Chicken Egg Chorioallantoic Membrane Research Articles

Chick Embryo Chorioallantoic Membrane as a Platform for Assessing the InVivo Efficacy of Chimeric Antigen Receptor T-cell Therapy in Solid Tumors.

The fertilized chicken egg chorioallantoic membrane (CAM), a highly vascularized membrane nourishing the developing embryo, also supports rapid growth of three-dimensional vascularized tumors from engrafted cells and tumor explants. Because murine xenograft models suffer limitations of time, cost, and scalability, we propose CAM tumors as a rapid, efficient screening tool for assessing anti-tumor efficacy of chimeric Ag receptor (CAR) T cells against solid tumors. We tested the efficacy of human epidermal growth factor receptor 2 (HER2)-specific CAR T cells against luminescent, HER2-expressing (FaDu, SCC-47) or HER2-negative (MDA-MB-468) CAM-engrafted tumors. Three days after tumor engraftment, HER2-specific CAR T cells were applied to tumors grown on the CAM. Four days post-CAR T cell treatment, HER2-expressing FaDu and SCC-47 tumors treated with CAR T showed reduced viable cancer cells as assessed by luciferase activity. This reduction in viable tumor cells was confirmed by histology, with lower Ki-67 staining observed in CAR T cell-treated tumors relative to T cell-treated controls. Persistence of CAR T in CAM and tumor tissue 4 days post-treatment was confirmed by CD3 staining. Altogether, our findings support further development of the chick CAM as an invivo system for rapid, scalable screening of CAR T cell efficacy against human solid tumors.

Enhancing bone tissue engineering using iron nanoparticles and magnetic fields: A focus on cytomechanics and angiogenesis in the chicken egg chorioallantoic membrane model.

To evaluate the potential of iron nanoparticles (FeNPs) in conjunction with magnetic fields (MFs) to enhance osteoblast cytomechanics, promote cell homing, bone development activity, and antibacterial capabilities, and to assess their in vivo angiogenic viability using the chicken egg chorioallantoic membrane (CAM) model. Experimental study conducted in a laboratory setting to investigate the effects of FeNPs and MFs on osteoblast cells and angiogenesis using a custom titanium (Ti) substrate coated with FeNPs. A custom titanium (Ti) was coated with FeNPs. Evaluations were conducted to analyze the antibacterial properties, cell adhesion, durability, physical characteristics, and nanoparticle absorption associated with FeNPs. Cell physical characteristics were assessed using protein markers, and microscopy, CAM model, was used to quantify blood vessel formation and morphology to assess the FeNP-coated Ti's angiogenic potential. This in vivo study provided critical insights into tissue response and regenerative properties for biomedical applications. Statistical analysis was performed using appropriate tests to compare experimental groups and controls. Significance was determined at P < 0.05. FeNPs and MFs notably improved osteoblast cell mechanical properties facilitated the growth and formation of new blood vessels and bone tissue and promoted cell migration to targeted sites. In the group treated with FeNPs and exposed to MFs, there was a significant increase in vessel percentage area (76.03%) compared to control groups (58.11%), along with enhanced mineralization and robust antibacterial effects (P < 0.05). The study highlights the promising potential of FeNPs in fostering the growth of new blood vessels, promoting the formation of bone tissue, and facilitating targeted cell migration. These findings underscore the importance of further investigating the mechanical traits of FeNPs, as they could significantly advance the development of effective bone tissue engineering techniques, ultimately enhancing clinical outcomes in the field.

Secretome from Magnetically Stimulated Muscle Exhibits Anticancer Potency: Novel Preconditioning Methodology Highlighting HTRA1 Action.

