MH was successfully loaded (MH-MSN) using the evaporation method, with a loading capacity of 28.3% and loading efficiency of 99.1%. In vitro release studies showed that morin release from MH-MSNs was enhanced at pH 5.2, indicating increased flavonoid solubility. The in vitro cytotoxicity of MH and MH-MSNs on human A375, MNT-1 and SK-MEL-28 melanoma cell lines was investigated. Exposure to MSNs did not affect the cell viability of any of the cell lines tested, suggesting that the nanoparticles are biocompatible. The effect of MH and MH-MSNs on reducing cell viability was time- and concentration-dependent in all melanoma cell lines. The A375 and SK-MEL-28 cell lines were slightly more sensitive than MNT-1 cells in both the MH and MH-MSN treatments. Our findings suggest that MH-MSNs are a promising delivery system for the treatment of melanoma.Melanoma incidence, a type of skin cancer, has been increasing worldwide. There is a strong need to develop new therapeutic strategies to improve melanoma treatment. Morin is a bioflavonoid with the potential for use in the treatment of cancer, including melanoma. However, therapeutic applications of morin are restrained owing to its low aqueous solubility and limited bioavailability. This work investigates morin hydrate (MH) encapsulation in mesoporous silica nanoparticles (MSNs) to enhance morin bioavailability and consequently increase the antitumor effects in melanoma cells. Spheroidal MSNs with a mean size of 56.3 ± 6.5 nm and a specific surface area of 816 m The incidence of cancer is rising worldwide and is one of the most common causes of mortality, being a major global public health concern ,2,3. MelTraditional melanoma treatments include surgery, mostly used in the early stages, and in metastatic stages, chemotherapy, radiotherapy, targeted therapy and immunotherapy are the chosen therapeutic strategies ,15. ThisA decline in death rates for melanoma was reported in the United States between 2013 and 2017 after the introduction of new therapies for metastatic melanoma [The use of novel compounds in melanoma treatment using a nanoscale approach represents a promising strategy to overcome the current limitations of conventional therapies. Flavonoids, a group of polyphenolic compounds mainly found in plants, fruits and vegetables, have demonstrated potential anticancer capacity ,21,22. SWhile morin has shown promising results, it shares some of the limitations of other flavonoids, including poor aqueous solubility, low chemical stability, low bioavailability and rapid excretion. These characteristics restrict its full potential for use in the biomedical field ,44. To aMesoporous silica nanoparticles (MSNs) are one of several delivery systems that have been used incorporate various bioactive compounds, including flavonoids . MSNs ha2 adsorption–desorption. The specific surface area estimated via BET modeling was 816 m2/g. The BJH average pore size was 6.8 nm, and the porosity was 1.64 cm3/g. The N2 adsorption–desorption isotherm (77 K) in Mesoporous silica nanoparticles (MSNs) were firstly synthesized and then loaded with morin hydrate (MH). The MSNs showed spheroidal shapes with a mean size of 56.3 ± 6.5 nm determined via TEM analysis a,b. The −1) of the synthesized MSNs is depicted in −1, in 2, namely at 1093 cm−1 and 804 cm−1 , 968 cm−1 (Si-OH stretching), 468 cm−1 (O-Si-O- bending) and 3445 cm−1 (-OH stretching) ; 1626 cm−1 (C=C stretching in aromatic ring); 1613, 1571, 1508 and 1460 cm−1 (C=C and C-C stretching); and 1380 and 1310 cm−1 (C-OH bending) [−1 to 1658 cm−1, which indicated an increase in the H-bonding of -C=O morin groups [The FTIR spectrum (1850 to 350 cmetching)  [57,66,7re of MH . Other ibending) ,75. Thesn groups , possibl2) nanoparticles was 10% [The MH was loaded onto the MSNs using the evaporation method. The loading procedure was selected based on a previous study that clearly revealed that the evaporation method was more efficient, in comparison with the conventional immersion method, for loading resveratrol, a non-flavonoid polyphenolic compound, onto the MSNs (resulting in a higher loading capacity) . The car was 10% , while i was 10% . In bothLoaded carriers (MH-MSNs) were further characterized via DTA , DSC and XRD. The DTA curve of bulk MH b showed Converting crystalline MH into amorphous state is expected to increase its solubility. This effect has been observed previously for morin encapsulated in polymeric carriers ,79, as wThe surface charge of both synthesized MSN particles and those loaded with MH particles was evaluated through zeta potential measurements at pH 5.2 and 7.4. In both cases, the particles exhibited a negative surface charge at the tested pH levels. Specifically, at physiological pH (pH 7.4), the surface charge decreased from −24.2 mV to −29.1 mV after loading with MH. At the acid pH environment of 5.2, the surface charge of the MSNs was less negative, as expected (−12.2 mV), and showed no marked variation after MH loading (−12.7 mV). Based on the FTIR spectra, it appeared that MH molecules could interact with silanol groups present on the surface of the MSNs. It has been previously reported that the pKa of morin in the acid range is 5.2 , which iAt pH 5.2, there was an enhancement in morin release from MH-MSNs compared to non-encapsulated MH. This enhancement resulted in an increase in release from 10.2% to 17.9% after 24 h and from 12.6% to 23.7% after 48 h. The increase in morin release was ascribed to an improvement in the solubility and dissolution rate of amorphous morin compared to the crystalline form ,79. At p2), chi-square (χ2) and average relative error (ARE). Respective equations are listed in 2, χ2 and ARE, as shown in To investigate the morin release kinetics and understand the release mechanism, the cumulative release data were fitted into various kinetic models: the Korsmeyer–Peppas model (K-P), the Weibull model and a model based on the Noyes–Whitney equation and applying Fick’s law (NWF) . The modThe Korsmeyer–Peppas (K-P) model is a semi-empirical model that correlates the release with the time through an exponential function ,85. The The cell lines A375 ,54,86,87The cell viability of A375 a and SK-50) for MH decreased with increased time exposure. The MH concentrations that inhibited 50% of the growth of A375 were 194.4, 140.3 and 105.0 µg/mL at 24, 48 and 72 h, respectively. The IC50 for MNT-1 for 24 h of exposure was 178.0 µg/mL, less than the A375 cell line, but for 48 and 72 h, it was 142.0 and 124.4 µg/mL, respectively, slightly higher when compared to A375 cells. Regarding SK-MEL-28, the IC50 values were 163.1, 115.1 and 107.4 µg/mL for 24, 48, and 72 h, respectively. Although there were not many differences, at the shortest exposure period (24 h), SK-MEL-28 was the most sensitive cell line, but at 72 h of exposure, A375 was revealed to be the most sensitive for MH.The time-dependent toxicity of MH is also evidenced in + melanoma MV3 cell subpopulation was significantly reduced by morin, and the in vivo study also showed the suppression of melanoma tumor growth [Previous studies using different cell lines have revealed the anticancer activity of flavonoids such as fisetin ,100, apir growth . Furtherr growth  used a mr growth ,106. Morr growth  and in Sr growth . Compari50 for 24 h was markedly above the range of the MH-MSN concentrations tested. The MH-MSNs at 507.7 and 414.0 µg/mL reduced the cell viability by 50% in A375 cells after 48 and 72 h of exposure, respectively (50) was achieved at 464.1 µg/mL for 48 h and 423.3 µg/mL for 72 h. The cytotoxicity of MH-MSNs increased with concentration, especially for 48 and 72 h, which agrees with our observations when using MH. Notably, a significant decrease in the cell viability of A375, MNT-1 and SK-MEL-28 cells was noticed between MH-MSN concentrations of 350 and 400 µg/mL after 48 and 72 h of exposure. We observed a significant decrease in the viability of A375, MNT-1, and SK-MEL-28 cells when exposed to MH-MSN concentrations between 350 and 400 µg/mL after 48 and 72 h. While the differences were minor, it appears that the A375 and SK-MEL-28 cell lines, which are amelanotic , were slightly more sensitive than the highly pigmented MNT-1 cells, particularly with prolonged exposure to both MH and MH-MSN treatments.The morin-loaded MSNs were then tested in the three melanoma cell lines. Exposure for 24 h with morin-loaded MSNs  did not ectively . MH-MSNs50 values for 48 and 72 h was estimated. For the A375 cell line, the entrapped MH concentration was 143.7 and 117.2 µg/mL for 48 and 72 h, respectively. Regarding MNT-1, it was 164.8 and 132.5 µg/mL for 48 and 72 h, respectively. Finally, the estimated MH