The vaccine microneedle patches were then evaluated in vivo in a murine model. The results from this study demonstrated that the vaccine nanoparticles effectively stimulated antigen-presenting cells in vitro resulting in enhanced autophagy, nitric oxide, and antigen presentation. In mice, the vaccine elicited M2e-specific antibodies in both serum and lung supernatants  and induced significant expression of CD4+ and CD8+ populations in the lymph nodes and spleens of immunized mice. Hence, this study demonstrated that polymeric particulates for antigen and adjuvant encapsulation, delivered using fast-dissolving microneedles, significantly enhanced the immunogenicity of a conserved influenza antigen.This study focused on developing an influenza vaccine delivered in polymeric nanoparticles (NPs) using dissolving microneedles. We first formulated an influenza extracellular matrix protein 2 virus-like particle (M2e VLP)-loaded with poly(lactic-co-glycolic) acid (PLGA) nanoparticles, yielding M2e5x VLP PLGA NPs. The vaccine particles were characterized for their physical properties and in vitro immunogenicity. Next, the M2e5x VLP PLGA NPs, along with the adjuvant Alhydrogel Each year, there are approximately three to five million severe cases of influenza worldwide, causing between 290,000 and 650,000 deaths . The infVaccination remains the most effective strategy against influenza . Since 2® and Sci-B-VacTM (hepatitis B virus), Gardasil® (human papillomavirus), and MosquirixTM  [The extracellular domain of the matrix protein 2 (M2e) of the influenza virus has gained much interest as a target for the development of a universal influenza A vaccine ,11,12,13malaria) . Therefomalaria) .To further improve the immunogenicity of an antigen, particulate delivery systems such as liposomes and polymeric microparticles can be used . Particu®, commonly referred to as alum, is one of the most widely used licensed adjuvants, specifically against influenza [® or MPL-A® is an emulsion-based low-toxicity derivative of lipopolysaccharide (LPS) [® is a toll-like receptor 4 (TLR-4) agonist and has been shown to skew the immune response towards Th1, which induces cell-mediated immunity [Previous studies have shown that microparticles , themselves, can confer adjuvant capabilities on their own ,24. Delinfluenza ,27. It pnfluenza . Monophode (LPS) . MPL-A® immunity . It has immunity .Lastly, the immunization route plays a significant role in vaccine-induced immunity. The most common route for vaccines is the intramuscular route. However, recent vaccine research has focused on alternate routes using non-injectable strategies. One of these immunization routes targets the skin, which is the first line of defense against pathogens . The epiIn this study, we found that M2e5x VLP encapsulated in PLGA nanoparticles induced a strong in vitro immunogenicity in antigen-presenting cells (APCs) through induction of autophagy and nitric oxide, and heightened antigen presentation. In vivo, the vaccine nanoparticles administered to mice using dissolving microneedles in the skin elicited M2e-specific antibody and cell-mediated responses. Hence, our proof-of-concept study demonstrated our influenza vaccine to be a suitable candidate for a potential universal pan-influenza A vaccine.®-loaded PLGA nanoparticles, the yield was 92.6% with an average particle size of 457.8 ± 1.76 nm, and a zeta potential of 12 ± 0.25 mV. Lastly, the product yield of MPL-A®-loaded PLGA nanoparticles was 92.8%, with an average size of 339 ± 5.3 nm, and a zeta potential of 0.338 ± 0.16 mV. Scanning electron microscopy images of the M2e5x VLP PLGA NPs are shown in The M2e5x VLP-loaded PLGA nanoparticles product yield was 91.7%, with an average particle size of 580.2 ± 20.2 nm, and zeta potential of −7.1 ± 0.44 mV. For Alhydrogel® + MPL-A® PLGA NPs also showed significantly higher levels of nitrite production compared to Fluzone (marketed flu vaccine) and M2e5x VLP suspension  and the study timeline are shown in subtypes C, M2e5x 2e5x VLP D. + T cells in lymph nodes and a higher expression of CD8+ cells in the lymph nodes and spleen  in 0.5 mL deionized water (W1) was added dropwise to the O1 phase. The production of M2e5x VLP has been described previously [2), which consisted of 2% w/v polyvinyl alcohol  in 20 mL of deionized water. The mixture was then homogenized at 17,000 rpm for a total of 1 min to form the double emulsion. To facilitate particle size reduction, the W1/O1/W2 double emulsion was probe sonicated at 30% power for a total of 15 s . The previously mentioned procedure was also used to produce MPL-A®  PLGA NPs. For Alhydrogel® (InvivoGen) PLGA NPs, probe homogenization steps were carried out at 31,000 rpm. All formulations were put under magnetic stirring for 4–5 h to evaporate the DCM and harden the particles. Next, the nanoparticles were washed twice at 17,000 rpm for 12 min at 4 °C. The supernatant was then discarded, and the nanoparticles were resuspended in 2 mL of deionized water containing 2% w/v trehalose. The formulations were then lyophilized for 33 h and stored at 4 °C.First, poly(lactic-co-glycolic acid) PLGA; 70:25) was purchased from Evonik Industries . The PLGA powder (20 mg) was dissolved in 2.5 mL of dichloromethane  to form the O was purceviously . In brieeviously . For forTo calculate the total product yield of PLGA NPs, the following equation was used: For determining particle size and charge , a Malvern Zetasizer Nano ZS  was used. The nanoparticles were visualized using scanning electron microscopy . To determine encapsulation efficiency, 10 mg of M2e5x VLP PLGA nanoparticles were dissolved in 1 mL of DCM. The sample was then centrifuged at 4000 rpm for 10 min at RT. Next, the DCM was evaporated under a fume hood and the precipitated M2e5x VLP was resuspended in 1 mL of PBS and quantified using a Micro BCA™ Protein Assay Kit . The integrity of the encapsulated antigen in PLGA nanoparticles was assessed using a sodium dodecyl sulfate (SDS) PAGE. M2e5x VLP PLGA NPs were first dissolved in DCM. The DCM was evaporated, and the extracted M2e5x VLP was resuspended in 30 μL of PBS, followed by the addition of 10 μL of sample loading buffer. M2e5x VLP suspension was used as a control. All samples were run on an 8% pre-cast gel . To determine M2e5x VLP release from the PLGA polymeric matrix, M2e5x VLP-loaded nanoparticles were dispersed in PBS (pH 7.4). Samples were taken at various time points over 6 days. ® in PLGA NPs were suspended in the solution. Next, sodium hyaluronate  was then added to the suspension to form a 10% w/v gel. The formulation was mixed thoroughly, after which 25 mg of gel was added to a 10 × 10 microneedle array mold . The molds were then centrifuged at 4000 rpm for 20 min at 15 °C, and dried overnight in a desiccator. The next day, a concentrated HA solution was added to the molds to form a backing, which was then dried overnight. The formulation of PLGA nanoparticle-loaded dissolving MNs has been described previously . In brie® Autophagy Detection Kit 2.0 . After 1 h, the cells were washed 2× twice with PBS, gently scraped, and analyzed using a flow cytometer (BD Accuri C6 flow cytometer).To evaluate the induction of autophagy in dendritic cells, DCs  were plated in 24-well plates at 50,000 cells/well. After reaching 70–75% confluency, the cells were pulsed with Fluzone (0.25 μg/well), M2e5x VLP (0.5 μg/well), Blank PLGA NPs (10 μg/well), and M2e5x VLP PLGA NPs (10 μg/well) overnight. The cells were washed 2× with PBS and stained according to protocol using a CYTO-ID® + MPL-A® PLGA NPs . This antigen:adjuvant ratio was selected as it would be the same ratio used for immunizations. The cells were incubated for 24 h after which the Griess’ Assay to assess nitric oxide production was performed. First, samples were centrifuged, and 50 μL of supernatant was transferred to a 96-well plate. Next, 50 μL of 1% sulphanilamide (Sigma Aldrich) was added to the wells and incubated at room temperature for 5 min in the dark. Next, 0.1% N-(1-naphthyl)ethylenediamine  in 5% phosphoric acid (Sigma Aldrich) was added to the wells and placed on a shaker for 5 min. The plate was then read at 540 nm using a plate reader . To assess the potential of M2e5x VLP PLGA NPs to induce the production of nitric oxide in immune cells, a Griess’ Assay was performed. First, dendritic cells were plated in a 24-well plate at a seeding density of 50,000 cells/well. After the cells reached approximately 70% confluency, various treatments were introduced to the cells: Cells Only, Lipopolysaccharide  Blank PLGA NPs (10 μg/well), M2e5x VLP (0.5 μg/well), M2e5x VLP PLGA NPs (10 μg/well), and M2e5x VLP + Alhydrogel® + MPL-A® PLGA NPs . The cells were incubated for 36 h. After the incubation, the cells were washed with PBS and gently scraped off the wells and resuspended in PBS and kept on ice. Next, anti-mouse MHC II and CD40 or MHC I and CD80 antibodies were added to the samples. Cells were incubated for 1 h and then washed 3× with PBS. The samples were analyzed using flow cytometry. To assess antigen presentation in vitro, DCs were seeded in 24-well plates at a density of 50,000 cells/well. After reaching confluency, the cells were pulsed with various treatments: no treatment, Fluzone (0.25 μg/well), Blank PLGA NPs (10 μg/well), M2e5x VLP (0.5 μg/well), M2e5x VLP PLGA NPs (10 μg/well), and M2e5x VLP + Alhydrogeln = 5): Naïve (no treatment), Fluzone (1.5 μg), Blank PLGA NPs (900 μg unloaded NPs), M2e5x VLP (20 μg), M2e5x VLP PLGA NPs (20 μg M2e5x VLP), and M2e5x VLP + Alhydrogel® + MPL-A® PLGA NPs . All groups, except Naïve and Fluzone, received treatment via the dissolving microneedles. Fluzone mice were immunized intraperitoneally. Mice were immunized using a prime-boost strategy in weeks 0 and 3. Blood was collected intermittently in weeks 1, 4, and 7. Whole blood was centrifuged at 1000× g to isolate the serum, which was then collected and stored at −80 °C until use. In week 8, mice were challenged intranasally with 50 μL of 0.5 × LD50 of A/Philippines/2/82 (H3N2). A sublethal challenge was used to assess M2e priming in immunized groups as well as having a surviving naïve control for T cell analysis. The mice were monitored for 14 days. In week 10, the mice were sacrificed. Organs including lymph nodes, spleens, and lungs were then harvested. For animal studies, 4–6-week-old female Swiss Webster (CFW) mice  were immunized as follows  and then blocked with 150 μL of 3% bovine serum albumin  in PBS for 2.5 h at RT. Next, plates were washed 3× with PBST, and diluted serum samples (50 μL/well) starting at a 1:10 dilution were added to plates and incubated at 37 °C for 1.5 h. The plates were then washed and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, IgG1, and IgG2a antibodies (Thermo Fisher) were added to the wells incubated at 37 °C for 1.5 h. The plates were then washed prior to the addition of 50 μL/well of 3,3′,5,5′-tetramethylbenzidine . The plates were then incubated at room temperature for 7 min followed by the addition of 50 μL/well of 0.3 M sulfuric acid (H2SO4). The absorbance was read at 450 nm. For assessing IgA in lungs, after sacrifice, lung tissue from mice was removed and homogenized. Tissue was then centrifuged at 1200 rpm, and supernatants were collected and frozen at −80 °C. For analysis, supernatants from lung homogenates were ten-fold serially diluted, starting at a 1:10 dilution. The same method for ELISA for serum samples was utilized for lung supernatants, with a 1:3000 dilution of HRP-conjugated IgA. M2e-specific serum antibody responses were evaluated using enzyme-linked immunosorbent assay (ELISA). First, M2e was coated on high-binding 96-well plates at a concentration of 0.2 μg/well. For the positive control, Fluzone, wells were coated with 0.2 μg/well of Fluzone. The plates were incubated at 4 °C overnight. The next day, plates were washed 3× with PBST  to form a single-cell suspension, and centrifuged at 1200 rpm for 10 min. Next, 1 mL of ACK lysis buffer was added to the splenocytes and incubated for 3 min. Next, the cells were centrifuged. Finally, the cell