NIH3T3 Cells Research Articles

CD52/FLAG and CD52/HA Fusion Proteins as Novel Magnetic Cell Selection Markers.

At present, the magnetic selection of genetically modified cells is mainly performed with surface markers naturally expressed by cells such as CD4, LNGFR (low affinity nerve growth factor receptor), and MHC class I molecule H-2Kk. The disadvantage of such markers is the possibility of their undesired and poorly predictable expression by unmodified cells before or after cell manipulation, which makes it essential to develop new surface markers that would not have such a drawback. Earlier, modified CD52 surface protein variants with embedded HA and FLAG epitope tags (CD52/FLAG and CD52/HA) were developed by the group of Dr. Mazurov for the fluorescent cell sorting of CRISPR-modified cells. In the current study, we tested whether these markers can be used for the magnetic selection of transduced cells. For this purpose, appropriate constructs were created in MigR1-based bicistronic retroviral vectors containing EGFP and DsRedExpress2 as fluorescent reporters. Cytometric analysis of the transduced NIH 3T3 cell populations after magnetic selection evaluated the efficiency of isolation and purity of the obtained populations, as well as the change in the median fluorescence intensity (MFI). The results of this study demonstrate that the surface markers CD52/FLAG and CD52/HA can be effectively used for magnetic cell selection, and their efficiencies are comparable to that of the commonly used LNGFR marker. At the same time, the significant advantage of these markers is the absence of HA and FLAG epitope sequences in cellular proteins, which rules out the spurious co-isolation of negative cells.

Engineering free-standing electrospun PLLCL fibers on microfluidic platform for cell alignment

Here, a PLLCL-on-chip platform was developed by direct electrospinning of poly (L-lactide-co-ε-caprolactone) (PLLCL) on polymethyl methacrylate (PMMA) microfluidic chips. Designed microchip provides the electrospinning of free-standing aligned PLLCL fibers which eliminates limitations of conventional electrospinning. Besides, aligned fiber structure favors cell alignment through contactless manipulation. Average fiber diameter, and fiber alignment was evaluated by SEM analyses, then, leakage profile of microchip was investigated. 3D cell culture studies were conducted using HeLa and NIH-3T3 cells, and nearly 85% cell viability was observed in PLLCL-on-chip for 15 days, while cell viability of 2D control started to decrease after 7 days based on Live dead and Alamar Blue analyses. These findings emphasize biocompatibility of PLLCL-on-chip platform for 3D cell culture and its ability to mimic extracellular matrix (ECM). Immunostaining results prove that PLLCL-on-chip platform favors the secretion of ECM proteins compared to control groups, and cytoskeletons of cells were in aligned orientation in PLLCL-on-chip, while they were in random orientation in control groups. Overall, these results demonstrate that the developed platform is suitable for the formation of various 3D cell culture models and a potential candidate for cell alignment studies.Graphical

Iron Nano Biocomposite-Infused Biopolymeric Films: A Multifunctional Approach for Robust Skin Repair.

Sodium alginate (SA) biopolymeric films have various limitations such as poor mechanical properties, high vapor permeability, lack of antibacterial activity, excessive burst release, and weak cell adhesion. To overcome these limitations, a strategy involving the integration of nanofillers into an SA film matrix is explored. In this context, a cost-effective iron-containing carbon nano biocomposite (FeCNB) nanofiller is developed using a solvent-free technique. This nanocomposite is successfully incorporated into the alginate film matrix at varying concentrations (0.05, 0.1, and 0.15%) aimed at enhancing its physicochemical and biological properties for biomedical applications. Characterization through FESEM and BET analyses confirms the porous nature of the FeCNB. EDX shows the FeCNB's uniform distribution upon its integration into the film matrix, albeit without strong chemical interaction with SA. Instead, hydrogen bonding interactions become apparent in the FTIR spectra. By incorporating the FeCNB, the mechanical attributes of the films are improved and the water vapor permeability approaches the desired range (2000-2500 g/m2day). The film's swelling ratio reduction contributes to a decrease in water permeability. The antibacterial activity and sustained release property of the FeCNB-incorporated film are established using tetracycline hydrochloride (TCl), a model drug. The drug release profile resembled Korsmeyer-Peppas's release pattern. In vitro assessments via the MTT assay and scratch assay on NIH-3T3 cells reveal that FeCNB has no adverse effects on the biocompatibility of alginate films. The cell proliferation and adhesion to the SA film are significantly enhanced after infusion of the FeCNB. The in vivo study performed on the rat model demonstrates improved wound healing by FeCNB-impregnated films. Based on the comprehensive findings, the proposed FeCNB-incorporated alginate films prove to be a promising candidate for robust skin repair.