concentration in the MH-MSNs for the SK-MEL-28 cell line was 131.3 and 119.8 µg/mL for 48 and 72 h, respectively. The MH concentrations that reduced the cell viability by 50% presented in Considering the loading capacity of 28.3% determined in Previous studies have shown that morin encapsulation significantly enhanced its anticancer properties by improving its intracellular delivery when compared to free morin, inducing cell death in different cancer cell lines, such as lung ,108,109,3CH2OH; >99%) were acquired from Fisher Scientific . Hydrochloric acid  was bought from VWR International . Deionized water obtained with a Milli-Q system comprising a 0.22 μm filter  was employed in all experiments.All chemicals were of analytical grade and utilized as received. Cetyltrimethylammonium bromide , tetraethoxysilane , morin hydrate , phosphate-buffered saline (PBS) pH 7.4  and cylindrical dialysis cellulose membranes (3.5 kDa MWCO) were acquired from Sigma-Aldrich . Triethanolamine  and ethanol (CHv/v) in ethanol under reflux conditions. The remaining HCl was cleaned out with one final ethanol wash. Lastly, MSNs were calcinated in a muffle furnace  for 5 h at 550 °C to completely eliminate the surfactant. After all experimental steps, the yield of MSN production was 37%.The MSNs were synthesized using a simple method adapted from the literature with some adjustments . SuccincMorin was loaded via the rotary evaporation technique. Briefly, 40 mg of morin hydrate (MH) was dissolved in a round-bottom flask with 10 mL of ethanol and placed in the ultrasonic bath for 2 min. After that, 100 mg of MSN was added to the flask and dispersed in the ultrasonic bath for 5 min. To obtain the morin-loaded MSNs, the solvent was then gradually evaporated in a rotary evaporator at 50 °C until all ethanol was removed. The final solids (MH-MSNs) were scraped from the flask, kept at room temperature, and covered with aluminum foil to prevent light exposure.2 adsorption-desorption isotherms were obtained using a Micrometrics Gemini V2.0  system at 77 K. Samples were pre-treated at 473 K for 5 h under N2 gas. The pore size and surface area were calculated from desorption branches via the Barrrett–Joyner–Halenda (BJH) method and the Brunauer–Emmett–Teller (BET) isotherm, respectively. Fourier-transform infrared spectroscopy (FTIR) spectra were collected on a Mattson 7000 spectrometer  using KBr pellets; the measurement parameters were 256 scans per sample in the range of 4000–300 cm−1 with a resolution of 2 cm−1. The carbon and hydrogen content were ascertained via elemental analysis on a Leco TruSpec-Micro CHNS 630-200-200 . The amount of morin loaded on the MSNs was calculated from carbon content and verified with thermogravimetric analysis (TGA). TGA was performed in a Hitachi STA300 instrument  heated from 30° to 800 °C at 5 °C/min under a N2:O2 atmosphere (3:1). The differential thermal analysis (DTA) of the materials was obtained simultaneously in the same equipment and conditions. Heating curves of the materials were obtained using a differential scanning calorimeter . Accurately weighed samples (2–5 mg) were loaded into a non-hermetically crimped aluminum pan and heated under a nitrogen purge at the rate of 50 mL/min, from 100 to 350 °C at a heating rate of 10 °C/min. Data were analyzed using TA Instruments’ Universal Analysis 2000 software V4.5. UV–VIS spectra were obtained in a SYNERGY HT microplate reader . The surface charge of the nanoparticles was assessed using zeta potential measurements, which were performed through electrophoretic light scattering performed using a Zeta Sizer Nano Plus equipment  by suspending the nanoparticles at different pH values. The equilibration time established was 120 s (for each sample), and 3 measurements were performed with 100 runs each.The morphology and size of the MSNs were evaluated via transmission electronic microscopy (TEM) using a Hitachi H-9000 instrument  operating at 300 kV. Samples were prepared through the immersion of a copper grid coated with an amorphous carbon film in diluted suspensions of the nanoparticles in ethanol. X-ray diffractograms were obtained using a PANalytical Empyrean diffractometer  with Kα(Cu) radiation and a curved graphite monochromator. The analysis in reflection geometry was performed in the range of 3.5° < 2θ < 50° (step 0.026°), while analysis in transmission geometry was performed in the range of 1° < 2θ < 5° (step 0.026°). NThe morin release was investigated in PBS at pH 7.4 and pH 5.2. Several suspensions were prepared by dispersing morin-loaded MSNs  and MH (for comparison) into 1 mL of PBS. The suspensions were then placed in a dialysis membrane (3.5 kDa cut-off) and immersed in 29 mL of PBS. The resulting solutions were gently stirred at 37 °C while being protected from light exposure. After specific periods of time, 1 mL aliquots of solution were withdrawn for analysis and immediately replaced with the same volume of PBS. These aliquots were used to monitor the amount of morin released during 48 h.2 = 0.9987 and r2 = 0.9993 for pH 7.4 and 5.2, respectively). The cumulative release of morin, in a percentage, was given via The aliquots were analyzed with a UV–VIS spectrophotometer . The concentration of morin in the aliquots was estimated according to the Lambert–Beer law, which correlates the absorbance with the concentration of the sample, using the calibration curves that were prepared for the morin solutions at pH 7.4 and 5.2. Several morin solutions of known concentrations were prepared, and their absorbance at λ = 390 nm for pH 7.4 and λ = 384 nm for pH 5.2 was measured. The absorbance vs. concentration was plotted  and follThe stability of morin in PBS was evaluated by following its concentration using UV-VIS spectroscopy. The solutions were prepared by dissolving MH in 50 mL of PBS (pH 5.2 and 7.4) and maintaining it at 37 °C under constant stirring while being protected from light. Aliquots were withdrawn for analysis at the following time intervals: 0 h, 3 h, 6 h, 12 h, 24 h, 36 h and 48 h.−1) were obtained from Gibco, Life Technologies . Penicillin–streptomycin  was purchased from Grisp .3--2,5-diphenyl tetrazolium bromide  and dimethyl sulfoxide  were acquired from Sigma-Aldrich . Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin-ethylendiaminetetraacetic acid EDTA (0.25% trypsin and 1 mM EDTA), fetal bovine serum (FBS), l-glutamine and fungizone  and 1% fungizone (250 U mL−1). Cells were monitored to ensure satisfactory confluence and morphology using an inverted phase-contrast Eclipse TS100 microscope . When 70–80% of confluence was reached, cells were trypsinized with trypsin-EDTA (0.25% trypsin and 1 mM EDTA) and used in the in vitro cell viability assays (A375 and SK-MEL-28 human melanoma cell lines were acquired from the European Collection of Authenticated Cell Cultures (ECACC). The MNT-1 melanoma cell line was kindly supplied by Dr. Manuela Gaspar . Cell lines were cultured at 37 °C under 5% COy assays .A morin hydrate stock solution was prepared by dissolving MH in 5% DMSO and culture medium to ensure complete dissolution. The final volume was adjusted, and the final concentration of DMSO at the highest concentration tested was 0.58%. Encapsulated MH was dispersed in culture medium. The three cell lines were seeded in 96-well plates. A375 cells were seeded at 40,000, 25,000 and 20,000 cells/mL for 24, 48 and 72 h of exposure, respectively. MNT-1 cells were seeded at 35,000, 25,000 and 15,000 cells/mL for 24, 48 and 72 h of exposure, respectively, and SK-MEL-28 at 50,000, 20,000 and 10,000 cells/mL for 24, 48 and 72 h of exposure, respectively. After 24 h, the medium was replaced with the exposure solutions containing (i) MH , (ii) MSNs , or (iii) MH-MSNs . As a negative control, cells exposed to the control medium were used. Cell viability was measured at 24, 48 and 72 h following exposure.2 humidified atmosphere. Afterwards, the culture medium containing MTT was withdrawn and changed with 150 µL of DMSO for formazan crystal solubilization. Samples’ absorbance was measured at 570 nm with a BioTek Synergy HT plate reader .Cell viability was assessed via the MTT colorimetric assay . At the p < 0.05) as well as via Dunnett’s test (p < 0.05).The results were presented as mean ± standard deviation. SigmaPlot version 14.0  was used for statistical analysis. Data were analyzed via one-way ANOVA  to act as carriers for morin, an anticancer drug. The MSNs were synthesized and morin hydrate (MH) was efficiently loaded onto them using the evaporation technique. The loading capacity was 28.3% and the loading efficiency was 99.1%. The encapsulation of MH facilitated its release in vitro, likely due to the conversion of crystalline MH into an amorphous state upon loading onto MSNs, which increased its solubility and dissolution rate. The enhancement of MH release in acidic pH suggests that morin-loaded MSNs could be a promising nano-delivery system to treat melanoma skin cancer. However, we detected MH degradation at pH 7.4, which is a point that should be improved in future studies. MH and MH-MSNs induced concentration- and time-dependent cytotoxicity in the A375, MNT-1 and SK-MEL-28 melanoma cell lines. Notably, the A375 and SK-MEL-28 cell lines were slightly more sensitive to both the MH and MH-MSN exposure treatments than the MNT-1 cells.We hope that this study will inspire further in vitro studies, such as using 3D models, and in vivo studies to confirm the efficacy of morin-loaded MSNs.


CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), CD276 (B7-H3), JAK2, and FoXO1 in different BC cell lines, derived mammospheres, and co-cultures with peripheral blood mononuclear cells (PBMCs) by real-time quantitative polymerase chain reaction (qRT-PCR). Our results showed that intrinsic CTLA-4, CD274 (PD-L1), and PDCD1LG2 (PD-L2) were highly expressed in triple-negative cell lines, while CD276 was predominantly overexpressed in luminal cell lines. In contrast, JAK2 and FoXO1 were under-expressed. Moreover, high levels of CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), and JAK2 were found after mammosphere formation. Finally, the interaction between BC cell lines and peripheral blood mononuclear cells (PBMCs) stimulates the intrinsic expression of CTLA-4, PCDC1 (PD1), CD274 (PD-L1), and PDCD1LG2 (PD-L2). In conclusion, the intrinsic expression of immunoregulatory genes seems very dynamic, depending on BC phenotype, culture conditions, and tumor-immune cell interactions.Advances in immunotherapy have increased interest in knowing the role of the immune system in breast cancer (BC) pathogenesis. Therefore, immune checkpoints (IC) and other pathways related to immune regulation, such as JAK2 and FoXO1, have emerged as potential targets for BC treatment. However, their intrinsic gene expression in vitro has not been extensively studied in this neoplasia. Thus, we evaluated the mRNA expression of tumor-cell-intrinsic  Breast cancer (BC) is the most common cancer in women and the leading cause of cancer-related death in this gender worldwide . AlthougHistorically, BC has been considered a poorly immunogenic neoplasm compared to others with a higher mutational load . HoweverICs are a set of proteins that modulate the duration and amplitude of the immune response. They are a set of proteins that maintain homeostasis and self-tolerance under physiological conditions. However, within a pathological context, tumor cells can express these molecules as an essential mechanism of immune resistance . The ICsCTLA-4 is mainly expressed on activated, memory, and regulatory T cells (Tregs)  and is rOn the other hand, PD1 is mainly expressed on the surface of activated T cells and B cells, natural killer (NK) cells, activated monocytes, dendritic cells, and thymocytes . Its priB7-H3 (CD276) is mainly expressed in APCs ,32, actiOf note, ICs are not the only proteins that regulate the immune response. Other molecules, such as JAK2 and FoXO1, also play an essential role. Thus, JAK2 is a tyrosine kinase protein that, together with signal transducer and activator of transcription (STATs) proteins, is responsible for initiating the transcription of different target genes involved in various cellular processes . In addiFurther, it has been directly involved in cell proliferation, migration and invasion, angiogenesis, resistance to treatment and apoptosis, and the maintenance of stemness and EMT in different neoplasms ,46,47. ITherefore, there is a need to analyze the role of ICs, JAK2, and FoXO1 in regulating the immune response. Currently, the potential involvement of these molecules in the pathogenesis of BC has not been extensively investigated in different phenotypes, and data from in vitro studies and stem cell conditions are limited. CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), CD276 (B7-H3), JAK2, and FoXO1 in neoplastic cells depends on BC immunophenotype, stem cell properties, and tumor microenvironment. Therefore, we conducted different in vitro studies on six BC cell lines, derived mammospheres, and co-cultures with peripheral blood mononuclear cells (PBMCs) to determine this question. We hypothesize that intrinsic expression of CTLA-4, CD274 (PD-L1), and PDCD1LG2 (PD-L2) were intrinsically overexpressed in triple-negative cell lines, while CD276 was increased in luminal cell lines, both in the absence of adaptative immunity. In contrast, JAK2 and FoXO1 were under-expressed in most of the cell lines analyzed. Moreover, the expression of CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), and JAK2 in aggressive phenotypes increased after mammosphere formation. Finally, we observed that the interaction between BC cell lines and PBMCs stimulates the intrinsic expression of CTLA-4, PCDC1 (PD1), CD274 (PD-L1), and PDCD1LG2 (PD-L2), which may vary depending on BC immunophenotype and time of interaction.Our data showed that CTLA-4 and PDCD1 (PD1) receptors, so the ΔCt value was used for the analysis. In this case, the ΔCt value acted as a relative gene expression value, where low values are indicative of higher gene expression. We observed a significant increase in CTLA-4 in MDA-MB-231 and MDA-MB-468 compared to MCF-7 (both p = 0.025) and BT-474 , but not against SK-BR-3 . In addition, CTLA-4 expression was not detected in T-47D (PDCD1 (PD1), the expression remained stable in all cell lines, with no significant differences found among them   A.CD274 (PD-L1) and PDCD1LG2 (PD-L2) were also increased in MDA-MB-231 compared to 184A1, which was only statistically significant for CD274 (PD-L1) (p = 0.019). In the other cell lines studied, both genes were found to be significantly under-expressed  (PDCD1LG2 (PD-L2) expression in BT-474. Among the tumor cell lines, MDA-MB-231 presented the highest levels of CD274 (PD-L1) and PDCD1LG2 (PD-L2) expression compared to all cell lines . Likewise, an under-expression of both genes was observed in MCF-7, T-47D, and BT-474 cell lines concerning SK-BR-3 .< 0.014) B. In addCD276 (B7-H3) expression was significantly higher in MCF-7 (p = 0.049) and BT-474 (p = 0.021) cell lines. A significant decrease was also observed in SK-BR-3 (p = 0.003) compared to normal cell line 184A1 (p < 0.028).ne 184A1 C. When cJAK2 was significantly under-expressed in MCF-7, T-47D, BT-474, and SK-BR-3 cell lines  compared with 184A1. A slight decrease was also seen for MDA-MB-231, although only as a trend towards significance (p = 0.055) (JAK2 expression was significantly decreased in BT-474 compared to MCF-7 (p = 0.022), SK-BR-3 (p = 0.012), and MDA-MB-468 (p = 0.021) cell lines. Finally, FoXO1 was significantly under-expressed in BT-474, SK-BR-3, and MDA-MB-231  cell lines compared with 184A1 (p < 0.05).On the other hand, = 0.055) C. Furtheth 184A1 C. Among high/CD24lowCD44 phenotype in BC [CD44 expression and normal or decreased CD24 expression compared to controls  expression, we observed that both genes were significantly overexpressed in the mammospheres of BT-474 and SK-BR-3 cell lines  compared to controls. This increase also occurred in the mammospheres of the MCF-7 cell line but was not statistically significant. In addition, an increase in CTLA-4 expression (p < 0.029) and a decrease in CD274 (PD-L1) expression (p < 0.05) were observed in MDA-MB-231 mammospheres. Finally, in T-47D mammospheres, CD274 (PD-L1) expression was significantly under-expressed (p < 0.05), while CTLA-4 was not detected . On the other hand, CD274 (PD-L1) was significantly under-expressed in T-47D and MDA-MB-231 mammospheres concerning the rest .Regarding detected A. After PDCD1 (PD1) and PDCD1LG2 (PD-L2) expression, there were statistically significant differences between mammospheres obtained from MCF-7 and MDA-MB-231 cell lines  and controls. However, the levels remained close to normal in the rest of the mammospheres (PDCD1LG2 (PD-L2) was not expressed in BT-474 mammospheres. When comparing the mammospheres, it was seen that PDCD1 (PD1) expression in MDA-MB-231 mammospheres was significantly higher than in the rest . At the same time, PDCD1LG2 (PD-L2) levels were significantly lower in SK-BR-3 mammospheres versus those derived from MCF-7 and MDA-MB-231 (both p < 0.018).Regarding ospheres B. FurtheCD276 (B7-H3) was under-expressed in mammospheres obtained from BT-474, T-47D, and SK-BR-3 cell lines, being significant in the latter two cases (both p < 