Chick Chorioallantoic Membrane Model Research Articles

Cytotoxic potential of Hemp seed oil and molecular docking studies with inflammatory markers of diabetic cardiomyopathy

Present study was to assess the potential cytotoxic effects of commercially available Hemp seed oil and the effectiveness of its compounds in inhibiting inflammatory markers associated with diabetic cardiomyopathy. The bioactive components of Hemp seed oil were analysed using gas chromatography coupled with mass spectrometry (GC–MS). To evaluate the oil's cytotoxic properties, a modified 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium (MTT) assay was conducted on L929 cells (a mouse fibroblast cell line). Furthermore, an in vivo antiangiogenic assessment of the oil was performed using the chick chorioallantoic membrane (CAM) model, following a laboratory-standardized protocol. Docking and simulation studies were carried out to examine the binding and stability of the oil's compounds with inflammatory markers relevant to diabetic cardiomyopathy. The GC–MS analysis revealed that hemp seed oil contains several compounds, including Cannabidiol, Benzenemethanaminium, Dronabinol, Cannabinol, and Squalene. The cytotoxicity assessment on L929 cells demonstrated that Hemp seed oil did not exhibit any harmful effects at a concentration of 2.5 - 5 mg/mL. The CAM model confirmed the presence of antiangiogenic properties in Hemp seed oil, as evidenced by the inhibition of vascular patterns and blood vessel dissolution in the eggs. Additionally, docking and molecular dynamics (MD) simulations indicated that Benzenemethanaminium and Cannabielsoin exhibited the highest inhibitory efficacy and stability when interacting with IL-1β and C-reactive protein, respectively. This study suggests that Hemp seed oil possesses anti-inflammatory and anti-angiogenic properties, making it a potential herbal remedy for patients with diabetic cardiomyopathy.

HIF1α Counteracts TGFβ1-Driven TSP1 Expression in Endothelial Cells to Stimulate Angiogenesis in the Hypoxic Tumor Microenvironment.

Emerging evidence suggests that transforming growth factor β1 (TGFβ1) can inhibit angiogenesis, contradicting the coexistence of active angiogenesis and high abundance of TGFβ1 in the tumor microenvironment. Here, we investigated how tumors overcome the anti-angiogenic effect of TGFβ1. TGFβ1 treatment suppressed physiological angiogenesis in chick chorioallantoic membrane and zebrafish models but did not affect angiogenesis in mouse hepatoma xenografts. The suppressive effect of TGFβ1 on angiogenesis was recovered in mouse xenografts by a hypoxia-inducible factor 1α (HIF1α) inhibitor. In contrast, a HIF1α stabilizer abrogated angiogenesis in zebrafish, indicating that hypoxia may attenuate the anti-angiogenic role of TGFβ1. Under normoxic conditions, TGFβ1 inhibited angiogenesis by upregulating anti-angiogenic factor thrombospondin 1 (TSP1) in endothelial cells (ECs) via TGFβ type I receptor (TGFβR1)-SMAD2/3 signaling. In a hypoxic microenvironment, HIF1α induced microRNA-145 (miR145) expression; miR145 abolished the inhibitory effect of TGFβ1 on angiogenesis by binding and repressing SMAD2/3 expression and subsequently reducing TSP1 levels in ECs. Primary ECs isolated from human hepatocellular carcinoma (HCC) displayed increased miR145 and decreased SMAD3 and TSP1 compared to ECs from adjacent non-tumor livers. The reduced SMAD3 or TSP1 in ECs was associated with increased angiogenesis in HCC tissues. Collectively, this study identified that TGFβ1-TGFβR1-SMAD2/3-TSP1 signaling in ECs inhibits angiogenesis. This inhibition can be circumvented by a hypoxia-HIF1α-miR145 axis, elucidating a mechanism by which hypoxia promotes angiogenesis.

Exploiting WEE1 kinase activity as FUS::DDIT3-dependent therapeutic vulnerability in myxoid liposarcoma.