Abolished clustering of MeCP2T158M can be partially reverted with small molecules

Rett syndrome (OMIM 312750) is a rare neurodevelopmental disorder caused by de novo mutations in the Methyl-CpG Binding Protein 2 (MeCP2) gene located on the X-chromosome, typically affecting girls. Rett syndrome symptoms, characterized by microcephaly and lack of motor coordination, first appear between 6 to 18 months of age. The disease continues to progress until adulthood at which point it reaches a stationary phase. Currently, available therapy for Rett Syndrome is only symptomatic. More than 800 mutations causing Rett syndrome have been described, the most common being T158M (9% prevalence) located in the Methyl-Binding Domain (MBD) of MeCP2. Due to its importance for DNA binding through recognition of methylated CpG, mutations in the MBD have a significant impact on the stability and function of MeCP2. MeCP2 is a nuclear protein and accumulates in liquid–liquid phase condensates visualized as speckles in NIH3T3 cells by microscopy. This speckled pattern is lost with MeCP2 mutations in the MBD such as T158M. We developed a high content phenotypic assay, detecting fluorescent MeCP2 speckles in NIH3T3 cells. The assay allows the identification of small molecules that stabilize MeCP2-T158M and phenotypically rescue speckle formation. To validate the assay, a collection of 3572 drugs was screened, including FDA-approved drugs, compounds in clinical trials and biologically annotated tool compounds. 18 hits showed at least 25% rescue of speckles in the mutant cell line while not affecting wild-type MeCP2 speckles. Primary hits were confirmed in a dose response assay, a thermal shift assay with recombinant MeCP2 and by testing the MeCP2 expression levels. One class of identified hits represents histone deacetylase inhibitors (HDACis) showing 25% speckle rescue of mutant MeCP2 without toxicity. This screening strategy can be expanded to additional compound libraries and will support novel drug discovery.

Development and assessment of novel pyrazole-thiadiazol hybrid derivatives as VEGFR-2 inhibitors: design, synthesis, anticancer activity evaluation, molecular docking, and molecular dynamics simulation.

Cancer remains a significant health challenge globally, requiring the development of targeted chemotherapeutics capable of specifically inhibiting cancer cell growth. Angiogenesis is one of the key features of tumor growth and metastasis and is, therefore, an important target for the treatment of many tumors. The vascular endothelial growth factor (VEGF) signaling pathway has proven to be a promising lead in anticancer therapy due to the central role it plays in tumor angiogenesis. Vascular endothelial growth factor receptor-2 (VEGFR-2) is a key mediator in the signaling pathway regulating angiogenesis. Targeting VEGFR-2 may disrupt angiogenesis, leading to a reduction in tumor blood supply and tumor progression. The design, synthesis, and assessment of novel VEGFR-2 inhibitor derivatives are the focus of this study, with particular emphasis on incorporating the pyrazole-thiadiazol pharmacophore into the molecular structure. Taking advantage of the pharmacophoric properties of pyrazole and 1,3,4-thiadiazol, compounds with different substituents in the main structure were designed and synthesized. The compounds were also evaluated for antiproliferative activity against cancer cell lines. Compound 4e demonstrated the highest activity among all compounds, with an IC50 of 9.673±0.399 μM against HT-29 cells and 23.081±0.400 μM against NIH3T3 cells. To further support the inhibitory activity of compound 4e, an in silico study was performed. Compound 4e demonstrated strong binding to the active site of VEGFR-2 in molecular docking studies, forming hydrogen bonds with key amino acid residues. The stability of the compound in the enzyme's active site was demonstrated through molecular dynamics simulations.