0.025) compared to controls. On the other hand, the remaining mammospheres showed CD276 (B7-H3) levels close to the controls. Likewise, it was observed that the mammospheres derived from the MDA-MB-231 cell line presented a significantly higher expression  than the rest, except those derived from MCF-7 (p = 0.513).As can be seen in JAK2 expression was remarkably under-expressed in T-47D and BT-474 mammospheres (both p = 0.014) and over-expressed in SK-BR-3 and MDA-MB-231 (both p = 0.014) compared to the controls . controls C. FurtheFoXO1 remained statistically underexpressed in most mammospheres  compared to controls. The only exception was the MDA-MB-231 mammospheres that maintained a near-normal expression (p < 0.033). In contrast, those derived from T-47D showed a significant underexpression .Finally, pression C. After CTLA-4 and PDCD1 (PD1) increased expression was seen in all co-cultures compared with control cell lines , the differences in expression observed between the second and third day in the T-47D cell line and those between the first and second day for the MDA-MB-468 cell line were statistically significant (both p = 0.043)  B.CD274 (PD-L1) and PDCD1LG2 (PD-L2), showing an increase (CD274 (PD-L1) levels during the first day of interaction with PBMCs in the SK-BR-3 cell line compared to the second and the third day on which the measurements were carried out  (PDCD1LG2 (PD-L2) was significantly elevated on the second day of interaction for the T-47D and MDA-MB-231 cell lines compared with the first day of measurements (both p = 0.043) (PDCD1LG2 (PD-L2) in BT-474 cells, with significant differences between the ΔCt values on the second and third day (p = 0.043). In contrast, CD276 (B7-H3) and JAK2 levels in co-cultures did not differ from those obtained in the cell lines used as controls (p < 0.027). Finally, FoXO1 expression remained at near-normal levels in all co-cultures. Nevertheless, we observed significant differences between the second and the third day of interaction in the co-cultures of the MCF-7 cell line (p = 0.030). We also found differences between the first and the second day (p = 0.002), as well as the second and the third day of interaction (p = 0.036) in the co-cultures of BT-474  C. On the= 0.043) D. In addcontrols E,F. Howef BT-474 G.CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), CD276 (B7-H3), JAK2 and FoXO1 in six BC cell lines, their derived mammospheres, and in co-cultures with PBMCs. Our data showed an intrinsic CTLA-4 mRNA increase in the triple-negative cell lines MDA-MB-231 and MDA-MB-468 compared to the luminal cell lines MCF-7 and BT-474. This increase was also observed in mammospheres derived from BT-474, SK-BR-3, and MDA-MB-231 cell lines. Moreover, as in the controls, no expression was detected in the luminal A T-47D cell line and derived mammospheres. In agreement with our results, previous studies have also demonstrated its expression in MCF-7 and MDA-MB-231 BC cell lines at both mRNA and protein levels in the absence of immune system cells [In the current study, we analyzed the intrinsic mRNA expression of several immunoregulatory genes, such as em cells ,61,62,63CTLA-4 expression has never been previously analyzed in mammospheres. According to the current results, CTLA-4 expression on neoplastic cells may be essential for the pathogenesis of BC and the modulation of the tumor microenvironment, especially in triple-negative BC. In addition, our results suggest that its expression might be implicated in acquiring and maintaining tumor stem cell characteristics in aggressive BC phenotypes. Intrinsic CTLA-4 expression has also been linked to BC cell proliferation, differentiation, survival , and thePDCD1 (PD1) maintained a similar mRNA level for all analyzed cell lines, with no significant differences. However, it was overexpressed in mammospheres derived from MCF-7 and MDA-MB-231 cell lines. This increase was significant in the rest of the MDA-MB-231 mammospheres. To our knowledge, this is the first study to characterize the intrinsic expression of PDCD1 (PD1) in BC cell lines and mammospheres without an immunological environment. Of note, its expression has been evaluated in other neoplasms, where in most cases, it has been described as a pro-oncogenic factor. In this regard, several independent studies have confirmed that the intrinsic expression of PD1 in melanoma, hepatocellular carcinoma, and bladder cancer cell lines promotes neoplastic growth without an adaptative immune response [We also demonstrated that response ,67,68,69response . In contresponse . These dCD274 (PD-L1) and PDCD1LG2 (PD-L2) were intrinsically overexpressed in the triple-negative MDA-MB-231 cell line, although only significant for CD274 (PD-L1). The rest of the cell lines showed underexpressed levels compared to controls. In addition, the absence of PDCD1LG2 (PD-L2) expression in the BT-474 line was noteworthy. In agreement with our results, numerous authors have observed a significant increase of both ligands in the MDA-MB-231 cell line compared with luminal cell lines, both at the mRNA and protein levels [PDCD1LG2 (PD-L2) has also been reported in MDA-MB-231, SK-BR-3, and MCF-7 compared to the non-malignant MCF10A cell line by others [n levels ,73,74,75y others . Therefoy others . CD274 (PD-L1) expression in neoplastic cells favors immune response evasion and increases neoplastic aggressiveness. Their gene expression levels have also been related to tumor stemness maintenance in different neoplasms [CD274 (PD-L1) was significantly overexpressed in mammospheres derived from HER2-expressing cell lines (BT-474 and SK-BR-3). In contrast, compared with controls, intrinsic PDCD1LG2 (PD-L2) was upregulated in those derived from MCF-7 and MDA-MB-231. In agreement with our results, previous investigators demonstrated intrinsic PD-L2 expression by flow cytometry in MCF-7-derived mammospheres with no significant differences concerning the control cell line [Concerning intrinsic functions, PD-L2 has been less studied in cell lines, whereas PD-L1 has been implicated in resistance to chemo- and radiotherapy in the MDA-MB-231 cell line  and apopeoplasms . We founell line .CD274 (PD-L1) and PDCD1LG2 (PD-L2) in maintaining BC’s tumor stem cell phenotype.In contrast, a high intrinsic PD-L1 expression at the mRNA and protein level has been described in mammospheres derived from MCF-7 and MDA-MB-231 cell lines ,78. FurtCD276 (B7-H3) was overexpressed in luminal cell lines (MCF-7 and BT-474) and underexpressed in HER2-enriched (SK-BR-3) compared to the control. In the remaining cell lines, the expression was close to normal. Conversely, BT-474, T-47D, and SK-BR-3-derived mammospheres showed decreased expression, while those derived from MCF-7 and MDA-MB-231 presented levels close to normal. The intrinsic expression of this immune checkpoint has been previously described in MCF-7 and T-47D cell lines in variable levels [We detected that intrinsic e levels . These re levels  and indue levels  and inhie levels  through CD276 (B7-H3) expression in neoplastic cells plays a crucial role in the induction of the EMT process and acquiring stem cell phenotype. For example, its intrinsic expression in colorectal cancer cell lines decreased E-cadherin expression and increased stem cell-associated markers such as N-cadherin, vimentin, CD133, CD44, and OCT4 [CD276 (B7-H3) is a critical factor in evading the immune response by tumor stem cells in head and neck carcinoma [Furthermore, recent studies have shown that intrinsic and OCT4 . Likewisand OCT4  and the and OCT4 . In addiarcinoma . In BC, arcinoma . Indeed,arcinoma . In contJAK2 was significantly under-expressed in the luminal , HER2-enriched (SK-BR-3), and triple-negative MDA-MB-231 cell lines, the latter only as a trend. However, in MDA-MB-468, cell line values were close to normal. Balko et al. suggest that the difference between the MDA-MB-231 and MDA-MB-468 cell lines could be explained by variations in the gene copy number [JAK2 in the MCF-7 cell line, in contrast to our data.Intrinsic y number . They alhigh/CD24low cells and inhibits the formation of mammospheres [JAK2 in triple-negative cell lines favored mammospheres’ formation and increased chemotherapy resistance [JAK2 expression is essential for mammosphere formation, maintenance of the stem cell phenotype in BC, and resistance to conventional treatments.Regarding its function, it is directly involved in the proliferation and aggressiveness of MDA-MB-231 and MDA-MB-468 cell lines . Likewisospheres ,91. Simiospheres . Howeverospheres . Moreoveospheres . In likesistance . This prsistance . Thus, tFoXO1 in the most aggressive cell lines . In addition, Liang et