pellets were resuspended in DMEM containing 70% fetal bovine serum  and stored at −80 °C until analysis. Similarly, lymph nodes were passed through a 40-μm cell strainer, centrifuged, and resuspended in DMEM containing 70% FBS before being frozen at −80 °C. To assess cell surface markers CD4 and CD8 (Thermo Fisher), and intracellular cytokines interferon-gamma (IFN-γ) and interleukin-4 (IL-4), cell suspensions were thawed and centrifuged at 1200 rpm for 10 min. Lymph node and spleens cells from M2e5x VLP antigen-immunized groups were pre-stimulated with 1 μg of M2e protein (provided by Dr. Sang-Moo Kang) in 24-well plates. Immune organs from mice immunized with Fluzone were pre-stimulated with 0.5 μg quadrivalent Fluzone. Next, cells were supplemented with 50 μM β-mercaptoethanol (Thermo Fisher) and incubated overnight at 37 °C. The following day, 100 μL of 1× protein inhibitor Brefeldin A (Thermo Fisher) was added to each well. The plates were incubated at 37 °C for 1 h after which the cells were stained with the CD4 and CD8 for 1 h. For intracellular markers, cells were fixed and permeabilized using the Fixation and Permeabilization kit (Thermo Fisher) following the manufacturer’s instructions. Next, cells were stained with fluorescently labeled anti-mouse IFN-γ and IL-4. The samples were then incubated on ice for 1 h. The cells were washed and analyzed using flow cytometry. p-value < 0.05 was considered to be statistically significant.Data analyses were performed on GraphPad Prism Version 9.2. and expressed as mean values with the standard deviation (SD). For multiple comparisons, one-way analysis of variance (ANOVA) was performed with Tukey’s test for post-hoc analysis. A ® + MPL-A® nanoparticles was shown to be an immunogenic M2e-based influenza vaccine. The nanoparticles induced significant autophagy and nitrite production, and antigen presentation in dendritic cells. Furthermore, the M2e5x VLP PLGA NPs along with adjuvants demonstrated enhanced M2e-specific IgG, IgG1, IgG2a, and IgA responses compared to the M2e5x VLP suspension. Additionally, the vaccine nanoparticles with adjuvants elicited a significant expression of CD4+ and CD8+ T cells in immunized mice. Hence, the incorporation of M2e5x VLP into a biodegradable PLGA polymeric matrix with adjuvants and delivered non-invasively through the transdermal route using a fast-dissolving microneedle shows the potential for an innovative pan-influenza A vaccine capable of inducing robust immunity against the influenza virus.The M2e5x VLP formulated into PLGA nanoparticles and combined with Alhydrogel


X-ray diffraction (XRD) results showed that Fe3O4, Fe0, and ZnO crystals were formed in the LSP-derived carbon matrix. Notably, transmission electron microscopy (TEM) images showed that the shapes of these components consisted of not only nanoparticles but also nanowires. Fe and Zn contents in MAC determined by atomic absorption spectroscopy (AAS) were 6.89 and 3.94 wt%, respectively. Moreover, SBET and Vtotal of MAC prepared by coactivation with ZnCl2 and FeCl3 were 1080 m2/g and 0.51 cm3/g, which were much higher than those prepared by single activation with FeCl3 (274 m2/g and 0.14 cm3/g) or ZnCl2 (369 m2/g and 0.21 cm3/g). MAC was subsequently applied as an oxidation catalyst for Fenton-like degradation of acid orange 10 (AO10). As a result, 0.20 g/L MAC could partially remove AO10 (100 ppm) with an adsorption capacity of 78.4 mg/g at pH 3.0. When 350 ppm H2O2 was further added, AO10 was decolorized rapidly, nearly complete within 30 min, and 66% of the COD was removed in 120 min. The potent catalytic performance of MAC might come from the synergistic effect of Fe0 and Fe3O4 nanocrystals in the porous carbon support. MAC also demonstrated effective stability and reusability after five consecutive cycles, when total AO10 removal at 20 min of H2O2 addition slightly decreased from 93.9 ± 0.9% to 86.3 ± 0.8% and minimal iron leaching of 1.14 to 1.19 mg/L was detected. Interestingly, the MAC catalyst with a saturation magnetization of 3.6 emu/g was easily separated from the treated mixture for the next cycle. Overall, these findings demonstrate that magnetic activated carbon prepared from ZnCl2 and FeCl3 coactivation of lotus seedpod waste can be a low-cost catalyst for rapid degradation of acid orange 10.Lotus seedpods (LSPs) are an abundant and underutilized agricultural residue discarded from lotus seed production. In this study, ZnCl Today, agricultural activities generate enormous amounts of solid wastes all over the world. These agricultural wastes are commonly disposed of by burning them in the fields. This activity can cause a variety of ecological and environmental problems . Hence, 0, Fe2O3, Fe3O4, and MnFe2O4 particles could be dispersed on BC, resulting in a new material known as magnetic biochar (MBC).Biochar (BC) is a carbon-rich material prepared from the pyrolysis of different biomass resources in oxygen-free environments –11. Desp3 into biomass resources and then pyrolyzing the obtained mixtures to yield MBC [3 produces MBC with well-dispersed iron-based nanoparticles and well-developed porosity. However, compared with other well-known activating agents, FeCl3 has limited activation efficiency. Porous systems of MBC grow slightly. For instance, specific surface areas (SBET) of MBC are obtained from municipal sludge (FeCl3/N2): 38 m2/g [3/N2): 8 m2/g [3/N2): 159 m2/g [3/O2-limited): 374 m2/g [3/biomass ratio could improve the activation process. However, an excessive ratio can affect the porous properties and application performance of MBC [3 should be well impregnated inside the natural holes of biomass. Therefore, high FeCl3 loading may form bigger Fe-based particles and clusters, decreasing the surface area of catalytic Fe sites. The bigger Fe-based particles may also block the pores of carbon bases, negatively affecting mass transfer. The interaction between MBC and organic pollutants mainly comes from surface porous carbon with functional groups rather than Fe-based particles. High Fe loading content may decrease adsorption sites, causing weaker adsorption. To expand the porous system of MBC effectively, a few recent reports propose the combination of FeCl3 with another activating agent during one-pot pyrolysis of biomass. By replacing N2 with CO2, the SBET of MBC obtained from the FeCl3-activation of spent coffee grounds increased remarkably from 8 to 512 m2/g [To synthesize MBC from biomass, magnetic precursors are commonly loaded onto carbon surfaces . This olield MBC , 27. Accield MBC , biomass 38 m2/g ; spent c: 8 m2/g ; peanut 159 m2/g ; and lot374 m2/g . Increase of MBC , 33. FeC512 m2/g . Hence, 2O or CO2 [2CO3, H3PO4, H2SO4, AlCl3, ZnCl2, and FeCl3, and then carbonized and activated in one-step pyrolysis. Those agents might theoretically activate MBC [2 is preferable [2-activated carbon possessed a greater surface area and a greater number of micropores. In addition, the aromatic structure of ZnCl2-activated carbon was enhanced [2 and FeCl3 when few studies have been found in the literature. Lee and Ahmad Zaini [2 and FeCl3 coactivation of palm kernel shell offered MAC with a very high SBET of 1775 m2/g. As a result, the obtained MAC demonstrated exceptional adsorption of rhodamine B at 371 mg/g. Similarly, Lou et al. [SBET of 1409 m2/g for significantly enhanced Cr (VI) removal of 185.8 mg/g. Based on the abovementioned results, it is possible to use both ZnCl2 and FeCl3 as an activating mixture in a facile one-pot preparation of MAC from biomass resources. In terms of adsorption, the superiority of MAC over MBC has been proven; however, its catalytic activity has been studied very little. Such reports [Physical and chemical activation are the two most common techniques used to activate carbon-based materials . PhysicaO or CO2 , 35. Forvate MBC , 37. Thevate MBC , 35. Theeferable , 39. Expenhanced . Hence, ad Zaini  demonstru et al.  prepared reports , 43 indi2O2) offers several advantages over other oxidizing agents, including its safety, environmental friendliness, versatility, cost-effectiveness, simple operation, and mild conditions [2O2 alone is less effective. To accelerate the treatment of organic pollutants by H2O2, catalysts can be used. As mentioned before, magnetic biochar has proven that it is an effective catalyst for the treatment of synthetic dyes by H2O2 owing to its effectiveness, stability, low cost, and environmental friendliness [Nowadays, numerous industrial processes, including food processing, papermaking, printing, leather, textiles, cosmetics, and pharmaceuticals, discharge vast quantities of dyes into the aquatic environment –46. Dye nditions , 58. Howndliness , 59.3 activation [2O2. Herein, LSP was continuously selected as a biomass resource for one-pot preparation of MAC using ZnCl2 and FeCl3 coactivation. To evaluate the catalytic performance of as-prepared MAC samples in a Fenton-like process, acid orange 10 (AO10), a synthetic azo dye with extensive usage, limited biodegradability, and potential toxicity [Lotus seedpods (LSP) are released from seed gathering in markets and factories, resulting in massive agricultural waste , 61. LSPtivation , 27. Thetoxicity –64 was s3.6H2O (≥99.0%), ZnCl2 (≥98.0%), H2SO4 (95.0–98.9%), NaOH (≥96.0%), Na2S2O3.5H2O (≥99.0%), H2O2 (≥30.0%), KH2PO4 (≥99.5%), Na2HPO4.12H2O (≥99.0%), and acid orange 10 were obtained from Xilong Scientific Co., Ltd., China. All analytical grade chemicals were used directly, without further refinement.Raw lotus seedpod residue was received from a factory for lotus seed production located in Thap Muoi District, Dong Thap Province, Vietnam. The pods were washed with tap and distilled water to remove all dirt before being dried in an electric drying oven at 105°C for 24 h. Then, the raw material was cut and milled to obtain a fine powder. To avoid moisture, the powder was stored in an airtight vessel for later use. FeCl2 and FeCl3-loaded lotus seedpod residue. First, 4.00 g of LSP powder, 0.80 g of FeCl3, and a certain amount (4x g) of ZnCl2 were added to 100 mL of distilled water. After 3.0 h of stirring, the mixture was dried in an oven at 105°C for 24 h. The dried sample was then added to a glass reaction tube in a vertical furnace. A constant nitrogen flow rate of 250 mL/min maintained the inert atmosphere inside the tube. To pyrolyze, the tube was heated from room temperature to 600°C at an average rate of 5°C/min and then held at that temperature for 60 min. The obtained solid was washed repeatedly to remove all residual FeCl3 and ZnCl2. Wastewater was tested with a pH meter, an electrical conductivity meter, an aqueous NaOH solution, and an aqueous AgNO3 solution to detect ion leaching . Lastly, the sample was dried at 80°C for 24 h to obtain MAC. Due to the mass ratio of ZnCl2/FeCl3/LSP being x/0.2/1.0, the as-prepared MAC samples were denoted as MAC-x. Moreover, biochar (BC), magnetic biochar (MBC), and activated carbon (ZAC), which served as reference samples, were prepared by the pyrolysis of LSP, FeCl3-loaded LSP, and ZnCl2-loaded LSP under the same procedure. These labels are presented in Magnetic activated carbon was prepared via the one-pot pyrolysis of ZnClθ = 10–80° range was measured on a Bruker AXS D8 diffractometer using CuK α radiation (λ = 1.5418 Å). Fe and Zn contents in MBC and MAC samples were analyzed by a Perkin Elmer Analyst 800 atomic absorption spectrophotometer (AAS). These metal elements were extracted from MBC and MAC samples in a HCl (6 M) solution at 60°C for 60 min. Nitrogen adsorption and desorption isotherms of MBC, ZAC, and MAC were measured at 77 K on a Micromeritics® TriStar II Plus. All samples were degassed at 250°C for 5 h. The specific surface area (SBET) was calculated from the Brunauer–Emmett–Teller equation. The total pore volume  was determined at P/Po = 0.995. The average pore size (daverage) was obtained from 4Vtotal/SBET. The pore size distribution was determined by the BJH method. The magnetic properties of MBC and MAC were examined with a vibrating sample magnetometer (VSM) at room temperature. Fourier transform infrared (FTIR) spectroscopy of MAC was performed using a Tensor 27 spectrometer. Scanning electron microscope (SEM) images, energy dispersive X-ray (EDX) spectroscopy, and elemental mapping of MAC were analyzed using a JEOL JSM-IT200 instrument. Transmission electron microscopy (TEM) images of BC, MBC, and MAC were recorded by a JEOL JEM-1010 instrument.Powder X-ray diffraction (XRD) in the 22O2 as an oxidizing agent at room temperature (30°C). In brief, 500 mL of AO10 (100 ppm) and a certain MAC dosage were added to a 1000 mL glass cylinder. The initial pH value of the mixture was adjusted using H2SO4 (0.5 M) and NaOH (0.1 M) solutions. The adsorption step was carried out within the first 20 min. The adsorption capacity (Q), therefore, was calculated from the following equation:CM (g/L) is the material dosage, and C0A and C20A (ppm) are the AO10 concentrations at the beginning and after 20 min of adsorption.The catalytic performance of MAC samples was explored through the degradation of acid orange 10 using H2O2 to the mixture. Samples taken were added immediately to a solution of phosphate buffer and Na2S2O3 (2.0 g/L) to adjust the pH to 7.0 and eliminate excess H2O2. AO10 concentrations were quantitatively examined at 480 nm with a UV-Vis spectrophotometer (Lovibond PC Spectro). The decolorization efficiency and total removal of AO10 were calculated as follows:C0O and C30O (ppm) are the AO10 concentrations at the beginning and after 30 min of oxidation.After the adsorption step, the oxidation step was initiated by the rapid addition of H2O2 on COD results, samples were mixed with a solution of 20.0 g/L Na2CO3 and incubated at 90°C for 60 min [The chemical oxidation demand (COD) was quantified using the closed-reflux titrimetric method (5220C) . To minir 60 min .To evaluate the stability and reusability of the MAC catalyst, a sample was used in five consecutive experiments. The used catalyst was recovered using a magnet, rinsed with distilled water and ethanol, and then placed in an oven at 110°C. The dried catalyst was weighed in preparation for the subsequent experiment. At the end of each cycle, the treated solution was analyzed with the previously mentioned AAS instrument to identify Fe leaching.3O4 crystals were found in MBC at 2θ = 18.3, 30.1, 35.4, 42.4, 52.4, 56.0, and 61.5°, respectively, corresponding to the (111), (220), (311), (400), (422), (511), and (440) planes (JCPDS 19-0629). The following reactions are proposed for the formation of Fe3O4 during the one-pot pyrolysis of FeCl3-loaded LSP:XRD was used to examine the development of crystals on MBC, ZAC, and MAC samples, as shown in 2 activation, ZAC possessed the peaks at 2θ = 31.7, 34.3, 36.1, 47.4, 56.3, 62.7, 67.7, and 68.9°, which respectively correspond to the (100), (002), (101), (102), (110), (103), (112), and (201) planes of hexagonal ZnO crystals (JCPDS 36-1451). Based on a report by Ma [With ZnClrt by Ma , the fol2 can promote dehydration processes to cleave polymer chains of lignocellulosic biomass, yielding H2O and a thermoplastic carbonaceous phase [2 can then combine with H2O to produce Zn2OCl2.2H2O. Subsequently, the decomposition of Zn2OCl2.2H2O can produce ZnCl2 vapor, and its diffusion can activate the thermoplastic phase to offer the last porous carbon system [At high temperatures, molten ZnClus phase . ZnCl2 cn system . Moreove2 and FeCl3, all MAC samples produced Fe3O4 and ZnO. Notably, the presence of zero-valent Fe crystals was demonstrated at 2θ = 44.6 and 64.9° (JCPDS 06-0696). Thus, Fe3O4, Fe0, and ZnO were the main products present in MAC. Compared to MBC and ZAC samples, MAC samples contained a greater amount of background noise. In MAC samples, strong activation could significantly reduce crystallinity and increase amorphous components, such as carbon base. Therefore, intense scattering may obscure the low peaks of the available crystals. In fact, several peaks of ZnO and Fe3O4 crystals in MAC samples were overlapped by background noise.By coactivation of LSP with ZnCl2/LSP mass ratio rose from 0.1 to 0.4, the peak intensities of Fe3O4 declined while those of Fe0 increased. These results demonstrate that higher ZnCl2-loading content could enhance the decomposition of LSP into carbon base and H2O, and more ZnO could be formed. As shown in 2/LSP mass ratio increased from 0.1 to 0.4. Faster carbonization boosted by ZnCl2 could provide more decomposition products  to accelerate equations , 2900 cm−1 (C-H stretching vibrations), 2300 cm−1 (O=C=O stretching vibrations), 1756 cm−1 (C=O stretching vibrations), 1570 cm−1 (C=C stretching vibrations), 1144 cm−1 (C-O stretching vibrations), and 826 cm−1 (C-H stretching vibrations) [−1 could be Fe-O bonds [−1 could be Zn-O bonds [FTIR spectroscopy of MAC-0.4 is presented in rations) –76. Nota-O bonds , 78, and-O bonds , 79. Mor2 and FeCl3 coactivation of LSP might affect micropores more than mesopores and macropores. Therefore, those macropores could come from the natural vascular bundles of LSP [The surface morphology of MAC-0.4 was observed by SEM images . Sharp-es of LSP , 81. Esps of LSP , 28. Of 3 was impregnated inside LSP. Hence, the distribution of Fe may be spread throughout the carbon structure, resulting in comparable Fe contents from EDX and AAS results. Unlike Fe, the minor surface Zn content (0.29 wt%) was much lower than the bulk Zn content (3.94 wt%). It reveals that Zn on the carbon surface may readily be removed during pyrolysis. As previously indicated, ZnCl2 vapor could be formed and diffused into porous carbon. Due to its high mobility, ZnCl2 vapor may escape off the MAC surface and be carried away by the flow of N2 gas. Then, only the inner carbon matrix may retain Zn better. Interestingly, the atomic ratio of O/Fe was approximately 5.0, which is much higher than that of Fe3O4. This comparison demonstrates that a considerable surface O content was present in the functional groups, as listed in the FTIR results.EDX spectroscopy and elemental mapping were used to determine the chemical composition and elemental distribution on the surface of MAC-0.4 . The pre3 and ZnCl2. As previously stated, MAC was cleaned until no Fe3+, Zn2+, or Cl− leaching was detected. Therefore, these elements could be firmly bound within the carbon matrix by strong mechanical or chemical linkages [3 and ZnCl2 in LSP. Following is a discussion on nanoscale TEM analysis for clarifying the interior structure of materials.For the remaining elements in MAC, Si and Cl were identified at 0.45 and 0.59 wt%, respectively. Several reports demonstrate that minor elements, including Si, can be present in LSP , 83. Howlinkages . Lastly,3O4. At the nanoscale, dust-like Fe3O4 particles were observed throughout the carbon matrix. These nanoparticles seem to group together in clusters. In addition to nanoparticles, MBC contained nanowires of Fe3O4. Intriguingly, the existence of magnetic nanowires in MBC is rare. It appears possible that Fe3O4 nanowires may be formed in nanopores that resemble tubes [3O4 but also Fe0 and ZnO crystals were present in MAC. It was suggested that the initial natural porous structure of LSP for ZnCl2 and FeCl3 loading played an important role in the morphology of Fe- and Zn-based products. LSP contains natural cellulose fibers [TEM images were used to observe the internal structures of BC, MBC, and MAC-0.4 . BC showle tubes . Similare fibers . Wire-lie fibers . These a2O2 was investigated. For complete oxidation of 100 ppm AO10, a minimum of 316 ppm H2O2 is theoretically required [SBET and Vtotal) in carbon-based materials could play an important role in AO10 removal. Furthermore, π-π, hydrogen, and electrostatic interactions between functional groups on the MAC surface and AO10 [The catalytic activity of MAC in AO10 degradation using Hrequired . Accordirequired , 350 ppmand AO10 , 85 may 2 activation, ZAC eliminated 10.0% of AO10 through adsorption, and then almost lacked catalytic AO10 degradation in the subsequent step. Conversely, all MBC and MAC catalysts showed certain catalytic activity toward AO10 degradation. These results indicated that Fe-based components rather than ZnO and carbon-based support were the active sites for these catalytic processes. The MBC sample contained Fe3O4 crystals, while the MAC samples contained both Fe3O4 and Fe0 crystals. These Fe sites could catalyze AO10 degradation as follows (C− denotes that the Fe sites were incorporated into the carbon matrix) [AO10 degradation was carried out with BC, ZAC, MBC, and MAC catalysts, as shown in  matrix) :(12)C−Fe2/LSP mass ratio improved the decolorization rate of AO10. As presented in 0 and Fe3O4 exhibited higher catalytic performance than each component [0 and Fe3O4 may facilitate electron transfer and •OH generation. MAC contained not only Fe0 but also Fe3O4, which may follow this synergic effect. Furthermore, Fe-based components were fixed in the carbon supports, which could affect the process indirectly. MBC and MAC samples had different porous properties  and crystal structures of Fe-based components. As mentioned before, the nanoscale Fe sites were well dispersed in the porous carbon system of MAC-0.4, which had a high SBET and a large Vtotal. Hence, mass transfer in these pores might become more convenient, and more catalytic sites with high residual energy might be accessible. These main advantages might explain the robust enhancement of the catalytic oxidation of AO10 by H2O2.Compared with MBC, all MAC samples showed much faster AO10 decolorization rates. In addition, increasing the ZnClomponent , 87. The− ions could be detrimental to AO10 degradation [− ions may be a result of their interaction with •OH. However, MAC was carefully rinsed to remove all water-soluble components. Consequently, trace quantities of Cl and Si may not exist as ions or be firmly bound within the carbon framework. It may be challenging to leach those elements into the treatment media. Due to the strong catalytic activity of MAC on AO10 degradation, the significance of these trace elements may be negligible.The presence of minor elements in MAC might impact its catalytic activity. According to such reports, Clradation , 89. The2O2 to eliminate AO10. In contrast, when MAC was applied, AO10 decolorization occurred rapidly. With MAC dosages between 0.20 and 0.60 g/L, AO10 was nearly completely decolored within 30 min. These results demonstrated that MAC catalyzed this decolorization effectively. In addition, the decolorization rate generally increased when the MAC dosage rose from 0.10 to 0.60 g/L. A high catalyst dosage may increase the number of active sites for H2O2 decomposition into •OH radicals. However, increasing the MAC dosage from 0.40 to 0.60 g/L did not result in a significant improvement. According to reports in the literature, the excess catalyst might deactivate the originally generated •OH radicals, as shown in the following equation [equation -91:(16)C3 can be formed from Fe(III) and cover the active sites of the catalyst, making H2O2 adsorbed and reducing the amount of free •OH radicals formed [2O2 addition, low pH could promote Fe leaching, leading to a homogeneous mechanism for catalytic decolorization [pH can be a crucial variable for AO10 degradation catalyzed by MAC on the basis of the Fenton-like mechanism. As shown in s formed , 93. Non2O2. In the same H2O2/AO10 mass ratio, 50 and 200 ppm of AO10 were investigated with 175 and 700 ppm of H2O2, respectively. Although the MAC dosage was kept at 0.20 g/L, AO10 was completely eliminated within 30 min of oxidation  with an adsorption capacity of 78.4 mg/g. When 350 ppm of H2O2 was added, AO10 decolorization occurred rapidly and was practically complete within 30 min. At 120 min, 66% of the COD was removed. Moreover, the catalytic performance remained stable, with total AO10 removal slightly decreasing from 93.9 ± 0.9% to 86.3 ± 0.8% after five consecutive cycles. The minimal iron leaching ranged from 1.14 to 1.19 mg/L. In conclusion, these results indicated that magnetic activated carbon derived from ZnCl2 and FeCl3 coactivation of lotus seedpod residue is an efficient catalyst for robust acid orange 10 decolorization.In summary, magnetic activated carbon was successfully prepared using one-pot pyrolysis of ZnCl