CRGD-modified hybrid lipopolymeric nanoplexes for gene editing in the posterior segment of the eye

Eye-related diseases, specifically retinal dystrophy (RD) conditions, are the leading cause of blindness worldwide. Gene addition, regulation, or editing could potentially treat such diseases through gene expression regulation. CRISPR/Cas9 gene editing is one of the most prominent and precise gene editing tools which could be employed to edit genes related to the dystrophic condition. However, CRISPR/Cas9 faces in vivo delivery challenges due to its high molecular weight, negative charge, prone to degradation in the presence of nucleases and proteases, poor cellular degradation, etc., which makes it challenging to adopt for therapeutic applications. We developed cRGD-modified lipopolymeric nanoplexes loaded with Cas9 RNPs with a particle size and zeta potential of 175 ± 20 nm and 2.15 ± 0.9 mV, respectively. The cRGD-modified lipopolymeric nanoplexes were stable for 194 h and able to transfect >70 % ARPE-19 and NIH3T3 cells with an Indel frequency of ~40 % for the VEGF-A gene. The cRGD-modified lipopolymeric nanoplexes found good vitreous mobility and could transfection retinal cells in vivo after 48 h of intravitreal injection in Wistar Rats. Moreover, in vivo VEGFA gene editing was ~10 % with minimal toxicities. Collectively, the cRGD-modified lipopolymeric nanoplexes were found to have extreme potential in delivering CRISPR/Cas9 RNPs payload to the retinal tissues for therapeutic applications.

Cell-Specific Impacts of Surface Coating Composition on Extracellular Vesicle Secretion.

Biomaterial properties have recently been shown to modulate extracellular vesicle (EV) secretion and cargo; however, the effects of substrate composition on EV production remain underexplored. This study investigates the impacts of surface coatings composed of collagen I (COLI), fibronectin (FN), and poly l-lysine (PLL) on EV secretion for applications in therapeutic EV production and to further understanding of how changes in the extracellular matrix microenvironment affect EVs. EV secretion from primary bone marrow-derived mesenchymal stromal cells (BMSCs), primary adipose-derived stem cells (ASCs), HEK293 cells, NIH3T3 cells, and RAW264.7 cells was characterized on the different coatings. Expression of EV biogenesis genes and cellular adhesion genes was also analyzed. COLI coatings significantly decreased EV secretion in RAW264.7 cells, with associated decreases in cell viability and changes in EV biogenesis-related and cell adhesion genes at day 4. FN coatings increased EV secretion in NIH3T3 cells, while PLL coatings increased EV secretion in ASCs. Surface coatings had significant effects on the capacity of EVs derived from RAW264.7 and NIH3T3 cells to impact in vitro macrophage proliferation. Overall, surface coatings had different cell-specific effects on EV secretion and in vitro functional capacity, thus highlighting the potential of substrate coatings to further the development of clinical EV production systems.

Enhancing cellular behavior in repaired tissue via silk fibroin-integrated triboelectric nanogenerators

Triboelectric nanogenerators (TENGs) have emerged as a promising approach for generating electricity and providing electrical stimuli in medical electronic devices. Despite their potential benefits, the clinical implementation of TENGs faces challenges such as skin compliance and a lack of comprehensive assessment regarding their biosafety and efficacy. Therefore, further research is imperative to overcome these limitations and unlock the full potential of TENGs in various biomedical applications. In this study, we present a flexible silk fibroin-based triboelectric nanogenerator (SFB-TENG) that features an on-skin substrate and is characterized by excellent skin compliance and air/water permeability. The range of electrical output generated by the SFB-TENG was shown to facilitate the migration and proliferation of Hy926, NIH-3T3 and RSC96 cells. However, apoptosis of fibroblast NIH-3T3 cells was observed when the output voltage increased to more than 20 V at a frequency of 2 Hz. In addition, the moderate electrical stimulation provided by the SFB-TENG promoted the cell proliferation cycle in Hy926 cells. This research highlights the efficacy of a TENG system featuring a flexible and skin-friendly design, as well as its safe operating conditions for use in biomedical applications. These findings position TENGs as highly promising candidates for practical applications in the field of tissue regeneration.