al. reported in the MDA-MB-231 cell line that it promotes cell proliferation but not migration or apoptosis [FoXO1 mRNA and protein levels, suggesting a robust translational control [We demonstrated an intrinsic underexpression of poptosis . However control .FoXO1 expression in all analyzed mammospheres, except those from the MDA-MB-231 cell line. Similar results have been previously reported in MCF-7, SK-BR-3, and MDA-MB-231 [We also detected a decrease in A-MB-231 ,98,99. DA-MB-231  and doxoA-MB-231  in TAMR-A-MB-231 .FoXO1 expression in MCF-7, T-47D, and BT-474 was directly related to progesterone receptor phosphorylation, inhibition of cell proliferation, and mammosphere formation [FoXO1 may play a critical role in BC pathogenesis and drug resistance development. Although the published data are not entirely clear, our results show the relevance of FoXO1 in the maintenance of the tumor stem cell phenotype. In contrast, Truong et al. demonstrated that ormation . These cormation . The preCTLA-4, PCDC1 (PD1), CD274 (PD-L1), and PDCD1LG2 (PD-L2) intrinsic expression after a direct interaction with PBMCs. Furthermore, T-47D and BT-474 cell lines recovered cell-intrinsic CTLA-4 and PDCD1LG2 (PD-L2) expression after tumor-immune cell interactions, respectively. However, CD276 (B7-H3), JAK2, and FoXO1 maintained their intrinsic expression levels close to normal, even after interaction with PBMCs. Similar results were already reported for PD-L1 by Hinterneder et al. [FoXO1 expression increased at 72 h of interaction compared to the first 48 h. Additionally, the T-47D cell line showed an increase in PDCD1 (PD1) and PDCD1LG2 (PD-L2) on the second day compared to the first and the third day, respectively. Likewise, the BT-474 cell line experienced an increase in PDCD1LG2 (PD-L2) levels at 72 h of interaction, while FoXO1 was overexpressed after the first 24 h. Regarding the MDA-MB-468 cell line, PDCD1 (PD1) was under-expressed at 48 h versus 24 h, while CD276 (B7-H3) and JAK2 were over-expressed at 72 h of interaction. Finally, the MDA-MB-231 cell line only manifested an increase in PDCD1LG2 (PD-L2) during the second interaction day compared to the first one. These temporal variations in intrinsic gene expression could be due to the inhibition of immune cells and thus less production of soluble factors and fewer cell-cell interactions. In support of this, Chen et al. demonstrated in BC cell lines that the intrinsic expression of CTLA-4 could inhibit the processes of antigenic presentation and expression of inflammatory cytokines, preventing the proper activation of type 1 T helper (Th1) and cytotoxic T lymphocytes [Finally, we observed in all co-cultures a significant increase in r et al. , who desr et al. . In thisr et al. . Furtherr et al. , althougr et al. . For itsr et al. . Despiter et al. . Here, wphocytes .Similarly, overexpression of PD-L1 and PD-L2 in neoplastic cells would directly inhibit effector T lymphocytes . Our exp2 flask  and Dulbecco Modified Eagle Medium (DMEM)/Ham F12 (1:1) with L-glutamine and 15 mM HEPES media (Biowest) supplemented with 10% fetal bovine serum (FBS) (Biowest), 50 U/mL of penicillin, and 50 mg/mL of streptomycin (Biowest). Cells were incubated at 37 °C in a humidified atmosphere with 5% of CO2 and were grown to around 80% confluence before RNA isolation.Six human BC cell lines, MCF-7 and T-47D , BT-474 , SK-BR-3 (HER2-enriched), MDA-MB-231 and MDA-MB-468 , and the normal epithelial breast cell line 184A1 were obtained from the American Type Culture Collection (ATCC)  . All celg for 15 min. Further, the PBMCs were collected from the interface between plasma and Biocoll, washed twice, and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium with glutamine  and supplemented with 10% FBS (Capricorn Scientific GmbH) and 1% antibiotics (50 U/mL penicillin and 50 mg/mL streptomycin) (Capricorn Scientific GmbH). PBMCs were incubated for 2 h at 37 °C and 5% CO2, allowing monocyte separation by adhering to the plastic of the culture flask [Peripheral blood was collected from 30 BC patients with informed consent to use their samples for research purposes and ethical approval from the ethics committee (ethic code PI2019/089 and PI2020-265) at our institution. The selected patients represented each BC immunophenotype . PBMCs were isolated by density gradient centrifugation. Briefly, 3 mL of Biocoll Separating Solution  and 6 mL of peripheral blood samples diluted in phosphate-buffered saline (PBS) were placed in sterile tubes centrifuged at 1300× re flask . After t2. Once cultures reached 70–80% confluence, tumor cells were seeded in duplicate at 100,000 cells/well in sterile 6-well plates (SPL Life Sciences). After 24 h, the isolated PBMC were co-cultured in the 6-well plates with the pre-seeded tumor cells at a concentration of 500,000 cells/well. The above was performed considering the phenotype of the cell lines and the BC patients who donated the peripheral blood sample. The co-cultures were then maintained in a humid environment at 37 °C and 5% CO2 , supplemented with 10% FBS (Biowest), 50 U/mL of penicillin, and 50 mg/mL of streptomycin (Biowest). The cell lines were seeded in sterile culture flasks and incubated in a humid atmosphere at 37 °C and 5% COd 5% CO2 . After c3 cells per well. Under these conditions, the cells grew as a non-adherent spherical cluster  supplemented with 10% proliferation supplements (Stemcell Technologies), 0.48 mg/mL of hydrocortisone (Stemcell Technologies), 4 mg/mL of heparin (Stemcell technologies), 50 U/mL of penicillin, and 50 mg/mL of streptomycin (Biowest). Single T-47D, MCF-7, BT-474, SK-BR-3, and MDA-MB-231 cells were seeded in six-well plates coated with poly-2-hydroxyethyl-methacrylate  to prevent cell attachment at a density of 5 × 10 cluster . Every tSingle-cell suspensions obtained from cell line cultures, co-cultures, and mammospheres were used for RNA isolation. Cells were lysed in 600 µL of RLT buffer  supplemented with 1% β-mercaptoethanol  through a 29G needle. Following the RNeasy Mini kit protocol , 70% ethanol was added to cell lysates, and the samples were mixed by pipetting before being transferred to the columns. Total RNA was extracted according to the manufacturer’s protocol with on-column DNase digestion. RNA concentration and purity were assessed using a NanoDrop spectrophotometer  and optical density ratios of 260/230 nm and 260/280 nm. Before the reverse transcription reaction, isolated RNAs were stored at −80 °C. For complementary DNA synthesis, 2 µg of total RNA was reverse-transcribed using random hexanucleotides and the High-Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) following the manufacturer’s instructions. The reaction product was diluted to a final concentration of 20 ng/µL and stored at −20 °C until gene expression analysis.® Fast Universal PCR Master Mix and assays based on hydrolysis probes , as they do not detect genomic DNA. Gene expression was performed for CTLA-4 (Hs00175480_m1), PDCD1 (PD1) (Hs00169472_m1), CD274 (PD-L1) (Hs00204257_m1), PDCD1LG2 (PD-L2) (Hs00228839_m1), CD276 (B7-H3) (Hs00987207_m1), JAK2 (Hs01078136_m1), and FoXO1 (Hs01054576_m1) for cell lines, mammospheres, and co-cultures samples. As reference genes to normalize gene expression, we used both β-ACTIN (Hs99999903_m1) and PUM1 (Hs00472881_m1). Depending on the assay type, a pool of RNA from the breast epithelial cell line 184A1 or the tumor cell lines was used as reference calibrator samples. No template controls were included in each reaction, and all experiments were duplicated. Relative changes in gene expression were calculated as fold change by the 2−ΔΔCt method [The qRT-PCR was carried out in a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) using a total volume of 10 µL according to the manufacturer’s instructions. We used TaqMant method . Resultst-test for paired data and the Wilcoxon test were used to compare parametric and non-parametric variables, respectively. For our in vitro study, we define increased or decreased levels of any gene expression as those above or below a relative quantification value equal to 1. In all cases, a p-value of <0.05 was considered statistically significant. Statistical analysis was performed using the SPSS version-23 statistical software package.The Kolmogorov–Smirnov test was performed to define the distribution of the variables. For the differences in gene expression, parametric variables were compared using Student’s t-test, while non-parametric variables were compared using the Mann–Whitney U-test. In the co-cultures, in which repeated measurements were performed over time, the Student’s PDCD1 (PD1), JAK2, and FoXO1 expression are highly variable in BC cell lines independently of adaptative immunity. In addition, intrinsic CTLA-4, CD274 (PD-L1), and PDCD1LG2 (PD-L2) expression could be essential for the pathogenesis of triple-negative BC cells, whereas CD276 (B7-H3) expression would be necessary for luminal cell lines. Moreover, we have demonstrated the intrinsic expression of CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), and JAK2 in mammospheres derived from aggressive BC phenotypes. However, CD276 (B7-H3) and FoXO1 expression seem not to be directly involved in the acquisition and/or maintenance of tumor stem cell characteristics in vitro. Finally, we have determined that neoplastic cell-intrinsic CTLA-4, PCDC1 (PD1), CD274 (PD-L1), and PDCD1LG2 (PD-L2) expression is dynamic and varies depending on BC immunophenotype and tumor-immune cell interactions. Therefore, these data confirm our hypothesis that the expression of some immunoregulatory genes depends on BC phenotype and stem cell properties. They further support the view that this expression is intrinsic to neoplastic cells and depends on the tumor microenvironment.In conclusion, we have found that


