Stem cell therapy has been extensively studied to improve heart function following myocardial infarction; however, its therapeutic potency is limited by low rates of engraftment, survival, and differentiation. Here, we aimed to determine the roles of the β-catenin/Oct4 signaling axis in the regulation of long-term survival and angiogenesis of peripheral blood mesenchymal stem cells (PBMSCs). These cells were obtained from rat abdominal aortic blood. We showed that β-catenin promotes the self-renewal, antiapoptotic effects, and long-term survival of PBMSCs by activating the Oct4 pathway through upregulation of the expression of the antiapoptotic factors Bcl2 and survivin and the proangiogenic cytokine bFGF and suppression of the levels of the proapoptotic factors Bax and cleaved caspase-3. β-Catenin overexpression increased Oct4 expression. β-Catenin knockdown suppressed Oct4 expression in PBMSCs. However, β-catenin levels were not affected by Oct4 overexpression or knockdown. Chromatin immunoprecipitation assays proved that β-catenin directly regulates Oct4 transcription in PBMSCs. In vivo, PBMSCs overexpressing β-catenin showed high survival in infarcted hearts and resulted in better myocardial repair. Further functional analysis identified Oct4 as the direct upstream regulator of Ang1, bFGF, HGF, VEGF, Bcl2, and survivin, which cooperatively drive antiapoptosis and angiogenesis of engrafted PBMSCs. These findings revealed the regulation of β-catenin in PBMSCs by the Oct4-mediated antiapoptotic/proangiogenic signaling axis and provide a breakthrough point for improving the long-term survival and therapeutic effects of PBMSCs. Boosting expression of a specific gene has allowed researchers to generate stem cells with increased capacity for tissue repair after a heart attack. Several studies have shown that treatment with a population of circulating cells known as ‘peripheral blood mesenchymal stem cells’ (PBMSCs) can regenerate cardiac tissue. These cells generally have a short lifespan when used therapeutically, but researchers led by Shaoheng Zhang at Jinan University in Guangzhou China have increased long-term survival and performance by boosting expression of the gene encoding β-catenin, a protein that promotes cell survival and proliferation. PBMSCs expressing increased levels of β-catenin preserved heart function in a rat model of heart attack, stimulating blood vessel growth and improving animal survival. This study also reveals proteins regulated by β-catenin, which could potentially be exploited for finer control of PBMSC function. Determining the mechanisms sustaining long-term proliferation and survival of MSCs is fundamental to developing new therapeutic treatments for MI.Coronary heart disease is one of the deadliest and most prevalent diseases worldwide. Acute myocardial infarction (MI) is commonly encountered in hospitals and has high short-term and long-term morbidity and mortality rates. Mesenchymal stem cells (MSCs) are a promising cell type being evaluated for patients with MI. Peripheral blood MSCs (PBMSCs) have biological functions similar to those of bone marrow MSCs. However, the therapeutic potency of transplanted MSCs is limited by low rates of engraftment, survival, and differentiation2, is related to the transcriptional coactivation of molecules in the Wnt signaling pathway3. β-Catenin is vital for cancer stem cell (CSC) maintenance and self-renewal5. Inhibition of Wnt/β-catenin decreased the proliferation, survival, and clonogenic activity of T-cell acute lymphoblastic leukemia cells6. However, given the effects of β-catenin on CSCs, the precise role of Wnt/β-catenin signaling in MSC regulation remains unclear.β-Catenin, an important factor in leukemia stem cell activation and glioma progression8. Oct4, a POU isotopic transcription factor, is commonly expressed and activated in CSCs9. As an important protective factor for survival, Oct4 promotes MSC proliferation and inhibits cell apoptosis10. Previously, we suggested that Oct4 promotes the release of cytoprotective factors, such as basic fibroblast growth factor (bFGF), survivin, and Bcl2 (B-cell lymphoma-2). Therefore, Oct4 signaling might be the main contributor to maintaining the survival and function of MSCs11. However, whether Oct4-promoted MSC growth and antiapoptotic effects are regulated by β-catenin remains to be confirmed.Wnt/β-catenin signaling might affect CSC biology by modulating the expression and activity of pluripotency and self-renewal-related transcription factors, namely, octamer-binding protein 4 (Oct4), Nanog, SRY box 2 (Sox2), and c-MycBased on the above considerations, we aimed to compare PBMSCs with β-catenin/Oct4 overexpression or knockdown. The protective effects of these PBMSCs in animal models of MI were also examined. Furthermore, we assessed the antiapoptotic and proangiogenic effects of β-catenin on PBMSCs by activating the Oct4 pathway.An expanded “Materials and methods” section containing details regarding the isolation, culture, purification, and characterization of PBMSCs; cell treatments and groups; analysis of cell proliferation and apoptosis; enzyme-linked immunosorbent assays (ELISAs); quantitative real-time reverse transcription-PCR (qRT‒PCR); immunoblotting; immunocytofluorescence; fluorescence-activated cell sorting (FACS); animal allocation; the animal model; the study design; gene transfer; cell transplantation; echocardiography; histology; terminal deoxynucleotidyl transferase nick-end-labeling (TUNEL) staining analysis; and statistics is available in the Online Data Supplement.Supplementary Table http://www.nap.edu/).Inbred Lewis rats were used. The Animal Care and Use Committee of GuangZhou Red Cross Hospital Medical College of Ji-Nan University approved all animal experiments , which were carried out in compliance with the Guide for the Care and Use of Laboratory Animals published by The National Academies Press : one group of rats underwent surgery on their backs, and the other group of rats received no surgery. PBMSCs were isolated from rat abdominal aortic blood and cultured via the adherent culture method, as described previously12. Fourth-generation cells were used for subsequent experiments and were assessed for purity, viability, characteristics, pluripotency, and gene transfection.Rats were randomly divided into two groups  markers CD34 and CD4514; and the endothelial progenitor cell (EPC) molecular markers CD31 and CD13315. Mouse IgG1, IgG2a, and IgG2b were used as isotype controls.The characteristics of PBMSCs were determined using FACS  to detect marker expression. The characteristics of the PBMSCs were confirmed using antibodies recognizing the MSC-associated cell surface markers CD44, SH3 [CD71], CD90, and SH2 [CD105]16. For chondrogenesis, PBMSCs were induced using a chondrogenic differentiation medium kit  and evaluated using alcian blue staining17. For adipogenesis, PBMSCs were cultured using an adipogenic differentiation medium kit . The formation of lipid vacuoles was assessed by oil red O staining18. For vascular differentiation, the growth factors bFGF  and vascular endothelial growth factor (VEGF)  were added. Angiogenesis was detected using immunofluorescence with double-positive staining for factor VIII and alpha smooth muscle actin (α-SMA)11.The PBMSCs underwent directed differentiation toward osteogenesis, chondrogenesis, adipogenesis, and angiogenesis by growth factor supplementation and growth on defined matrices. For osteogenesis, PBMSCs were induced with an osteogenic differentiation medium kit , and alizarin red staining was performed to evaluate osteogenic productsoe) β-catenin (encoding β-catenin) or Oct4 were transfected together with pReceiver-LV233 lentiviral vector  into PBMSCs using Fugene HD reagent  as directed by the manufacturer’s instructions. A pSi-LVRU6GP vector with a puromycin resistance cassette  was used to express short hairpin (sh) RNAs to knock down β-catenin and Oct4 expression.Retroviral plasmid vectors, pMXs, overexpressing (β-catenin overexpression group (oeβ-catenin), the β-catenin knockdown group (shβ-catenin), and the β-catenin overexpression+β-catenin knockdown group (oeβ-catenin plus shβ-catenin). To determine the effect of Oct4 on the proliferation and survival of PBMSCs, we divided the cells into the no intervention group, the Oct4 overexpression group (oeOct4), the Oct4 knockdown group (shOct4), and the Oct4 overexpression+Oct4 knockdown group (oeOct4 plus shOct4). To evaluate the effects of β-catenin and Oct4 on cell growth and apoptosis, we added oeβ-catenin+shOct4 and shβ-catenin+oeOct4. After transfection, all groups of PBMSCs were cultured for 70 days for subsequent experiments to analyze the growth and apoptosis of PBMSCs.To determine the effect of β-catenin on the proliferation and survival of PBMSCs, we randomly divided the cells into the following four groups according to different intervention methods: the no intervention group (serving as the control group), the oeβ-catenin or a β-catenin agonist , and β-catenin inhibition was carried out by shβ-catenin or a β-catenin inhibitor .To investigate the mechanisms of β-catenin-mediated cytoprotective effects against apoptotic cell death, we performed β-catenin activation in PBMSCs using 3 cells/well on a 96-well plate at 37 °C and cultured for 48 h. Cell viability was determined by the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method in accordance with the CellTiter 96 Aqueous One Solution Viability assay manual .For the cell viability assay, PBMSCs were seeded at 2 × 103 cells/well onto 24-well plates in a 5% CO2 atmosphere at 37 °C. According to the number of cells corresponding to each time point , the growth curve was drawn, and the population doubling (PD) of PBMSCs was determined.PBMSCs were seeded at a density of 2 × 10For each group, 500 cells were transferred to 6-well plates after transfection. A cell mass containing more than 50 cells was identified as a cell colony, and the number of cell colonies was counted under a phase contrast microscope .Cells on slides were sealed with parafilm and stained with X-gal staining solution . The stained and unstained cells were counted using a phase contrast microscope (Olympus). The percentage of senescent cells (blue) among the total cells in the field of view was calculated.Cells for cell cycle analysis were stained with Annexin V-APC and propidium iodide (PI) and analyzed using flow cytometry with a flow cytometer . Data were analyzed using BD ModFit LT version 3.3 (BD Biosciences).6 cells were used in each ChIP experiment. ChIP assays were performed according to the manufacturer’s protocol from a ChIP assay kit . The DNA samples were detected using real-time PCR analysis. To amplify the β-catenin binding site in the Oct4 promoter, we used the primer sequences shown in Supplementary Table Approximately 2.0 × 10Cells or tissues were fixed with 4% paraformaldehyde (Sigma). Immunofluorescence staining was performed by incubation with antibodies recognizing β-catenin, Oct4, Factor VIII, α-SMA, Angiopoietin 1 (Ang1), or VEGF  as the control19.Total mRNA was extracted according to standard protocols provided by Invitrogen  and served as a template for reverse-transcription (RT)-PCR, following standard protocols from Promega. cDNA levels representing gene expression were then calculated using quantitative real-time PCR. The primer sequences used are shown in Supplementary Table Cells or hearts were collected and pulverized to extract protein for immunoblotting. Supplementary Table oeβ-catenin, shβ-catenin, oeOct4, shOct4, or control vectors, cells were cotransfected with a lentiviral vector containing enhanced green fluorescent protein (EGFP) cDNA, as described previously20. More than 70% of PBMSCs were GFP-positive, as determined by flow cytometry.At 24 h after transfection with the oeβ-catenin, shβ-catenin, oeOct4, shOct4, or control vectors. Cell transplantation was achieved by injection into the infarct and peri-infarct regions . To minimize postoperative pain, we sprayed 2.5% bupivacaine at the point of incision immediately before closure, and buprenorphine hydrochloride (0.03 mg/kg) was administered intramuscularly. After the final layer of skin was closed, triple antibiotic ointment  was applied to the wound. Cyclosporin A  was administered daily  from the first day after MI until the animals were sacrificed on Day 90. Finally, twenty animals were studied in each subgroup.Inbred Lewis rats were used. After anesthesia with an intraperitoneal injection of 3% sodium pentobarbital, MI was induced in the rats by ligating the left anterior descending coronary artery. Animals with an ejection fraction (EF) <70% and fractional shortening (FS) <35%, as evaluated using echocardiography after induction of MI, were selected. After establishment of the MI model, the animals randomly received an injection of phosphate-buffered saline (PBS) or PBMSCs pretreated with Under general anesthesia, as described above, rats underwent echocardiography in a 7.5-MHz phased-array transducer  operated by an experienced technician blinded to the treatment group identity. Two-dimensional images were obtained at the midpapillary and apical levels. The left ventricular end-diastolic volume (LVEDV) and internal diameter at diastolic phase (LVEDD) were measured using the biplane area-length method. LV FS was calculated according to the modified Simpson method: FS (%) = [(LVIDd-LVIDs)/LVIDd] × 100, where LVID is the LV internal dimension, s is systole, and d is diastole. All measurements were averaged for three consecutive cardiac cycles.At the end of each study (90 days after cell transplantation), rats were sacrificed by cervical dislocation, and then, the hearts were perfused with 4% buffered formalin, harvested, and sectioned into 2 transverse slices parallel to the atrioventricular ring. Myocardial tissue sections were randomly chosen from five animals for triphenyltetrazolium chloride (TTC) staining. The infarct size was determined by calculating the percentage of the infarcted area against the whole LV area using Scion ImageJ . The peri-infarct regions from the MI model rats and cell therapy rats were embedded in paraffin, sectioned, and stained with Masson trichrome staining, hematoxylin and eosin (H&E), and immunofluoroscence. In each case, five independent images from each area were analyzed from each section.Blood samples were collected from rats by cardiac puncture under anesthesia. The levels of Ang1, bFGF, hepatocyte growth factor (HGF), and VEGF in the supernatant of heart tissues and the plasma of rats were measured using an ELISA kit according to the manufacturer’s instructions.11. The collected cells were washed with PBS and analyzed with flow cytometry (Becton Dickinson). Engraftment was evaluated by determining the proportion of cells that expressed EGFP relative to all isolated ventricular cells. Vascular differentiation was evaluated by calculating the proportion of cells that expressed both EGFP and CD31 relative to all EGFP-positive cells. Immunofluorescence staining was performed to evaluate the expression levels of factor VIII, Ang1, and VEGF on all EGFP-positive cells using the appropriate antibodies.Heart tissues were immunostained for EGFP-PBMSCs, and the cardiomyocyte marker protein troponin T was detected to accurately verify the retention of transplanted PBMSCs. Cells were collected from the left ventricles of five randomly selected hearts per experimental group, as described previouslyP < 0.05) was considered significant.Data are presented as the mean ± standard error of the mean (SEM). Discrete variables are presented as the frequency and proportion. After performing a normality test (Shapiro–Wilk test) and homogeneity test of variance, we used the data that satisfied a normal distribution and equal variance assumptions for one-way analysis of variance (ANOVA) of these variables. When the data conformed to a normal distribution but had nonhomogeneity of variance, Welch ANOVA was performed. Comparisons were performed using the chi-squared or Fisher’s exact test for discrete variables. A 95% confidence interval (CI)  had a homogeneous and fibroblast-like morphology Fig.  and wereTo perform a more comprehensive analysis of the multipotent differentiation of PBMSCs, we performed direct differentiation toward adipocytes, chondrocytes, osteoblasts, and vascular cells by growth factor supplementation and growth on defined matrices. PBMSCs cultured under adipogenic conditions presented cytoplasmic lipid droplets under light microscopy. The culture was stained with oil red O to confirm that the contents of the droplets were lipids Fig. . Aggrecaoeβ-catenin), β-catenin shRNA (shβ-catenin), or oeβ-catenin plus shβ-catenin or vehicle (control). After 7 days of culture, the colony number in the oeβ-catenin-treated PBMSCs was 1.5-fold and 1.8-fold greater than that in the vehicle- and the shβ-catenin-treated PBMSCs, respectively -β-galactosidase  staining. The number of SA-β-Gal-stained cells was markedly smaller in the ups Fig. . The ratoup Fig. . The antoup Fig. . However21. To determine the underlying mechanism of Oct4 in β-catenin-mediated survival of PBMSCs, we analyzed key TFs related to stem cell stemness. We found that β-catenin overexpression markedly increased Oct4 mRNA expression in the oeβ-catenin-treated PBMSCs, whereas Oct4 mRNA was almost undetectable in the β-catenin-deficient PBMSCs , is essential for bone marrow MSC self-renewaloeOct4) or Oct4 shRNA (shOct4) or vehicle or cotransfected with oeOct4 plus shOct4. After 7 days of culture, we observed that Oct4 overexpression markedly increased colony formation in the oeOct4 group and Oct4 knockdown significantly decreased colony formation in the shOct4 group compared with the vehicle group  overexpression of Oct4 led to a substantial increase in Oct4 expression, while β-catenin expression was similar in the groups receiving oeOct4 or shOct4  or lentiviruses carrying targeted β-catenin/Oct4 shRNA (shβ-catenin/shOct4).To provide further mechanistic insights into β-catenin/Oct4-mediated cytoprotective effects against apoptotic cell death, we assessed antiapoptotic and proapoptotic molecules. First, PBMSCs were treated with a β-catenin agonist or inhibitor. The addition of the β-catenin agonist to the culture media strongly enhanced the expression of β-catenin, Oct4, and the antiapoptotic molecules bFGF, survivin, and Bcl2 and decreased the expression of the proapoptotic molecules Bax and caspase-3. Suppression of β-catenin by its inhibitor had the opposite effects in PBMSCs. However, the β-catenin agonist or inhibitor did not significantly change glycogen synthase kinase 3 beta (GSK3β) expression  or in combination with shOct4 or oeOct4 lentiviral vectors, only shOct4 or only oeOct4 was randomly transplanted into the infarcted hearts. The rats with MI were also randomly assigned to receive PBS injection as a control group. The 160 animals were randomly divided into eight groups and underwent serial echocardiography studies. Thereafter, all animals were followed-up for 90 days, during which 46 rats died. The surviving 114 rats were then sacrificed and subjected to pathology and molecular biology tests. After 90 days, Kaplan–Meier survival analysis showed a higher survival rate in the rats receiving oeβ-catenin- or oeOct4-treated PBMSC transplantation than in the rats receiving PBS injection and PBMSC therapy alone , and this effect was eliminated in the rats receiving shβ-catenin or shOct4. Moreover, the inhibition resulting from β-catenin knockdown was rescued by Oct4 overexpression (shβ-catenin+oeOct4). However, no significant differences were found between the animals receiving oeβ-catenin- or oeOct4-treated PBMSCs and between those receiving PBMSCs pretreated with shβ-catenin or shOct4 . The indices in the rats receiving PBS indicated sustained exacerbation, with decreased LVFS, dilation of LVEDD and LVEDV, and thinning of the LVAWd. There was no significant difference between the animals receiving oeβ-catenin- or oeOct4-treated PBMSCs and those receiving PBMSCs pretreated with shβ-catenin or shOct4. Thus, overexpression of β-catenin/Oct4 in PBMSCs enhanced their ability to improve cardiac remodeling after MI.Echocardiographic studies showed that on Day 1 post-infarct, the animals in all eight groups developed typical changes of acute heart failure and LV early remodeling compared with baseline levels. These changes included decreases in the cardiac function indices left ventricular fractional shortening (LVFS), dilated LVEDD and LVEDV and thinning of the left ventricular end-diastolic anterior wall thickness (LVAWd) Fig. . Althougoeβ-catenin or oeOct4 groups than in the PBS group and the PBMSC alone group , and adding oeOct4 rescued the beneficial effects (shβ-catenin+oeOct4). Taken together, these data suggest that the β-catenin/Oct4 axis in ischemic hearts improves PBMSC-mediated restoration of cardiac function and structure.Postmortem morphometry revealed that TTC staining at Day 90 following MI showed transmural infarct scars, mostly in the apex, anteroseptal, and anterior wall in the PBS-treated rats Fig. . The infoup Fig. . Multiploup Fig. . Transploup Fig. , accompaSCs Fig. . Microscion Fig. . However+ cells were highly enriched in the ischemic hearts receiving PBMSCs transfected with oeβ-catenin or oeOct4 but were sparse in the control vector-treated (−) ischemic hearts. However, β-catenin or Oct4 deficiency significantly reduced PBMSC engraftment. EGFP+ PBMSCs with oeβ-catenin or oeOct4 exhibited much higher engraftment in the whole infarcted heart than PBMSCs with control vectors or shβ-catenin . Consistently, β-catenin transfection significantly increased the retention of EGFP+ PBMSCs compared with transfection with control vectors, but this effect was abolished by Oct4 knockdown . The levels of the proapoptotic molecules Bax and caspase-3 decreased markedly in the rats with MI that received PBMSCs pretreated with β-catenin/Oct4 overexpression . In addition, the overexpression or knockdown of β-catenin or Oct4 caused corresponding changes in the expression