Controlled anticancer 5-Fluorouracil release from functionalized 5-FU/PLGA/CaP coating on titanium implants: Characterization, in vitro drug delivery and cytotoxicity

Work presents a novel approach for creating anticancer coatings on titanium implants using 5-Fluorouracil (5-FU) loaded in calcium phosphate which is encapsulated in poly(lactic-co-glycolic acid) (PLGA/CaP) for controllable drug release. Our method involves the hybrid ultrasound-assisted micro-arc oxidation (UMAOH) technique, which make use of an ultrasound as an additional means of energy input resulting in increased pore size, drug deposition, and layer-by-layer self-assembly of PLGA. The multilayer coatings exhibit amorphous-crystalline structure with dicalcium phosphate anhydrous (DCPA) and β-tricalcium phosphate (β-TCP) phases, Ca/P ratio of 0.3, surface roughness (Ra) of 2.3–3.5 µm, and thickness between 52–60 µm. Drug release kinetics revealed a burst release for 5FU/CaP in the initial 10–20 min and sustained release over 2–4 h. 5FU/PLGA/CaP displayed a smooth, sustained release over 5 days. Biocompatibility studies showed that extracts from drug-free CaP and CaP/PLGA samples were nontoxic to HeLa, NIH-3Т3, and MCF-7 cell lines (≥ 80 % cell viability) for 0.2–24 h. In contrast, extracts from drug-loaded carriers were cytotoxic to NIH-3T3 cells (> 60 % cell death). Notably, 5-FU impregnation in the CaP coting demonstrated varying cytotoxicity, with the following cell death order: 75 % HeLa > 68 % NIH-3Т3 > 59 % MCF-7. PLGA modification reduced cytotoxicity with the following cell death order: 61 % HeLa > 52 % NIH-3Т3 > 46 % MCF-7. This work presents a promising strategy for localized, controlled drug delivery 5FU/PLGA/CaP system, with reduced cytotoxicity compared to PLGA-free 5-FU/CaP coating, advancing implant-based cancer treatment.

Enhanced synthesis of antibody-functionalized gold nanoparticles for multiplexed exosome detection via mass signal amplification in LDI-TOF MS.

We present a novel method for sensitive exosomal protein detection using organic matrix-free laser desorption/ionization time-of-flight mass spectrometry (LDI-TOF MS) and gold nanoparticles (AuNPs) functionalized with mass tags for signal amplification (Am-tags). Target exosomes were captured by specific antibodies on AuNPs and a biochip, where the antibody-presenting AuNPs (Ab/Am-tag@AuNPs) contained excess Am-tags. LDI-TOF MS analysis revealed the mass signal of Am-tags on Ab/Am-tag@AuNPs, indicating the presence of target exosomes. Thus, the target signal was amplified by a large number of Am-tags, resulting in enhanced sensitivity. We optimized the protocol to prepare stable Ab/Am-tag@AuNPs, focusing on parameters such as the concentration and ratio of thiol molecules for AuNP functionalization, suitable solvents for the coupling reaction, and amount of antibodies conjugated to the AuNPs. Subsequently, we evaluated the ability of our method to detect exosomes isolated from three cell lines, NIH3T3, MCF7, and HeLa, using an anti-Rab5 immobilized gold chip and anti-CD63/Am-tag@AuNPs with LDI-TOF MS analysis. Calibration curves constructed for the three cell lines showed a linear relationship with an excellent limit of detection. Finally, we emphasized the versatility of our method for the quantitative detection of exosomal proteins CD63 and mucin 1 (MUC1) using two types of Am-tags. LDI-TOF MS analysis revealed the presence of CD63 and MUC1 at different expression levels in HeLa and MCF7 cancer cells. Our findings clearly indicate the potential of Ab/Am-tag@AuNPs as a sensitive and reliable approach for identifying biomarkers in exosomes, providing valuable insights into their utility in biomedical research and clinical settings.

High-Throughput Microfluidic Particle Counter Based on Optical Absorption.

