besides, P-M/epoxy composites exhibit a very high order of mechanical properties. Further, Mg0.5VOPO4 has the greatest effect on the anti-corrosion properties of epoxy coating followed by Zn0.5VOPO4 and Ni0.5VOPO4. All these properties lead to developing effective anti-corrosion coatings. Thus, the net result from this research work is highly promising and provides a potential for future works on the anti-corrosion coating.Epoxy nanocomposite coatings are an essential way to protect petroleum storage tanks from corrosion. For this purpose, the new nanocomposite epoxy coatings (P-M/epoxy composites) have been successfully designed. The P-M/epoxy composites are based on the metal vanadium oxy-phosphate M The tank coatings are considered the first bumper wall to protect storage tanks from corrosion9. Despite this fact, many coatings suffer from several faults such as weak adhesion and high permeability12.The corrosion in the petroleum storage tanks causes heavy economical and environmental damages in the petroleum field15. Though epoxy resin displays better anti-corrosion properties, it still lacks enough barrier layer owed to the high resin permeability, which leads to hinder the anti-corrosion performance16.Nanocomposite coatings have received worthy concern for future using as anti-corrosion barriers for petroleum storage tanks. As we know, epoxy resin exhibits high anti-corrosion and mechanical properties than that of other resin material19. However, we noted that there are very few works on the influence of phosphates materials on the anti-corrosion properties of the coating.Indeed, nano-phosphate materials are essential components in nanocomposite epoxy coatings due to their ability to recover the epoxy damages because of the corrosive solution17 have shown that α-zirconium phosphate has a great role in increasing the anti-corrosion properties of phosphate coating. Morozov et al.20 reported that cerium particles and organophosphate groups improved the corrosion resistance of the epoxy coating. They indicated that the integration between cerium and phosphates leads to a synergistic anti-corrosion effect on both anodic and cathodic sites. Deayb et al.21 indicated that the permeability of the epoxy resin was significantly improved by the incorporation of hydrogen-phosphate particles into the epoxy matrix and this led to the creation of a good anti-corrosion coating layer.Tian et al.0.5VOPO4 . In this work, we combine electrochemical and mechanical assays to investigate how the title compounds influence the anti-corrosion properties of epoxy coating nanocomposites.Recently, there is an urgent need in developing the performance of epoxy resin using phosphate compounds to protect the metal structures from corrosion. According to this, we develop new epoxy nanocomposites based on new vanadium oxy-phosphate compounds M0.5VOPO4  compounds were successfully prepared using a single step via a solvo-thermal route without employing any heat treatments according to the previously reported procedures23. Stoichiometric mixtures of NH4VO3 , acetic acid CH3COOH , M(NO3)2·6H2O , and NH4H2PO4  were used in the synthesis. First, NH4VO3 and acetic acid CH3COOH with a mole ratio of 1:1 were dissolved in 20 ml of H2O to form a clear green solution (Solution A). M(NO3)2·nH2O was added into solution A and then was stirred at 70 °C for 30 min. NH4H2PO4 was then dissolved in 10 ml of H2O (Solution B). After stirring at 70 °C for 30 min, solution B was added to solution A drop-wise to form a new mixture, which was stirred at 70 °C for an additional 1 h. The solution was finally poured in a 100 ml autoclave which was then heated at 200 °C for 24 h. After filtering the solution, the obtained green powder was dried at 100 °C for 12 h under vacuum.M0.5VOPO4 . All the ingredients were homogenized using a speed mixer for 3.0 h. The final formula was grounded for 2.0 h to achieve adequate fineness.The nanocomposite coatings  were prepared by blending epoxy resin (type Bisphenol-based—Ciba Co.), poly-amidoamine hardener (Arkema Co.), xylene and 1.0% of M25. The film applicator was used to apply a very thin layer on the steel surface. The coated electrodes were placed in the oven at 333 K to get a complete cure coating surface. The coating micro-meter (Mitutoyo) was used to measure the coating layer thickness. It was approximately 38 ± 5 μm.Carbon steel sheets (from petroleum storage tank source) were utilized as coated working electrodes. The electrode dimension is 12 mm × 16 mm × 0.50 mm). The preparation of working electrodes before the coating was conducted using the standard method ASTM G1-03EIS measurements were used to explore the anti-corrosion performance of new nanocomposite coatings. The adequate 3-electrodes  and Pt electrodes) glass cell was used for EIS measurements. All experiments were conducted using Potentiostat//Galvanostat system type Gill-AC-947.Frequency range = 0.01 Hz to 100 kHz,Amplitude = 10 mV,Operation potential = open circuit potential (OCP),Immersion time = 7 days,Temperature = 303 K.The EIS experiments conditions are:Ø%) of the nanocomposite coatings was calculated using the coating capacitance from EIS experiments at initial (C0) and after 7 days (Ct) of the immersion time. Brasher–Kingsbury relation was used to get Ø% for different nanocomposite coatings27.H2O is the dielectric constant of H2O (εH2O = 80).Water