of Oct4, but alteration of Oct4 resulted in no significant change in β-catenin expression. All these findings suggest that β-catenin overexpression promotes the long-term engraftment of PBMSCs in ischemic hearts by activating Oct4-related antiapoptotic signaling.Then, we tested whether activated β-catenin/Oct4 signaling was associated with increased expression of its related apoptotic signaling molecules. Under conditions of β-catenin overexpression, the mRNA and protein levels of β-catenin, Oct4, and antiapoptotic molecules (Bcl2 and survivin) increased in cardiac tissue from the rats receiving ion Fig. , suggest22; therefore, we investigated the roles of β-catenin/Oct4 signaling in regulating angiogenesis of PBMSCs to provide mechanistic insights into myocardial repair. The blood vascular density was markedly higher in the rats receiving the oeβ-catenin-treated MSCs than in the animals receiving PBS injection or PBMSCs alone; however, deficiency of β-catenin or Oct4 (shβ-catenin or oeβ-catenin+shOct4) significantly decreased the blood vascular density . This finding indicated that treatment of PBMSCs with β-catenin overexpression before cell transplantation markedly enhanced their angiogenic potential by activating Oct4.Angiogenesis is a major determinant of myocardial repair induced by MSCsity Fig. . In addioeβ-catenin/oeOct4 group compared with the control vector group, and shβ-catenin or shOct4 appeared to significantly decrease the levels of these proteins  and showed low expression of HSC markers (CD34 and CD45) and EPC markers (CD133 and CD31), which was consistent with that described for rat bone marrow MSCs31. The results showed that these cells mainly consisted of MSCs, excluding HSC and EPC contamination of the primary PBMSCs. Third, regarding vascular differentiation, PBMSCs produce vascularization, which is a critical feature of angiogenesis in the ischemic myocardium32. The identification of factors that enhance cardiac repair by the restoration of the vascular network is important33. Therefore, we concluded that PBMSCs have potential as grafted cells for myocardial repair.In this study, we proposed peripheral blood as an alternative, noninvasive source of rat MSCs on the basis of the following observations. First, PBMSCs were successfully isolated from peripheral arterial blood, which was consistent with our previous study in which MSCs were collected from the peripheral vein and the culprit coronary artery of MI patients34. However, a potential drawback of using PBMSCs is poor survival, which could limit their efficacy for ischemic myocardial repair35. In our in vitro experiments, PBMSCs revealed gradual senescence and apoptosis over culture time. Therefore, we developed a novel strategy to promote their long-term survival.Autologous applications for regenerative therapies are an attractive approach because PBMSCs provide an autologous, effective, alternative, and readily available cell source to treat patients with MI36. Recent studies have suggested that Wnt/β-catenin signaling maintains the self-renewal and tumorigenicity of head and neck CSCs by activating Oct437. In this study, PBMSCs overexpressing β-catenin showed increased viability/survival and decreased senescence/apoptosis even after 70 days of culturing. These findings were in line with observations in other stem cells40. Hoffmann et al. found that agonism of Wnt/β-catenin signaling promotes MSC expansion41. Moreover, β-catenin deficiency caused the opposite effect, and simultaneous overexpression and knockdown of β-catenin resulted in no significant change. All these data confirm that β-catenin overexpression has long-term advantages for stem cells and can be considered a feasible alternative to improve the viability/survival of PBMSCs in vivo.β-catenin, the prime effector of canonical Wnt signaling, expresses numerous genes responsible for cell proliferation, differentiation, and survival42. We found that following β-catenin activation in PBMSCs, only Oct4 was produced at high levels. Other stem cell factors showed no significant change in response to altered β-catenin expression, suggesting that β-catenin specifically activates Oct4 expression. Notably, whether the Oct4 levels were high or low, β-catenin levels did not change following alteration of Oct4 expression. However, modification of β-catenin/Oct4 expression caused significant changes in Oct4 expression. The ChIP assay showed that β-catenin directly activated Oct4 transcription in PBMSCs. These findings provide evidence that Oct4 functions as a downstream effector of β-catenin signaling in PBMSCs. In addition, Oct4 was revealed to function as a downstream target of β-catenin/T-cell factor (TCF) in Wnt-activated mouse embryonic stem cells43.Based on the antiapoptotic effect of β-catenin, activation of β-catenin expression showed a more dramatic response to β-catenin overexpression than no intervention (vehicle). β-Catenin signaling maintains the growth of CSCs and directly modulates the expression of Nanog and Oct444. In the present study, Oct4 overexpression resulted in increased expression levels of the antiapoptotic proteins Bcl2 and survivin and decreased expression levels of the apoptosis-related proteins Bax and caspase-3. Moreover, high expression of Oct4 increased proliferation and decreased apoptosis of PBMSCs. The higher levels of Oct4, Bcl2, and survivin after β-catenin overexpression would explain the long-term engraftment of PBMSCs in the ischemic hearts. In addition, Oct4 overexpression ameliorated the reduced engraftment and apoptosis of PBMSCs induced by β-catenin deficiency, similar to the long-term survival role for Oct4 in embryonic stem cells45. Taken together, these findings provide mechanistic insights into the promotive effect of β-catenin/Oct4 signaling on PBMSC long-term survival, thereby illustrating the possibility of activating β-catenin/Oct4 signaling to improve the poor survival of PBMSCs for myocardial repair after MI.As a transcription factor, Oct4 activates the expression of several downstream genes involved in cell proliferation and survival46. A similar finding in the rats with MI receiving PBS injection revealed a progressive decline in cardiac ejection fraction, progressive enlargement of the left ventricle, gradual thinning of the ventricular wall, and expansion of the infarct size. The infarcted hearts clearly showed inflammation and fibrosis. Although transplantation of PBMSCs alone transiently prevented ischemia-induced aggravation of cardiac function and structure at 30 d post-MI, these benefits were not sustained at 90 d post-MI. PBMSC therapy alone did not significantly reduce the infarct size, MI-induced fibrosis, myocyte loss, or inflammation. However, using a lentivirus encoding β-catenin, we demonstrated that β-catenin, via Oct4 signaling, persistently maintained cardiac function and ameliorated cardiac remodeling by improving PBMSC engraftment and decreasing PBMSC apoptosis, which resulted in increased 90-day survival of the rats with MI receiving β-catenin/Oct4-treated MSCs compared with that of the PBS or vehicle control groups. Correspondingly, high β-catenin/Oct4 expression increased the levels of antiapoptotic proteins, supporting the view that β-catenin/Oct4 signaling is associated with long-term engraftment and myocardial repair of PBMSCs in infarcted hearts. Importantly, we showed that Oct4 knockdown by transfection of an Oct4 shRNA completely eliminated the cardiac effects of both β-catenin overexpression and PBMSC therapy (oeβ-catenin+shOct4), as shown by worsening cardiac function, augmented fibrosis, myocardial infarct size and loss, and decreased myocardial engraftment of PBMSCs. Stimulating β-catenin also helped to sustain somatic cell survival and proliferation by activating Oct447. Collectively, these findings indicated that β-catenin plays a crucial role in rescuing stem cell fate and maintaining cardiac function by inducing an Oct4-dependent antiapoptotic mechanism.Therefore, we further investigated the effects of β-catenin/Oct4 signaling on myocardial repair induced by PBMSC therapy. The hearts of people with MI undergo cardiac remodeling, characterized by an expanded myocardial infarct size and a dilated left ventricle48. Our results in ischemic hearts revealed that high β-catenin expression resulted in high levels of proangiogenic cytokines and stimulated PBMSC vascular differentiation, which contributed to the formation of blood vessels, indicating the contribution of mesenchymal-endothelial transition (MEndoT) to angiogenesis49. In the rats receiving oeβ-catenin-treated PBMSCs, Oct4 was upregulated in ischemic cardiomyocytes to an optimal extent, which promoted the secretion of proangiogenic cytokines . This phenomenon increased angiogenesis, improved cardiac function, and inhibited cardiac remodeling. Furthermore, Oct4 overexpression alleviated β-catenin knockdown-induced injury in PBMSCs, as shown by preserved paracrine function and MEndoT. In contrast, Oct4 deficiency completely eliminated PBMSC angiogenesis induced by β-catenin overexpression, as evidenced by decreased vascularization, reduced vascular density, and decreased β-catenin-mediated proangiogenic cytokines. Our previous study demonstrated that Oct4-dependent FoxC1 activation improves the survival and neovascularization of MSCs under myocardial ischemia50. All these data might reveal a novel mechanism: β-catenin ameliorated ischemia-related impairment of PBMSC-mediated angiogenesis by inducing MEndoT via an Oct4-dependent mechanism. This issue will be an important subject for future investigation. Our findings provide a rationale for the ongoing clinical evaluation of combinatorial therapies comprising genetic modification and stem cell transplantation51.In addition, the β-catenin pathway controls the maintenance of CSCs in many tissues and organs by regulating angiogenesis, epithelial-mesenchymal transition, and the microenvironment52. Yan et al. demonstrated that N-cadherin overexpression enhances adipose tissue-derived MSC (ADSC)-protective effects against ischemic heart failure via β-catenin-mediated MMP-10/MMP-13/HGF expression and production, promoting ADSC/cardiomyocyte adhesion and ADSC retention53. These conflicting reports suggest that the combination of β-catenin overexpression with other strategies  may have better benefits for cell therapy and warrants further dedicated investigation. Second, compared with those from uninjured rats, PBMSCs from the peripheral blood of the traumatized rats showed greater proliferation, but they grew slowly and underwent an ~2-fold increase in PD on the 2nd-day post-culture. The average population doubling time of MSCs from adipose, placenta, bone marrow, umbilical cord, and amnion was less than 2 days under appropriate culture conditions28. This phenomenon might greatly hinder their clinical application, given that cultures must be expanded to obtain a sufficient number of cells for transplantation. However, cell doubling rapidly increased, by over 2-fold of PD, in less than 2 days after transfection of β-catenin or Oct4. This finding suggests that in vitro culture in combination with genetic engineering can be expanded to generate a sufficient number of PBMSCs for clinical application.Two limitations exist in the present study. First, we observed that β-catenin agonists or inhibitors mainly affected β-catenin/Oct4-specific antiapoptotic/proapoptotic protein expression but did not significantly change GSK-3β expression. These findings are inconsistent with those of previous studies. A GSK inhibitor or N-cadherin antibody treatment successfully enhanced β-catenin signaling and resulted in significant increases in mature cardiomyocyte proliferationFinally, we showed that β-catenin can improve the long-term survival and angiogenesis of PBMSCs via Oct4 signaling, which considerably promoted the effects of stem cell therapy in ischemic hearts, opening up further options for therapeutic rescue experiments.Supplemental material