The pathognomonic FUS::DDIT3 fusion protein drives myxoid liposarcoma (MLS) tumorigenesis via aberrant transcriptional activation of oncogenic signaling. Since FUS::DDIT3 has so far not been pharmacologically tractable to selectively target MLS cells, this study investigated the functional role of the cell cycle regulator WEE1 as novel FUS::DDIT3‑dependent therapeutic vulnerability in MLS. Immunohistochemical evaluation of the cell cycle regulator WEE1 was performed in a large cohort of MLS specimens. FUS::DDIT3 dependency and biological function of the G1/S cell cycle checkpoint were analyzed in a mesenchymal stem cell model and liposarcoma cell lines in vitro. WEE1 activity was modulated by RNAi‑mediated knockdown and the small molecule inhibitor MK-1775 (Adavosertib). An established MLS cell line-based chicken chorioallantoic membrane model was employed for in vivo confirmation. We demonstrate that enhanced WEE1 pathway activity represents a hallmark of FUS::DDIT3‑expressing cell lines as well as MLS tissue specimens and that WEE1 is required for MLS cellular survival in vitro and in vivo. Pharmacologic inhibition of WEE1 activity results in DNA damage accumulation and cell cycle progression forcing cells to undergo apoptotic cell death. In addition, our results uncover FUS::DDIT3-dependent WEE1 expression as an oncogenic survival mechanism to tolerate high proliferation and resulting replication stress in MLS. Fusion protein-driven G1/S cell cycle checkpoint deregulation via overactive Cyclin E/CDK2 complexes thereby contributes to enhanced WEE1 inhibitor sensitivity in MLS. Our preclinical study identifies WEE1-mediated replication stress tolerance as molecular vulnerability in FUS::DDIT3-driven MLS tumorigenesis that could represent a novel target for therapeutic intervention.

Polyoxometalate inhibition of SOX2-mediated tamoxifen resistance in breast cancer

BackgroundIncreased cancer stem cell (CSC) content and SOX2 overexpression are common features in the development of resistance to therapy in hormone-dependent breast cancer, which remains an important clinical challenge. SOX2 has potential as biomarker of resistance to treatment and as therapeutic target, but targeting transcription factors is also challenging. Here, we examine the potential inhibitory effect of different polyoxometalate (POM) derivatives on SOX2 transcription factor in tamoxifen-resistant breast cancer cells.MethodsVarious POM derivatives were synthesised and characterised by infrared spectra, powder X-ray diffraction pattern and nuclear magnetic resonance spectroscopy. Estrogen receptor (ER) positive breast cancer cells, and their counterparts, which have developed resistance to the hormone therapy tamoxifen, were treated with POMs and their consequences assessed by gel retardation and chromatin immunoprecipitation to determine SOX2 binding to DNA. Effects on proliferation, migration, invasion and tumorigenicity were monitored and quantified using microscopy, clone formation, transwell, wound healing assays, flow cytometry and in vivo chick chorioallantoic membrane (CAM) models. Generation of lentiviral stable gene silencing and gene knock-out using CRISPR-Cas9 genome editing were applied to validate the inhibitory effects of the selected POM. Cancer stem cell subpopulations were quantified by mammosphere formation assays, ALDEFLUOR activity and CD44/CD24 stainings. Flow cytometry and western blotting were used to measure reactive oxygen species (ROS) and apoptosis.ResultsPOMs blocked in vitro binding activity of endogenous SOX2. [P2W18O62]6− (PW) Wells-Dawson-type anion was the most effective at inhibiting proliferation in various cell line models of tamoxifen resistance. 10 µM PW also reduced cancer cell migration and invasion, as well as SNAI2 expression levels. Treatment of tamoxifen-resistant cells with PW impaired tumour formation by reducing CSC content, in a SOX2-dependent manner, which led to stem cell depletion in vivo. Mechanistically, PW induced formation of reactive oxygen species (ROS) and inhibited Bcl-2, leading to the death of tamoxifen-resistant cells. PW-treated tamoxifen-resistant cells showed restored sensitivity to tamoxifen.ConclusionsTogether, these observations highlight the potential use of PW as a SOX2 inhibitor and the therapeutic relevance of targeting SOX2 to treat tamoxifen-resistant breast cancer.

Glucose Oxidase-Coated Calcium Peroxide Nanoparticles as an Innovative Catalyst for In Situ H2O2-Releasing Hydrogels.