absorption (28.The cathodic disbanding experiments were conducted according to ASTM G8-96(2019)32.All the mechanical experiments  were conducted in accordance with ASTM D522, ASTM D 3359-17, ASTM D7334, and ASTM D2794, respectivelyDifferential scanning calorimetry (DSC) and Glass Transition Temperature (Tg) measurements were recorded by DSC 3 METTLER TOLEDO (Heating rate = 20 °C/min—nitrogen flow rate = 20 ml/min).0.5VOPO4  compounds were analyzed using the powder X-Ray diffraction technique. The diffractogram of the materials was recorded in the 2-theta range of 10°–80° as illustrated in Fig. 0.5VOPO4 materials without the presence of any crystallized impurities.The M0.5VOPO4 on the anti-corrosion properties of the epoxy coating was confirmed by the EIS studies. The Nyquist plots for carbon steel electrodes coated with neat epoxy, P–Ni/epoxy nanocomposite, P–Zn/epoxy nanocomposite and P–Mg/epoxy nanocomposite in 3.5% NaCl solution at 303 K are presented in Fig. 33. While the second peak at the low frequency is due to the corrosion process under the coating layer34.The influence of new synthesis phosphate compounds M35. This leads to steel corrosion process at the liquid/metal interface (Fe(s) = Fe(aq)2+  + 2e)37. According to this situation, the most suitable equivalent electric circuit that fits the Nyquist plots for neat epoxy, P–Ni/epoxy nanocomposite and P–Zn/epoxy nanocomposite is shown in Fig. Cc), pore resistance (Rpo), polarization resistance (Rp), solution resistance (Rs) and the capacitance of double layer (Cdl). All these elements are listed in Table Rpo and Rp values were significantly increased by using P–Ni/epoxy and P–Zn/epoxy composites comparing with Rpo and Rp values in the case of pure epoxy. Interestingly, P–Mg/epoxy composite is able to heal the coating defect and form one time constant. In this case, the second peak at the low-frequency disappears  penetrates the coating texture and forms solution/metal interface under the coatingCc and Cdl are linked to the barrier performance of composites coatings. The low Cc and Cdl values of new P-M/epoxy composites indicate their good barrier performance against corrosive solution39. It is worth noting that Mg0.5VOPO4 has the greatest effect on the anti-corrosion properties of epoxy coating followed by Zn0.5VOPO4 and Ni0.5VOPO4.Following the same EIS data, it was clear that 40. The cathodic de-lamination data for neat epoxy, P–Ni/epoxy, P–Zn/epoxy and P–Mg/epoxy nanocomposites in 3.5% NaCl solution at 303 K are exhibited in Fig. 0.5VOPO4 into the epoxy resin has a slight impact on the cathodic disbonded area. On the other side, the incorporation of Mg0.5VOPO4 and Zn0.5VOPO4 into the epoxy resin has a great impact on the cathodic disbonded area. This means that the new synthesis phosphate compounds could result in a strong adhesion between the epoxy resin and the steel substrate. Our study also confirms that Mg0.5VOPO4 has the greatest impact on the cathodic disbonded area followed by Zn0.5VOPO4.The cathodic de-lamination tests for the new nanocomposite coatings are critical to investigate the strength of coatings adhesion with metal subtractØ%  for nea0.5VOPO4. As illustrated in Table -M/epoxy composites. We also observed a significant increase in the impact resistance of the coatings from 65 kg cm−2 in the case of pure epoxy to 85, 88 and 93 kg cm−2 in the cases of P–Ni/epoxy, P–Zn/epoxy and P–Mg/epoxy composites, respectively  further reveal the various mechanical features of epoxy coating acquired by incorporation by new synthesis phosphate compounds M44.Epoxy coating permeability represents the vital defect in the coating layer leading to the failure in preventing the corrosive ions from transferring causing metal surface corrosion0.5VOPO4 inside the epoxy matrix is able to heal the epoxy coating layer. According to the above data, the phosphate particles M0.5VOPO4 were distributed inside the pore of the epoxy matrix, leading to very low pore size. This action makes the zigzagging route for moving the corrosive ions is longer45, leading to the low possibility of the corrosion of steel surface and formation of iron oxide46.Here, the incorporation of a small size of M0.5VOPO447. DSC curves  comparing with Zn (− 1.1 V)51. This higher electronegative potential supplies more cathodic protection for steel surfaces resulting in higher anticorrosion properties.The type of metal atoms M = Mg, Ni and Zn in the structure of phosphate particles M0.5VOPO4  was successfully developed the anti-corrosion properties of epoxy coating nanocomposites were clearly investigated by electrochemical and mechanical measurements. In summary, the anti-corrosion properties of epoxy were improved by incorporating vanadophosphates inside the epoxy resin. This was clearly detected from the high values of pore resistance and polarization resistance. Our study also confirms that Mg0.5VOPO4 has the greatest impact on the cathodic disbonded area followed by Zn0.5VOPO4. The formation of epoxy nanocomposites containing vanadium oxy-phosphate was decisive for achieving excellent mechanical properties such as cross-cut adhesion, impact resistance, bend test and contact angle). The changing of the metal atoms M = Mg, Ni and Zn in the structure of M0.5VOPO4 particles is the main factor in determining the anti-corrosion performance difference between epoxy nanocomposites. This work establishes the great potential of the vanadophosphates/epoxy nanocomposites for the development of high-performance anti-corrosion coatings.In this study, the new P-M/epoxy composites based on the vanadium oxy-phosphate M