The G protein–coupled receptor melanocortin-4 receptor (MC4R) and its associated protein melanocortin receptor–associated protein 2 (MRAP2) are essential for the regulation of food intake and body weight in humans. MC4R localizes and functions at the neuronal primary cilium, a microtubule-based organelle that senses and relays extracellular signals. Here, we demonstrate that MRAP2 is critical for the weight-regulating function of MC4R neurons and the ciliary localization of MC4R. More generally, our study also reveals that GPCR localization to primary cilia can require specific accessory proteins that may not be present in heterologous cell culture systems. Our findings further demonstrate that targeting of MC4R to neuronal primary cilia is essential for the control of long-term energy homeostasis and suggest that genetic disruption of MC4R ciliary localization may frequently underlie inherited forms of obesity.  MC4R are the most common cause of monogenic obesity in humans , a member of the MRAP family composed of single-pass transmembrane proteins that interact with melanocortin receptors  and othe–/– mice , which b–/– mice , 22.Here, we find that the central mechanism of MRAP2-associated obesity is the critical role for MRAP2 in targeting MC4R to cilia.Mrap2–/– mice, leads to obesity, although to a lesser extent than that observed in Mc4r–/– mice (Mrap2-KO–first (“tm1a”) allele and confirmed that, as previously reported (–/–Mrap2) (https://doi.org/10.1172/jci.insight.155900DS1) were obese and hyperphagic at 12 weeks of age but did not differ from their WT littermates at 4 weeks of age   . Mrap2fltermates .Mc4r-t2a-Cre (fl/flMrap2 mice (hereafter referred to as t2aCre/t2aCreMc4rfl/flMrap2). t2aCre/t2aCre Mrap2fl/flMc4r mice fed ad libitum regular chow developed early-onset obesity with significantly higher body weight   . In thesb-GFPtg) .gfpMc4r), allowing for the assessment of the subcellular localization of the endogenous MC4R. In the PVN of gfpMc4r mice, most MC4R localized at primary cilia  was decreased in  with WT , and MC4 with WT . Thus, MMrap2–/–gfpMc4r mice could be secondary, resulting from developmental effects. We therefore determined whether acute deletion of Mrap2 in the PVN of adult mice would affect MC4R ciliary localization. We injected fl/fl Mc4rgfpMrap2 mice unilaterally with an adeno associated virus (AAV) encoding mCherry-IRES-Cre. Mrap2 was deleted from the infected PVN by Cre-mediated recombination, and the contralateral uninfected PVN served as an internal control  in the presence or absence of MRAP2. SSTR3 is a cilia-localized GPCR that is also expressed in the PVN , includiIft88 in adult mice leads to obesity — either when deleted ubiquitously, specifically in neurons, or only in the PVN — directly implicating PVN cilia in the control of feeding behavior . IMCD3 cells were transfected using X-tremeGENE 9 DNA Transfection Reagent . The transfection reagent was diluted in OptiMEM (Invitrogen) and incubated at room temperature for 5 minutes. Then, the mixture was added to the diluted plasmids in a 6:1 ratio (6 μL transfection reagent to 1 μg DNA) and incubated at room temperature for 20 minutes. In total, 50,000 cells in suspension were added to the transfection mixture in a 24-well plate. Transfected cells were switched to starvation media after 24 hours and fixed 16 hours afterward. Double plasmid transfections were performed by diluting equal mass of each vector. Three independent transfections were carried out per condition.In total, 50,000 cells were seeded for transfection on acid-washed 12 mm #1.5 cover glass  in a 24-well plate. Starved cells were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde (Electron Microscopy Sciences) for 15 minutes at room temperature and permeabilized in ice-cold 100% methanol (Thermo Fisher Scientific) for 5 minutes. Cells were then further permeabilized in PBS containing 0.1% Triton X-100 , 5% normal donkey serum , and 3% BSA  for 30 minutes. Permeabilized cells were incubated with specified antibodies  for 1 hour, washed with PBS, and incubated with dye-coupled secondary antibodies (Jackson ImmunoResearch) for 30 minutes. Cells were then washed with PBS, stained with Hoechst DNA dye, and washed with PBS before mounting with Fluoromount G (Electron Microscopy Sciences).ΔTATA:MRAP2-3xFLAG-T2A-IgK-HA-Halo-MC4R-3xmNeonGreen). The parental line, IMCD3 FlpIn, was used as negative control. Both cell lines were stained as mentioned above with rabbit anti-MRAP2  and mouse anti–acetylated tubulin . MC4R-3xNeonGreen was detected by endogenous fluorescence.The anti-MRAP2 antibody was validated in a stable clonal cell line expressing MC4R-3xNeonGreen and MRAP2-3xFlag. The 2 proteins are produced in stoichiometric amount as they are encoded within a single mRNA and separated by a self-cleaving T2A peptide (pEFα×/1.40NA objective lens (Olympus), a pco.edge 4.2 sCMOS camera, a solid state illumination module (Insight), and a Quad polycroic (Chroma). Z stacks with 0.2 μm separation between planes were acquired using SoftWoRx. The illumination settings were: 140 μW 390 nm wavelength for 0.15 seconds to image Hoechst, 222 μW 475 nm wavelength for 0.3 seconds to image Alexa Fluor 488–stained MCRs, 123 μW 543 nm wavelength for 0.3 seconds to image Cy3-stained acetylated tubulin, and 115 μW 632 nm wavelength for 0.15 seconds to image Cy5-stained FLAG-labeled MRAPs. Images were flat field corrected, background subtracted, and maximally projected using Fiji. Ciliary intensity measurements were also taken in Fiji as previously published (Cells were imaged in a widefield fluorescence DeltaVision microscope (Applied Precision) equipped with a PlanApo 60ublished . To compMice were housed in a barrier facility and maintained on a 12:12 light cycle (from 7 a.m. to 7 p.m.) at an ambient temperature of 23°C ± 2°C and relative humidity 50%–70%. Mice were fed with rodent diet 5058 (Lab Diet) and group housed up to 5. Experiments were performed with weight-matched littermates.tm1(egfp)VaiMc4r mice have an EGFP tag inserted in frame at the C-terminus of the endogenous Mc4r locus Vai mice have a -t2a-Cre sequence inserted in frame at the C-terminus of the endogenous Mc4r locus  mice carry an frt-flanked β-gal gene and neo cassette preventing widespread expression of the Mrap2 gene WtsiMrap2/Jsbgj, The Jackson Laboratory). When mice harboring this allele are crossed into an actin-FLpE background, the frt-flanked cassette is excised and MRAP2 WT function is restored . After Flip-mediated excision, a loxP-flanked Exon 4 remains, which allows for MC4R cell-specific deletion when crossed to Mc4r-t2a-Cre–knock-in mice (t2aCre/t2aCre Mrap2tm1c/tmc1Mc4r or t2aCre/t2aCre Mrap2fl/flMc4r). We obtained t2aCre/t2aCre Mrap2fl/flMc4r and t2aCre/t2aCre Mrap2+/+Mc4r littermates by crossing t2aCre/t2aCre Mrap2fll+Mc4r mice. All mice were maintained on a mixed background.EUCOMM fl/flMrap2gfpMc4r females mice (n = 4) were injected unilaterally with pAAV-Ef1a-mCherry- IRES-CRE . Animals were anesthetized with an initial flow of 4% isoflurane (Dechra), maintained under anesthesia using 2% isoflurane, and kept at 30°C–37°C using a custom heating pad. The surgery was performed using aseptic and stereotaxic techniques. Briefly, the animals were put into a stereotaxic frame (KOPF Model 1900), the scalp was opened, the planarity of the skull was adjusted, and a hole was drilled . A volume of 300 nL was injected at a rate of 0.1 μL/min. Animals were given preoperative analgesic  and postoperative antiinflammatory meloxicam  and were allowed to recover at least 10 days, during which time they were single housed and handled frequently. The mice were calorie restricted at 75% for 2 weeks prior to perfusion.Eight-week-old Mice were perfused transcardially with PBS, followed by 4% paraformaldehyde fixation solution. Brains were dissected and postfixed in fixation solution at 4°C overnight, soaked in 30% sucrose solution overnight, embedded in OCT (Tissue-Tek), frozen, and cut into 20–35 μm coronal sections before being stored at –80°C until staining.After washing, sections were blocked for 1 hour in 50% serum , 50% antibody buffer , 1% BSA , 1.8 % L-Lysine , and 0.04% sodium azide (Millipore Sigma), followed by incubation with primary antibody overnight at 4°C . After washing, sections were incubated with secondary antibodies for 1 hour at room temperature , washed, and stained with Hoechst . They were then washed and mounted with Prolong Diamond antifade Mountant .Mrap2 WT mice compared with MRAP2-KO sections . In In Z stack, 0.5 μm steps). The images used to assess the colocalization of MC4R-GFP and MRAP2 in P6 mice were acquired with a Leica SP8 with resonant scanner. The SSTR3 imaging and MRAP2 immunofluorescence in MC4R-GFP adult mice were acquired with a Nikon W1 wide field-of-view spinning disk confocal with Andor Zyla sCMOS camera . Images in Z stack, 0.2 μm steps).The images used for the assessment of MC4R-GFP at the cilium of MRAP2 WT versus KO were acquired with a Leica SP5  or ACTB+ (in vitro) signal. This 2D space was then used to the pixel intensity of the other channels (integrated density [IntDen]). Ciliary intensity of MC4R-GFP was then calculated as the IntDen of MC4R-GFP in the cilium, subtracting adjacent background (measured as IntDen of same defined area near the cilium). To calculate relative cilia enrichment, the IntDen (cilium) was divided by the IntDen (cell body), measured in the closest cell body (as defined by the presence of a Hoechst+ nucleus). Enrichment > 1, therefore, indicates higher localization of the receptor at the primary cilium compared with the cell body. n = 20–40 ciliated cells per condition were imaged and analyzed. Matched 2. It was analyzed with CalR app software , except for EE data that was analyzed using the lm function of R software . P ≤ 0.0All animal procedures were approved by the IACUC of the UCSF.A Bernard, JFR, MVN, and CV designed the research study and wrote the manuscript. A Bernard, ION, and XY conducted experiments and acquired and analyzed data. JC, EM, FBG, A Blake, KK, and SZ conducted experiments. FM analyzed data.