We evaluated the impact of S30+LGA on body weight, estrogen markers, uterine and bone health, vascular markers, and neurotransmitter levels. The results revealed that treatment with S30+LGA1 significantly improved body weight and uterine and bone health. Moreover, S30+LGA1 demonstrated promising effects on lipid profile, liver function, and vascular markers and positively impacted serotonin and norepinephrine levels, indicating potential mood-enhancing effects. In conclusion, S30+LGA1, possessing anti-menopausal effects in vitro and in vivo, can be recommended as a soy-based diet, which offers various health benefits, especially for menopausal women.Menopause is a significant phase in a woman’s life. Menopausal symptoms can affect overall well-being and quality of life. Conventionally, hormone replacement therapy (HRT) is used to alleviate menopausal symptoms; however, depending on the conditions, HRT may lead to side effects, necessitating the exploration of alternative therapies with fewer side effects. In this study, we investigated the effects of a combination of soybean germ extract (S30) containing 30% ( Menopause results from a gradual decline in estrogen levels. It is associated with an increased risk of experiencing emotional disorders, such as anxiety and depression . MoreoveHormone replacement therapy (HRT), or estrogen and progesterone supplementation, is commonly used to alleviate menopausal symptoms ,7. The aSoybeans are common in Asian diets and are rich in phytoestrogens. A correlation between soybean consumption and reduced occurrence of menopausal symptoms, osteoporosis, cardiovascular diseases, and hormone-dependent cancers has been demonstrated . These bGut microbiota actively participate in food metabolism via the expression of various enzymes . Gut micLactobacillus gasseri were investigated using in vitro and in vivo models of menopause.This study primarily aimed to identify, via screening, LAB with high β-glucosidase activity, which can convert soy isoflavone glucosides into their active aglycone forms to enhance their bioavailability and health benefits. In addition, the potential therapeutic effects of soybean germ extract and The S30 was prepared by dissolving 40 g of soybean germ extract  supplied by Mirae Biotech Co. , in sterilized water. The pH of the S30 was adjusted to 6.7 using 5 M sodium hydroxide, and it was then sterilized via autoclaving at 121 °C for 15 min.Lactobacillus rhamnosus CBT LR5 , L. gasseri CBT LGA1 , and Lactococcus lactis subsp. Lactis CBT SL6  were cultured in De Man, Rogosa, and Sharpe medium  at 37 °C for 18 h aerobically. Bifidobacterium breve CBT BR3  and B. longum CBT BG7  were cultured in BL® medium  at 37 °C for 18 h anaerobically. After incubation, the probiotic cultures were centrifuged, and the resulting pellets containing 1 × 109 CFU/mL were obtained. Then, the pellets were washed thrice using a 100 mM potassium phosphate buffer (pH 7.4) and used for the β-glucosidase enzyme activity test.p-nitrophenyl-β-d-glucopyranoside (pNPG). The pellets were resuspended with 1 mL of a 5 mM pNPG solution in 100 mM sodium phosphate buffer (pH 7.0) and incubated at 37 °C for 15 min [g for 30 min. Finally, the amount of p-nitrophenol released from pNPG was measured at 420 nm using a spectrophotometer . One unit of β-glucosidase activity was defined as the amount of enzyme required to produce 1 μmole of p-nitrophenol from the pNPG per minute under the reaction conditions described above.After the final wash, the bacterial cells were resuspended in 1 mL of sterilized water, and the optical density (OD) at 600 nm was adjusted to the same level for each probiotic species. Subsequently, the pellets were inoculated into 20 mL of S30 solution. The pellets were collected at specific time points: 1, 2, 4, 8, 12, 24, and 48 h. The ODs of all species were adjusted to match the same lowest OD value. Subsequent time points were measured based on the OD value in the first hour, and OD values were standardized for each species. The enzyme activity in S30 was determined by measuring the hydrolysis rate of r 15 min . The rea2.MCF-7, the human breast adenocarcinoma cell line, was purchased from the American Tissue Culture Collection  and cultured in Dulbecco’s Modified Essential Medium supplemented with 10% fetal bovine serum (FBS), 100 IU/mL of penicillin G, and 100 µg/mL of streptomycin. The human osteosarcoma cell line, MG-63, was obtained from ATCC and cultured in a minimal essential medium supplemented with 10% FBS and the same concentrations of penicillin G and streptomycin. Cultures in exponential growth were maintained at 37 °C in a humidified atmosphere with 5% CO® 480 Real-Time PCR System . The primers used in this study are listed in −ΔΔCt method was used to calculate the relative expression of target genes, using β-actin as the endogenous gene for normalization.After a 24 h treatment with fermentation products obtained from filtered probiotics, the MCF-7 and MG-63 cells were immediately collected, frozen in liquid nitrogen (LN), and stored at −80 °C. Then, these frozen cells were homogenized using an LN-cooled mortar, and RNA was extracted using Trizol  according to the manufacturer’s instructions. The obtained RNA underwent reverse transcription using HyperScript reverse transcription reagents . After adding SYBR Green Supermix  to the reaction mixture, real-time quantitative PCR (qPCR) was performed using a Roche LightCyclerThirty-six female Sprague Dawley rats, aged 6 weeks, were obtained from Orient Bio . The rats were maintained in a separate cage in a controlled environment with a temperature of 24 ± 2 °C, relative humidity of 40–60%, and a 12 h light/dark cycle.The rats were provided ad libitum access to food and water during the 1-week adaptation period. Subsequently, the rats underwent bilateral ovariectomies (OVX) under anesthesia . Sham-operated rats (sham) underwent the same surgical procedure, except without ovariectomy. One week post-operatively, the rats were randomly divided into four groups based on their weight, with 10 animals in each group. The rats were fed a modified version of the AIN-93G diet, where corn oil was replaced with soybean oil.9 CFU/head; and (6) OVX + S30 + LGA1 group, administered both S30 at a dose of 10 mg/kg and LGA1 at a dose of 1 × 109 CFU/head. LGA1 was in a lyophilized powder form. S30 and LGA1 administration was continued for 8 weeks.The rats were further assigned randomly to one of six groups, with six rats in each group A: (1) shThe animal study protocol was reviewed and approved by the Institutional Animal Care and Use Committee board (IACUC) at CellBiotech , following the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).Micro-computerized tomography (micro-CT) images of the fixed left femur samples and a standard phantom were acquired using a micro-CT scanner . The scanning parameters were set as follows: X-ray voltage, 50 kV; current, 445 μA; resolution, 18 μm; camera pixel size, 18.02 mm; aluminum filter size, 1 mm; thick aluminum filter was used; scanning process rotation step, 0.5°; and rotation angle, 360°. The acquired images were reconstructed using the image analyzer . Subsequently, the reconstructed images were analyzed using the same software. Various bone parameters of the left femur head, such as bone volume/total volume (BV/TV), bone mineral density (BMD), trabecular number (Tb. N), trabecular thickness (Tb. Th), and trabecular separation (Tb. Sp), were analyzed.Abdominal visceral adiposity parameters of the live animals, including total abdominal fat (TAF), visceral abdominal fat (VAF), and subcutaneous abdominal fat (SAF) volume, were measured non-invasively at 12 weeks using three-dimensional (3D) Scanco VivaCT 80 scanner , following the manufacturer’s instructions.During the scanning process, the rats were anesthetized with 1% isoflurane  and positioned on their back with their face up and head toward the front. Both hind limbs were extended and secured to a specimen holder, forming a 90° angle between the femur and spine, with the legs fully extended. The scanning area covered the region between the proximal end of the L1 and the distal end of the L6 lumbar vertebrae. Scanning was performed with an isotropic voxel size of 18 μm, using parameters of 70 kVp energy, 114 μA intensity, 31.9 mm field of view (FOV)/diameter, and 200 ms integration time. The scanning energy and voxel size were determined based on optimizing scanning time, tissue detail, and minimizing radiation exposure, following the manufacturer’s guidelines.The acquired micro-CT scans were processed using the imaging software , and the TAF, VAF, and SAF were analyzed using the generated 3D reconstructed images.Sections of the uterus were stained with hematoxylin and eosin and prepared for light microscopy analysis. The radius of the uterine endometrium  and myometrium was determined via optical micrometry using a ×10 objective. Additionally, the height of the luminal epithelium was measured using a ×20 objective. Measurements were calibrated using the image of a stage micrometer at the same magnification to ensure standardization. Four areas within each of the three transverse sections of the uterus were analyzed for each animal. Regarding epithelial cell height, four measurements were taken within the aforementioned areas.C-telopeptide of type I collagen (CTX-1), N-telopeptide of type I collagen (NTX-1), endothelin-1, and endothelial nitric oxide (eNOS) were analyzed using the corresponding specific ELISA kits for rats: rat Estradiol (E2) ELISA Kit , rat 5-hydroxy tryptamine (5-HT) ELISA Kit , rat norepinephrine (NE) ELISA Kit , rat osteocalcin (OC) ELISA Kit , rat bone alkaline phosphatase ELISA kit , rat deoxypyridinoline (DPD) ELISA Kit , rat pyridinoline (PYD) ELISA Kit , rat cross-linked C-telopeptide of type I collagen (CTX-I) ELISA Kit , rat cross-linked N-telopeptide of type I collagen, NTX-1 ELISA Kit , rat endothelin 1 ELISA Kit , and rat endothelial nitric oxide synthase ELISA Kit . The serum samples were processed following the manufacturer’s instructions. The absorbance of samples obtained from each assay was measured using the VersaMaxTM tunable microplate reader .Serum levels of estradiol, serotonin, norepinephrine, osteocalcin (OC), bone alkaline phosphatase (ALP), deoxypyridinoline (DPD), pyridinoline (PYD), Biochemical parameters, including cholesterol, triglycerides, high-density lipoprotein (HDL), low-density lipoprotein (LDL), aspartate aminotransferase (AST), alanine transaminase (ALT), albumin, and calcium levels, were measured using the XL-100 automatic analyzer .p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001).The data were presented as mean ± standard error of the mean (SEM). Statistical analysis was conducted using GraphPad Prism 9 software . The significance of differences was evaluated using a one-way analysis of variance (ANOVA) with Sidak multiple comparisons tests among multiple groups. In the figures, asterisks denote statistical significance , B. longum CBT BG7 (BG7), L. rhamnosus CBT LR5 (LR5), L. gasseri CBT LGA1 (LGA1), and L. lactis subsp. lactis CBT SL6 (SL6) were compared for their β-glucosidase activities. These LAB strains were incubated in the S30 over 48 h at 37 °C, and their β-glucosidase activities were measured using whole cells without disrupting cells with pNPG as the substrate , whereas it decreased to 0.28 U/mL after 48 h of incubation (lowest level). The β-glucosidase activity of other strains after 1 h of incubation was as follows: BG7, 2.02 U/mL; LR5, 1.53 U/mL; BR3, 1.41 U/mL; and SL6, 0.33 U/mL. Overall, LGA1 exhibited the highest β-glucosidase activity against During menopause, the estrogen level decreases . Therefop < 0.0001) was 2.7-fold higher than that without treatment (1.00 ± 0.07). Additionally, the mRNA expression level of ESR 1 with S30+LGA1  was 2.9-fold higher than that with S30 (0.91 ± 0.17). Similarly, the mRNA expression level of ESR 2 with S30+LGA1  