Klotho is an antiaging protein, and its levels decline with age and chronic stress. The exogenous administration of Klotho can enhance cognitive performance in mice and negatively modulate the Insulin/IGF1/PI3K/AKT pathway in terms of metabolism. In humans, insulin sensitivity is a hallmark of healthy longevity. Therefore, this study aimed to determine if exogenous Klotho, when added to neuronal and astrocytic cell cultures, could reduce the phosphorylation levels of certain insulin signaling effectors and enhance antioxidant strategies in these cells. Primary cell cultures of cortical astrocytes and neurons from mice were exposed to 1 nM Klotho for 24 h, with or without glucose. Klotho decreased pAKT and mTOR levels. However, in astrocytes, Klotho increased FOXO-3a activity and catalase levels, shielding them from intermediate oxidative stress. In neurons, Klotho did not alter FOXO-3 phosphorylation levels but increased proteasome activity, maintaining lower levels of PFKFB3. This study offers new insights into the roles of Klotho in regulating energy metabolism and the redox state in the brain. Within the central nervous system (CNS), the choroid plexus is regarded as the primary site of Klotho synthesis4. However, it is also found in lower concentrations in other regions, including the cortex, hippocampus, cerebellum, striatum, substantia nigra, olfactory bulb, and medulla8.The Klotho protein was first identified in 1997, when animal studies linked its absence to an accelerated aging phenotype. Consequently, Klotho has been recognized as an anti-aging protein3 and in the cerebrospinal fluid. These forms exert biological actions in distant organs and across multiple systems5. Conversely, the secreted form originates from alternative splicing, leading to the creation of a single domain (KL1). This form functions as an endocrine regulator of various cell surface glycoproteins5.Klotho protein exists in two primary isoforms: membrane and soluble. The membrane Klotho comprises two domains, KL1 and KL2. These domains can be cleaved, resulting in the primary soluble forms found in circulation6, chronic stress7, and neurodegenerative diseases8. Interestingly, a decrease in Klotho expression has been linked to the onset of cognitive deficits9 and increased senescence in neural progenitor cells10. Conversely, Klotho overexpression seems to prolong the lifespan of animals11, enhance cognitive function, and improve learning and memory, regardless of age12. A recent study involving mice demonstrated that peripheral administration of Klotho enhanced cognitive performance13. Although the precise role of Klotho in the CNS is yet to be clearly defined, research has underscored its potential in boosting cognitive performance13.Scientific evidence indicates a decline in Klotho levels with age15. In humans, insulin sensitivity is a hallmark of healthy longevity16. Moreover, soluble Klotho has a negative modulatory effect on IGF-118, which results in a decrease in the activity of downstream signaling cascades, including the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB or AKT) pathways20.The Insulin/Insulin-like Growth Factor 1(IGF-1) is a crucial pathway that influences aging and lifespan in animals21. When less active, it can contribute to lifespan extension22. The activation of PI3K, and consequently AKT, is triggered by the binding of insulin to the insulin receptor (IR), primarily through phosphorylation at Thr-308 induced by phosphoinositide-dependent kinase-1 (PDK-1). At this juncture, AKT can phosphorylate the mechanistic target of rapamycin (mTOR), a Ser/Thr kinase23. Full activation of AKT is achieved through its phosphorylation at Serine 473 by mTOR21. Once activated, AKT phosphorylates the Forkhead box O (FOXO) proteins, inhibiting FOXO nuclear translocation and its role as a transcriptional factor24. Thus, the Insulin/PI3K/AKT signaling pathway is instrumental in regulating members of the FOXO family.The PI3-K/AKT signaling pathway plays a crucial role in integrating physiological responses associated with healthy aging27. A potential mechanism is that, upon activation, FOXOs enhance the expression of catalase and manganese superoxide dismutase (MnSOD or SOD2), which detoxify harmful reactive oxygen species (ROS) derived from hydrogen peroxide and superoxide, respectively28. In the human retinal pigment epithelium, Klotho can reinstate the SOD2 gene expression levels under conditions of oxidative stress29. This finding aligns with a prior study that reported similar observations in HeLa cells18. Notably, the FOXO-3a member plays a pivotal role in oxidative stress resistance, autophagy, and apoptosis31.Several studies have identified FoxO forkhead transcription factors (FOXOs) as significant regulators of longevity32. In addition, in hepatocarcinoma cells, the increase in exogenous klotho levels downregulates the phosphorylation levels of the IGF-1 receptor and the downstream AKT, ERK, and p70S6K proteins33. A similar effect was observed in a model of diabetic kidney disease, where Klotho gene deficiency enhanced the phosphorylation of the mammalian target of rapamycin (mTOR) and protein p70 ribosomal protein S6 kinase (p70S6K) in the kidney. However, when rat mesenchymal cells were incubated with exogenous Klotho prior to normal or high glucose 24 h exposure, Klotho inhibited mTOR phosphorylation stimulated by high glucose34. Therefore, we decided to verify whether Klotho was able to modulate these signaling pathways in neurons and astrocytes.Klotho has an important effect as an autophagy regulator in many types of cells owing to its influence on the IGF-1/PI3K/Akt/mTOR signaling pathway35. Neurons directly use the lactate released by astrocytes to fulfill their energy demands during periods of high activity or indirectly by coupling the transamination of pyruvate to alanine with the conversion of glutamate to α-ketoglutarate. Lactate also plays a crucial role in lipid synthesis, which is essential for dendritic branching and synaptic plasticity37. Our recent findings also indicate that hypomorphic transgenic mice with the Klotho gene exhibit enhanced insulin signaling, resulting in increased AKT and mTOR activation and reduced FOXO activity in the hippocampus (unpublished data).Furthermore, data from our group indicate that secreted Klotho (sKlotho) from neurons rapidly enhances aerobic glycolysis in astrocytes, which results in lactate release through the activation of fibroblast growth factor receptor 1 (FGFR1) and extracellular signal-regulated kinase 1/2 (ERK 1/2)38. Klotho mitigates insulin signaling in 3T3-L1 adipocytes, leading to a decrease in the phosphorylation of AKT, Glycogen Synthase Kinase-3 (GSK3β), and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3)39.Mice deficient in Klotho exhibit extreme sensitivity to insulin and hypoglycemia. In contrast, transgenic mice overexpressing Klotho do not develop diabetes, despite showing moderate resistance to insulin and IGF-140. In neurons, PFKFB3 is ubiquitinated and degraded by the proteasome to maintain lower levels of fructose-2,6-bisphosphate and reduced PFK1 activity41. This process ensures that the pentose phosphate pathway (PPP) predominates in glucose metabolism, thereby elevating the levels of NADPH, which is instrumental in oxidative damage repair.PFKFB3 serves as a regulator of cellular metabolism within the CNS. It controls the concentration of fructose-2,6-bisphosphate, a crucial activator of the phosphofructokinase-1 enzyme (PFK1) and glycolysisOur observations