In situ forming and hydrogen peroxide (H2O2)-releasing hydrogels have been considered as attractive matrices for various biomedical applications. Particularly, horseradish peroxidase (HRP)-catalyzed crosslinking reaction serves efficient method to create in situ forming hydrogels due to its advantageous features, such as mild reaction conditions, rapid gelation rate, tunable mechanical strength, and excellent biocompatibility. Herein, a novel HRP-crosslinked hydrogel system is reported that can produce H2O2 in situ for long-term applications, using glucose oxidase-coated calcium peroxide nanoparticles (CaO2@GOx NPs). In this system, CaO2 gradually produced H2O2 to support the HRP-mediated hydrogelation, while GOx further catalyzed the oxidation of glucose for in situ H2O2 generation. As the hydrogel is formed rapidly is expected and the H2O2 release behavior is prolonged up to 10 days. Interestingly, hydrogels formed by HRP/CaO2@GOx-mediated crosslinking reaction provided a favorable 3D microenvironment to support the viability and proliferation of fibroblasts, compared to that of hydrogels formed by either HRP/H2O2 or HRP/CaO2/GOx-mediated crosslinking reaction. Furthermore, HRP/CaO2@GOx-crosslinked hydrogel enhanced the angiogenic activities of endothelial cells, which is demonstrated by the in vitro tube formation test and in ovo chicken chorioallantoic membrane model. Therefore, HRP/CaO2@GOx-catalyzed hydrogels is suggested as potential in situ H2O2-releasing materials for a wide range of biomedical applications.

Raman spectroscopy for classification of neoplastic and non-neoplastic CAM colon tumors

This paper demonstrates the potential of Raman spectroscopy for differentiating neoplastic from non-neoplastic colon tumors, obtained with the CAM (chicken chorioallantoic membrane) model. For the CAM model two human cell lines were used to generate two types of tumors, the RKO cell line for neoplastic colon tumors and the NCM460 cell line for non-neoplastic colon tumors. The Raman spectra were acquired with a 785 nm excitation laser. The measured Raman spectra from the CAM samples (n = 14) were processed with several methods for baseline correction and to remove artifacts. The corrected spectra were analyzed with PCA (principal component analysis). Additionally, machine learning based algorithms were used to create a model capable of classifying neoplastic and non-neoplastic tumors. The principal component scores showed a clear differentiation between neoplastic and non-neoplastic colon tumors. The classification model had an accuracy of 93 %. Thus, a complete methodology to process and analyze Raman spectra was validated, using a rapid, accessible, and well-established tumor model that mimics the human tumor pathology with minor ethical concerns.

(SiFA)SeFe: A Hydrophilic Silicon-Based Fluoride Acceptor Enabling Versatile Peptidic Radiohybrid Tracers.

The radiohybrid (rh) concept to design targeted (and chemically identical) radiotracers for imaging or radionuclide therapy of tumors has gained momentum. For this strategy, a new bifunctional Silicon-based Fluoride Acceptor (SiFA) moiety (SiFA)SeFe was synthesized, endowed with improved hydrophilicity and high versatility of integration into rh-compounds. Preliminary radiolabeling and stability studies under different conditions were conducted using model bioconjugate peptides. Further, three somatostatin receptor 2 (sstR2)-targeted rh-compounds ((SiFA)SeFe-rhTATE1-3, TATE = (Tyr3)-octreotate) were developed. Compound (SiFA)SeFe-rhTATE3, enables labeling with 18F for PET imaging or chelation of 177Lu for therapy. The rh-compounds possess comparable receptor binding affinity and in vitro performance as good as the clinically proven gold standards. SstR2-specificity was further shown for (SiFA)SeFe-rhTATE2 using the chicken chorioallantoic membrane (CAM) model. The biodistribution of two compounds in mice showed high accumulation in tumors and excretion via the kidneys, demonstrating the clinical applicability of the (SiFA)SeFe moiety.

Photoangiolysis with the 445-nm Blue Laser and the Potassium-Titanyl-Phosphate Laser: A Comparison.