The events occurring from signal to transcription are evolutionarily conserved, and their final output orchestrates countless processes during embryonic development and tissue homeostasis. Importantly, a dysfunctional Wnt/β‐catenin pathway causes developmental malformations, and its aberrant activation is the root of several types of cancer. A rich literature describes the multitude of nuclear players that cooperate with β‐catenin to generate a transcriptional program. However, a unified theory of how β‐catenin drives target gene expression is still missing. We will discuss two types of β‐catenin interactors: transcription factors that allow β‐catenin to localize at target regions on the DNA, and transcriptional co‐factors that ultimately activate gene expression. In contrast to the presumed universality of β‐catenin's action, the ensemble of available evidence suggests a view in which β‐catenin drives a complex system of responses in different cells and tissues. A malleable armamentarium of players might interact with β‐catenin in order to activate the right “canonical” targets in each tissue, developmental stage, or disease context. Discovering the mechanism by which each tissue‐specific β‐catenin response is executed will be crucial to comprehend how a seemingly universal pathway fosters a wide spectrum of processes during development and homeostasis. Perhaps more importantly, this could ultimately inform us about which are the tumor‐specific components that need to be targeted to dampen the activity of oncogenic β‐catenin.Cancer > Molecular and Cellular PhysiologyCancer > Genetics/Genomics/EpigeneticsCancer > Stem Cells and DevelopmentThis article is categorized under: The Wnt/β‐catenin dependent transcription is executed via a complex network of dynamically interacting co‐factors whose reciprocal requirements and timely modes of action we do not fully understand. In the following years the structure and sequence of the gene were elucidated, and its homology with the Drosophila melanogaster segment‐polarity gene wingless (wg) exposed, suggesting a signaling function in the development of multicellular organisms  gene in patients with colorectal cancer (CRC)  was sensitive to the Wingless  ligands secreted from nearby cells  is a family of four transcription factors (TFs) named TCF7, LEF1, TCF7L1, and TCF7L2 . They had been formerly discovered as important regulators of gene expression in immune cells, neural crest, tooth germs, and whisker follicles during embryogenesis  or tighter (heterochromatin) via post‐translational modifications  of the histone proteins. As a consequence, DNA is more or less accessible to transcriptional regulators, respectively. When accessible, transcription factors can make contact with the regulatory regions of specific genes, such as promoters, enhancers, and insulators, and turn genes ON and OFF   secreted from adjacent cells in developing Drosophila embryos  and time .Epistasis refers to a genetic phenomenon that reveals functional relationships between genes. If a loss‐of‐function mutation in gene adherens junction formation and the ability of mESC to differentiate into endoderm and neuroepithelium, but not into mesoderm  lacking β‐catenin maintain pluripotency but fail to generate meso‐endodermal and neuroepithelium layers. A signaling‐defective β‐catenin rescues the 2.4The central role of β‐catenin in the assembly of the nuclear transcriptional complex downstream of WNT signals, recently referred to as Wnt enhanceosome  nuclear translocation of β‐catenin, (ii) activation of target genes, and (iii) body axis duplication—a sign of aberrantly activated Wnt pathway ‐box DNA‐binding domain that displays remarkable amino‐acid sequence conservation . β‐catenin binds their N‐terminus, which is also considerably conserved . It is therefore not surprising that TCF/LEF sustain overlapping functions , which binds to the region of β‐catenin spanning from repeat 10 to repeat 12 . RNF14 was found to interact with TCF/LEF and promote Wnt signaling both in vivo in zebrafish and in human CRC cells, where it is required for their survival  increases in abundance and competes with TCF7L2 for direct binding to β‐catenin. Here, β‐catenin‐HIF1α regulates classical HIF1 target genes that ultimately promote cell survival and adaptation to hypoxia. β‐catenin physically associates with HIF‐1α on target loci, and enhances HIF‐1α‐mediated transcription by a mechanism not entirely elucidated  is a type of intracellular receptor activated by the binding of androgenic hormones, such as testosterone and dihydrotestosterone. When found by its ligand in the cytoplasm, AR translocates to the nucleus where it acts as TF: it binds DNA and regulates gene expression  allowing cell propagation and resistance to apoptosis‐inducing stimuli. These observations might entail that this ternary complex could be essential for transformation and survival of several others Wnt/β‐catenin‐driven tumors.YAP1/TBX5—In a powerful screen for identifying essential genes in human cancer cell lines whose survival depends on β‐catenin, Rosenbluh et al.  found thOCT4—The Doble group identified a mechanism that regulates the balance between differentiation and renewal in mESCs. Under conditions where the signaling pool of β‐catenin is stabilized, a fraction of it associates with the pluripotency TF OCT4 as an alternative to TCF/LEF. While β‐catenin coactivates the expression of different sets of genes in concert with either TCF/LEF or OCT4, it is the β‐catenin‐OCT4 partnership that sustains mESC pluripotency. Consistently, attenuation of the β‐catenin/TCF branch, via overexpression of a dnTCF protein variant, impairs differentiation into all germ layer lineages but enhances retention of pluripotency markers  immunoprecipitated β‐catenin in human CRC cells  is an orphan nuclear receptor expressed in endodermal tissues and is involved in the control of cholesterol homeostasis. LRH‐1 induces cell proliferation in pancreatic and hepatic cells via the concomitant activation of both SOX17—Another class of TFs interacting with WNT signaling is the SOX family of TFs  or pan (TCF) the transcriptome‐wide gene expression pattern normally occurring upon Wg/WNT stimulation was dramatically amputated. Also the genomic Wg/Wnt‐responsive enhancer elements showed full dependency on the presence of Pan, as indicated by STARR (self‐transcribing active regulatory region)‐sequencing, a method to identify novel enhancers based on the activity of millions of candidates from a genomic DNA library  even in the absence of TCF/LEF. These target regions (by definition TCF/LEF‐independent) exhibited enriched consensus motifs of the Forkhead box‐containing (FOX) TFs, and genes in their vicinity were transcriptionally sensitive to FOXO4 downregulation. FOXO TFs (FOXO3a and FOXO4 in particular) have been previously implicated as partners of β‐catenin  that occurred upon β‐catenin stabilization only when all the four TCF/LEF proteins are mutated. Of note, the GHOST response was in part mediated by FOXO4  appears in the effective absence of all TCF/LEF TFs , seemed to provide a conclusive explanation. Pontin52 exhibited simultaneous binding to β‐catenin and TATA‐box binding protein (TBP), a basic component of the eukaryotic transcriptional machinery associated with RNAPII  CREB‐binding protein (CBP) and the closely related p300  in Drosophila suppressed the rough eye phenotype caused by activated Arm/β‐catenin , which includes several MLL players, TRRAP, and β‐catenin , slightly after but similarly to loss of β‐catenin  reinstated expression of the target gene engrailed  was simultaneously identified in four independent genetic screens carried out in Drosophila   complex. Hyrax is an important addition to the equation describing how the β‐catenin complex translates to the RNAPII‐dependent execution of transcription. PAF1 is in fact a conserved multifunctional complex that regulates all stages of the RNAPII transcription cycle—initiation, elongation, and termination—across the animal kingdom  revealed that also BCL9 associates directly to ChiLS via the HD3 domain, while simultaneously connecting with TLE/Groucho factors via the C‐terminus. This emphasizes the central role of Lgs/BCL9 as a core component of the complex in providing a scaffolding function, both in Wnt‐ON and Wnt‐OFF states. According to this model, Lgs/BCL9 would facilitate the complex assembly via three functionally separate interactions: with Pygo, ChiLS, and Groucho/TLE. These interactions precede the appearance of WNT signals, and a BCL9 conformational rearrangement accommodates the HD2‐dependent recruitment of β‐catenin to start transcriptional activation , a segment‐specific homeotic TF . This is interesting, as Tsh is a trunk‐specific modulator of cell fate and is exclusively expressed in the Drosophila embryonic trunk segments where it is required for segment identity in cooperation with other HOX proteins. Of note, Tsh is not expressed in the head and in the tail. The Tsh‐Arm interaction could therefore explain the transactivation potential of Arm/β‐catenin only in trunk cells, but not in other structures where Tsh is not expressed—but Arm/β‐catenin required. As early as 1999, the authors speculated that “the transactivating domain of Arm could bind several different and localized factors allowing tissue‐specific output” . Ebd1 and its human orthologue Jerky share sequence similarity and function, and both interact directly with Arm/β‐catenin in a ternary complex with TCF. The problem is that, in contrast with the ubiquitous expression of other core pathway components, Ebd1 is only expressed in a restricted subset of Wg/Wnt responsive cells, including primarily myocytes, neurons, and glial cells, but is notably absent in ectoderm‐derived epithelia which are highly Wnt responsive ‐type called Jerky  is a Wnt target gene in intestinal neoplasia , SSDP, and the LIM‐domain proteins LMO3 and LHX2  with current technology.While this remains a theoretical proposition, β‐catenin protein structure itself offered a first hint into its function as a hub of combinatorial and noncompetitive multiprotein complexes. A broad central region (from residues 141–664 in human β‐catenin) that consists of 12 imperfect Armadillo repeats forms a rigid scaffold required for the simultaneous anchoring of several interaction partners  how different β‐catenin‐dependent factors that possess DNA‐binding capacity cooperate by contacting correctly spaced adjacent binding sites within the WRE; (ii) how chromatin modifying enzymes are called up in a temporal fashion to assist the access of RNAPII to promoters; and (iii) how nonubiquitous co‐factors and TFs contribute to the establishment of cell‐specific gene regulation.A number of questions relevant to understand these points could be experimentally addressed by existing technology. For example, are all β‐catenin co‐factors simultaneously present on the WRE? If not, in what temporal/epistatic order are they brought together? What is the genomic distribution of all the TFs and co‐factors before the WNT stimulus triggers the pathway, and how is their rearrangement adjusted upon β‐catenin arrival? Is the Wnt/β‐catenin‐dependent transcriptional machinery equal in all cell types? Could its assembly even be different on each WRE within the same cell?We are far from providing the answer to each of these questions. Nevertheless, we are confident that future work will address them. But this effort is much needed, and below we show why.5.8—but comforting—that this search produced a steeply rising curve displaying the annual increment of original articles in the field  to scout all publications containing the term “Wg/Wnt” in the title and/or in the abstract. We also restricted the search to publications annotated by MEDLINE as “journal article” . It is unsurprisingd Figure . This sh66.1CCND1  that are relevant in the corresponding context.APC mutations are present in ca. 80% of CRC, mutations in AXIN is most prevalent in hepatocellular carcinomas, while activating mutations in CTNNB1 have been found with high frequency in melanoma and thyroid tumors , identified in a screening for oncogenes that modulate β‐catenin‐dependent transcription  is amplified in several cancers  significantly more than Pygo1/2 genetic recombination  recapitulated the full loss of the protein, instilling hope that the proverbial magic bullet should be developed to precisely hit this protein–protein interaction surface , made possible the study of genetics and molecular systems in a holistic manner, revolutionizing the research in many areas of the natural world (Hasin, Seldin, & Lusis, As a consequence of the Wnt signaling ductility, also cancer cells have numerous strategies at their disposal to exploit the Wnt/β‐catenin machinery for tumor growth and metastatic progression. This complex pathophysiological relationship, together with the fundamental role that Wnt signaling plays in many biological contexts, makes therapeutic targeting of Wnt components problematic. No single magic bullet will likely be discovered to cure all types of Wnt‐driven cancers, each of which will need to rely on a distinct attack strategy. The realization that “canonical” Wnt/β‐catenin signaling is a set of highly diverse routes is an important first step to recognize the complexity underlying Wnt‐driven cancers. Mapping and discovering the details of this diversity will subsequently be a crucial step toward the successful design of small inhibitory molecules that act in each specific context with minimal adverse effects on the other Wnt‐mediated functions. In this endeavor, increased knowledge of Wnt signaling and its components, especially regarding the β‐catenin/TCF transcriptional complex—which can be considered by and large the main route that Wnt signals undertake—is still necessary. The past and future discoveries about how Wnt signaling is mechanistically executed in each context will be integrated in an incremental pool of information that will likely be difficult to depict as a simplified linear series of events. In this regard, system biology comes to the rescue, and offers several ways to analyze and visualize complex systems by integrating omics data with methods from statistics and graph theory (Barabási, Gulbahce, & Loscalzo, The authors declare the absence of any conflict of interest.Simon Söderholm: Conceptualization; data curation; formal analysis; investigation; project administration; resources; software; writing‐original draft; writing‐review and editing. Claudio Cantù: Conceptualization; funding acquisition; project administration; software; supervision; visualization; writing‐original draft; writing‐review and editing.Mechanisms of Wnt signaling and control