With the utilization of advanced microfluidic techniques, the microfluidic particle counter demonstrates significant potential due to its high efficiency, precise manipulation, and portability. This work focuses on a photodetection counter based on optical absorption. To achieve precise particle detection, a Christmas tree-like structure was implemented to separate a single particle from a cluster, which was then detected in independent multiple parallel channels. The system exhibits a high degree of reliability, as evidenced by a linear correlation coefficient over 0.99 obtained during testing with gradient-concentrated beads. Furthermore, when the calculated density of NIH 3T3 cells is compared with that of a traditional flow cytometer, the system achieves a substantial agreement percentage ranging from 87.5 to 99.9%. The system's ability to perform high-throughput analysis with a high acquisition rate positions it as a promising tool for real-time point-of-care testing.

Fabrication and characteristics of multifunctional hydrogel dressings using dopamine modified hyaluronic acid and phenylboronic acid modified chitosan.

The healing of damaged skin is a complex and dynamic process, and the multi-functional hydrogel dressings could promote skin tissue healing. This study, therefore, explored the development of a composite multifunctional hydrogel (HDCP) by incorporating the dopamine modified hyaluronic acid (HA-DA) and phenylboronic acid modified chitosan (CS-PBA) crosslinked using boric acid ester bonds. The integration of HA-DA and CS-PBA could be confirmed using the Fourier transform infrared spectrometer and 1H nuclear magnetic resonance analyses. The fabricated HDCP hydrogels exhibited porous structure, elastic solid behavior, shear-thinning, and adhesion properties. Furthermore, the HDCP hydrogels exhibited antibacterial efficacy against Gram-negative Escherichia coli (E. coli) and Gram-positive Staphylococcus aureus (S. aureus). Subsequently, the cytocompatibility of the HDCP hydrogels was verified through CCK-8 assay and fluorescent image analysis following co-cultivation with NIH-3T3 cells. This research presents an innovative multifunctional hydrogel that holds promise as a wound dressing for various applications within the realm of wound healing.

Exosomes isolated from IMMUNEPOTENT CRP, a hemoderivative, to accelerate diabetic wound healing.

The increasing risk of amputation due to diabetic foot ulcer calls for new therapeutic options; for that, we determined the role of IMMUNEPOTENT CRP (ICRP) and its parts in the wound healing process of superficial wounds in diabetic BALB/c mice. A potency test was performed to confirm the batch of ICRP, and then its parts were separated into pellets, supernatants, and exosomes, and another group of exosomes loaded with insulin was added. Viability and scratch healing were assessed in NIH-3T3, HUVEC, and HACAT cell lines. Diabetes was induced with streptozotocin, and wounds were made by dissecting the back skin. Treatments were topically applied, and closure was monitored; inflammatory cytokines in sera were also evaluated by flow cytometry, and histological analysis was performed by Masson's staining and immunohistochemistry for p-AKT, p-FOXO, p-P21, and p-TSC2. ICRP pellets and exosomes increased cellular viability, and exosomes and exosome-insulin accelerated scratch healing in vitro. Exosome-insulin releases insulin constantly over time in vitro. In vivo, treatments accelerated wound closure, and better performance was observed in pellet, exosome, and exosome-insulin treatments. Best collagen expression was induced by ICRP. P-AKT and p-FOXO were overexpressed in healing tissues. Inflammatory cytokines were downregulated by all treatments. In conclusion, IMMUNEPOTENT CRP components, especially exosomes, and the process of encapsulation of exosome-insulin accelerate diabetic wound healing and enhance cellular proliferation, collagen production, and inflammation modulation through the phosphorylation of components of the AKT pathway.

LINC02535 + miR-30a-5p combination enhances proliferation and inhibits apoptosis in metastatic breast Cancer cells

Current clinical therapies for metastatic breast cancer (MBC) have limited therapeutic efficacy and induce significant systemic side effects, leading to poor patient compliance. To address this challenge, this investigation focuses on the design of LINC02535 + miR-30a-5p for treating breast cancer. In vitro cytotoxicity studies confirmed that LINC02535 + miR-30a-5p was more effective in 4 T1 cells, with reduced toxicity in NIH3T3 cells. Further verification of cellular morphology was achieved through various biochemical staining methods. Additionally, the antimetastatic attributes of LINC02535 + miR-30a-5p have been evaluated using both migration scratch and Transwell migration assays, which have collectively revealed excellent antimetastatic potential. The DNA fragmentation of the 4 T1 cells was assessed using a comet assay. LINC02535 + miR-30a-5p improved ROS levels and caused mitochondrial membrane potential alterations and DNA damage, which resulted in apoptosis. Therefore, we propose that LINC02535 + miR-30a-5p could be an alternative therapeutic strategy for breast cancer therapy.