The Wnt/β-catenin signaling pathway regulates many aspects of tumor biology, and many studies have focused on the role of this signaling pathway in tumor cells. However, it is now clear that tumor development and metastasis depend on the two-way interaction between cancer cells and their environment, thereby forming a tumor microenvironment (TME). In this review, we discuss how Wnt/β-catenin signaling regulates cross-interactions among different components of the TME, including immune cells, stem cells, tumor vasculature, and noncellular components of the TME in hepatocellular carcinoma. We also investigate their preclinical and clinical insights for primary liver cancer intervention, and explore the significance of using Wnt/β-catenin mutations as a biomarker to predict resistance in immunotherapy. In HCC, most patients diagnosed at early or intermediate stages can potentially be cured by surgery, including liver resection, transplantation, and ablation or palliative locoregional therapies such as transarterial chemoembolization. However, the disease recurrence rate is high. Approximately 40% of patients diagnosed with advanced disease can be palliatively treated with systemic therapy when curative therapies and transarterial chemoembolization are no longer available2.Globally, primary liver cancer has been upgraded to the third leading cause of cancer-related mortality according to the latest data released by International Agency for Research on Cancer. Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer, accounting for about 70%–85%3. With the development of immunotherapy, immune checkpoint inhibitors against PD-1 and CTLA-4 have also been approved by the FDA for the second-line treatment of HCC4. However, the limited median survivals of these patients after treatment with 12–13 months using first-line treatments and 9–11 months using second-line treatments are still an outstanding issue5. Identifying more feasible targets and using predictive biomarkers to screen suitable patients is therefore necessary. Furthermore, comprehending mechanisms underlying drug response or resistance and exploring combinational therapies are imperative to improve the efficacies of HCC immunotherapies and the patients’ survival.The multi-tyrosine kinase inhibitor (TKI) sorafenib is the first drug to be granted approval by U.S. Food and Drug Administration (FDA) for treating advanced HCC. Recently, other TKIs like lenvatinib commonly used in first-line treatment, and regorafenib, cabozantinib, and ramucirumab used in second-line treatment are also approved because of survival advantages in phase III clinical trials6. Therefore, effective treatment strategies need to understand both factors.A tumor is a type of organoid structure consisting of malignant cells and their environment. The occurrence and development of liver tumors have been attributed to altered signaling pathways and tumor microenvironments (TMEs) involving vasculature, inflammatory cells, stromal cells, and the extracellular matrix (ECM)7. Abnormal Wnt/β-catenin signaling induces the occurrence and/or progress of various liver diseases, including cancer. In the 2 most common types of primary liver cancer, HCC and cholangiocarcinoma, the Wnt/β-catenin signaling is often hyperactivated and promotes the initiation and progression of tumors8. Activating mutations of CTNNB1, which codes β-catenin, have been detected in 11%–37% of HCC patients, and inactivating mutations of Axin1 or APC exist in approximately 5%–15% or 1%–2% of liver cancer specimens, respectively, acting as negative regulators9.The Wnt/β-catenin signaling pathway is extremely conserved and tightly regulated. It plays a vital role in liver biology. Despite inactivity in most mature and healthy livers, this signaling pathway can be reactivated during cell renewal and/or regeneration processes, some pathological conditions, also diseases and cancerIn this review, we describe the complex interactions involving the Wnt/β-catenin signaling pathway in the TME of HCCs. We also describe the clinically targeted therapies currently designed for this pathway as well as preclinical and clinical applications encompassing Wnt/β-catenin signaling in HCC treatments with sorafenib or immune checkpoint inhibitors. Understanding the role of the Wnt/β-catenin signaling pathway in TME can help us understand the initiation and progression of liver cancer, and provide new avenues for targeted treatment of this disorder.10.The Wnt signaling pathway participates in many basic processes of embryonic development and homeostasis. It is commonly classified into canonical or Wnt/β-catenin signaling pathways and non-canonical pathways, including planar cell polarity and the Wnt/calcium signaling pathway12. The Wnt pathway has been shown to participate in cell proliferation, survival, self-renewal or differentiation, and tissue patterning of multicellular organisms13.Wnts belong to a highly evolutionally conserved, secreted protein family, which is necessary for triggering the Wnt signaling pathway. The human Wnt family containing 19 gene-encoding cysteine-rich glycoproteins bind to more than 15 receptors