Briefly (10 min) exposing C2C12 myotubes to low amplitude (1.5 mT) pulsed electromagnetic fields (PEMFs) generated a conditioned media (pCM) that was capable of mitigating breast cancer cell growth, migration, and invasiveness in vitro, whereas the conditioned media harvested from unexposed myotubes, representing constitutively released secretome (cCM), was less effective. Administering pCM to breast cancer microtumors engrafted onto the chorioallantoic membrane of chicken eggs reduced tumor volume and vascularity. Blood serum collected from PEMF-exposed or exercised mice allayed breast cancer cell growth, migration, and invasiveness. A secretome preconditioning methodology is presented that accentuates the graded anticancer potencies of both the cCM and pCM harvested from myotubes, demonstrating an adaptive response to pCM administered during early myogenesis that emulated secretome-based exercise adaptations observed in vivo. HTRA1 was shown to be upregulated in pCM and was demonstrated to be necessary and sufficient for the anticancer potency of the pCM; recombinant HTRA1 added to basal media recapitulated the anticancer effects of pCM and antibody-based absorption of HTRA1 from pCM precluded its anticancer effects. Brief and non-invasive PEMF stimulation may represent a method to commandeer the secretome response of muscle, both in vitro and in vivo, for clinical exploitation in breast and other cancers.

Design of Lysozyme‐Templated Biocompatible Fluorescent Nanocomposites and Their Potential Applications for Cell Imaging, Hydrogen Peroxide Sensing, and Cancer Therapy

AbstractRecently, multifunctional metal‐based nanocomposites gained immense importance in various biomedical applications. Here, a simple approach is reported to synthesize fluorescent bimetallic nanocomposites (NCs) of silver‐gold (Ag@Au NC) and silver‐platinum (Ag@Pt NC) by reducing silver nitrate (AgNO3) on gold nanoparticles and platinum nanoparticles (AuNPs, PtNPs), respectively, in the presence of lysozyme that acts as capping agent. Various in vitro (in normal cells including Chinese hamster ovarian and human umbilical vein endothelial cells) and in vivo (in mouse and chicken egg chorioallantoic membrane model) studies reveal the biocompatibility of these nanocomposites. However, these NCs show inhibition of cancer cell proliferation when treated to various cancer cells (MCF‐7, MDA‐MB‐231, and B16F10), indicating their anticancer property. Intraperitoneal administration of these NCs resulted in the regression of melanoma tumor in C57BL/6J mouse model. Further, these NCs displayed green fluorescence inside MCF‐7 cells facilitating in vitro live‐cell imaging and sense hydrogen peroxide (H2O2) in cellular level (B16F10 and MCF‐7) using their fluorescence enhancement property in presence of H2O2.The results (in vitro and in vivo) altogether demonstrate the promising multifunctional applications of bimetallic nanocomposites (NCs) that can be utilized as promising transformative drugs in cancer theranostics applications.

Targeting IRS-1/2 in Uveal Melanoma Inhibits In Vitro Cell Growth, Survival and Migration, and In Vivo Tumor Growth.

Uveal melanoma originating in the eye and metastasizing to the liver is associated with poor prognosis and has only one approved therapeutic option. We hypothesized that liver-borne growth factors may contribute to UM growth. Therefore, we investigated the role of IGF-1/IGF-1R signaling in UM. Here, we found that IRS-1, the insulin receptor substrate, is overexpressed in both UM cells and tumors. Since we previously observed that IGF-1R antibody therapy was not clinically effective in UM, we investigated the potential of NT157, a small molecule inhibitor of IRS-1/2, in blocking this pathway in UM. NT157 treatment of multiple UM cell lines resulted in reduced cell growth and migration and increased apoptosis. This treatment also significantly inhibited UM tumor growth in vivo, in the chicken egg chorioallantoic membrane (CAM) and subcutaneous mouse models, validating the in vitro effect. Mechanistically, through reverse phase protein array (RPPA), we identified significant proteomic changes in the PI3K/AKT pathway, a downstream mediator of IGF-1 signaling, with NT157 treatment. Together, these results suggest that NT157 inhibits cell growth, survival, and migration in vitro, and tumor growth in vivo via inhibiting IGF-1 signaling in UM.