Photoangiolytic lasers have yielded significant innovation in laryngeal surgery in the last 25 years. After the discontinuation of the potassium titanyl phosphate (KTP) laser, a novel 445-nm blue laser was developed. The optimal balance between a laser's desired tissue effects and collateral tissue damage is a major determinant of laser selection in microlaryngeal surgery. The shell-less incubation system for the chick chorioallantoic membrane (CAM) simulates the microvasculature of the human vocal fold and is useful for testing effects of laser settings and in simulated surgery. The aim of this study is to compare the tissue effects of the KTP and blue lasers using the shell-less CAM model. The shell-less incubation system contains: polymethylpentene film (used as a culture vessel), calcium lactate and distilled water supplementations. By using this system, the chick chorioallantoic membrane (CAM) can be fully exposed with a good field for surgery simulation. The effects of the 2 lasers (532 nm KTP and 445 nm blue) were quantified at clinically relevant energy settings and laser distances from target. Measures included imaging real-time vascular reactions in the CAM model, post-procedure histologic analysis of CAM tissue and temperature changes. Vessel coagulation and rupture rates were less common with the blue laser compared with the KTP laser. Histologic analysis demonstrated less tissue disruption with the blue laser. Temperature changes were less with the blue laser. In this CAM model with specific conditions, the blue laser reveals less tissue damage than the KTP laser. Suitable working distance and power setting of the laser are necessary for desired tissue effects.Level of Evidence: Level 3.

Anti-angiogenic Potential of Trans-chalcone in an In Vivo Chick Chorioallantoic Membrane Model: An ATP Antagonist to VEGFR with Predicted Blood-brain Barrier Permeability.

Glioblastoma multiforme (GBM) is characterized by massive tumorinduced angiogenesis aiding tumorigenesis. Vascular endothelial growth factor A (VEGF-A) via VEGF receptor 2 (VEGFR-2) constitutes majorly to drive this process. Putting a halt to tumordriven angiogenesis is a major clinical challenge, and the blood-brain barrier (BBB) is the prime bottleneck in GBM treatment. Several phytochemicals show promising antiangiogenic activity across different models, but their ability to cross BBB remains unexplored. We screened over 99 phytochemicals having anti-angiogenic properties reported in the literature and evaluated them for their BBB permeability, molecular interaction with VEGFR-2 domains, ECD2-3 (extracellular domains 2-3) and TKD (tyrosine kinase domain) at VEGF-A and ATP binding site, cell membrane permeability, and hepatotoxicity using in silico tools. Furthermore, the anti-angiogenic activity of predicted lead Trans-Chalcone (TC) was evaluated in the chick chorioallantoic membrane. Out of 99 phytochemicals, 35 showed an efficient ability to cross BBB with a probability score of > 0.8. Docking studies revealed 30 phytochemicals crossing benchmark binding affinity < -6.4 kcal/mol of TKD with the native ligand ATP alone. Out of 30 phytochemicals, 12 showed moderate to low hepatotoxicity, and 5 showed a violation of Lipinski's rule of five. Our in silico analysis predicted TC as a BBB permeable anti-angiogenic compound for use in GBM therapy. TC reduced vascularization in the CAM model, which was associated with the downregulation of VEGFR-2 transcript expression. The present study showed TC to possess anti-angiogenic potential via the inhibition of VEGFR-2. In addition, the study predicted TC to cross BBB as well as a safe alternative for GBM therapy, which needs further investigation.

Denosumab Induces Neoplastic Stromal Cell Apoptosis Via p62 Downregulation Dependent on Autophagy Pathway in Giant Cell Tumour of Bone.

As the only humanized monoclonal antibody against receptor activator of nuclear factor-κB ligand (RANKL) for giant cell tumour of bone (GCTB) therapy, denosumab has limited antitumour effect on neoplastic stromal cells. Nevertheless, its mechanism of action has not yet been clarified. A previous study has revealed that p62 may play an important role in the antitumour activity of denosumab. The study aimed to investigate if the mechanism by which denosumab inhibits GCTB neoplastic stromal cells growth is via p62 modulation and other related mechanisms. p62 expression before and after denosumab therapy was analysed by RT‒qPCR, western blot, ELISA, and immunohistochemical assays. Two primary neoplastic stromal cells were isolated from fresh GCTB tumour tissue (L cell) and metastatic tissue (M cell). Cell proliferation, migration, apoptosis, and autophagy were investigated in p62 knockdown neoplastic stromal cells transfected by short hairpin RNA lentivirus in vitro. Tumor growth was evaluated in the chick chorioallantoic membrane model in vivo. p62 expression was found to be downregulated following denosumab therapy. The patients with a decrease in p62 expression had lower recurrence-free survival rates. The proliferation of M cells was not inhibited by denosumab therapy, but it was restored by p62 knockdown. Moreover, p62 knockdown inhibited tumour growth in vivo. Denosumab induced M cell apoptosis and arrested the cell cycle at the G1/G0 transition and these effects were also enhanced by p62 knockdown. Autophagic flux assays revealed p62 modulation to be dependent on autophagy following denosumab incubation. Denosumab induced neoplastic stromal cells apoptosis via p62 downregulation dependent on autophagy pathway. The combination of p62 and RANKL knockdown might be a better strategy than RANKL knockdown alone for GCTB targeted therapy.