and co-receptors14. The β-catenin is composed of 3 domains: the N-terminal region containing the phosphorylation sites for CK1α and GSK-3β, the armadillo repeat including 12 copies, and the C-terminal domain interacting with TCF/LEF. When Wnt ligands are absent, most β-catenin combines with E-cadherin in adherence junctions at the plasma membrane. The β-catenin in the cytoplasm is degraded by a degradation complex consisting of adenomatous polyposis coli protein (APC), the Axin scaffolding protein, and 2 kinases, casein kinase I isoform-α (CK1α) and glycogen synthase kinase 3β (GSK-3β)15. CK1α and GSK-3β can separately phosphorylate Ser45 and Thr41, Ser37, and Ser33 at the N-terminus of β-catenin. Then, β-transducin repeat-containing protein (β-TrCP), a component of the E3 ubiquitin ligase complex, recognizes phosphorylated β-catenin. Finally, β-catenin is polyubiquitinated and undergoes proteasome degradation16 . However, when the Wnt ligand binds to the frizzled protein receptor cooperative with LRP5/6, phosphorylation of Dishevelled protein (DVL) and recruitment of Axin and other degradation complex components are induced, leading to dissociation of the degradation complex, inhibition of β-catenin phosphorylation, and nuclear transportation17. Axin proteins are rapidly degraded by tankyrase enzymes via ubiquitin-mediated proteasomal degradation. In the nucleus, β-catenin acts with the transcription factors of T cell Factor/Lymphoid Enhancer Factor (TCF/LEF) family. P300, CREB-binding protein (CBP), Pygo, BCL9, and other transcriptional co-activators are recruited for initiating transcription of Wnt target genes mediating the formation of the extracellular matrix, anti-apoptosis, cell proliferation  and angiogenesis (VEGF and c-MET)19 . Mutations of key genes in this process can prevent β-catenin phosphorylation and then abrogate its degradation. The consequence of increasing β-catenin protein levels in both cytoplasm and nucleus results in the constitutive activation of Wnt/β-catenin pathway.In the canonical pathway, the Wnt ligands  bind to corresponding Frizzled receptors (10 kinds found in human and mice) and low-density lipoprotein receptor-related proteins 5/6 (LRP5/6) co-receptors on the cell surface, to initiate intracellular signal transduction through nuclear translocation of β-cateninFigure 2 and Table 1).The immune infiltrate refers to the entire encompassing immune cells in the TME. They have been thought to be involved in the regulation of tumor development. There is a great need for more comprehensive knowledge of tumor immunity. We have therefore summarized current findings of the manipulation of tumor infiltrates by Wnt/β-catenin signaling, which has allowed us to better understand multi-component antitumor responses to guide us in the development of more effective immunotherapies for HCC patients 21. It has been reported that Wnt ligands from macrophages can activate the Wnt signal pathway in oncocytes. In chronic liver disease, after phagocytosis of hepatocyte debris, hepatic macrophages secrete Wnt-3a, as an activator of Wnt/β-catenin signaling in hepatic progenitor cells (HPCs) to promote their differentiation into hepatocytes22 . Macrophages are crucial sources of Wnts in the liver during steatosis. Steatosis-induced activation of Wnt/β-catenin promotes the propagation of tumor initiating cells and progression of tumorigenesis23 . One study reported that liver regeneration was initiated through β-catenin activation in hepatocytes after partial hepatectomy. This kind of β-catenin activation is regulated by Wnt secreted from endothelial cells and Kupffer cells24. Moreover, Wnt/β-catenin can also be triggered inside macrophages by secreted Wnt ligands from cancer cells. The hepatic tumor cells can activate the Wnt/β-catenin signaling pathway by a paracrine function to stimulate M2 macrophage polarization, which promotes protumor activity25. In the tumor mouse model, orthotopically inoculated hepatic Hepa1-6 and Wnt3a secreted from Hepa1-6 can activate Wnt/β-catenin signaling and also function as a mitogen for Kupffer cells like TAMs (kclTAMs). However, whether there are other secreting Wnt ligands or mitogens playing roles in the modulation of kclTAM propagation needs further investigation26. It was also verified that one kind of long noncoding RNA, LINC00662, could activate the Wnt/β-catenin pathway in an autocrine manner by increasing Wnt3a expression and secretion, and also in a paracrine manner to promote M2 macrophage polarization, leading to HCC tumorigenesis and metastasis in vivo27 . Therefore, in future work, efficient strategies for HCC treatment involve inhibiting secretion of Wnts from hepatoma cells or macrophages, as well as inhibiting activation of Wnt/β-catenin signaling.Tumor-associated macrophages (TAMs) exert their effects on the development of inflammation-related cancers, including HCC, as the main components of the TME. Macrophages can be polarized into M1 macrophages, which have anti-tumor functions induced by IFN-γ alone or with lipopolysaccharides and M2 macrophages, which exhibit pro-tumor function induced by IL-4 and IL-1328. Expressions of β-catenin and GSK-3β are higher in DCs than in monocytes. Crosstalk of the Wnt and MAPK signaling pathways has been implicated in DC differentiation, maturation, and function29. It has been reported that tumor-intrinsic