Characterizing treatment resistance in muscle invasive bladder cancer using the chicken egg chorioallantoic membrane patient-derived xenograft model

BackgroundNon-metastatic muscle invasive urothelial bladder cancer (MIBC) has a poor prognosis and standard of care (SOC) includes neoadjuvant cisplatin-based chemotherapy (NAC) combined with cystectomy. Patients receiving NAC have at best <10% improvement in five-year overall survival compared to cystectomy alone. This major clinical problem underscores gaps in our understanding of resistance mechanisms and a need for reliable pre-clinical models. The chicken embryo chorioallantoic membrane (CAM) represents a rapid, scalable, and cost-effective alternative to immunocompromised mice for establishing patient-derived xenografts (PDX) in vivo. CAM-PDX leverages an easily accessible engraftment scaffold and vascular-rich, immunosuppressed environment for the engraftment of PDX tumors and subsequent functional studies. MethodsWe optimized engraftment conditions for primary MIBC tumors using the CAM-PDX model and tested concordance between cisplatin-based chemotherapy response of patients to matching PDX tumors using tumor growth coupled with immunohistochemistry markers of proliferation and apoptosis. We also tested select kinase inhibitor response on chemotherapy-resistant bladder cancers on the CAM-PDX using tumor growth measurements and immuno-detection of proliferation marker, Ki-67. ResultsOur results show primary, NAC-resistant, MIBC tumors grown on the CAM share histological characteristics along with cisplatin-based chemotherapy resistance observed in the clinic for matched parent human tumor specimens. Patient tumor specimens acquired after chemotherapy treatment (post-NAC) and exhibiting NAC resistance were engrafted successfully on the CAM and displayed decreased tumor growth size and proliferation in response to treatment with a dual EGFR and HER2 inhibitor, but had no significant response to either CDK4/6 or FGFR inhibition. ConclusionsOur data suggests concordance between cisplatin-based chemotherapy resistance phenotypes in primary patient tumors and CAM-PDX models. Further, proteogenomic informed kinase inhibitor use on MIBC CAM-PDX models suggests a benefit from integration of rapid in vivo testing of novel therapeutics to inform more complex, pre-clinical mouse PDX experiments for more effective clinical trial design aimed at achieving optimal precision medicine for patients with limited treatment options.

Abstract 2998: Targeting resistance mechanisms in muscle invasive bladder cancer using the chicken egg chorioallantoic membrane patient-derived xenograft model

Abstract Introduction: Non-metastatic muscle invasive urothelial bladder cancer (MIBC) has a poor prognosis and standard of care (SOC) consists of neoadjuvant cisplatin-based chemotherapy (NAC) combined with cystectomy. Patients receiving SOC have at best &amp;lt;10% improvement in five-year overall survival. Residual invasive cancer following NAC has no SOC and has high mortality risk. This major clinical problem underscores gaps in our understanding resistance mechanisms and a need for reliable pre-clinical models. The chicken embryo chorioallantoic membrane (CAM) represents a rapid, scalable, and cost-effective alternative in vivo patient-derived xenograft (PDX) platform. It leverages an easily accessible engraftment scaffold and vascular-rich, immunosuppressed environments for the engraftment of PDX tumors and subsequent functional studies. Our previous proteogenomic data on MIBCs with known chemo-resistant outcomes obtained prior to NAC (pre-NAC) suggests a benefit from treatment with kinase inhibitors (KI). Use of the CAM model in this study aims to address the MIBC pre-clinical model deficit and allow for the rapid screening of therapeutics against NAC-resistant PDXs using selected KI. Methods: We employed the CAM model to expand primary MIBC tumors and tested concordance between cisplatin-based chemotherapy response of patients to matching PDX tumors. We also tested selected KI response on chemotherapy-resistant bladder cancers. PDX growth was assessed by tumor surface area quantification following a week of growth with chemotherapy and KI treatment. Hematoxylin and eosin staining coupled with in situ hybridization, immunohistochemistry for Ki67, and cleaved PARP helped identify tumor cells on the CAM as well as their proliferative and apoptotic indexes. Results: Our initial results show pre-NAC primary MIBC tumors grown on the CAM phenocopy cisplatin-based chemotherapy resistance observed in parent tumor specimens. Histological analysis confirmed engraftment and growth of bladder tumors on the CAM. Patient tumor specimens acquired after chemotherapy treatment (post-NAC) and exhibiting NAC resistance were engrafted successfully on the CAM. Such CAM PDX tumors displayed decreased tumor growth size and proliferation in response to treatment with a dual EGFR and HER2 inhibitor, but had no significant response to either CDK4/6 or FGFR inhibition. Conclusion: Our data suggests concordance between Cisplatin-based chemotherapy resistance phenotypes in primary patient tumors and CAM PDX models. Further, proteogenomic informed KI use on MIBC CAM PDX models suggests a benefit from integration of rapid in vivo testing of novel therapeutics to inform more complex, pre-clinical mouse PDX experiments for more effective clinical trial design aimed at achieving optimal precision medicine for patients with limited treatment options. Citation Format: Hugo Villanueva, Patricia Castro, Andrew G. Sikora, Seth P. Lerner. Targeting resistance mechanisms in muscle invasive bladder cancer using the chicken egg chorioallantoic membrane patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2998.