have led us to hypothesize that Klotho may activate FOXO-3a and enhance the expression of catalase and SOD2 in neurons and astrocytes, possibly owing to the inhibition of insulin/IGF-1 signaling. This process could facilitate the removal of reactive oxygen species and bolster cellular resistance to oxidative stress. Furthermore, we sought to determine whether Klotho could regulate PFKFB3 levels in the CNS as an additional cellular protective strategy. Our findings indeed suggest that Klotho functions as a potent antioxidant in astrocytes, augmenting FOXO-3a activity and catalase expression, thereby protecting them from death. However, Klotho at 1 nM was unable to prevent neuronal death following severe oxidative insult, but it did reduce PFKFB3 levels, thereby enhancing proteasome activity.Cell culture reagents were purchased from Thermo Fisher Scientific . The protein assay kit, obtained from Bio-Rad  was utilized in accordance with the manufacturer´s instructions. Routine reagents were purchased from Sigma Chemicals , while recombinant α-Klotho was acquired from R&D Systems . All solutions were freshly prepared prior to use.https://arriveguidelines.org), which are grounded in the Ethical Principle in Animal Research endorsed by the Brazilian College of Animal Experimentation (CONCEA). These procedures received approval from the Ethical Committee for Animal Research (CEUA) at the Biomedical Sciences Institute of the University of São Paulo, São Paulo, São Paulo State, Brazil. The protocol was officially registered under the number 12/2016 CEUA for animals utilized in experimentation.All procedures adhered to the ARRIVE guidelines . The tissue was then dissociated using 0.25% trypsin at 37 °C for 5 min, after which Dulbecco's Modified Eagle Medium (DMEM)  was added. The cells were further dissociated using a Pasteur pipette, filtered, and plated at a density of 1.5 × 104 cells/cm2 in a T75 flask. The medium was refreshed every 3 days. The cultures were incubated until they achieved confluency, typically within 10–14 days. The cells were then digested with 0.25% trypsin at 37 °C for 10 min to facilitate detachment from the flask. Digestion was halted by adding DMEM with 10% fetal bovine serum (FBS), after which the cells were centrifuged for 2 min at 2000 rpm. The resulting pellet was resuspended in DMEM 10% FBS and counted for plating. Each well in a 6-well plate received 1 × 106 cells. For a 24-well plate, cells were seeded at a density of 1 × 104 per well. The flasks were then incubated in an orbital shaker at 37 °C and 180 r/min for 15 h, following a previously described method to separate astrocytes from other glial cells35. The resulting culture, used for the experiments, was deemed an astroglial-enriched culture, as it contained 91.2% glial fibrillary acidic protein-labeled cells. Representative photomicrographs of the culture phenotype are provided in the supplementary material  mice of both sexes2 incubator. Following the removal of the trypsin solution, the tissue was rinsed twice with HBSS. The tissue was then dissociated in HBSS containing 0.1 mg/mL DNAse through mechanical trituration using a glass pipette. The cells were counted and seeded at a density of 1 × 105 cells/cm2 on dishes pre-coated with polyethyleneimine (Sigma-Aldrich). The neurons were maintained for 2 weeks in Neurobasal medium (GIBCO) supplemented with B27 (GIBCO), 2 mM L-glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin, and 0.25 mg/mL amphotericin B. A representative photomicrograph of the culture phenotype is presented in Supplementary Fig. Primary cortical neuronal cultures were prepared from both male and female C57BL/6 mouse embryos on gestational days 16th–17th (E16-17). The meninges were excised from the brain, and the cortices were sectioned into small fragments and incubated in a 2 mg/mL trypsin solution for 20 min at 37 °C in a 5% CO43, which incorporates three iterations of FOXO-responsive promoters and encodes firefly luciferase. Concurrently, the pRL-TK plasmid (Promega), expressing renilla luciferase constitutively, was transfected as an internal control. Astrocytes were seeded in 60 mm diameter plates (1 × 106 cells/plate) and transfected after 48 h (approximately 60% confluence) using Fugene 6 (Promega) with 1 µg DBE DNA, 0.2 µg pRL-TK DNA, and a DNA: Fugene ratio of 1:6. Following 48 h of transfection, the cells were treated by washing twice with phosphate buffered saline (PBS) and incubating for 24 h in DMEM containing either 4.5 g/L or 1 g/L glucose concentration, with or without 1 nM Klotho. Post-treatment, the cells were lysed as per the Dual-Luciferase® Reporter Assay system (Promega) guidelines. Equal sample volumes were used, and the luminescence was developed and read according to the manufacturer's instructions (Promega) on opaque white plates using a Synergy H1 Hybrid Multi-Mode plate reader (BioTek). The results are presented as the ratio of firefly luciferase fluorescence to renilla luciferase, normalized to the mean of the control group.The activity of the FOXO transcription factor was assessed using the pGL-3 × DBE plasmid (Promega)45. This method relies on the ability of viable cells to convert tetrazolium salt (MTT) into formazan, a colored compound. Cells were pretreated with either PBS or Klotho for a duration of 24 h, followed by a 30-min exposure to H2O2. Cell viability was ascertained by incubating cells with MTT (12 mM) in the cell culture medium for 2 h at 37 °C, 24 h post the final treatment. The dark crystals that formed were solubilized with DMSO, and the absorbance was measured at a wavelength of 570 nm using a microplate reader. The MTT (%) was calculated using the formula: (absorbance of the sample − absorbance of DMSO)/(absorbance of the control − absorbance of DMSO) × 100.The MTT assays were conducted as previously described2EDTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate, and mini protease inhibitor cocktail). The Bradford method was used to determine protein concentration. Equal protein amounts (15 µg) were separated on 4–12% bis–tris NuPAGE gels (Invitrogen) and transferred to a nitrocellulose membrane. The membrane was then incubated with a 5% bovine serum albumin (BSA) solution in Tris-buffered saline with tween (TBS-T)  for 2 h. The membranes were incubated overnight at 4 °C in primary antibody solution  with pAKT Ser473 , AKT pan , p-mTOR Ser2448 , mTOR , FOXO3 , p-FOXO3 SER253 , Catalase , PFKFB3 , and Beta-actin . After three washes with TBS-T, the membranes were incubated for 2 h with a secondary antibody solution  containing horseradish peroxidase-conjugated antibodies anti-rabbit or anti-mouse IgG-Fc (1:4000). Following three additional washes with TBS-T, proteins recognized by the antibodies were revealed using an ECL system . The photo documentation system DP-001-FDC  and N.I.H. ImageJ software (http://rsb.info.nih.gov/ij) were used to standardize and quantify the immunoblots. Multiple exposure times were analyzed to ensure the linearity of the band intensities.Total protein extracts (nuclear and cytoplasmic) were obtained by collecting and pelleting cells, which were then homogenized in RIPA lysis buffer containing protease and phosphatase inhibitors . Neurons were plated into a 96-well black microplate with a flat and translucent bottom. Cells were cultured under normal conditions for 14 days and then subjected to different conditions: (i) 24 h with complete medium (control group); (ii) 24 h with medium without insulin, supplemented with B27 minus insulin ; (iii) 24 h with 1 nM of Klotho, and (iv) with the proteasome inhibitor MG132 at 50 µM for 2 h before performing the assay. The assay relies on the proteosome complex performing the proteolytic breakdown of the fluorogenic substrate, LLVY-R110. Cleavage of LLVY-R110 by the proteasome generates the R110 product, which shows intense green fluorescence (λex = 480–500 nm and λem = 520–530 nm). Following treatments, the culture medium was immediately replaced with an LLVY-R110 solution, and incubation lasted for 1 h. Fluorescence reading was conducted using a Synergy H1 Hybrid Multi-Mode plate reader (BioTek). Fluorescence values were corrected for the blank (cell-free well) and are presented as a function of the mean values of the control group. Three independent neuronal cultures were analyzed, and the final value expressed in the resulting graph represents an average of 24 wells per group/ per culture. The control group had 22 wells in each plate.6 cells). The cells were gathered and homogenized in non-denaturing lysis buffer  using ultrasonication on ice. A total of 100 µg of protein (diluted in 400 µL of buffer) was incubated with 2 µg of antibody