constitutive activation of β-catenin can affect antitumor immunity by defective recruitment of DCs into the tumor microenvironment in melanomas30, and the failure of DC recruitment is partly due to decreased chemokine CCL4 and high IL-10 production resulting from β-catenin activation within tumor cells32. In HCC, it was also found that the CTNNB1 mutation correlated with reduced DCs33. The activation of β-catenin promotes immune evasion, which can be ascribed to failing recruitment of DCs, which in turn influences antigen-specific T cell activity34.DCs function as specialized cells that recognize and present antigens to T cells, with antigenic specificity to modulate T cell functions. DCs are crucial in regulating adaptive immunity. Notch signaling triggers expression of more than 8 Frizzled receptors, to activate the downstream canonical Wnt pathway in hematopoietic progenitor cells, thereby favoring differentiation of DCs35. For many tumors, increased CD8+ T lymphocytes have been associated with a better prognosis. Nonetheless, the genetic and epigenetic mechanisms underlying the differences of T cell infiltration among tumors are not clear. For HCC, about one-third of patients with gain-of-function mutations of CTNNB1 are relevant to scarce intratumoral T cell content36. HCC patients with CTNNB1 mutations have significantly reduced CD8+, CD4+ T cells, Th2 cells, Tfh cells, and B cells33. Luke et al.37 concluded by analyzing solid tumors in TCGA that tissue inflammation lacking T cells occurred frequently in primary cancers including HCC, and they found a correlation between tumor intrinsic activation of Wnt/β-catenin and non-T cell-inflamed TMEs. Mei et al.38 revealed that DVL1 expression and the infiltration levels of CD4+ T, CD8+ T, and B cells in HCC were negatively correlated. Blockade of Wnt signaling activation in HCC-bearing mice increased CD4+ and CD8+ T cells39. By employing a novel HCC mouse model with the hydrodynamic tail vein delivery of exogenous antigens, and the CRISPR technique aiming at antioncogenes and genetic elements encoding oncogenes, it has been functionally demonstrated that β-catenin activation results in immune escape and tolerance to anti-PD-1 therapy. This can be partially interpreted by abortively producing chemokine CCL5, which affects the supplementation of CD103+ DCs, further influencing antigen-specific CD8+ T cells in the liver, leading to decreased immune surveillance34. Treg cells, as important immunosuppressive cells, have been proven to be regulated by β-catenin through activation of CCL28 in gastric cancer40. Inhibition of the β-catenin/BCL9 interaction can suppress Treg cell infiltration through the TGF-β-CCL20/CCL22 axis in colorectal cancer41. In HCC-bearing mice, inhibition of Wnt activation lead to reduced CD25+Foxp3+ Treg cells39. Based on these observations, we speculate that there is a connection between Wnt signaling and Treg cells. However, whether and how Wnt/β-catenin signaling directly regulates Treg cells remains unclear in studies of HCC.The lymphocyte infiltration density of most malignant cancer tissues is heterogenous. Based on the cytotoxic T cell infiltrate within the tumor, it was called a cold or hot tumor in immunotherapy terminology42. NKT cells are divided into 2 major subsets having opposing roles in immune responses by secreting proinflammatory or antiinflammatory chemokines and cytokines43. Invariant NKT cells and the chemokine-like factor, LECT2, co-regulate the progression of tumors at the cellular and molecular levels, respectively, in β-catenin-induced HCCs44. A study using mouse model also revealed the complex functions of Wnt/β-catenin signaling in regulating hepatic NKT cell activation. The imposing effects of β-catenin on NKTs are dependent or independent on Wnt45.Natural killer T (NKT) cells are important innate-like hepatic lymphocytes implicated in immune responses during infection, cancer, and autoimmunity47. There is emerging evidence that CSCs within the TME are associated with aggressive tumor progression, chemoresistance, and recurrence in HCC patients. Hepa1-6 CSC spheroids with activating β-catenin have a stronger in vivo tumorigenic ability in the immunocompetent liver microenvironment of a HCC orthotopic mouse model48. EPCAM, LGR5, CD44, and CD133 are representative cell surface markers of CSCs51. A subset of “superpotent CSCs”-Wnt-activityhighALDH1+EpCAM+ triple-positive cells with the highest tumorigenesis capacity among all HCC cells promotes Wnt/β-catenin signaling by increased DVL152. HCC is believed to originate from CSCs. The existence of autocrine Wnt signaling, particularly Wnt3 in EpCAM+ CSCs from HCC, was reported in 201753. LGR5 is the target gene of the canonical Wnt signaling. In LGR5+ circulating stem/progenitor cells, Wnt, and R-spondin receptor encoded by LGR5 further upregulate the expressions of CD44 and CD13354. Wnt signaling can also be activated in liver CSC by a long noncoding RNA called lncTCF7, which attracts the SWI/SNF compound to the TCF7 promoter, thus inducing CSC self-renewal and tumor propagation55. Besides, Fan et al.56 discovered that CSC-like traits, tumorigenicity, and sorafenib-resistance in HCC were enhanced by active Wnt/β-catenin pathway stimulated by the nonreceptor protein tyrosine kinase 2. Activation of the Wnt/β-catenin signaling pathway induced by Ring1 also promotes