An alternative in vivo model to evaluate pluripotency of patient-specific iPSCs.

The generation of autologous human induced pluripotent stem cells (hiPSCs) from a patient’s somatic cells and the sub­sequent differentiation of these cells into desired cell types offer innovative treatment options for tissue regeneration. The hiPSCs obtained are usually implanted in immunodeficient mice, and teratoma formation is analyzed after 4 to 6 weeks to assess the cells’ pluripotency. In this study, an alternative in vivo model based on chicken egg chorioallantoic membrane (CAM) was established to analyze the pluripotency of newly created hiPSCs. 0.5, 1, 2, 4 x 106 hiPSCs gen­erated from urine-derived renal epithelial cells were seeded on CAM and incubated for 9 days. Teratoma formation was detected in 70% of eggs inoculated with 2 x 106 hiPSCs and in 100% of eggs inoculated with 4 x 106 hiPSCs. All teratomas exhibited vascular structures. The robustness of the CAM model was confirmed using two additional hiPSC lines derived from human fibroblasts (NuFFs) or jaw periosteal cells. The presence of all three germ layers within the teratomas was successfully verified by histochemical and immunofluorescence staining and gene expression analysis of germ layer-specific markers. Urine-derived renal epithelial cells were used as negative control and showed no teratoma formation. The CAM-based in vivo model provides an optimal in vivo test environment for the pluripotency evaluation of newly generated hiPSC lines. This simple, fast, inexpensive and reproducible method reduces the suffering of animals and thus implements the principles of the 3Rs (replacement, reduction, and refinement).

An alternative in vivo model to evaluate pluripotency of patient-specific iPSCs_suppl

The generation of autologous human induced pluripotent stem cells (hiPSCs) from a patient’s somatic cells and the sub­sequent differentiation of these cells into desired cell types offer innovative treatment options for tissue regeneration. The hiPSCs obtained are usually implanted in immunodeficient mice, and teratoma formation is analyzed after 4 to 6 weeks to assess the cells’ pluripotency. In this study, an alternative in vivo model based on chicken egg chorioallantoic membrane (CAM) was established to analyze the pluripotency of newly created hiPSCs. 0.5, 1, 2, 4 x 106 hiPSCs gen­erated from urine-derived renal epithelial cells were seeded on CAM and incubated for 9 days. Teratoma formation was detected in 70% of eggs inoculated with 2 x 106 hiPSCs and in 100% of eggs inoculated with 4 x 106 hiPSCs. All teratomas exhibited vascular structures. The robustness of the CAM model was confirmed using two additional hiPSC lines derived from human fibroblasts (NuFFs) or jaw periosteal cells. The presence of all three germ layers within the teratomas was successfully verified by histochemical and immunofluorescence staining and gene expression analysis of germ layer-specific markers. Urine-derived renal epithelial cells were used as negative control and showed no teratoma formation. The CAM-based in vivo model provides an optimal in vivo test environment for the pluripotency evaluation of newly generated hiPSC lines. This simple, fast, inexpensive and reproducible method reduces the suffering of animals and thus implements the principles of the 3Rs (replacement, reduction, and refinement).

EGFR-Binding Peptides: From Computational Design towards Tumor-Targeting of Adeno-Associated Virus Capsids.