SLC15A3 is transcriptionally regulated by HIF1α and p65 to worsen neuroinflammation in experimental ischemic stroke.

Systemic inflammatory stimulus is a risk factor for the incidence of ischemic stroke and contributes to poorer clinical outcomes. Solute carrier 15A3 (SLC15A3) is a peptide/histidine transporter that is implicated in regulating inflammatory responses. However, whether SLC15A3 affects the progression of ischemic stroke associated with systemic inflammation is unclear. The transient middle cerebral artery occlusion (tMCAO) mice with LPS administration (LPS/tMCAO) were prepared as an in vivo model, and LPS-induced BV2 cells under oxygen-glucose deprivation (OGD) exposure were utilized as an in vitro model. We found that SLC15A3 was highly expressed in the ischemic penumbra of LPS/tMCAO mice, and its inhibition reduced infarct area, attenuated neurological deficit, recovered motor function, and mitigated apoptotic neurons. Knockdown of SLC15A3 suppressed the proinflammatory M1-type markers and promoted the levels of M2-associated genes. The in vitro results confirmed that SLC15A3 overexpression promoted microglia polarizing towards M1 subtypes, while SLC15A3 inhibition exerted an opposite effect. In addition, we demonstrated that the p65 signaling pathway and HIF1α were activated by LPS/OGD. Luciferase reporter assay showed that inhibiting p65 using its specific inhibitor BAY 11-7082 or silencing HIF1α using siRNAs reduced the transcriptional activity of SLC15A3in LPS/OGD-induced BV2 cells. Results in NIH 3T3 cells also confirmed that p65 and HIF1α directly bound to the SLC15A3 promoter to activate SLC15A3 transcription. In conclusion, this work shows that SLC15A3, transcriptionally activated by p65 and HIF1α, contributes to poor outcomes in ischemic stroke associated with systemic inflammation by promoting microglial cells polarizing towards M1 types.

Icariside II modulates pulmonary fibrosis via PI3K/Akt/β-catenin pathway inhibition of M2 macrophage program

BackgroundIdiopathic pulmonary fibrosis (IPF) is a debilitating interstitial lung disorder characterized by its limited therapeutic interventions. Macrophages, particularly the alternatively activated macrophages (M2 subtype), have been acknowledged for their substantial involvement in the development of pulmonary fibrosis. Hence, targeting macrophages emerges as a plausible therapeutic avenue for IPF. Icariside II (ISE II) is a natural flavonoid glycoside molecule known for its excellent anti-tumor and anti-fibrotic activities. Nevertheless, the impact of ISE II on pulmonary fibrosis and the intricate mechanisms through which it operates have yet to be elucidated. ObjectiveTo scrutinize the impact of ISE II on the regulation of M2 macrophage polarization and its inhibitory effect on pulmonary fibrosis, as well as to delve deeper into the underlying mechanisms of its actions. MethodsThe effect of ISE II on proliferation and apoptosis in RAW264.7 cells was assessed through the use of EdU-488 labeling and the Annexin V/PI assay. Flow cytometry, western blot, and qPCR were employed to detect markers associated with the M2 polarization phenotype. The anti-fibrotic effects of ISE II in NIH-3T3 cells were investigated in a co-culture with M2 macrophages. Si-Ctnnb1 and pcDNA3.1(+)-Ctnnb1 plasmid were used to investigate the mechanism of targeted intervention. The murine model of pulmonary fibrosis was induced by intratracheal administration of bleomycin (BLM). Pulmonary function, histopathological manifestations, lung M2 macrophage infiltration, and markers associated with pulmonary fibrosis were evaluated. Furthermore, in vivo transcriptomics analysis was employed to elucidate differentially regulated genes in lung tissues. Immunofluorescence, western blot, and immunohistochemistry were conducted for corresponding validation. ResultsOur investigation demonstrated that ISE II effectively inhibited the proliferation of RAW264.7 cells and mitigated the pro-fibrotic characteristics of M2 macrophages, exemplified by the downregulation of CD206, Arg-1, and YM-1, Fizz1, through the inhibition of the PI3K/Akt/β-catenin signaling pathway. This impact led to the amelioration of myofibroblast activation and the suppression of nuclear translocation of β-catenin of NIH-3T3 cells in a co-culture. Consequently, it resulted in decreased collagen deposition, reduced infiltration of profibrotic macrophages, and a concurrent restoration of pulmonary function in mice IPF models. Furthermore, our RNA sequencing results showed that ISE II could suppress the expression of genes related to M2 polarization, primarily by inhibiting the PI3K/Akt and β-catenin signaling pathway. In essence, our findings suggest that ISE II holds potential as an anti-fibrotic agent by orchestrating macrophage polarization. This may have significant implications in clinical practice. ConclusionThis study has provided evidence that ISE II exerts a significant anti-fibrotic effect by inhibiting macrophage M2 polarization through the suppression of the PI3K/Akt/β-catenin signaling pathway. These findings underscore the potential of ISE II as a promising candidate for the development of anti-fibrotic pharmaceuticals in the future.