Sevofluran Maruziyetinin Civciv Embriyo Gelişimi Üzerindeki Ex-Ovo Değerlendirilmesi: Anjiyogenez Etkilerinin Araştırılması

Objective: Angiogenesis, defined as the formation of new blood vessels from pre-existing ones, plays a vital role in both physiological and pathological conditions. Understanding agents that can influence angiogenesis is crucial in managing diseases where angiogenesis is dysregulated. This study focuses on sevoflurane, a commonly used inhalation anesthetic, whose effects on angiogenesis and embryonic development are not well understood. Our objective was to assess the impact of sevoflurane exposure on angiogenesis using the ex-ovo chick chorioallantoic membrane model. Methods: In this model, fertilized chicken eggs were exposed to sevoflurane at concentrations of 2% and 4%, for varying durations of 1, 2, and 4 hours. The embryos were then divided into control and experimental groups for quantitative angiogenesis assessment using Image J software, followed by statistical analysis with one-way analysis of variance. Results: The results indicate that sevoflurane exposure has a dose-dependent positive effect on angiogenesis, with significant increases in vascular density observed in embryos exposed to both concentrations compared to the control group. Additionally, the length of exposure was found to further enhance these angiogenic effects. Conclusion: Despite the dose and duration-dependent impact of sevoflurane on angiogenesis, the existing literature presents mixed findings, highlighting the need for additional research to elucidate sevoflurane’s role in angiogenesis. This is particularly important for understanding its implications in various medical conditions, such as cancer, wound healing, and fetal development. Future investigations into sevoflurane’s effects on placental angiogenesis could also provide valuable insights into its potential consequences on intrauterine growth. Keywords: Angiogenesis, sevoflurane, chicken embryo, model organisms, ex-ovo

Anti-angiogenic and anti-tumour activities of Lignosus rhinocerus (Cooke) Ryvarden water extracts on HCT116 human colorectal carcinoma cells implanted in chick embryos

Ethnopharmacological relevanceThe sclerotium of Lignosus rhinocerus (Cooke) Ryvarden is used by the local communities in Southeast Asia and China to treat cancer, asthma, fever, and other ailments based on traditional knowledge. The sclerotial water extracts were previously reported to exhibit cytotoxic, apoptotic, and immunomodulatory activities – providing a scientific basis for its use in treating cancer; however, there is still a lack of evidence on its potential anti-angiogenic activity. Aim of the studyThis study aimed to investigate the toxicity, anti-angiogenic, and anti-tumour activities of the hot-water and cold-water extracts of L. rhinocerus using HCT116 human colorectal carcinoma cells implanted in the chick chorioallantoic membrane (CAM) model. Materials and methodsThe toxicity of L. rhinocerus extracts towards the chick embryos was determined 24 h post-treatment. The anti-angiogenic activity of the extracts was then investigated at 0.1–10 μg/embryo (6.7–670 μg/mL) at targeted blood vessels. The anti-tumour effect of selected extracts against the HCT116 human colorectal carcinoma cells xenografted onto the chick embryos was also studied. ResultsThe cold-water extracts of L. rhinocerus displayed strong in ovo toxicity (LC50: 1.2–37.7 μg/mL) while the hot-water extracts are non-toxic up to 670 μg/mL. Among the extracts, the hot-water extracts demonstrated the highest anti-angiogenic activity with 44.0 ± 17.7% reduction of capillary diameter (relative to the saline-treated control). Moreover, treatment of the HCT116 cells xenografted onto the chick embryos with the hot-water extracts resulted in smaller tumour size and lower number of blood vessels compared to the saline-treated control. ConclusionsThe hot-water extracts of L. rhinocerus sclerotium demonstrated anti-angiogenic and anti-tumour activities but most of the cold-water extracts at similar concentrations were devoid of that. Our findings provide further scientific validation of the medicinal use of the sclerotium in treating cancer and thus, expanding our knowledge on the possible mechanism of its anti-cancer effect apart from direct cytotoxicity, induction of apoptosis and immunomodulation that have been studied thus far.