The epidermal growth factor receptor (EGFR) plays a central role in the progression of many solid tumors. We used this validated target to analyze the de novo design of EGFR-binding peptides and their application for the delivery of complex payloads via rational design of a viral vector. Peptides were computationally designed to interact with the EGFR dimerization interface. Two new peptides and a reference (EDA peptide) were chemically synthesized, and their binding ability characterized. Presentation of these peptides in each of the 60 capsid proteins of recombinant adeno-associated viruses (rAAV) via a genetic based loop insertion enabled targeting of EGFR overexpressing tumor cell lines. Furthermore, tissue distribution and tumor xenograft specificity were analyzed with systemic injection in chicken egg chorioallantoic membrane (CAM) assays. Complex correlations between the targeting of the synthetic peptides and the viral vectors to cells and in ovo were observed. Overall, these data demonstrate the potential of computational design in combination with rational capsid modification for viral vector targeting opening new avenues for viral vector delivery and specifically suicide gene therapy.

Ferulic Acid Prevents Angiogenesis Through Cyclooxygenase-2 and Vascular Endothelial Growth Factor in the Chick Embryo Chorioallantoic Membrane Model.

This study was designed to verify the antiangiogenic activity of ferulic acid (FA) and its potency to inhibit cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) expression in the chorioallantoic membrane (CAM) model. Moreover, we verified its mechanism of action by docking the molecule on COX-2, tyrosine kinase, and VEGF-2 proteins in silico. An antiangiogenesis assay of FA at doses of 30, 60, and 90 μg was performed using the CAM of chicken eggs that were 9 days old and stimulated by 60 ng of basic fibroblast growth factor. Celecoxib (60 μg) was used as the reference drug. The inhibitory activity on VEGF and COX-2 expression was determined by immunohistochemistry assay. Molecular docking of FA was accomplished by Molegro Virtual Docker program ver. 5.5 on COX-2 enzyme (PDB ID 1CX2), tyrosine kinase receptor (PDB ID 1XKK), and VEGF-2 receptor (PDB ID 4ASD). FA at doses of 30, 60, and 90 μg significantly prevented angiogenesis in the CAM model, which was represented as inhibitory activity against endothelial cells of blood vessels (42.6-70.7%) and neovascularization (43.0-86.6%). The inhibitory activity of FA against VEGF expression was stronger than its action on COX-2 expression. Molecular docking on VEGF-2 receptor resulted in an RS value of FA of -73.844 kcal/mol and for celecoxib it was -94.557 kcal/mol. The RS value on tyrosine kinase of FA was -84.954 kcal/mol, while on celecoxib it was -93.163 kcal/mol. Docking on COX-2 receptor gave an RS value of FA of -73.416 kcal/mol, while for celecoxib it was -118.107 kcal/mol. Reductions in VEGF-2 and COX-2 expression due to treatment with FA at the dose range 30-90 μg appeared to be related to angiogenesis inhibition, which was shown by two parameters, namely inhibition of neovascularization and endothelial cell growth in blood vessels. It was concluded that FA is a promising antiangiogenic therapeutic agent especially at the early stage, and this activity can arise from inhibitory action on COX-2 and VEGF-2 proteins.

Antiproliferative and caspase-mediated apoptosis inducing effects of Murraya koenigii seeds against cancer cells