transformation of HPCs into liver cancer stem-like cells57. In addition, β-catenin is involved in Shp2-promoted liver CSC expansion connected with patient responses to chemotherapeutics58. In mouse hepatic stellate cells and liver tumor-initiating stem cell-like cells, the Wnt/β-catenin pathway regulates stearoyl-CoA desaturase expression to stabilize LRP5 and LRP6 mRNA, further augmenting Wnt signaling to result in liver fibrosis and tumor development59. The epithelial-mesenchymal transition (EMT) has been confirmed to be closely related to activation of CSC in tumor tissues62. It has also been suggested that the EMT is regulated by the Wnt/β-catenin pathway in HCC64.CSCs refer to a subpopulation of poorly differentiated cells that are qualified with the same self-renewing and pluripotent potentials as common somatic stem cells (SSCs)65. Small molecular inhibitors targeting these associated pathways have been implicated in preclinical and clinical studies67. However, the specific mechanism of how the Wnt signaling pathway orchestrates interactions between CSCs and the TME needs further investigation.Understanding the pathways that regulate CSC self-renewal, differentiation, and tumorigenicity are important for the development of anti-cancer therapies. Conserved signaling pathways like Notch, Hedgehog, and Wnt pathways play important roles in the function of normal stem cells and are also implicated in cancer68. HCC has properties of hypervascularization and vascular abnormalities. Tumor vascularity can expand by enrolling existing vessels into the tumor zone in HCC69. The Wnt/β-catenin signaling pathway regulates angiogenic factors. Enhanced Wnt/β-catenin-mediated FGF18 expression and secretion are induced by ribosomal protein, S15a, which promotes angiogenesis and tumor growth in HCC cells70. Yang et al.71 reported that miR-1301 restrained HCC cell transplantation, aggression, and vascularization by reducing Wnt/β-catenin signaling by targeting BCL9, a coactivator of β-catenin transcription. Endotheliocytes express many Wnt pathway-associated elements, and the activation of Wnt1 can induce endotheliocytes expansion and capillary-like network arrangements 72. Functional studies of HCC by Hu et al.73 also showed that Wnt signaling was necessary for angiogenesis. Wnt antagonists inhibit tube formation and decrease microvessel density and angiogenic factors. The study clarified the antitumor potential of Wnt antagonists in increasing cell apoptosis and in attenuating tumor angiogenesis.Tumor angiogenesis is a key factor in maintaining tumor growth. Angiogenic factors take part in tumor angiogenesis, infiltration, and metastasis. At the molecular level, angiogenesis is a consequence of competition between proangiogenic factors and inhibitors. Many key angiogenic factors have been discovered, including vascular endothelial growth factor, matrix metalloproteinase, fibroblast growth factor (FGF), platelet-derived growth factors, angiopoietins, hepatocyte growth factor, and hypoxia-inducible factor74. The ECM participates in the initiation and progression of tumors, and also affects cancer therapy75. Cysteine-rich 61, a member of the CCN growth factor family and also an ECM protein, is a target gene of β-catenin and promotes the progression of HCC through regulation of hepatic stellate cells77. Dermatopontin is an ECM protein implicated in intercellular adhesion and ECM development. Liu et al.78 reported that dermatopontin repressed HCC proliferation by inactivating pro-oncogenic Wnt/β-catenin signaling through inhibition of CXX4. As for the growth factors in the TME, the intricate crosstalk of Wnt/β-catenin and the epidermal growth factor receptor, FGF pathways have been discussed in the context of HCC80. Upregulation of FGF15/19 signaling activates canonical Wnt signaling and the EMT to facilitate HCC in a metabolic disorder background81. Overexpression of platelet-derived growth factor B enhances expression of pro-oncogenic factors, including β-catenin, VEGF, and platelet endothelial cell adhesion molecule-182. MMPs are a family of endoproteases, which degrade the ECM including collagen, gelatin, and elastin. It is thought that MMP-2 and MMP-9 are direct targets of Wnt/β-catenin signaling83. Activation of the Wnt/β-catenin pathway promotes the EMT, migration, and malignant development of HCC by regulating MMP85. Exosomes are small extracellular vesicles ranging in size from 30 to 150 nm, and are increasingly regarded as important carriers of information in the TME86. It has been shown that exosomes contributed to HCC development and progression88. Yu et al.89 showed that hypoxia-induced exosomes activated the Wnt/β-catenin pathway and promoted proliferation, migration, and the EMT in normoxic HCC cells.In addition to various cell types, the TME also includes noncellular components involving the ECM, growth factors, proteolytic enzymes, and their inhibitors90. A study reported that in liver cancer stem cells, Notch negatively regulated active β-catenin protein expression, and Notch1 was downstream of Wnt/β-catenin signaling91. In mice KclTAM, activation of Notch signaling impeded CTNNB1 mRNA. This negative correlation was also confirmed in CD68+ macrophages of patient-derived HCC biopsies92.The interaction between the Wnt and Notch signaling pathways participates in many developmental processes. Cells can use the