Abstract PO4-28-11: Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells

Abstract Several studies in the past have demonstrated the role of cell-to-cell interaction (crosstalk) between tumor cells and normal (nontumorigenic) cells in cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have a potential to transform the phenotypes and the behaviors of normal cells. Application of the 2D in-vitro cultures has been limited due to its inability to replicate the complex in-vivo TME. Conditioned Medium (CM) obtained from cultured cancer cells contains secreted growth factors that potentially regulate the phenotype and the functionality of normal cells. In this study, a culture of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells with conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in an altered phenotype with increased cell viability. 4T1CM treated NIH3T3 and RAW 264.7, in comparison with the respective control cells, resulted in an upregulation of the genes including- α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF). In addition, 4T1.CM treated NIH3T3 showed an EMT phenotype as indicated by the regulation of EMT markers such as, E-cadherin, β-catenin, N-cadherin, and Vimentin. Interestingly, an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1) was observed in 4T1CM treated RAW 264.7, a tendency towards exhibiting an inhibitory immune response. Intriguingly, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers including- sex determining region Y-box 2, and Aldehyde dehydrogenase. Collectively, our study suggested a role for 4T1CM in transforming the normal NIH3T3 and RAW 264.7 into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). RNAseq experiments are underway to map differentially expressed genes (DEGs) that potentially regulate the 4T1CM induced transformation of NIH3T3 and RAW 264.7 cells. Furthermore, invitro drug testing in 3D model of these transformed cells to target the pathway intermediates may provide novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Joseph Francis. Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-11.

Tanshinone IIA alleviates pulmonary fibrosis by modulating glutamine metabolic reprogramming based on [U-13C5]-glutamine metabolic flux analysis