was 3.2-fold higher than that without treatment (1.01 ± 0.14). Moreover, the mRNA expression level of ESR 2 with S30+LGA1  was 4-fold higher than that with S30 (0.79 ± 0.28). Similarly, the mRNA expression level of pS2 with S30+LGA1  was 3.9-fold higher than that without treatment (1.00 ± 0.11) and 3.3-fold higher than that with S30 (1.17 ± 0.24). Thus, S30+LGA1 exhibited a significant effect on the upregulation of the genes of ESR1, ESR2, and pS2 in MCF-7 cells.Regarding the effect of treatment with S30+LGA1 and S30 and no treatment (control) on MCF-7 cells, S30+LGA1 exhibited significantly higher mRNA expression levels of ESR 1, ESR 2, and pS2 than the others . The mRNBone loss, or osteoporosis, occurs during menopause . The hump < 0.0001) was 2.8-fold higher than that without treatment (1.01 ± 0.12) (p < 0.001) was 2.2-fold higher than that with S30 (1.27 ± 0.39).The carboxylated form of OC binds calcium directly, thus concentrating in bone ,41,42,43 ± 0.12) . The mRNp < 0.0001) was 3-fold higher than that without treatment (0.98 ± 0.23) and 2.8-fold higher than that with S30 (1.06 ± 0.27) (Osteoprotegerin (OPG) or osteoclastogenesis inhibitory factor regulates bone density . The mRN ± 0.27) .p < 0.0001) was 2.4-fold higher than that without treatment (1.00 ± 0.08) (p < 0.001) was 2.1-fold higher that with S30 (1.14 ± 0.32).Bone morphogenetic protein-2 (BMP 2) induces bone generation and regeneration via extraskeletal and skeletal organogenesis . The mRN ± 0.08) . The mRNp < 0.0001) was 3.4-fold higher than that without treatment (1.05 ± 0.16) (p < 0.0001) was 2.4-fold higher than that with S30 (1.49 ± 0.27).Morphogenetic protein 4 (BMP 4) is known to be involved in bone and cartilage development . The mRN ± 0.16) . The mRNp < 0.0001) was 4.15-fold higher than that without treatment (1.00 ± 0.08) (p < 0.0001) was 3.6-fold higher than that with S30 (1.16 ± 0.28).Collagen type I (Col1) is the most abundant protein in mammals, contributing to 90% of the total organic component of bone matrix . The mRN ± 0.08) . The mRNp < 0.0001) was 2.5-fold higher than that without treatment (1.01 ± 0.12) (p < 0.0001) was 2.3-fold higher than that with S30 (1.10 ± 0.22).Alkaline phosphatase (ALP) contributes to bone growth and fracture healing . The mRN ± 0.12) . The mRNp < 0.0001) was 3.6-fold higher than that without treatment (1.01 ± 0.15). The mRNA expression of ESR1 with S30+LGA1  was 3.1-fold higher than that with S30 (1.19 ± 0.36) (p < 0.001) was 3-fold higher than that without treatment (1.01 ± 0.14) (p < 0.01) was 2.4-fold higher than that with S30 (1.25 ± 0.60).The mRNA expression of ESR1 with S30+LGA1  . The mRN ± 0.14) . The mRNp < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in body weight gain compared with the sham group. However, the OVX+S30 group did not exhibit any significant differences compared with the OVX group.The effect of S30+LGA1 on the body weight of OVX rats was investigated . The bod 0.0001) B. Howevep < 0.0001) (p < 0.0001) exhibited a significant increase in 17β-estradiol compared with the OVX group (19.3 ± 9.0 pg/mL). The other groups did not exhibit any significant differences compared with the OVX group.The serum level of 17β-estradiol in the OVX group (19.3 ± 9.0 pg/mL) was significantly decreased by 21% compared with that in the sham group  C. Howeve3) was increased by 134% compared with that in the sham group  , OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in the total abdominal volume compared with the OVX group. The other groups did not exhibit any significant differences compared with the OVX group.The effect of S30+LGA1 on the abdominal fat volume in the OVX rats was investigated using micro-CT images. We observed significant increases in the abdominal fat volume in the OVX group compared with that in the sham group A. Howeve 0.0001) B. Howeve3, p < 0.0001) was significantly increased by 157% compared with that in the sham group  , OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in the total abdominal fat volume compared with the OVX group. The other groups did not exhibit any significant differences compared with the OVX group.Similarly, the total abdominal fat volume in the OVX group  C. Howeve3, p < 0.0001) was significantly increased by 170% compared with that in the sham group  , OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in the abdominal visceral fat volume compared with the OVX group. The other groups did not exhibit any significant differences compared with the OVX group.The abdominal visceral fat volume in the OVX group  D. Howeve3, p < 0.0001) was significantly increased by 137% compared with that in the sham group  , OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases compared with the OVX group. The other groups did not exhibit any significant differences compared with the OVX group.The abdominal subcutaneous fat volume in the OVX group  E. Howevep < 0.0001), indicating the successful establishment of ovarian hormone loss (p < 0.001) was significantly increased compared with that in the OVX group (0.17 ± 0.01 g). The uterine weight in the other groups did not differ significantly from that in the OVX group.To investigate the effect of S30+LGA1 on uterine weight and tissues in the OVX rats, the animals in the six study groups were euthanized, and the uterine weights were subsequently measured. The uterine weight was decreased in the OVX group compared with that in the sham group A. Moreovone loss B. The utp < 0.0001) (p < 0.0001) and OVX+S30+LGA1  groups demonstrated marginal increases in epithelial thickness compared with the OVX group (24.87 ± 2.23 μm). The OVX+S30 and OVX+LGA1 groups did not exhibit any significant differences compared with the OVX group.Furthermore, significant differences were observed in the vaginal epithelial height among the groups. The effects on uterine histomorphology revealed that the sham and OVX groups exhibited tall columnar and low cuboidal epithelia, respectively C. Howeve 0.0001) D. Howevep < 0.0001). However, both the OVX+E2  and OVX+S30+LGA1  groups exhibited increases in BMD compared with the OVX group. The OVX+S30 and OVX+LGA1 groups did not exhibit any significant differences. Moreover, the bone volume fraction (BV/TV) ratio in the OVX group (10.72 ± 1.63%) was significantly decreased by 27% compared with that in the sham group   C. Howevep < 0.0001) (p < 0.0001) and OVX+S30+LGA1  groups exhibited a significant decrease, while the Tb. Sp of the OVX+S30 and OVX+LGA1 groups did not differ significantly compared with those in the OVX group. Overall, these results suggest the positive impact of S30+LGA1 on bone health, as evidenced by the improved BMD and other bone morphometric parameters.Similarly, other bone parameters, such as the trabecular number (Tb. N) and trabecular thickness (Tb. Th), were significantly increased following supplementation with E2 and S30+LGA1 D,E. Howe 0.0001) F. The TbThe effects of S30+LGA1 on the bone-related biochemical markers, such as OC and ALP, and bone resorption markers, including deoxypyridinoline (DPD), pyridinoline (PYD), amino-terminal cross-linked telopeptide of collagen (NTX), and carboxy-terminal cross-linked telopeptide of collagen (CTX), were investigated .p < 0.0001) (p < 0.0001) and OVX+S30+LGA1  groups exhibited significant increases in OC levels compared with the OVX group. The OVX+S30 and OVX+LGA1 groups did not exhibit any significant differences compared with the OVX group.Regarding bone formation markers, the serum OC level in the OVX group (3586.4 ± 894.5 pg/mL) was significantly decreased by 24% compared with that in the sham-operated group  A. Howevep < 0.0001) (p < 0.0001) and OVX+S30+LGA1  groups exhibited significant increases in ALP levels compared with the OVX group. The OVX+S30 and OVX+LGA1 groups did not exhibit any significant differences compared with the OVX group.Similarly, the serum ALP level in the OVX group (66.2 ± 4.7 U/L) was significantly decreased by 44% compared with that in the sham group  B. Howevep < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in DPD level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.Regarding bone resorption markers, the serum level of DPD in the OVX group (146.1 ± 21.5 nmol/L) was significantly increased by 171.5% compared with that in the sham group  C. Howevep < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in PYD level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.Similarly, the serum level of PYD in the OVX group (1423.7 ± 203.7 nmol/L) was significantly increased by 171.5% compared with that in the sham group  D. HoweveX level in the OVX group (1074.6 ± 121.2 nmol/L) was significantly increased by 330.7% compared with that in the sham group  (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in NTX level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.The NT 0.0001) E. HoweveX level in the OVX group (134.0 ± 9.5 ng/L) was significantly increased by 208.4% compared with that in the sham group  (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in CTX level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group. Overall, these results indicate the potential beneficial effects of S30+LGA1 on bone health, as evidenced by its influence on bone-related biochemical markers.The CT 0.0001) F. Howevep < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in ET-1 level compared with the sham group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.The effect of S30+LGA1 on vascular homeostasis in OVX rats was investigated . The ser 0.0001) A. Howevep < 0.001) (p < 0.001) and OVX+S30+LGA1  groups exhibited significant increases in eNOS level compared with the sham group. The OVX+S30 and OVX+LGA1 groups did not exhibit any significant differences compared with the OVX group.The eNOS level in the OVX group (2.4 ± 0.7 ng/mL) was significantly decreased compared with that in the sham group  B. Howevep < 0.01) (p < 0.0001) group exhibited a significant increase in serotonin level compared with the sham group. The OVX+E2, OVX+S30, and OVX+LGA1 groups did not exhibit any significant differences compared with the OVX group.To assess the effects of S30+LGA1 on neurotransmitters in OVX rats, the serum levels of serotonin and norepinephrine were measured. The serotonin level in the OVX group (98.5 ± 4.3 ng/mL) was significantly decreased compared with that in the sham group  A. Howevep < 0.001) (p < 0.01) exhibited an increase in norepinephrine level compared with the sham group. The OVX+S30+LGA1 group  exhibited a significant increase in norepinephrine level compared with the sham group. However, the OVX+S30 and OVX+LGA1 groups did not exhibit any significant differences compared with the OVX group.Similarly, the serum levels of norepinephrine in the OVX group (2795.8 ± 143.5 pg/mL) were significantly decreased compared with those in the sham group  B. HoweveThe effects of S30+LGA1 on the lipid profile and AST, ALT, albumin, and calcium levels in the OVX rats were investigated. ELISA kits were used to measure the serum concentrations of these markers.p < 0.01) (p < 0.01), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in cholesterol level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.The serum cholesterol level in the OVX group (140.3 ± 10.2 mg/dL) was significantly increased compared with that in the sham group  A. Howevep < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant increases in triglycerides level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.Similarly, the serum triglycerides level in the OVX group (115 ± 11.7 mg/dL) was significantly decreased compared with that in the sham group  B. Howevep < 0.0001) (p < 0.0001) and OVX+S30+LGA1  groups exhibited significant increases in HDL level compared with the OVX group. The OVX+S30 and OVX+LGA1 groups did not exhibit any significant differences compared with the OVX group.The serum HDL level in the OVX group (40.7 ± 1.9 mg/dL) was significantly decreased compared with that in the sham group  C. Howevep < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in LDL level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.The