pattern Notch-ON/Wnt-OFF or Wnt-ON/Notch-OFF to regulate signaling. These 2 pathways modulate each other at different signal transduction levels93. Canonical Wnt signaling activation induces KIF2C expression, then enhances mTORC1 signaling and HCC development by KIFC2 binding to TBC1D794. In HepG2 hepatoma cells, Wnt/β-catenin activation and Snail expression are dependent on ERK1/2 signaling to regulate the EMT95. TGFβ signaling also induces Wnt and Sonic hedgehog signaling activation in development of HCC EMT96. Zhang et al.97 discovered that β-catenin interacted with hif-1α to promote hif-1α signaling and to suppress Wnt/β-catenin signaling, together leading to an exaggerated EMT in hypoxic HCC. These findings further addressed the complexity of the signaling pathway interaction network.Hippo, Notch, and Wnt/β-catenin signaling interact to maintain liver size and inhibit HCC. The positive interaction of Notch signaling and Hippo signaling effector YAP/TAZ promotes severe hepatomegaly and accelerates initiation and development of HCC. Unexpectedly, canonical Wnt signaling activation inhibits the interaction between YAP/TAZ and Notch signaling. The positive feedback loop was repressed and thus inhibited the formation of HCC98; ii) targeting Wnt signaling by extracellular components like Frizzled and LRP5/LRP6; iii) aiming at cytoplasmic molecules of signaling such as tankyrase and CK1α; and iv) impeding the intranuclear Wnt signaling, including β-catenin and the co-factors involved in the transcription of Wnt target genes99. However, there is currently no approved therapeutic agent for clinical use that can specifically and effectively target this pathway (Table 2). One of the major concerns is that inhibition of the Wnt signal pathway may affect Wnt-dependent stem cells and the regeneration of tissues and organs. Some modulators targeting different key steps of the Wnt/β-catenin pathway have been evaluated for their depressor effect and safety in clinical trials100. Among these trials, one study aiming at hepatocellular carcinoma using a humanized monoclonal antibody against Dickkopf-1 (DKK1), DKN-01, is involved in phase I and II trials . DKK1 is a secreted glycoprotein inhibiting the canonical Wnt pathway, by binding to the LRP5/6 co-receptor. The role of DKK1 is controversial. Qin et al.101 found inhibition of DKK1 expression facilitated β-catenin to translocate into nuclei, which may influence the proliferation and metastasis of HCC cells. Contrary to the hypothesis that DKK1 is supposed to function as a tumor suppressor, recent studies showed its oncogenic role in activating Wnt/β-catenin signaling pathway, thus promoting the invasion, metastasis, and tumorigenicity of HCC cell lines103. DKK1 has been shown to have protumor effects and was correlated with poor prognosis, rendering DKK1 an attractive anticancer target104. Moreover, Liang et al.105 injected tumorigenic β-catenin and MET into β-catenin-deficient livers to induce cancer, and unexpectedly found that Sox9+ cells with stem cell properties expanded inflammatory, upregulated cytokines, and several pro-tumorigenic pathways such as Akt, Erk, and Wnt/β-catenin were also enhanced to exacerbate HCC progression. Restraining β-catenin in the liver startingly creates a pro-tumorigenic microenvironment to aggravate HCC driven by β-catenin/MET, further adding to the complicacy of this pathway. It is also worth noting that as traditional Chinese medicines have gained constant attention because of potential anticancer abilities, several of them have been used in the treatment of HCC by targeting the immunosuppressive TME106. Some traditional Chinese medicines were found to suppress proliferation, induce apoptosis, and impair invasion of liver cancer cells by blocking the Wnt/β-catenin signaling pathway, such as agkihpin (a snake venom arginine esterase)107, Dendrobium candidum extract108, ethyl acetate extract from Jiedu Xiaozheng Yin109, and Zanthoxylum avicennae110. Further animal studies and clinical trials are warranted.Because of the essential and multiple roles of canonical Wnt signaling in the TME, novel molecular therapies targeting this pathway have shown unlimited prospects. Therapeutic drugs that directly target the Wnt signaling cascade are currently divided into the following categories: i) inhibiting secretion of Wnt ligands using porcupine (PORCN) inhibitors. PORCN is a membrane-bound O-acyltransferase acylating Wnt ligands during the posttranslational modification stage111. In turn, β-catenin can diminish apoptosis and cell growth inhibition induced by sorafenib treatment. Up-regulation of β-catenin contributes to HCC resistance112. Lin et al.113 confirmed that suppression of canonical Wnt signaling could improve the therapeutic effect of sorafenib alone in a HCC cell line and mouse xenograft model. In a preclinical pediatric HCC model, inhibition of β-catenin activity using the ICG001inhibitor combined with sorafenib led to lower cell viability and showed synergistic effects, indicating that blockade of Wnt/β-catenin signaling may attenuate sorafenib resistance114. Combinatory treatment using FH535, an artificial inhibitor of Wnt/β-catenin signaling with sorafenib on HCC cells, significantly decreased the autophagic flux and increased apoptosis115. Immunotherapy with checkpoint inhibitors is also an important emerging treatment option for