Abstract 1274: Investigating PLOD2 as a therapeutic target to overcome metastasis in radiorecurrent prostate cancer

Abstract Prostate cancer (PCa) is the second most common cancer in males, with 1 in 8 men developing the disease in their lifetime. For PCa, a common treatment strategy is external beam radiation therapy to the prostate. However, when cancer recurs (radiorecurrent PCa), it often behaves aggressively by invading into surrounding organs or spreading distantly. Radiorecurrent PCa metastasis is therefore a significant cause of morbidity and mortality that must be overcome to improve survival of advanced PCa patients. Proteomic analysis revealed the procollagen enzyme Lysyl Hydroxylase 2 (PLOD2) to be upregulated in our radiorecurrent, and highly aggressive, conventionally fractionated DU145 (DU145-CF) PCa cell line. Given its established function as a mediator of invasion in various other cancers, we sought to characterize the role of PLOD2 in the aggressive phenotype of our radiorecurrent PCa cells. Bioinformatic analysis of clinical data revealed PLOD2 genomic amplification to be significantly associated with biochemical recurrence in PCa patients. Upon further examination in vitro, it was revealed that PLOD2 knockdown significantly reduces matrigel invasion and migration in our radiorecurrent DU145-CF cell line, in addition to numerous other PCa cell lines, including primary cells we derived directly from PCa patients. Using the in vivo chick Chorioallantoic Membrane (CAM) model, we confirmed that PLOD2 knockdown significantly reduces the ability of DU145-CF cells to extravasate from the CAM vasculature into the surrounding stroma, a critical step of the metastatic cascade. To explore mechanisms of metastatic cellular reprogramming downstream of PLOD2, RNA sequencing of DU145-CF cells was conducted; a total of 681 genes were discovered to be dysregulated by PLOD2 knockdown, with their functional analysis suggesting changes in cellular metabolism and respiration. Finally, since PLOD2 is known to be regulated by hypoxia-induced protein HIF1α, we explored whether PLOD2 expression could be inhibited by the HIF1α inhibitor, PX-478. Treatment with PX-478 reduced both HIF1α and PLOD2 protein expression, and significantly reduced invasion, migration, and in vivo extravasation in DU145-CF cells, thereby indicating its potential as a pharmacological inhibitor of HIF1α-associated PLOD2 in radiorecurrent PCa. Together, our results demonstrate for the first time the role of PLOD2 in radiorecurrent PCa invasiveness, and point towards its potential as a therapeutic target to reduce metastasis and improve survival outcomes in PCa patients. Citation Format: Gavin Frame, Hon Leong, Roni Haas, Xiaoyong Huang, Jessica Wright, Paul C. Boutros, Thomas Kislinger, Stanley K. Liu. Investigating PLOD2 as a therapeutic target to overcome metastasis in radiorecurrent prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1274.

Biofabrication of nanocomposite-based scaffolds containing human bone extracellular matrix for the differentiation of skeletal stem and progenitor cells

Autograft or metal implants are routinely used in skeletal repair. However, they fail to provide long-term clinical resolution, necessitating a functional biomimetic tissue engineering alternative. The use of native human bone tissue for synthesizing a biomimetic material ink for three-dimensional (3D) bioprinting of skeletal tissue is an attractive strategy for tissue regeneration. Thus, human bone extracellular matrix (bone-ECM) offers an exciting potential for the development of an appropriate microenvironment for human bone marrow stromal cells (HBMSCs) to proliferate and differentiate along the osteogenic lineage. In this study, we engineered a novel material ink (LAB) by blending human bone-ECM (B) with nanoclay (L, Laponite®) and alginate (A) polymers using extrusion-based deposition. The inclusion of the nanofiller and polymeric material increased the rheology, printability, and drug retention properties and, critically, the preservation of HBMSCs viability upon printing. The composite of human bone-ECM-based 3D constructs containing vascular endothelial growth factor (VEGF) enhanced vascularization after implantation in an ex vivo chick chorioallantoic membrane (CAM) model. The inclusion of bone morphogenetic protein-2 (BMP-2) with the HBMSCs further enhanced vascularization and mineralization after only seven days. This study demonstrates the synergistic combination of nanoclay with biomimetic materials (alginate and bone-ECM) to support the formation of osteogenic tissue both in vitro and ex vivo and offers a promising novel 3D bioprinting approach to personalized skeletal tissue repair.Graphic abstract