As an effective combination chemotherapy, FOLFIRINOX regimen  has shown definite antitumor efficacy for treating pancreatic cancer (PC) nowadays. However, the traditional systematic administration of these chemotherapeutics limits the drug targeting and causes unwanted effects. Herein, we present a novel Christmas tree-shaped adhesive microneedle (MN) patch coloading fluorouracil, leucovorin, irinotecan, and oxaliplatin simultaneously to realize spatiotemporal FOLFIRINOX therapy in situ. Such MN patch was fabricated by using a layer-by-layer mold replication method, in which oxaliplatin and leucovorin are encapsulated in the top MNs, while irinotecan and fluorouracil are encapsulated in the bottom MNs. The multilayer structure imparts the MNs with enhanced adhesive ability and spatiotemporal drug release property, contributing to the antitumor effect on PC organoid models. Therefore, our Christmas tree-shaped MN patch represents an innovative approach for spatiotemporal multiple-drug delivering and realizes the combination chemotherapy for PC in a single platform. Pancreatic cancer (PC) is a refractory disease with an increasing morbidity , 2. The Herein, we present a novel Christmas tree-shaped microneedle (MN) patch coloading L-OHP, LV, CPT-11, and 5-FU simultaneously to realize spatiotemporal FOLFIRINOX therapy in a single regimen for the PC treatment, as schemed in Thus, inspired by the hierarchical architecture of Christmas tree, we fabricated a multilayer MN patch with L-OHP and LV encapsulated in the top MNs, while CPT-11 and 5-FU encapsulated in the bottom MNs, realizing the spatiotemporal administrated FOLFIRINOX therapy in a single platform to treat PC. Benefitting from the biocompatibility and maneuverability of GelMA and PEGDA, we testified the drug loading efficiencies, release kinetics, and mechanical properties, thus optimizing ideal concentrations of GelMA and PEGDA to fabricate the two layers of our MNs. It was demonstrated that the drugs encapsulated in the top and bottom MNs could be released sustainably in batches, imitating the administration of FOLFIRINOX in clinical practice. Additionally, compared with the traditional single-layer MN patch, our Christmas tree-shaped MN patch showed a significantly enhanced adhesive property, contributing to the stable adhesiveness to the moist and irregular surface of abdominal tumors. Furthermore, we illustrated the cytotoxicity of the Christmas tree-shaped MNs on PC organoids. By employing an organoid derived xenograft model established on mice, our MNs showed significant advantages over the traditional administration of FOLFIRINOX. Therefore, the proposed multidrug spatiotemporal delivery Christmas tree-shaped MNs suggested an effective stratagem for treating PC.In a typical experiment, the FOLFIRINOX-loaded Christmas tree-shaped MN patch was fabricated based on the mold replication process . The preSince the hierarchical morphologies of the Christmas tree-shaped MNs have been replicated, it is of great significance to evaluate the tissue adhesion property of the Christmas tree-shaped MN patch. First, we applied a piece of porcine skin to assess the tissue penetration of the MNs. After the successful fabrication of the two-layer MN patch, it was placed on the surface of the porcine skin with the needle tips facing the skin. Then, the MN patch was pressed with finger for at least 1 min before the removal . As illuThe evaluation of the spatiotemporal drug loading and release kinetics was then carried out. In clinical practice, the FOLFIRINOX regime consisted of 4 parts, including a 2-hour intravenous infusion of L-OHP, a 2-hour intravenous infusion of LV, a 90-minute intravenous infusion of CPT-11, and a 46-hour intravenous infusion of 5-FU. Thus, the initial sudden release of L-OHP, LV, and CPT-11 and the sustained release 5-FU are required to achieve in the MN platform. In the typical experiment, water-soluble red and blue molecular fluorescent dyes were loaded in the top and bottom MNs, respectively. As shown in We evaluated the biocompatibility of the MN patch on 3T3 cells first Figure . The celIn recent years, in vitro organoid culturation has shown broad potential in imitating in vivo physiological functions , 28. SpeEncouraged by the results of in vitro experiments, we applied the FOLFIRINOX-loaded Christmas tree-shaped MN patch to in vivo experiments. To establish the pancreatic tumor organoid xenograft, the patient-derived PC organoid suspension was injected into the envelope of the pancreatic tail in mice. After the tumor grew to approximately 4 mm in diameter, the mice were randomly divided into 4 groups, namely, control group, unloaded MN group, intraperitoneally injecting (I.P.) FOLFIRINOX group, and FOLFIRINOX-loaded Christmas tree-shaped MN (FOLFIRINOX MN) group. For the control group, normal saline was injected intraperitoneally. For the unloaded MN group and FOLFIRINOX MN group, the MN patches were pressed onto the tumor surface after the laparotomy operation once a week . The micThrough the immunohistology staining of MUC1 and EGFR, the successful establishment of PC organoid xenograft models can be proved . The masTherefore, inspired by the hierarchical microarchitecture of the stings of wasps, we have presented a Christmas tree-shaped MN patch. This MN patch, compared with traditional single-layer MN patch, has shown enhanced adhesive property. By loading L-OHP and LV encapsulated in the top MNs and loading CPT-11 and 5-FU encapsulated in the bottom MNs simultaneously, the Christmas tree-shaped MNs could realize the FOLFIRINOX therapy in a single regimen. Through the in vitro experiments, we elucidated that the drugs loaded in different layers constructed with different hydrogels could release in batches, simulating the clinically employed FOLFIRINOX treatment. Noteworthy, the local application of the FLOFIRINOX-loaded MN patch avoided the systematical adverse effects which can be caused by the intravenous injection. In addition to the excellent biocompatibility of PEGDA hydrogel, our FLOFIRINOX-loaded Christmas tree-shaped MN patch could achieve an improved therapeutic method. Thus, it is convinced that MNs with Christmas tree-shaped structures could be promising candidates in biomedical application.Poly (ethylene glycol) diacrylate (PEGDA), gelatin methacryloyl (GelMA), 2-hydroxy-2-methylpropiophenone (HMPP), oxaliplatin (L-OHP), and CellTiter-Glo Luminescent Cell Viability Assay Kit was purchased from Sigma-Aldrich. Fluorouracil (5-FU), irinotecan (CPT-11), and leucovorin (LV) were purchased from Aladdin. The porcine skin was purchased from the market. Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin (P/S), fetal bovine serum (FBS), Trypsin-EDTA solution, and phosphate buffer saline (PBS) were purchased from Gibco. The Live & Dead Viability/Cytotoxicity Assay Kit was purchased from Keygen Biotech Company. Pancreatic cancer surgical specimens were collected at the Department of General Surgery, Affiliated Drum Tower Hospital of Nanjing University Medical School. Male NSG mice aged 6-7 weeks were obtained from the Model Animals Research Center of Nanjing University. All animal treatments were operated in strict accordance with the guidelines approved by the Animal Ethics Committee of Nanjing Drum Tower Hospital.v/v) GelMA and 0.5% (v/v) HMPP were mixed together with L-OHP and LV solution to serve as the pregel solution (solution A). For the bottom MNs, 20% (v/v) PEGDA and 0.5% (v/v) HMPP were mixed with CPT-11 and 5-FU solution to serve as the pregel solution (solution B). For the MN patch, 20% (v/v) PEGDA and 0.5% (v/v) HMPP were mixed together to serve as the pregel solution (solution C). The abovementioned solutions were used right after they were prepared. A negative mold , the tip depth of which was 500 μm and the side length of the base patch of which was 1 cm, was applied. Solution B was first added into the mold. After 5 times of the vacuum process, the air in the tips was removed and the pregel solution filled into the tips. Then, the residue solution B was removed, and ultraviolet (UV) light was applied 30 s to polymerize PEGDA, followed by the readding and 30 s UV polymerization of solution C. Thus, a one-layer PEGDA MN patch can be obtained by squeezing the MN patch out of the negative mold. To fabricate the second layer, solution A was added into the mold and vacuumed for 5 times. After removing the residue solution A, the one-layer PEGDA MN patch was gently placed into the mold. By polymerizing solution A with 60 s UV, the double-layer MN patch can be obtained. The morphologic features of the Christmas tree-shaped MN was characterized with a stereomicroscope  equipped with a CCD camera .For the top MNs, 10% . The release amount of LV was measured by detecting the absorbance at 281 nm, and the release amount of 5-FU was measured by detecting the absorbance at 265 nm. The measured solution was returned into the testing centrifuge tube. This experiment replicated three times.To investigate the multi-drug-loading capacity of the Christmas tree-shaped MNs, 0.1 mg/mL red fluorescent dye was mixed with the PEGDA pregel solution to fabricate the top MNs, and 0.1 mg/mL blued fluorescent dye was mixed with PEGDA pregel solution to fabricate the bottom MNs. Through the bright-field, red, and blue fluorescent microscope  observation, the different and well-defined red and blue fluorescence verified the multidrug spatial delivery property of the Christmas tree-shaped MNs. To investigate the temporal drug release capacity of the Christmas tree-shaped MNs, 0.1 mg/mL LV was mixed with the PEGDA pregel solution to fabricate the top MNs, and 1 mg/mL 5-FU was mixed with PEGDA pregel solution to fabricate the bottom MNs. Then, the fabricated MN patches loaded with merely LV in the top part or 5-FU in the bottom part were immersed into 37°C PBS. At certain time intervals, 200 μmol/L Gastrin I, 500 nmol/L A83-01, 50 ng/mL EGF, 100 ng/mL Noggin, 100 ng/mLFGF-10, B27 supplement, and Glutamax.Organoids were established from fresh tumor tissue resected from PC patients. This experiment was approved by the Ethics Committee of Nanjing Drum Tower Hospital (No. 2020-072-01). Tumor tissues were minced into pieces and then digested with collagenase and DNAse mixture for 30 min. After washing with DMEM twice, the cells were centrifuged. Then, the cells were resuspended with Matrigel and seeded into a 6-well plate. The organoid culture medium was added to the wells after the Matrigel was fully solidified in 37°C. The organoid culture medium was composed of 50% Wnt3A conditioned media, 10% R-spondin1 conditioned media, 36% DMEM/F12 media, 0.01 μL Matrigel were seeded into each well of 24-well plates. The organoids were growing in organoid culture medium. 7 days later, when the cells formed the balloon morphology and reached a fast-growing state, MNs with or without FOLFIRINOX were used to treat the organoids by gently inserting the Christmas tree-shaped MN patch onto the surface of organoid-containing Matrigel. After another 24 h, the organoids were stained a live/dead staining kit. Also, the viability of organoids was examined using a CellTiter-Glo kit after 24, 48, and 72 h.Pancreatic cancer organoids were digested into single cells, and 4000 cells in 50 For organoid-derived xenograft model, 6- to 8-week-old male NSG mice were used in our experiment. Organoids were digested into single cells, and 106 cells were implanted orthotopically into the pancreas of each mouse. Two months later, the tumor volume reached about 4 mm and the mice were divided into four groups: (1) control group; (2) unloaded MN group; (3) I.P. FOLFIRINOX group; and (4) FOLFIRINOX MN group. For the I.P. FOLFIRINOX group, 24 mg/kg LV, 2 mg/kg L-OHP, 20 mg/kg 5-FU, and 20 mg/kg CPT-11 were mixed in 0.9% NaCl solution and injected through the tail vein immediately. The injection was performed once a week. For the MN groups, the mice were anesthetized, and the abdominal cavity was opened to press the MN patch onto the tumor. The surgery was also performed once a week. The loading amount of drugs in the FOLFIRINOX MN group was the same with that in the I.P. FOLFIRINOX group. After another 3 weeks, the mice were sacrificed, and the tumor weight was measured. The tumor tissues were also collected, and the immunohistochemistry analyses were performed to examine the proliferation index.