HCC. Nivolumab in first-line trials and pembrolizumab in second-line trials showed promise of efficacy, but neither reached statistical significance116. Clinical trials have shown that only about 15%–20% of HCC patients showed an objective response to these therapies. Harding et al.117 found that activating mutations of the Wnt pathway partly contributed to innate resistance to immune checkpoint blockade, and clinical findings displayed shorter median progression-free survival and median overall survival. Immune exclusion HCC class, characterized by mutations in the Wnt/β-catenin, are refractory to immune checkpoint blockers5. Du et al.118 found that EGFR activation and a Wnt ligand up-regulated PD-L1 expression by binding the β-catenin/TCF/LEF transcriptional complex to the CD274 gene promoter in glioblastoma immune evasion. Consistent with this finding, Deng et al.119 identified β-catenin as a transcriptional factor for PD-L1. An innate immune effector, ISG12a, inhibits Wnt/β-catenin signaling by inhibiting proteasomal degradation of Axin, thereby repressing immune checkpoint PD-L1 expression and sensitizing cancer cells to NK cell-mediated killing in HCC and gastric cancer. There are 20%–30% of advanced HCC patients carrying gain-of-function mutations in β-catenin, which is the third most frequent mutation following the hTERT promoter and TP53. This group of patients is unlikely to benefit from immune checkpoint inhibitors34. However, it is promising to utilize alterations in the β-catenin pathway as a biomarker to direct therapy for HCC patients. Checkmate459, a randomized phase III clinical test comparing nivolumab and sorafenib in first-line HCC therapy, failed to meet its primary endpoint, which clearly emphasized the urgent need for patient selection36. Detailed studies of marker candidates can be used to stratify clinical trials and improve guidance for therapeutic decisions for HCC patients.Sorafenib is the standard therapy for patients with advanced HCC. Sorafenib has been shown to suppress Wnt/β-catenin signalingThis review highlights the roles of the Wnt/β-catenin pathway in the TME of HCC, including its complicated roles in immune infiltration, cancer stem cell tumor vascularization, and its interaction with noncellular components in the TME. What increases the difficulty of studying this pathway is that the Wnt/β-catenin pathway also crosstalks with other pathways like Notch, Hedgehog, and Hippo. Furthermore, because of the tumor heterogeneity in HCC, it is reasonable that the diversified activation of the Wnt pathway could exist in tumor nodules, which results in more intricate Wnt signaling in response to variant stress, TKI treatment, and immunotherapy. These heterogeneities may lead to tumor resistance and treatment failure. Thus, designing specific targeted drugs and individualized therapies for different patients according to their tumor molecular typing and risk factor is warranted.CTNNB1 are involved in liver tumor development. Precise modulation of Wnt/β-catenin is therefore essential to equilibrize anticancer effectiveness and adverse events, to address the challenges for advancing and prospective clinical tests. In spite of these fears, new modulators of the Wnt/β-catenin signaling provide us with opportunities to enhance our understanding of this extremely complex pathway, and may be used to treat Wnt-related diseases including tumors. Although several traditional Chinese medicines have been shown to inhibit Wnt/β-catenin signaling in HCC cells, relevant clinical trials and data are still lacking. The efficacy of most Chinese herbs is often dependent on a variety of components directed to multiple targets, including altering the TME and the systemic immune system. It is therefore challenging to develop a single active ingredient from traditional Chinese medicine. Considering that Wnt/β-catenin mutations exert effects on the therapy of HCC patients, including sorafenib and immune checkpoint inhibitors, they can be used as biomarkers to predict resistance to therapies and applications of Wnt modulators in combination with immunotherapy regimens for the development of novel cancer treatments. It is also worth using traditional Chinese medicine combined with immunotherapy to treat HCC, in view of their inhibitory effects on Wnt signaling. The application of Wnt modulators in preclinical and clinical studies remains to be corroborated and deserves further exploration in advanced HCC. A precise and detailed understanding of the contributions of Wnt/β-catenin signaling to diverse compositions in the TME in HCC is a prerequisite to thoroughly comprehend data from preclinical and clinical studies, to provide a foundation for conceiving better therapeutic regimens targeting Wnt/β-catenin signaling in HCC. It is promising that the emerging single-cell-based omics technology, including but not limited to scRNA-seq, scATAC-seq, single-cell mass cytometry, and spatial transcriptome analysis, have enabled microscopic interpretation of cellular behaviors and immune mechanisms. These new techniques have helped us to understand tumor heterogeneity, immune characteristics of tumors, the TME, tumor recurrence, and cancer treatment more comprehensively.Tumors with mutations in the Wnt/β-catenin pathway are characterized by immune-exclusion in the TME. Both the activation and inactivation mutations of