Antiangiogenic Effects of Infliximab in Chick Chorioallantoic Membrane Model

Introduction: TNF-α plays a key role in initiating pathologic angiogenesis, however, literature on the effects of infliximab on angiogenesis is limited.&#x0D; Objective: The study aimed to investigate the effects of infliximab on angiogenesis in an in vivo chick chorioallantoic membrane (CAM) model.&#x0D; Methods: The study was approved by the local ethics committee on animal experimentation. Thirty fertilized specific pathogen-free eggs were incubated and kept under appropriate temperature and humidity control. On the third day of the incubation, infliximab (1 µmol) dissolved in phosphate-buffered saline in the first group, phosphate-buffered saline (negative control) (0.1 ml) in the second group, anti-VEGF (positive control) (1 µmol) in the third group were administered by injection. On the eighth day of incubation, the vascular structures of the CAMs were macroscopically examined, and standard digital photographs were taken. The digital images were analyzed, and data including mean vessel density, thickness, and number were compared between groups. P &lt; 0.05 was considered statistically significant.&#x0D; Results: Angiogenesis was significantly reduced in the anti-VEGF and infliximab groups compared to the saline-only group. Vessel thickness, vessel number, and vessel density were significantly less in the infliximab and anti-VEGF groups compared to the saline-only group (p=0.034, 0.029, 0.024, respectively).&#x0D; Conclusions: Infliximab showed promising antiangiogenic effects in the chick CAM model. Thus, infliximab could be a treatment agent in pathological processes in which angiogenesis is responsible. The antiangiogenic effect of infliximab could be due to the inhibition of various angiogenesis-related cytokines and adhesion molecules. Further studies are needed to clarify the exact mechanisms.

Evaluation of chicken chorioallantoic membrane model for tumor imaging and drug development: Promising findings.

The chicken chorioallantoic membrane (CAM) model is a potential alternative to the mouse model based on the 3R principles. However, its value for determination of the invivo behaviors of radiolabeled peptides through positron emission tomography (PET) imaging needed investigation. Herein, the chicken CAM tumor models were established, and their feasibility was evaluated for evaluating the imaging properties of radiolabeled peptides using a 68 Ga-labeled HER2 affibody. Two human breast cancer cell lines were inoculated into chicken CAM and mice, respectively. The tumor-targeting potential and pharmacokinetic profile of a 68 Ga-labeled affibody, 68 Ga-MZHER, in both tumor models were also determined. The tumor-formation time in chicken CAM model was shorter than that of mouse model. The uptake values of human epithelial growth factor receptor-2 (HER2)-positive Bcap37 tumors in chicken CAM and mouse models were 5.36 ± 0.26% ID/g and 5.26 ± 0.43% ID/g at 30 min postinjection of 68 Ga-MZHER, respectively. At the same time points, the uptake values of HER2-negative MDA-MB-231 tumors in the chicken CAM models and mouse models were 1.57 ± 0.15% ID/g and 1.67 ± 0.25% ID/g, respectively. Exvivo biodistribution confirmed that more radioactivity accumulated in Bcap37 tumors than in MDA-MD-231 tumors in both CAM and mouse models. In this study, the CAM tumor model was successfully prepared. The chicken CAM model is a novel tool for quickly determining the invivo properties of radiolabeled peptides targeting biomarkers. It may be beneficial for early monitoring of the therapeutic effect of a new drug through PET imaging with specific peptides.

New retinoblastoma (RB) drug delivery approaches: anti-tumor effect of atrial natriuretic peptide (ANP)-conjugated hyaluronic-acid-coated gold nanoparticles for intraocular treatment of chemoresistant RB.