serum LDL level in the OVX group (71.32 ± 7.7 mg/dL) was significantly increased compared with that in the sham group  D. Howevep < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in AST level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.The serum AST level in the OVX group (131.3 ± 4.2 IU/L) was significantly increased compared with that in the sham group  E. Howevep < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in AST level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.The serum ALT level in the OVX group (56.5 ± 3.7 IU/L) was significantly increased compared with that in the sham group  F. Howevep < 0.0001) (p < 0.0001), OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant increases in albumin level compared with the OVX group. The OVX+S30 group did not exhibit any significant differences compared with the OVX group.The serum albumin level in the OVX group (2.5 ± 0.2 g/dL) was significantly decreased compared with that in the sham group  G. Howevep < 0.0001) (p < 0.0001), OVX+S30 , OVX+LGA1 , and OVX+S30+LGA1  groups exhibited significant decreases in calcium level compared with the OVX group.The serum calcium level in the OVX group (14.0 ± 0.3 mg/dL) was significantly increased compared with that in the sham group  H. HoweveConventionally, HRT supplements are used to relieve menopausal symptoms ,50. HoweSeveral probiotic LAB strains have demonstrated potential in converting isoflavone glucosides to aglycones . IsoflavL. gasseri, which enhances the bioavailability of isoflavones from soy-based products [Estrogen deficiency causes rapid bone loss in post-menopausal women . Estrogeproducts .This study evaluated the effect of S30+LGA1 on various menopause-related aspects in OVX rat models. Menopausal women commonly tend to gain weight. Estrogen regulates body fat production because estrogen suppresses lipoprotein lipase at both mRNA and protein levels ,68,69. TThe results of our study demonstrate the potential of S30+LGA1 in reducing abdominal fat volume. The administration of S30+LGA1 led to a notable reduction in total abdominal fat volume in the treated groups. Furthermore, we observed significant reductions in abdominal visceral and subcutaneous fat volumes in the OVX+E2, OVX+LGA1, and OVX+S30+LGA1 groups compared with the OVX group. These results provide strong evidence of the potential anti-obesity effects of S30+LGA1. Our findings are in line with previous studies, which investigated the effects of estradiol and soybean isoflavones and aglycones on fat metabolism . These cIn the OVX rats, estradiol and soybean isoflavones and aglycones were found to alleviate uterine atrophy caused by the significantly decreased estrogen levels . The mosIn this study, we investigated the effects of S30+LGA1 on bone loss in OVX rats using micro-CT images. In the sham group, no significant changes were observed in the five bone indices. However, the OVX group exhibited significant decreases in BMD, BV/TV, Tb. N, and Tb. Th, and an increase in Tb. Sp , indicatX, and CTX, which are the markers of bone resorption, compared with the sham group. Physiological bone changes are typically triggered by decreased estrogen levels after ovariectomy [Regarding serum bone markers, the OVX group exhibited decreased levels of OC and ALP, which are the markers of bone formation, and increased levels of DPD, PYD, NTriectomy . Osteoperiectomy . HoweverPrevious studies demonstrated that estrogen supplementation may protect against hypertension by activating vasodilation pathways, such as the nitric oxide (NO) system, and inhibiting vasoconstricting systems, such as the endothelin (ET) system ,87,88,89Estrogen and phytoestrogen treatment can increase serotonin and norepinephrine levels, leading to the alleviation of depressive symptoms ,92,93. IMenopausal women often experience alterations in lipid metabolism and liver function, which can lead to increased risks of cardiovascular diseases and other health complications ,95. TherLiver enzymes, such as AST and ALT, are essential indicators of liver function . ElevateIn summary, our study investigated the effects of S30+LGA1—a soy-derived compound combined with a probiotic LAB—on menopausal conditions in OVX rats. Supplementation with S30+LGA1 yielded promising results, such as improved lipid profile and hepato- and osteo-protective effects. Additionally, S30+LGA1 demonstrated a positive impact on uterine health and mood regulation. These findings suggest that S30+LGA1 may be a natural, effective, and beneficial intervention for menopausal symptoms. To further assess the efficacy of S30+LGA1, a clinical trial needs to be conducted in the future.Our study highlights the potential benefits of S30+LGA1 for menopausal symptoms. S30+LGA1 exhibited positive effects on various aspects, including body weight, uterine and bone health, vascular markers, neurotransmitter levels, lipid profile, and liver enzymes. These findings suggest that S30+LGA1 could be a promising intervention for alleviating menopausal symptoms and addressing various menopause-related health aspects in the OVX model. Therefore, S30+LGA1 can be recommended as a soy-based diet as an alternative or supplement to HRT in menopausal women.


Reprogramming of tumor-associated macrophages (TAMs) is a promising strategy for cancer immunotherapy. Several studies have shown that cancer cells induce/support the formation of immunosuppressive TAMs phenotypes. However, the specific factors that orchestrate this immunosuppressive process are unknown or poorly studied. In vivo studies are expensive, complex, and ethically constrained. Therefore, 3D cell interaction models could become a unique framework for the identification of important TAMs programming factors. In this study, we have established and characterized a new in vitro 3D model for macrophage programming in the presence of cancer cell spheroids. First, it was demonstrated that the profile of cytokines, chemokines, and surface markers of 3D-cultured macrophages did not differ conceptually from monolayer-cultured M1 and M2-programmed macrophages. Second, the possibility of reprogramming macrophages in 3D conditions was investigated. In total, the dynamic changes in 6 surface markers, 11 cytokines, and 22 chemokines were analyzed upon macrophage programming (M1 and M2) and reprogramming (M1→M2 and M2→M1). According to the findings, the reprogramming resulted in a mixed macrophage phenotype that expressed both immunosuppressive and anti-cancer immunostimulatory features. Third, cancer cell spheroids were shown to stimulate the production of immunosuppressive M2 markers as well as pro-tumor cytokines and chemokines. In summary, the newly developed 3D model of cancer cell spheroid/macrophage co-culture under free-floating conditions can be used for studies on macrophage plasticity and for the development of targeted cancer immunotherapy. Tumor-associated macrophages (TAMs) are a major leukocyte population in the tumor microenvironment (TME). TAMs support tumor progression by inhibiting the anti-tumor immune response, induction of tumor growth, and angiogenesis . ConditiMacrophage reprogramming toward the M1 phenotype is a promising immunotherapy approach for cancer treatment . ReprogrTraditionally used cell co-cultivation in monolayers does not represent tumor architecture, cell–cell and cell–matrix contacts, or nutrient and oxygen gradients, and therefore is insufficiently appropriate for cancer cell–macrophage interaction studies . FurtherThis study aimed to investigate macrophage interactions with cancer cells and to evaluate reprogramming abilities towards the M1 phenotype in vitro under free-floating 3D conditions. For this purpose, we established a murine mammary cancer 3D model with macrophages at different polarization states . The model was characterized by determining the changes in cell surface marker expression and the secretome composition. We hypothesized that cancer cells switch macrophage polarization towards the M2 phenotype. M2 was expected to be involved in cancer cell migration as well as in the direct promotion of tumor growth. Reprogramming toward the M1 phenotype was expected to reverse these processes. This study represents the first attempt to culture macrophages under 3D free-floating conditions with the comprehensive quantitative characterization of surface markers and cytokine/chemokine profiles. This novel approach allows for the examination of cellular behavior and programming properties within a more physiologically relevant environment, deviating from traditional 2D culture systems. The assessment of the macrophage phenotypes co-cultured with cancer cell spheroids potentially will facilitate the modeling of spatial cellular interactions for therapeutic TME programming.4 cells per well) to achieve an undifferentiated M0 phenotype. A total of 50 ng/mL IFNγ and 100 ng/mL TLR2/1 ligand Pam3SCK4 were added to achieve the M1 phenotype, and 20 ng/mL IL-4 was used to achieve the M2 phenotype. Changes in the cell morphology were detectable after 48 h: M1 macrophages formed net-like structures, while M2 macrophages formed cell clumps, compared to M0  does not represent in vivo cell–cell interactions and architecture ,18. Furted to M0 a. When 7ed to M0 b–d. hi expressing cells . MHC II was upregulated in both M1 and M2 types of macrophages with predominant expression in M1 (M1 macrophages were found to express higher levels of CD38 96%), secrete NO (230 μM), and a high amount of the following pro-inflammatory cytokines: IL-12 (>4.0 ng/mL), TNFα (0.6 ng/mL), and IL-6 (9.8 ng/mL), as described in previous studies of macrophages cultured in monolayers %, secret. Althougon in M1 c.All macrophages were found to secrete IL-1β, IL-2, and IL-16 . Interesp = 0.0070). Previous studies showed that IFNβ has a growth inhibitory effect on cancer cells . Fu. Fup = 0w/o) and not reprogrammed were stated as reprogramming-negative controls (w/o) M1 and (w/o) M2). The reprogramming continued for 72 h, and on Day 6 of the experiment, the cells were visualized by microscopy, and the surface markers and secretome were analyzed using flow cytometry, ELISA, or Luminex, respectively. The reprogramming of M2 macrophages towards the M1 phenotype is a promising cancer immunotherapy method. To perform reprogramming in a 3D free-floating model, BMDMs were activated for 48 h as described above, and then the NO test of cell media was performed to control M1 polarization (w/o) M1 and (w/o) M2 were spread evenly in the wells. The cultivation of M1 without IFNγ/Pam3SCK4 ((w/o) M1) decreased the NO levels significantly  M2 showed a decrease in the CD206 marker, indicating possible depolarization in the absence of an IL-4 stimulus.Reprogramming led to morphological changes in 3D cultured macrophages b: M1→M2 hi compared to (w/o) M2  and CD38hi  (w/o) M1 = 13% vs. 0.2%, p = 0.0086) (Reprogramming was achieved as M2→M1 started to produce NO (330 μM) and express less CD11b 0.0001) . Further 0.0086) c. hi), representing a characteristic feature of M2 macrophages; the proportions of non-reprogrammed (w/o) M1 and (w/o) M2 Arg1hi-positive cells were 4% and 5%, respectively, while reprogrammed M1→M2 and M2→M1 were 27% and 33% (p = 0.0251 and p = 0.0720). Furthermore, the reprogrammed phenotypes began to secrete higher amounts of IFNβ (w/o) M1, and 1.9 ng/mL in the medium of reprogrammed M1→M2 macrophages (p = 0.0062). Similarly, in non-reprogrammed (w/o) M2 samples, the IFNβ was undetectable (<62.5 pg/mL), but for M2→M1, it was 1.36 ng/mL (p < 0.0001). Moreover, both reprogrammed phenotypes secreted higher amounts of GM-CSF (w/o) M1 and (w/o) M2, while in the medium of reprogrammed M1→M2 and M2→M1, the amounts were 0.6 pg/mL and 0.8 pg/mL (p = 0.0091 and 0.0026). IFNα and IL-10 were not detected  control macrophages had lower levels of secreted cytokines, except for IL-1β. Similar findings were previously described by Tarique et al. [The induction of pro-inflammatory cytokines and metabolic markers (Arg1) may be the result of cellular overstimulation during reprogramming. On the other hand, as macrophages possess