Our findings uncover a novel mechanism that KAT8 acetylation-controlled lipolysis affects invasive and migratory potential in colorectal cancer cells.Epigenetic mechanisms involved in gene expression play an essential role in various cellular processes, including lipid metabolism. Lysine acetyltransferase 8 (KAT8), a histone acetyltransferase, has been reported to mediate de novo lipogenesis by acetylating fatty acid synthase. However, the effect of KAT8 on lipolysis is unclear. Here, we report a novel mechanism of KAT8 on lipolysis involving in its acetylation by general control non-repressed protein 5 (GCN5) and its deacetylation by Sirtuin 6 (SIRT6). KAT8 acetylation at K168/175 residues attenuates the binding activity of KAT8 and inhibits the recruitment of RNA pol II to the promoter region of the lipolysis-related genes  ColoCP2 axis . In addiCP2 axis . Also, wCP2 axis . HoweverLysine acetyltransferase 8 (KAT8), also called MOF , is a histone acetyltransferase that belongs to MYST family. KAT8 is a chromatin regulatory protein that mainly functions by acetylating H4K16 –13. KAT8Lipolysis is a catabolic branch of the fatty acids (FA) cycle that provides FAs to meet metabolic need and removes them when in excess. Lipolysis hydrolyses the ester bonds of the triglyceride (TG) and plays a critical role in cell metabolism homoeostasis. Neutral hydrolysis of TGs to FAs and glycerol requires three consecutive steps and at least three different enzymes are involve, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol lipase (MGL) –26. ATGLde novo biosynthesis and fatty acid β-oxidation [ATGL and HSL, subsequently downregulating their expression to mediate lipolysis. Taken together, our data reveals that KAT8 acetylation plays a critical role in lipolysis to further affect invasive and migratory potential of CRC cells, and it may serve as a potential target for colorectal cancer therapy.Our previous study has reported that histone deacetylase SIRT6 plays a key role to regulate Cardiolipin xidation , 10. HerKAT8 is essential for cell proliferation and plays a critical role in multiple physiological process , 33. HowGiven that KAT8 involves in the process of lipolysis, we next detected the mRNA and protein levels of KAT8 after PA treatment. No significant difference was found in both KAT8 mRNA and protein levels after PA treatment Fig. . RemarkaSince KAT8 acetylation might play a critical role in regulating lipolysis in CRC cells, we next aimed to identify the specific acetyltransferase by screening a series of acetyltransferases. We co-transfected KAT8 with different histone acetyltransferases (HATs) and found that only GCN5 dramatically increased the acetylation level of KAT8 Fig. . KAT8 acNext, we detected whether there is a molecular link between GCN5 and KAT8. Exogenous, semi-exogenous and endogenous co-immunoprecipitation showed the interaction of GCN5 with KAT8 Fig. . We thenWe next sought to identify the deacetylase of KAT8 acetylation. HDACI, II family inhibitor TSA and the Sirtuin family inhibitor nicotinamide were used and we found that nicotinamide significantly increased the acetylation level of KAT8, suggesting that Sirtuins might be the major deacetylase of KAT8 Fig. . SubcellTo clarify which is the key mediator of KAT8 acetylation, we firstly monitored the interaction between GCN5/SIRT6 and KAT8 after PA treatment. The interaction between SIRT6 and KAT8 was sharply reduced accompanied by the increased interaction between GCN5 and KAT8 after PA treatment, showing that KAT8 acetylation might be dynamically regulated by the balance of GCN5 and SIRT6 Fig. . RemarkaHaving established that SIRT6 is a major deacetylase of KAT8, we aimed to clarify the molecular link between SIRT6 and KAT8. Exogenous, semi-exogenous and endogenous co-immunoprecipitation presented the interaction of SIRT6 with KAT8 Fig. . GST pulEQUUS to humans . The MS data indicated that KAT8 is acetylated at lysine 168 and lysine 175 residues after PA treatment Fig. . These tans Fig. . To furtans Fig. . At the ans Fig. . These dATGL and HSL genes and found that both genes reduced after PA stimulation   Fig. . RemarkaII) Fig. . Also, wlls Fig. . CollectWe further demonstrated the functional significance of KAT8 acetylation in CRC progression. The results of CCK-8 viability assay and colony formation assay showed that KAT8 acetylation is not affected the proliferation of CRC cells Fig. . Next, aFinally, we explored the mechanism insight into KAT8 acetylation-dependent cell migration and invasion. The expression of E-cadherin and N-cadherin, the main marker of epithelial-mesenchymal transition (EMT), were firstly examined and no significant changes were found in both KAT8-WT and KAT8-2KR transfected cells, implying that KAT8 acetylation-dependent cell migration and invasion is not driven by an EMT process Fig. . Lipase ATGL and HSL promoters to decrease the expression of these genes and further affect invasive and migratory potential in CRC cells  gene, showing that KAT8-dependent histone H4K16ac was important in the maintenance of TMS1 gene activity [ATGL and HSL genes was not through H4K16ac  was purchased from SIGMA  and then prepared to a stock solution and stored in room temperature.Human colon cancer HCT116 and SW480 cells, human liver cancer HepG2 cells, human pancreas cancer PANC1 cells, human gastric carcinoma MGC803 cells, human embryonic lung diploid fibroblasts 2BS were cultured by Dulbecco Modified Eagle Medium  supplemented with 10% heat-inactivated fetal bovine serum . Human colon cancer RKO cells was cultured by RPMI-1640 supplemented with 5% FBS. All the cells were cultured with penicillin/Streptomycin in a 37 °C incubator with a humidified, 5% COAll the genes open reading frame were amplified from a cDNA library of HCT116 cells by PCR  and cloned into 3x Flag CMV10, pCMV-myc, TF-pcold-His. The mutations of these genes were generated using a site-directed mutagenesis kit . Plasmids were transfected into cell lines by Hieff TransTM Liposomal Transfection Reagent  according to the manufacturer’s protocol.HCT116 cells were treated as required and staining with the Oil Red O working solution for 5 min. The stained lipid droplets were monitored under a microscope .HCT116, SW480, RKO, HepG2, MGC803 and PANC1 cells were treated as required and then lysed with the glycerol colorimetric assay kit . The lysates were then heated to 70 °C for 10 min to inactivate residue lipase activity. Glycerol in the lysates was determined by the enzyme-coupled GPO-Trinder reaction from the absorption of 550 nm.Total RNA was extracted by Trizol agent . cDNA was synthesized using Quantscript RT Kit  according to the instruction. RT-qPCR assay was performed by 7500 Fast RT-PCR machine with SYBR PCR mix agent . The primers sequences used for RT-qPCR are available upon request.RNA interference was performed as described . Cells wDifferent cells were harvested after treatment and proteins were extracted to detect the expression by Western blotting as previously described with minor modifications . Equal aAfter treatment, different cells were harvested and lysed in different lysis buffer to perform Co-immunoprecipitation as described before . ProteinGST fusion proteins or His-tagged proteins were purified as described before . His-tag2, 0.1 mM EDTA, 1 mM dithiothreitol (DTT), and 10% glycerol) with or without acetyl-CoA (5 mM) for 1 h at 30 °C. The reactions were stopped by adding 5 x protein sample buffer and the samples were boiled at 100 °C for 5 min before SDS-PAGE and immunoblotting.KAT8 and His-GCN5 were purified and incubated in acetylation buffer  for 1 h at 30 °C. The reactions were stopped by adding 5 x protein sample buffer and the samples were boiled at 100 °C for 5 min before SDS-PAGE and immunoblotting.SIRT6 and KAT8 were purified and incubated in deacetylation buffer  to establish a cell proliferation curve. For the colony formation assay, cells were treated as above and fixed by 4% formaldehyde and stained with 0.25% Coomassie brilliant blue after culturing normally for 2 weeks. All the experiments were performed in triplicate. The number of colonies was calculated using Photoshop .HCT116 or RKO cells were seeded and gently scraped with a 10 μl sterile pipette tip when the cells achieved 90% confluence. The wounded cells were continuously cultured with FBS free DMEM for 2 days. The healing width were recorded at the 0 h, 24 h and 48 h under inverted Microscope. The migration rate was measured by the ratio of the day 1 and day 2 width to original width. All the experiments were performed in triplicate.HCT116 or SW480 cells were cultured in 24-well plate with 8-μm polyethylene terephthalate membrane filters separating the lower and upper culture chambers . The membrane filter was coated with Matrigel . Then, cells were seeded and cultured by serum-free DMEM. The bottom chamber contained DMEM with 10% FBS. Cells were allowed to invade for 48 h. Before fixing, non-invasion cells on the upper side of the filter were detached using a cotton swab. Cells were fixed and stained with 0.1% crystal violet. Cells were counted in three random fields. All the experiments were performed in triplicate.t-tests were applied to compare data between two groups, while one-way analysis of variance (ANOVA) was used to compare data among multiple groups. The statistical information of each experiment were shown in the figures and corresponding legends.Measurement data analysis was conducted using PRISM and SPSS statistical analysis software . Unpaired Supplemental DataOriginal Data Fileaj-checklist