IntroductionGlutamine metabolic reprogramming, mediated by glutaminase (GLS), is an important signal during pulmonary fibrosis (PF) progression. Tanshinone IIA (Tan IIA) is a naturally lipophilic diterpene with antioxidant and antifibrotic properties. However, the potential mechanisms of Tan IIA for regulating glutamine metabolic reprogramming are not yet clear. ObjectivesThis study aimed was to evaluate the role of Tan IIA in intervening in glutamine metabolic reprogramming to exert anti-PF and to explore the potential new mechanisms of metabolic regulation. MethodsFibrotic characteristics was detected via immunofluorescence and western blotting analysis. Cell proliferation was examined with EdU Assay. Cell metabolites were labeled by using stable isotope [U-13C5]-glutamine. By utilizing 100% 13C glutamine tracers and employing network analysis to investigate the activation of metabolic pathways in fibroblasts, as well as evaluating the impact of Tan IIA on these pathways, we accurately quantified the absolute flux of glutaminolysis, proline synthesis, and the TCA cycle pathway using isotopomer network compartmental analysis (INCA), a user-friendly software tool for 13C metabolic flux analysis (13C-MFA). Molecular docking was used for identifying the binding of Tan IIA with target protein. ResultsTan IIA ameliorate TGF-β1-induced myofibroblast proliferation, reduce collagen I and III and α-SMA protein expression in MRC-5 and NIH-3T3 cells. Furthermore, Tan IIA regulate mitochondrial energy metabolism by modulating TGF-β1-stimulated glutamine metabolic reprogramming in NIH-3T3 cells and inhibiting GLS1 expression, which reduced the metabolic flux of glutamine into mitochondria in myofibroblasts, and also targeted inhibited the expression of Δ1-pyrroline-5-carboxylate synthase (P5CS), P5C reductase 1 (PYCR1), and phosphoserine aminotransferase 1 (PSAT1), and reduced proline hydroxylation and blocked the collagen synthesis pathway. ConclusionTan IIA reverses glutamine metabolic reprogramming, reduces mitochondrial energy expenditure, and inhibits collagen matrix synthesis by modulating potential targets in glutamine metabolism. This novel perspective sheds light on the essential role of glutamine metabolic reprogramming in PF.

Aaptamine-rich Fraction from the Indonesian Marine Sponge, Aaptos suberitoides, Exhibits a Cytotoxic Effect on DLD-1 Colorectal Cancer Cells.

This study aimed to investigate the cytotoxicity effect of the ethyl acetate extract of Aaptos suberitoides on colorectal cancer cells (DLD-1) and murine fibroblast cells (NIH-3T3). A. suberitoides was collected from Putus Island, Bunaken National Park, North Sulawesi, Indonesia, and was processed with maceration and ethyl acetate extraction. The sponge extract was characterized based on Thin Layer Chromatography (TLC) and then identified by using LCMS/MS analysis. DLD-1 and NIH-3T3 cells were treated with the ethyl acetate extract and then followed by 3- [4, 5-dimethylthiazol-2-yl] -2.5 diphenyl tetrazolium bromide (MTT) assay to assess their cytotoxicity effect. LCMS/MS analysis showed that the most abundant compounds in this extract were identified as aaptamine (1). Furthermore, this study revealed that the active ethyl acetate fraction of A. suberitoides has cytotoxic effects in colorectal cancer DLD-1 cells with an IC50 value of 9.597 µg/mL, higher than NIH-3T3 cells with an IC50 value of 12.23 µg/mL Thus, the active ethyl acetate fraction of A. suberitoides is considered more toxic to cancer cells than normal cells. This study provides the first evidence to support the role of the ethyl acetate extract of A. suberitoides sponge extracts to be developed as a colorectal anticancer agent.

All-aqueous droplets-templated tailorable core-shell alginate microspheres for constructing vascularized intestinal mucosa in vitro models

Recently, in vitro models of intestinal mucosa have become important tools for drug screening and studying the physiology and pathology of the intestine. These models enable the examination of cellular behavior in diseased states or in reaction to alterations in the microenvironment, potentially serving as alternatives to animal models. One of the major challenges in constructing physiologically relevant in vitro models of intestinal mucosa is the creation of three-dimensional microstructures that accurately mimic the integration of intestinal epithelium and vascularized stroma. Here, core–shell alginate (Alg) microspheres were generated to create the compartmentalized extracellular matrix microenvironment needed to simulate the epithelial and vascularized stromal compartments of the intestinal mucosa. We demonstrated that NIH-3T3 and human umbilical vein endothelial cells embedded in the core of the microspheres can proliferate and develop a vascular network, while human colorectal adenocarcinoma cells (Caco-2) can form an epithelial monolayer in the shell. Compared to Caco-2 monolayer encapsulated within the shell, the presence of the vascularized stroma enhances their proliferation and functionality. As such, our core–shell Alg microspheres provide a valuable method for generating in vitro models of vascularized intestinal mucosa with epithelial and vascularized stroma arranged in a spatially relevant manner and demonstrating near-physiological functionality.