Intraocular drug delivery is a promising approach for treatment of ocular diseases. Chemotherapeutic drugs used in retinoblastoma (RB) treatment often lead to side effects and drug resistances. Therefore, new adjuvant therapies are needed to treat chemoresistant RBs. Biocompatible gold nanoparticles (GNPs) have unique antiangiogenic properties and can inhibit cancer progression. The combination of gold and low-molecular-weight hyaluronan (HA) enhances the stability of GNPs and promotes the distribution across ocular barriers. Attached to HA-GNPs, the atrial natriuretic peptide (ANP), which diminishes neovascularization in the eye, is a promising new therapeutic agent for RB treatment. In the study presented, we established ANP-coupled HA-GNPs and investigated their effect on the tumor formation potential of chemoresistant RB cells in an in ovo chicken chorioallantoic membrane model and an orthotopic in vivo RB rat eye model. Treatment of etoposide-resistant RB cells with ANP-HA-GNPs in ovo resulted in significantly reduced tumor growth and angiogenesis compared with controls. The antitumorigenic effect could be verified in the rat eye model, including a noninvasive application form via eye drops. Our data suggest that ANP-HA-GNPs represent a new minimally invasive, adjuvant treatment option for RB.

GRB2 is a BECN1 interacting protein that regulates autophagy

GRB2 is an adaptor protein of HER2 (and several other tyrosine kinases), which we identified as a novel BECN1 (Beclin 1) interacting partner. GRB2 co-immunoprecipitated with BECN1 in several breast cancer cell lines and regulates autophagy through a mechanism involving the modulation of the class III PI3Kinase VPS34 activity. In ovo studies in a CAM (Chicken Chorioallantoic Membrane) model indicated that GRB2 knockdown, as well as overexpression of GRB2 loss-of-function mutants (Y52A and S86A-R88A) compromised tumor growth. These differences in tumor growth correlated with differential autophagy activity, indicating that autophagy effects might be related to the effects on tumorigenesis. Our data highlight a novel function of GRB2 as a BECN1 binding protein and a regulator of autophagy.

Decellularized Bone Matrix/45S5 Bioactive Glass Biocomposite Hydrogel-Based Constructs with Angiogenic and Osteogenic Properties: Ex Ovo and Ex Vivo Evaluations.

Decellularized extracellular matrix is often used to create an in vivo-like environment that supports cell growth and proliferation, as it reflects the micro/macrostructure and molecular composition of tissues. On the other hand, bioactive glasses (BG) are surface-reactive glass-ceramics that can convert to hydroxyapatite in vivo and promote new bone formation. This study is designed to evaluate the key properties of a novel angiogenic and osteogenic biocomposite graft made of bovine decellularized bone matrix (DBM) hydrogel and 45S5 BG microparticles (10 and 20wt%) to combine the existing superior properties of both biomaterial classes. Morphological, physicochemical, mechanical, and thermal characterizations of DBM and DBM/BG composite hydrogels are performed. Their in vitro biocompatibility is confirmed by cytotoxicity and hemocompatibility analyses. Ex vivo chick embryo aortic arch and ex ovo chick chorioallantoic membrane (CAM) assays reveal that the present pro-angiogenic property of DBM hydrogels is enhanced by the incorporation of BG. Histochemical stainings (Alcian blue and Alizarin red) and digital image analysis of ossification on hind limbs of embryos used in the CAM model reveal the osteogenic potential of biomaterials. The findings support the notion that the developed DBM/BG composite hydrogel constructs have the potential to be a suitable graft for bone repair.

Facilitated Transport across Glycocalyceal Barriers in the Chick Chorioallantoic Membrane.

Targeted drug delivery to visceral organs offers the possibility of not only limiting the required dose, but also minimizing drug toxicity; however, there is no reliable method for delivering drugs to the surface of visceral organs. Here, we used six color tracers and the chick chorioallantoic membrane (CAM) model to investigate the use of the heteropolysaccharide pectin to facilitate tracer diffusion across the glycocalyceal charge barrier. The color tracers included brilliant blue, Congo red, crystal violet, indocyanine green, methylene blue, and methyl green. The direct application of the tracers to the CAM surface or embedding tracers into linear-chain nanocellulose fiber films resulted in no significant diffusion into the CAM. In contrast, when the tracers were actively loaded into branched-chain pectin films, there was significant detectable diffusion of the tracers into the CAM. The facilitated diffusion was observed in the three cationic tracers but was limited in the three anionic tracers. Diffusion appeared to be dependent on ionic charge, but independent of tracer size or molecular mass. We conclude that dye-loaded pectin films facilitated the diffusion of color tracers across the glycocalyceal charge barrier and may provide a therapeutic path for drug delivery to the surface of visceral organs.