high plasticity, we suppose that they might lose phenotype features when the activation factors are removed ,31. Thuse et al. .hi, CD38, secreting IFNβ, IL-1β, IL-2, and IL-16  and GM-CSF (30 pg/mL). Furthermore, low levels of GM-CSF (7 pg/mL) were detectable even in a non-stimulated spheroid medium (S), which was previously described in the 4T1 cell line [To distinguish the cytokines produced by cancer cells, controls without macrophages were performed. The control spheroids were incubated without activation factors (S), with M1 activation factors IFNγ/Pam3SCK4 (S IFNγ Pam3), and with M2 activation factor IL-4 (S IL-4). We evaluated the secretion of IL-1β, IL-2, IL-6, IL-10, IL-12, IL-16, GM-CSF, IFNβ, and TNFα . It was ell line . It was hi (44%), and secreted NO (270 μM), TNFα (1.5 ng/mL), IL-6 (10.2 ng/mL), and IL-12 (>4.0 ng/mL). S + M2 macrophages were marked by the expression of CD206 (10%) and Arg1hi (50%). Similar to the macrophage 3D monoculture, both S + M1 and S + M 2 secreted IFNβ  and IL-1β   demonstrated that macrophages can be programmed by respective IFNγ/Pam3SCK4 and IL-4 stimuli . After 70 pg/mL) d, but no0 pg/mL) . The obtw/o) M1 and S + (w/o) M2, respectively, and used as controls.Here, we investigated the potential macrophage reprogramming abilities in the presence of cancer cell spheroids. Initially, macrophages were programmed without 4T1 spheroids in a 3D monoculture to acquire M1 and M2 phenotypes. Then, the macrophages were washed and transferred to the cancer cell spheroids, incubated for 24 h, and the reprogramming factors were added a. Brieflhi  and IFNβ  , while S + M2→M1 began to express the M1 marker CD38 (76%), fewer CD11bhi 40%; c, and prhi 40%; b, TNFα  c,d, but 2 ng/mL) . In addi2 ng/mL) c,d. Inte2 ng/mL) b. Chemokines are important for immune cell recruitment and immune control. In this study, macrophage reprogramming resulted in new, previously undescribed macrophage phenotypes, M1→M2 and M2→M1. Furthermore, we observed cancer cell (4T1/GFP) migration out of the spheroids in the presence of M0 and M2, but not M1 macrophages. To explain the cancer cell migration and formation of spheroid satellites, as well as to further characterize the reprogrammed macrophage phenotypes, we assessed the secreted chemokine profiles under 3D free-floating conditions. First, the chemokines secreted by cancer cell spheroids without macrophages and activation factors were determined a. The 4TChemokines secreted by the cancer cell spheroids without macrophages under the influence of activating factors were then determined. Upon stimulation with IFNγ/Pam3SCK4 (S IFNγ Pam3), the spheroids started to secrete chemokines induced by IFNγ—CCL5 (0.6 ng/mL), CXCL10 (3.4 ng/mL), and CXCL11 (190 pg/mL) a. IncreaAfter stimulation with IL-4, the spheroids began to secrete CCL5 (0.17 ng/mL), CCL17 (20 pg/mL), CCL11 (5 pg/mL), and CCL19 (7 pg/mL) a. Importhi TAMs in vivo [Then, the chemokines secreted by M1 macrophages in a free-floating monoculture (M1) and co-culture with cancer cell spheroids (S + M1) were determined b. M1 rev in vivo . In addip = 0.0004). CCL12 was also upregulated in M2-like MHC IIlow TAMs [Continuing the characterization of programmed macrophages, the chemokines secreted by M2 macrophages in 3D monoculture (M2) and co-culture with cancer cell spheroids (S + M2) were determined b. M2 seclow TAMs  and in tlow TAMs , suggestw/o M), the expression of some chemokines was remarkably stable (w/o) continued to secrete CCL5 (2.3 ng/mL), CXCL1 (0.6 ng/mL), CXCL5 (80 pg/mL), CXCL16 (0.8 ng/mL), and VEGF-A (2.7 ng/mL). The levels of the other tested chemokines are illustrated in Although the macrophages lost several phenotypic characteristics after the elimination of activation factors (y stable . Pre-actw/o) M1 continued to secrete the following M1-specific chemokines: CCL5 (2.2 ng/mL), CXCL1 (0.34 ng/mL), CXCL16 (0.63 ng/mL), VEGF-A (0.2 ng/mL), and CCL1 (1.9 pg/mL). After M1→M2 reprogramming, the M2-associated chemokine CCL22 (0.72 ng/mL) began to be secreted. Unlike M2 macrophages, M1→M2 began to secrete certain IL-4-induced chemokines only after co-culture with cancer cell spheroids, for instance, CCL17  and CCL24   .w/o M2) (The expression of M2-associated chemokines CCL17 (24 pg/mL), CCL22 (0.6 ng/mL), and CCL24 (2.7 ng/mL) remained stable in M2 macrophages incubated without factors (w/o M2) . Also, aw/o M2) . Becausew/o M2) .w/o) M1, (w/o) M2: without factors IFNγ/Pam3SCK4 and IL-4, respectively). For example, CD38 and CCL5 are stably expressed by M1 macrophages, including (w/o) M1 and M1→M2, and are slightly affected by the presence of cancer cell spheroids. In contrast, IL-12 and CXCL10 in M1 are highly dependent on the presence of IFNγ/Pam3SCK4 in the medium and are completely downregulated in (w/o) M1. This study represents a comprehensive evaluation of cytokine, chemokine, and surface marker profiles of macrophages co-cultured with cancer cell spheroids, as well as an assessment of their reprogramming potential. In this Section, we have highlighted the data demonstrating the influence of 4T1/GFP cancer cell spheroids on macrophage surface/intracellular markers and secretome profiles. Markers. How do 4T1/GFP spheroids affect the BMDMs phenotype? After incubation of M0 with cancer cell spheroids (S + M0) for 96 h  and MHC II   had decreased levels of CD11b 0.0242) c. In addhi . S + M1→M2 had downregulated levels of MHC II from 68% to 50%, p = 0.0007, and CD38 from 75% to 57%, p = 0.0007, but upregulated levels of CD206 from 13% to 22%, p = 0.0734 , while the levels of NO were downregulated compared to M0 cultivated in the 3D monoculture  ( 0.0081) .p = 0.0206) (+) cancer cell line T47D also expressed more TNFα [p = 0.0026; p = 0.0021) . M2→M1 macrophages had statistically significant downregulation in NO expression  and TNFα  after interaction with cancer cell spheroids had increased IL-1β (from (w/o) M2 90 pg/mL to S + (w/o) M2 136 pg/mL, p = 0.0009), IFNβ , and TNFα  cytokine secretion  (p = 0.0102), CXCL16 , and CCL22 , demonstrating the inhibitory effect of the cancer cell spheroids  b. Similapheroids b.p = 0.0377), CCL20 , and CX3CL1 , CCL12 , CCL22 , CCL24 , and CXCL11   b. Previop = 0.0013) and CCL12 . Increased levels of CX3CL1  and CCL11  were also observed in S + M2 (M2 macrophages polarized in the presence of spheroids (S + M2) had increased levels of chemokines CXCL1  after interaction with the cancer cell spheroid showed increased CCL20 (from (w/o) M1 3 to S + (w/o) M1 15 pg/mL, p = 0.0013) and CXCL1  levels, decreased CCL3  and CXCL13  (w/o) M2 macrophages showed increased levels of CXCL1 (from (w/o) M2 0.13 ng/mL to S + (w/o) M2 0.32 ng/mL, p = 0.0175) and CCL24 . It is theoretically possible that a leaky amount of activation factors  are present in the (w/o) macrophage environment and may interact with the cancer cell spheroid. However, taking into account the loss of the macrophage phenotypic characteristics, we suppose that the levels of the remaining activation factors are too small to affect chemokine production.M1 macrophages cultured without activation factors ( 0.0144) . A decrep = 0.0002), CCL17 , CCL20 , CCL22 , CCL24 , and CX3CL1   . The inc 0.0060) a. Increap = 0.0021) and CCL24  (p = 0.0010), CX3CL1 , CXCL1 , and CXCL10  in the S + M2→M1 culture medium can be attributed to the interaction of 4T1/GFP cancer cells with IFNγ/Pam3SCK4 because an increase in these chemokines was observed in spheroids cultured in the presence of IFNγ/Pam3SCK4 without macrophages  . The incrophages a.Changes in cell surface markers and secretome profiles after the co-culture of macrophages with cancer cell spheroids are summarized in The size of the cancer cell spheroids was measured using fluorometry and by counting total cell fluorescence in microscopy images. Relative spheroid sizes were determined using flow cytometry . The results of all three methods were similar, supporting the same tendency a.p = 0.0026 and p < 0.0001, respectively). As IFNγ has a direct antiproliferative effect on cancer cells [p < 0.0001). Furthermore, the spheroids cultivated with IFNγ/Pam3SCK4 had lower viability than the spheroids cultivated in the absence of factors (p = 0.0136) and spheroids cultivated with IL-4 (p = 0.0151), which can be explained by the toxic effect of these factors . Although macrophages were functionally programmed (or reprogrammed) to express pro-inflammatory markers and cytokines, they were not capable of decreasing the size of the cancer cell spheroids in this model.The effect of M1 and M2→M1 macrophages on spheroid size in the presence of IFNγ/Pam3SCK4 was not significantly different from the spheroids with IFNγ/Pam3SCK4 factors without macrophages . Xuan et al. showed that CXCL10 induces chemotaxis in M1 macrophages [Next, we examined the ability of M0, M1, and M2 macrophages to migrate toward 4T1/GFP cancer cells. For this, cancer cells were seeded in a 24-well plate, while BMDMs were added to the inserts with M0, M1, or M2 activation medium. Macrophages were able to migrate to cancer cells through an 8.0 µm pore membrane, but not to the 4T1/GFP culture medium d. M1 androphages . As we frophages , which iIn this study, we have developed and characterized a macrophage programming and reprogramming model in the presence of cancer cell spheroids under 3D free-floating conditions. In total, 6 surface/intracellular markers, 11 cytokines, and 22 chemokines were analyzed, providing a comprehensive characterization of the phenotypic and functional properties of BMDMs. Many studies on macrophage profiling are focused on the specific markers, cytokines, or chemokines ,56,57,58hi and the chemokines CCL17 and CCL24  change their chemokine profile. Upon stimulation with IFNγ/Pam3SCK4, the spheroids secrete increased levels of IL-6, IL-16, CCL5, CXCL10, and CXCL11 a; and aler model , indicat[+ cells . CXCL10 ot clear . Therefoot clear ,75,76. Tot clear . The tumot clear . The cooThe developed 3D free-floating cell system represents a useful tool for the analysis and modeling of the cytokine/chemokine profiles of immune cells in the presence/absence of cancer cells. In the established model, macrophages stimulated the migration of 4T1/GFP cancer cells out of the spheroid. In the M2 group, the formation of satellite GFP-positive spheroids was observed. Cancer cells were found to migrate to M0 and M2 macrophages, but not to M1, whereas M2 and M1 macrophages migrated to cancer cells c,d. ThusAlthough macrophages were functionally programmed/reprogrammed and expressed inflammatory markers and cytokines, they did not significantly affect the size of the cancer cell spheroids. Furthermore, IFNγ in combination with the TLR2/1 ligand Pam3SCK4 significantly reduced the size of the spheroid without the presence of macrophages. On the other hand, when these factors are removed, macrophages start to lose their phenotypic features. This makes potential therapeutic reprogramming challenging.+Ly6ChiCX3CR1low monocytes [Although the established model represents TME better than conventional 2D cultivation, it has certain limitations. First, the volume of the wells is limited and the model does not supply nutrients. As a result, the developed model is not suitable for long-term cell cultivation. The use of microfluidic devices can potentially solve the problem; however, it can be difficult to accomplish under free-floating conditions. Second, the traditional macrophage cultivation protocol requires the use of a medium supplemented with L929-CM, in which monocyte and eosinophil chemotactic factors CCL2, CCL7, VEGF-A, CCL11, and CXCL1 have been found . In thisonocytes . As TAMsonocytes ,80,81. Oonocytes . The speIn summary, a new spheroid-based cancer cell model for macrophage programming under 3D conditions was established and characterized. Unlike matrix-based 3D models, the use of an ultra-low attachment plate allows for the easy manipulation of cells and secreted factors. This model can be used for in vitro immunotherapy studies to develop a tumor micromodel with other cancer-associated cell types, such as fibroblasts or immune cells. The co-culture allowed us to evaluate how cancer cell spheroids affect macrophages in different polarization states, and how macrophages affect spheroid growth. The described model can be used to study TME cell–cell interactions and can be potentially applied as a screening tool for TAM-targeted therapies. 