Notwithstanding immune checkpoint blocking (ICB) therapy has made eminent clinical breakthroughs, overcoming immunologically “cold” tumors remains challenging. Here, a cascade potentiated nanomodulator AuPtAg‐GOx is engineered for boosting immune responsiveness. Upon 1064 nm laser irradiation, AuPtAg‐mediated mild photothermal therapy (PTT) activates cytotoxic T lymphocytes and reverses the immunogenic “cold” tumor microenvironment. Further, to amplify the thermal sensitivity of tumor cells, glucose oxidase (GOx) is introduced to suppress the production of heat shock proteins, thereby promoting mild photothermal therapy. Complementarily, AuPtAg nanozymes with catalase‐like activity can ameliorate tumor hypoxia, significantly improving the GOx activity. As a result, the combination of AuPtAg‐GOx with self‐augmented photothermal ability and PD‐L1 antibody can further escalate the antitumor efficacy. The AuPtAg‐GOx‐based synergistic starvation therapy, mild PTT, and immunotherapy cascade enhancement therapy strategy can be a favorable tool to effectively kill cancer cells. A cascade promoted AuPtAg‐glucose oxidase (GOx) nanomodulator is designed for synergistic starvation/photothermal/immunotherapy. The GOx‐mediated starvation therapy inhibits heat shock protein production for enhanced AuPtAg mediated mild photothermal therapy, which would sequentially activate the systemic immune response, prompting the tumor microenvironment to become “hot.” Therefore, AuPtAg‐GOx combined with ɑ‐PD‐1 ligand 1 could obviously inhibit tumor growth. Since 2011, FDA‐approved ipilimumab (anti‐CTLA4) has been used for the treatment of metastatic melanoma, monoclonal antibodies targeting programmed cell death 1 (PD‐1) and PD‐1 ligand 1 (PD‐L1) axis, indoleamine 2,3‐dioxygenase inhibitors, CD47 antibodies, etc. were successively developed for cancer immunotherapy. Among them, anti‐PD‐L1/anti‐PD‐1 antibodies have become the most widely used immune checkpoint inhibitors. However, the efficacy of monomodal ICB therapy is frequently modest due to immunologically “cold” tumors and low tumor‐infiltrating lymphocytes (TILs). Fortunately, the inadequacy of ICB‐based immunotherapy can be remedied by synergistic therapy, which has brought an incredible revolution in cancer therapy. For example, Yan et al. designed an aggregation‐induced emission reagent (TPA‐BT‐DPTQ)‐mediated PTT to eliminate primary tumors and boost immunogenicity. Synergistic PTT and PD‐L1 antibody successfully avoided metastasis and recurrence of cancer. Additionally, Liang et al. designed a nanoaircraft carrier to deliver miscellaneous therapeutic agents, achieving modulation of the immune response to facilitate ICB therapy. Therefore, it is a promising strategy to design a synergistic therapy that can reshape the tumor microenvironment (TME) and improve the TILs level in solid tumors.Immunotherapy has emerged as an excellent cancer treatment strategy, which stimulates and maintains the systemic immune response through the host's own immune system to resist the spread of tumor cells.1 Amon During PTT, photothermal conversion agents can transform light into hyperthermia to ablate the tumor cells. Importantly, hyperthermia can improve the immunogenicity of the tumor and create a favorable niche for TILs recruitment, thereby facilitating ICB therapy. However, to realize ideal tumor damaging effect, the hyperthermia over 50 °C is essential, which inevitably injures the healthy tissues around the tumors. As an alternative, mild PTT (40–43 °C) is more preferable for clinical applications. Nevertheless, the up‐regulated protective heat shock proteins (HSPs) within cancer cells and PD‐L1 expressed on the surface of cancer cells induced by heat stress often cause suboptimal PTT effect. To shrinking the thermoresistance of cancer cell, a number of small molecule HSPs inhibitors  have been exploited. Regrettably, these HSPs inhibitors can only inhibit the activity of the already generated HSPs after heat stimulus rather than before therapy, resulting in delayed therapeutic effect. Furthermore, these small molecular inhibitors lack generalizability, because one inhibitor can only target one specific HSP. Therefore, inhibiting all kinds of HSPs production at the source is more beneficial to improve the effect of mild PTT.Photothermal therapy (PTT), a spatiotemporally controllable noninvasive modality, has attracted increasing attention.13 Dur2 to produce H2O2 and gluconic acid. By consuming glucose, the GOx can effectively inhibit the production of intracellular adenosine triphosphate (ATP). As we all know, the expression of HSP was highly positively correlated with ATP content. Therefore, inhibiting the production of intratumoral ATP by GOx‐mediated starvation therapy can be a promising method to overcome HSPs‐induced tumor thermoresistance. However, the hypoxic TME severely constrains the activity of GOx. As a consequence, ameliorating hypoxia is crucial to optimize the effects of GOx. To solve the encountered limitations, noble metal nanozymes with extraordinary catalase (CAT)‐like activity and impressive photothermal conversion efficiency is an ideal candidate. Therefore, integrating GOx with noble metal nanozymes is an intelligent strategy for synergistically starvation/mild photothermal/immunotherapy.Glucose oxidase (GOx), a prominent endogenous oxidoreductase for cancer starvation therapy, can deplete intracellular glucose and Onic acid.20 By 2 as a bridge. After the nanoparticles were endocytosed into tumor cells, i) AuPtAg‐GOx with CAT‐like activity can catalyze intratumoral overexpressed H2O2 into O2. Then, AuPtAg‐GOx can realize a remarkable starvation therapy effect by consuming the glucose under the assistance of O2. What is more, the cutoff of nutrients cause a reduced intratumoral ATP level. ii) Upon 1064 nm laser irradiation, mild heat is generated by AuPtAg‐GOx in the tumor area. Meanwhile, the limited ATP level can inhibit the synthesis of HSPs, thus realizing the significant therapeutic effect of mild PTT. iii) Mild‐PTT can intensify the recruitment of TILs to reprogram the “cold” TME, sensitizing the tumor to ICB therapy. Therefore, by integrating AuPtAg‐GOx with ɑ‐PD‐L1, both primary and distant tumors can be effectively inhibited. In a nutshell, AuPtAg‐GOx can achieve mild PTT augmented by starvation therapy, and further promotes immunotherapy effects through mild PTT. Such cascade promoting synergistic strategies may be generally applicable for high‐efficient cancer therapy.Based on the above considerations, a noble AuPtAg‐GOx nanozyme was constructed for synergistic tumor immunotherapy induced by starvation therapy‐augmented mild photothermal therapy. The AuPtAg was first prepared by a simple one pot method and then GOx was covalently connected to its surface with SH‐PEG‐NH2 and the major experimental procedures were shown in Scheme 62−, Ag+ and AuCl4−) were gradually reduced by L‐ascorbic acid. A series of AuPtAg with different molar ratios of Au, Pt, and Ag were synthesized, and the optimal ratio of AuPtAg (Au:Pt:Ag = 1:1:1.22) was selected for subsequent tests  culture medium, providing a possibility for further in vivo applications  for 5 min, a significant concentration‐dependent temperature rise was observed , which also indicated the strong laser‐power‐dependent photothermal effect of AuPtAg . The photothermal conversion efficiencies of AuPtAg at 808 nm and 1064 nm were calculated to be 47% and 41% . In the case of little difference in photothermal conversion efficiency at 808 nm and 1064 nm, NIR‐II light (1064 nm) with inherent advantages, such as deeper tissue penetration and higher maximum permissible exposure (1 W cm−2), was selected for cancer treatment . In addition, the photothermal properties of AuPtAg did not change significantly in the process of four laser switches, indicating that it has good photothermal stability , the cell viabilities were still as high as 85%, indicating that AuPtAg‐PEG has low cytotoxicity  achieved high viability (>98%), indicating that light alone cannot kill cells. While 47% of 4T1 cells were killed in the AuPtAg‐GOx (200 µg mL−1) group. Notably, 89% cancer cells growth inhibition was achieved in the AuPtAg‐GOx (200 µg mL−1) + 1064 nm group  was attached on AuPtAg‐GOx for the labeling of AuPtAg‐GOx, as shown in the p Figure . This isp Figure . Then, tp Figure . The abo Subsequently, the ability of AuPtAg‐GOx to reprogram “cold” tumors into “hot” tumors was researched. Tumors are “cold” in immunology, which largely depends on the infiltration of immunosuppressive M2 phenotype tumor‐associated macrophages (TAMs). Hence, whether AuPtAg‐GOx could reprogram the macrophage polarization from M2 to M1 was measured in vitro , and high mobility group box 1 (HMGB‐1). Compared with the control group, the AuPtAg‐GOx + 1064 nm group showed significant ATP release (Figure It has been reported that photothermal can effectively reverse the immunosuppressive TME.27 SubFigure + T cells increased by 9.86% and 12.06% (Figure + T cells elevated by 14% and 16.3% (Figure α were examined by ELISA, and both AuPtAg + 1064 nm and AuPtAg‐GOx + 1064 nm treatment groups promoted IL‐6 and TNF‐α secretion compared to the control group (Figure Encouraged by the excellent ability to reverse immunosuppressive TME in vitro, the capacity of AuPtAg‐GOx to strengthen immunoreaction was further measured in 4T1 tumor‐bearing Balb/C mice. As expected, both AuPtAg + 1064 nm group and AuPtAg‐GOx + 1064 nm group could remarkably downregulate the proportion of M2 macrophages and increase the proportion of M1 macrophages in tumors. Furthermore, the effect was more obvious in the group of AuPtAg‐GOx + 1064 nm due to the synergistic effect of starvation therapy, the ratio of M2 macrophages shortened from 57.4% to 30.8% Figure 5a, M1 ma% Figure , and CD8Figure α‐PD‐L1 was selected for antitumor immunotherapy. Because solid tumors present an immune cold state, ɑ‐PD‐L1 alone was injected into mice with only 11% and 22% inhibition of the primary tumor and distal tumors (Figure α‐PD‐L1 group compared with the control group, indicating that the synergistic therapy could effectively inhibit cancer metastasis (Figure Inspired by the excellent ability to ablate 4T1 cells and immune activation property of AuPtAg‐GOx, the in vivo antitumor performance of AuPtAg‐GOx was evaluated in bilateral tumor‐bearing mice Figure 6a. To des Figure . The AuPs Figure  and thers Figure  and the 32O2. Then, the decrease in ATP concentration could hinder HSP production to render tumors less thermoresistance. Importantly, the coalescence of mild PTT with starvation therapy significantly enhances the efficacy of mild PTT with more excellent recruitment of TILs and making tumors “hot”, which could respond to immune checkpoint blockers. In vivo experiments also showed that ɑ‐PD‐L1 combined with AuPtAg‐GOx mediated mild PTT, starvation therapy and immunotherapy could better inhibit primary and distal tumors. In summary, starvation therapy‐augmented mild PTT can effectively reverse the immunosuppressive TME, indicating this strategy holds promise as a powerful tool for synergistic cancer immunotherapy.All in all, we designed an AuPtAg‐GOx nanozyme with mild photothermal properties and the ability to diminish the heat resistance of tumor cells to potentiate the anti‐tumor immune response. Upon 1064 nm laser irradiation, the tumor part would generate mild heat resulting from the AuPtAg‐GOx. Prior to this, AuPtAg‐GOx entering into tumor cells would firstly realize starvation therapy by consuming intratumoral glucose, which would suppress the content of ATP and produce HThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.