against phosphotyrosine  or against mono- and polyubiquitin K29, K48, K63 . For their respective negative controls, 2 µg of rabbit IgG or mouse IgG were used. The samples were incubated overnight with light orbital shaking. Subsequently, 20 µL of the suspension containing 50% agarose microspheres with protein A/G covalently immobilized was added, followed by incubation under light agitation for 3 h. The samples were then centrifuged and washed five times with ice-cold PBS. The final precipitate was resuspended in 20 µL of three-fold concentrated SDS sample buffer, homogenized, and centrifuged again, followed by heating at 70 °C for 10 min. After an additional centrifugation, the samples were loaded onto an SDS PAGE gel and analyzed by western blotting against PFKB3  for FK2 and PKM2 . This assay was conducted on three independent cell cultures.To assess protein phosphorylation and ubiquitination levels, astrocyte and neuron samples were examined through immunoprecipitation. These samples were obtained from pools of six wells from six-well plates, ensuring sufficient protein content  of the Institute of Biomedical Sciences, protocol registered under number 63 on page 07 of book 03, approved on May 29, 2013, and extended on May 12, 2017 (Of. CEUA. 014.2017) until May 29, 2021. The use of animals and all experimental procedures were deemed appropriate and in accordance with the Ethical Principles of Animal Experimentation adopted by the Brazilian Society of Laboratory Animal Science (SBCAL).35. Consequently, to ascertain whether 1 nM Klotho could modulate the Insulin/AKT/mTOR and FOXO3a signaling pathways in neurons, we treated primary cortical neurons with 1 nM Klotho for 24 h and subsequently analyzed them using western blotting. For the purpose of a positive control, cells were exposed to 100 nM insulin for the same duration. This assay involved the treatment and analysis of four independent cultures.In a prior study conducted by our group, we observed that treating astrocytes with 1 nM Klotho led to a rapid increase in aerobic glycolysis and lactate release via ERK activation, coupled with a simultaneous reduction in AKT phosphorylation6 cells. Each dot represents an independent cell culture.As anticipated, insulin treatment increased AKT phosphorylation at Ser473 Fig. A and mTO6 cells. Each data point in the graphs corresponds to a lysate derived from an independent cell culture.Following 24 h of treatment with either 1 nM Klotho or 100 nM insulin, FOXO3a phosphorylation levels in astrocytes were assessed using western blot analysis. Lysates were obtained from either a single well or two wells of a 6-well plate, yielding a total of 2 × 10Our findings indicate that the application of Klotho prevented the inhibitory phosphorylation of the FOXO3a transcription factor in astrocytes, as depicted in Fig. To evaluate the transcriptional activity of the FOXO transcription factor directly, astrocytes were transfected with a plasmid housing copies of (i) the FOXO-responsive sequence (pGL-3xDBE), serving as the promoter unit of a gene encoding firefly luciferase and (ii) plasmids carrying a gene encoding promoter-regulated renilla luciferase constitutive thymidine kinase (pRL-TK), which served as an internal control for the transfection rate. Following transfection, astrocytes were maintained for 24 h under varying culture conditions (in media with high or low glucose concentrations), with or without 1 nM Klotho. The relative luminescence was then measured to determine the transcription rate of the luciferase gene and, by extension, the activity of the FOXO transcription factor. The results indicated that a lower glucose concentration stimulated FOXO activity. Interestingly, Klotho augmented transcription factor activity in both high and low glucose concentrations, implying a potential role for Klotho in modulating FOXO activity in astrocytes   and catalase, has been previously described2O2 was assessed using the MTT assay. Our results indicated a significant reduction in astrocyte viability across all tested H2O2 concentrations  at varying concentrations for 24 h prior to a 30-min exposure to H2O2. Our findings demonstrated that all tested Klotho concentrations effectively mitigated astrocyte death induced by H2O2   for 2 h. Each dot on the graph represents the average of values measured in 22–24 wells of a 96-well plate from a single cell culture. We conducted this analysis of proteasome activity across three independent cell cultures.Based on the transcriptome results from a neuronal microarrayFigure To verify whether the observed decrease in PFKFB3 in neurons was a result of Klotho’s modulatory action on ubiquitination, we assessed the levels of polyubiquitinated PFKFB3 through immunoprecipitation. We employed an antibody against polyubiquitin (FK2) Fig. c, follow48. Neurons possess a limited capacity to metabolize energy substrates other than glucose, which accounts for the abundance of mitochondria and the intense oxidative phosphorylation observed49. Given that optimal neuronal function and, consequently therefore, healthy cognitive performance necessitate proper metabolic cooperation between astrocytes and neurons, this study aimed to determine whether exogenous Klotho could inhibit insulin/IGF-1 signaling in neurons and activate FOXO-3a. As previously noted, FOXOs enhance the expression of catalase, which detoxifies ROS from hydrogen peroxide. Additionally, we aimed to investigate whether Klotho could modulate PFKFB3 levels in the CNS. PFKFB3, a crucial regulator of cellular metabolism40, should be maintained at low levels in neurons to improve antioxidant defenses.The brain requires substantial amounts of oxygen and glucose to sustain high levels of neuronal activity50. Klotho can mitigate the suppressive effect of AKT on FOXO transcription factors by negatively modulating the PI3K/AKT pathway. This allows the transcription factors to relocate to the nucleus and become active. Consistent with our previous findings from astrocytes, Klotho has also been observed to reduce AKT and mTOR phosphorylation levels in cortical neurons  for survival significantly distinguishes astrocytic metabolism from neuronal metabolism. In astrocytes, OXPHOS inhibition triggers glycolysis, a response not mirrored to the same extentPFKFB3, a crucial enzyme, plays a significant role in managing energy metabolism and antioxidant defenses in neurons. This enzyme belongs to the PFKFB family, which is tasked with controlling the concentration of fructose-2,6-bisphosphate  within cells. F2,6BP serves as a powerful activator for the glycolytic enzyme PFK-1, the primary enzyme in the glycolytic pathway responsible for the conversion of glucose into pyruvate.41. Maintaining low PFKFB3 levels in neurons can promote the PPP, a crucial process for the production of NADPH and ribose-5-phosphate. These compounds are essential for nucleotide and fatty acid synthesis, as well as for antioxidant defenses58.The levels of PFKFB3 messenger RNA in neurons and astrocytes are identical. However, owing to ubiquitination leading to proteasomal degradation, the protein level of PFKFB3 in neurons is virtually null due to its ubiquitination and consequent proteasomal degradation59. A reduction in PFKFB3 levels within neurons can enhance PPP activity and NADPH production60. In adipocytes, PFKFB3 activity is decreased by Klotho39.Conversely, glutamate-induced excitotoxicity stabilizes neuronal PFKFB3. This stabilization subsequently diminishes the activity of the PPP and augments glycolysis, leading to oxidative damage and, ultimately, neuronal death. These detrimental effects can be mitigated by the overexpression of glucose-6-phosphate dehydrogenase, the enzyme that catalyzes the rate-limiting step in the PPP61.In this study, we demonstrated that Klotho can diminish PFKFB3 levels in neurons Fig. B by promIn conclusion, our research offers novel insights into the functions of Klotho in controlling energy metabolism and the redox state within the brain. The data we have gathered indicates that Klotho inhibits insulin/IGF-1 signaling in neurons and functions as an antioxidant in astrocytes by increasing FOXO activity and promoting catalase expression. Additionally, Klotho reduces PFKFB3 content in neurons, a crucial factor in the regulation of NADPH levels and the overall cellular reducing potential. Our study highlights the interaction between energy metabolism and redox balance, proposing that Klotho could be instrumental in preserving this equilibrium Fig. .Figure 6Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S3.Supplementary Figure S4.Supplementary Figure S5.Supplementary Figure S6.Supplementary Figure S7.