2 in a humidified atmosphere. Fibroblast-like L929 cells were obtained from ATCC® (CCL-1™) and cultivated in RPMI-1640 medium supplemented with 10% FBS, 1% PEST, 2 mM L-glutamine at 37 °C, and 5% CO2 in a humidified atmosphere.Murine triple-negative breast cancer 4T1-Fluc-Neo/eGFP-Puro cells (4T1/GFP) expressing green fluorescent protein (GFP) and firefly luciferase (Luc) were obtained from Imanis Life Sciences. The 4T1/GFP cells were cultivated in Roswell Park Memorial Institute (RPMI) in 1640 medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine , 1% penicillin/streptomycin (PEST) , 0.1 mg/mL G418 , 2 µg/mL puromycin  at 37 °C, and 5% CO2 incubator at 37 °C and passaged using 0.05% trypsin solution .Cells were incubated in a humidified 5% CO2 incubator at 37 °C. After cultivation, the conditioned medium (CM) of L929 cells was collected, centrifuged at 400× g for 10 min, transferred to a new tube, and centrifuged again at 13,000× g for 20 min at 4 °C to remove the cell pellets. The CM of L929 cells was aliquoted and stored at −20 °C until use.Mouse macrophages were differentiated from bone marrow cells according to an established protocol using an L929 cell-conditioned medium as a source of macrophage colony-stimulating factor (MCSF) . The L922 and MgCl2 for 30 min at 4 °C and by repeated gentle flushing of the dish, and then the BMDMs were collected by centrifugation. BMDMs were then frozen in Recovery™ Cell Culture Freezing Medium  and stored at −150 °C for future experiments.Bone marrow from the femurs and tibia of the hind legs of 8–12 weeks old female BALB/c mice was flushed with RPMI-1640 medium containing 10% FBS under sterile conditions and passed through a 70-µm cell strainer . Red blood cells (RBC) were removed by incubation in an ammonium–chloride–potassium (ACK) lysing buffer  for 10 min at room temperature. After centrifugation and washing, the remaining cells were cultured in 10 cm non-tissue culture-treated dishes  in RPMI-1640 medium (10% FBS) containing 30% L929-cell-CM. Bone marrow cells (Passage 0) were cultured for 5 days, after which non-adherent cells were washed off using phosphate-buffered saline (PBS), and the adherent macrophages were cultured for an additional day. Differentiated BMDMs were detached by incubation in PBS without CaCl4 cells were plated in a 96-well black round bottom ultra-low attachment plate  in 200 μL of the macrophage cultivation medium: RPMI-1640 medium containing 10% FBS, 50 U/mL penicillin, 50 µg/mL streptomycin, 2 mM L-glutamine, and 10% L929-CM. These cells (Passage 2) were used for polarization and reprogramming experiments. After thawing, BMDMs (Passage 1) were incubated for 4 days, detached, and filtered through a 70 μM cell strainer. For experiments under 3D conditions, 3.5 × 10The non-polarized or M0 phenotype was achieved by cultivating cells in RPMI-1640 medium supplemented with 10% FBS , 10% MCSF (L929-CM), 1% PEST, and 1% L-glutamine for 72 h. The media was supplemented either with 50 ng/mL recombinant mouse IFNγ  and 100 ng/mL Pam3SCK4  to achieve M1 phenotype, or with 20 ng/mL IL-4  to achieve M2 phenotype.4 cells in 100 μL per well in a fresh macrophage medium. After 24 h, 50 μL of macrophage cultivation medium supplemented with 50 ng/mL recombinant mouse IFNγ and 100 ng/mL Pam3SCK4 was added to the M2 pre-polarized macrophages and 20 ng/mL IL-4 was added to the pre-polarized M1. The samples (w/o) M1 and (w/o) M2 received 50 μL of macrophage cultivation medium to keep the total volumes equal in all wells. BMDMs were polarized to the M0/M1/M2 phenotype for 48 h, as described above. Then, cells were collected, washed with PBS, counted, and plated 2.87 × 103 cells in 100 μL per well) and incubated at 37 °C in a humidified 5% CO2 incubator.4T1-Fluc-Neo/eGFP-Puro (4T1/GFP) spheroids were generated using a 96-well black round bottom ultra-low attachment plate . Cells were collected from monolayers by trypsin treatment, filtered through a 40 μM cell strainer , counted, and resuspended in RPMI-1640 medium supplemented with 10% FBS, 1% PEST, and 1% L-glutamine. The suspension was added to a 96-well ultra-low attachment plate  were washed, counted, and 2.87 × 10g for 5 min and washed with PBS. Spheroids were treated with 0.05% trypsin solution for 5 min. Then, the cells were washed with PBS supplemented with 10% FBS (PBS-FS10%) and centrifuged at 410× g for 10 min. For flow cytometry, cells from 4 wells were combined, the suspension was centrifuged at 410× Cells were then washed with PBS and stained with Fixable Viability Stain 660  to determine cell viability. Briefly, cells were suspended in FVS 660 in PBS , incubated for 15 min at room temperature (RT), and then cells were washed twice with PBS.g for 10 min. Then, the cell pellet was resuspended in 50 μL PBS-FS10% with fluorophore-labeled monoclonal antibodies diluted as recommended by the manufacturers. The following monoclonal antibodies were used: anti-CD11b-APC-eFluor™ 780 ; anti-MHC II-PE ; anti-CD206-Briliant Violet 421 ; anti-CD38-PerCP-eFluor™ 710 . Cells were incubated for 1 h at 4 °C, after which 800 μL of PBS-FS10% was added, and the suspension was centrifuged for 10 min at 410× g. The cell pellet was washed once with PBS-FS10%.Then, 50 μL of PBS-FS10% supplemented with 12.5 μg/mL mouse IgG  was added to the cell pellet and incubated for 30 min at 4 °C. After blocking, the cells were washed with 800 μL PBS-FS10% and the suspension was centrifuged at 410× 2O) was added to the suspension, and it was centrifuged for 10 min at 410× g. The cell pellet was resuspended in 200 μL of PBS-FS10%. The experiment was repeated twice, and each staining was performed in duplicate. The data were obtained by a FACSAria BD Hardware flow cytometer using FACSDiva Software . To create the compensation matrix, UltraComp eBeads™  was used. The data were analyzed using FlowJo v10.0.7 software and presented as the mean of two independent experiments.For intracellular staining, PerFix-nc Kit  was used. Briefly, cells were resuspended in 25 μL FBS, and 15 μL of the fixative reagent was added. The suspension was mixed and incubated for 15 min at RT, and then 150 μL of permeabilization reagent and anti-Arginase1-APC  antibodies were added in appropriate amounts. The suspension was incubated in the dark at RT 40 min. Then, 1.2–1.8 mL of the final reagent , Mouse IFNα ELISA Kit , Mouse IFNβ ELISA Kit , and Mouse VEGF-A ELISA Kit . Supernatants of macrophages programmed and reprogrammed in the presence and absence of cancer cell spheroids were subjected to cytokine and chemokine analyses using ELISA and Luminex assays. The media was combined from at least 4 wells, centrifuged for 5 min at 500× For the determination of chemokines, Mouse Chemokine Panel 31-Plex  was used. Briefly, the beads were washed twice, and then 50 μL of the samples and standards were added to the beads. To ensure proper mixing, the plate was covered with foil and incubated at RT on a shaker at 850 rpm for 30 min. Following the incubation, the plate was washed three times before the addition of the detection antibodies. The plate was incubated on a shaker in the dark. After 30 min, the plate was washed three times. Then, streptavidin-phycoerythrin (SA-PE) was added to the plate. The plate was incubated in the dark at 850 rpm for 10 min. After the incubation, the plate was washed three times to remove any excess SA-PE. The beads were resuspended in an assay buffer and shaken for 30 s prior to analysis with Luminex 100/200™ and xPONENT 3.1.971.0 software package for the data acquisition system .A nitric oxide assay kit was used  to determine the level of NO in the cell culture medium. NO is measured by the determination of nitrites. Briefly, 50 µL of the cell culture medium was collected from each well, centrifuged, and used for NO quantification. The standards provided in the kit were utilized to generate a standard curve and quantify the nitrites. A spectrophotometer was employed to measure optical density at 540 nm.4 4T1/GFP cells in 300 μL complete medium were added to the upper inserts. Then, the inserts were placed into wells containing the differently activated macrophages. The 8.0 µm pore polycarbonate membrane inserts  were used to investigate the migration of cancer cells and the differently activated macrophages. To investigate the migration of cancer cells towards macrophages, BMDMs were plated on the bottom of the 24-well plate . When the macrophages reached confluency, they were programmed towards the M0/M1/M2 phenotype, as described above. After 4 h, 4 × 104 4T1/GFP cells were plated on the bottom of the 24-well plate. After 24 h, the BMDMs were stained with CellTracker™ CM-DiI Dye , and 300 μL of 7.5 × 104 cells were added into the upper inserts. The inserts were then inserted into the wells. To investigate the migration of BMDMs towards cancer cells, 5 × 10After 18 h, the non-migrated cells were removed using a cotton swab. Migrated cells localized on the opposite membrane site were counted using a Leica DM-IL fluorescent microscope. Cell migration against media  was used as a negative control.Fluorometry. The spheroid growth was measured by a GFP fluorometry assay using Victor3V 1420-040 Multilabel HTS Counter (PerkinElmer): emission filter 485 nm, detection filter 535 nm. Microscopy. Fluorescent microscopy images were obtained using a Leica DM-IL microscope. Total fluorescence was calculated by ImageJ using the following formula: Flow cytometry. The number of GFP-positive cells per sample was determined by a FACSAria II BD Hardware flow cytometer using FACSDiva 6.1.3 Software.t-test and one-way ANOVA. An unpaired one-tailed Student’s t-test was used to compare groups. Each experimental group in this study consisted of two biological replicates. To increase the statistical power, each biological replicate was created by pooling four independently treated samples, which were processed under the same experimental conditions. p < 0.05 was considered statistically significant . Statistical analysis of larger groups (n ≥ 3) was performed using one-way ANOVA. Statistical analysis was performed using the GraphPad Prism 8.02 software. Data were expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using the Student’s Heatmaps were generated using a normalization approach depending on the type of data being analyzed. For macrophage markers, the scale was based on the percentage of maximal expression, ranging from 0% to 100%. In the case of cytokines and chemokines, a normalized scale ranges from 0 to 1 and represents the relative amount of secreted cytokines.In this study, we created a 3D macrophage programming model in the presence of 4T1 breast cancer cell spheroids. The model was characterized using surface markers, secreted cytokines, and chemokines. Unlike many previous studies that focused on specific markers or cytokines, this research offers a global assessment of macrophage signatures.Macrophage reprogramming was performed, and it was established that reprogramming causes a heterogeneous phenotype that expresses both M1 and M2 markers. In a 3D spheroid model, cancer cells were demonstrated to alter macrophage characteristics, promoting M2-like phenotype features. This novel cancer cell spheroid-based model for macrophage programming in 3D settings can be exploited for in vitro immunotherapy studies. It offers opportunities for more complex heterotypic cultures resembling the native tumor composition and facilitates studies on immune cell programming in a clinically relevant context. Although the established model represents a step forward in mimicking the tumor microenvironment, it has certain limitations. Addressing these limitations, such as nutrient supply and the use of synthetic media, will further enhance the relevance and applicability of the model.