Murraya koenigii (Rutaceae), a tropical to the sub-tropical tree is well disseminated in the eastern region of India and known for its curative potential against ample diseases and disorders. In this study, the methanolic extract of Murraya koenigii seeds (MEMS) was screened for the first time to determine the in vitro cytotoxicity and caspase-mediated apoptosis-inducing effects against breast and colon cancer cells. Both in vitro assay and in silico molecular docking study were performed to screen the anti-proliferative potential of MEMS. Cicer arietinum seeds germination and Allium cepa root tip inhibition assay were used to study the anti-mitotic effect of the MEMS. Angiogenesis inhibition and cytotoxicity of MEMS were determined using chicken egg chorioallantoic membrane (CAM) assay and brine shrimp lethality bioassay respectively. MTT assay was used to exhibit the cell viability while apoptotic cells were determined using annexin V-fluorescein isothiocyanate assay. The activity of caspase-3 and caspase-7 was performed using the luminometric assay. SeDeM diagram and Heckel plot were generated for MEMS to predict their compression suitability. The MEMS significantly reduced the growth of Cicer arietinum seeds sprout (P<0.05) compared to control and on a par with methotrexate treated groups. Cell division in Allium cepa roots was diminished by 7.73 fold compared to control at the dose of 1000 µg/mL. MEMS showed moderate anti-angiogenic activity (14.81%) on CAM and considerable cytotoxicity (LD50= 40.860 μg/mL) to brine shrimps. MEMS elicited the prominent cytotoxic effect against DLD-1 colorectal cancer cells compared to MCF-7 breast cancer cells. It showed caspase-mediated apoptosis with reduced mitochondrial membrane potential. Early and late apoptotic rates were observed to be 7.13 and 2.57 fold higher respectively, than control. In silico docking analysis corroborated that neplanocin A and girinimbine interacted with proteolytic enzymes (caspase-3 and caspase-7) mainly by hydrogen bonding, aromatic, hydrophobic, and Van der Waals interactions. SeDeM diagram and Heckel plot revealed the poor compression behavior of MEMS. However, MEMS is having enormous potential and can be processed for further in vivo experiments.

Anti-angiogenic effects of mangiferin and mechanism of action in metastatic melanoma.

Advanced metastatic melanoma, one of the most aggressive skin malignancies, is currently without reliable therapy. The process of angiogenesis is crucial for progression and metastasis of the majority of solid tumors including melanomas. Therefore, new therapies are urgently needed. Mangiferin is a naturally occurring glucosylxanthone which exerts many pharmacological activities against cancer-inflammation. However, the effect of mangiferin on metastasis and tumor growth of metastatic melanoma remains unclear. In this study, we demonstrate that mangiferin interferes with inflammation, lipid and calcium signaling which selectively inhibits multiple NFkB target genes including interleukin-6, tumor necrosis factor, interferon gamma, vascular endothelial growth factor receptor 2, plasminogen activator urokinase, matrix metalloprotease 19, C-C Motif Chemokine Ligand 2 and placental growth factor. This abrogates angiogenic and invasive processes and capillary tube formation of metastatic melanoma cells as well as human placental blood vessel explants in-vitro and blocks angiogenesis characteristic of the chicken egg chorioallantoic membrane assay and in melanoma syngeneic studies in vivo. The results obtained in this research illustrate promising anti-angiogenic effects of the natural glucosylxanthone mangiferin for further (pre)clinical studies in melanoma cancer patients.

Natural compound Tetrocarcin-A downregulates Junctional Adhesion Molecule-A in conjunction with HER2 and inhibitor of apoptosis proteins and inhibits tumor cell growth

Overexpression of the tight junction protein Junctional Adhesion Molecule-A (JAM-A) has been linked to aggressive disease in breast and other cancers, but JAM-targeting drugs remain elusive. Screening of a natural compound library identified the antibiotic Tetrocarcin-A as a novel downregulator of JAM-A and human epidermal growth factor receptor-2 (HER2) protein expression in breast cancer cells. Lysosomal inhibition partially rescued the downregulation of JAM-A and HER2 caused by Tetrocarcin-A, and attenuated its cytotoxic activity. Tetrocarcin-A treatment or JAM-A silencing reduced AKT and ERK phosphorylation, inhibited c-FOS phosphorylation at Threonine-232 (its transcriptional regulation site), inhibited nuclear localization of c-FOS, and downregulated expression of the inhibitor of apoptosis proteins (IAP). This was accompanied by Tetrocarcin-A-induced caspase-dependent apoptosis. To begin evaluating the potential clinical relevance of our findings, we extended our studies to other models. Encouragingly, Tetrocarcin-A downregulated JAM-A expression and caused cytotoxicity in primary breast cells and lung cancer stem cells, and inhibited the growth of xenografts in a semi-in vivo model involving invasion across the chicken egg chorioallantoic membrane. Taken together, our data suggest that Tetrocarcin-A warrants future evaluation as a novel cancer therapeutic by virtue of its ability to downregulate JAM-A expression, reduce tumorigenic signaling and induce apoptosis.

Abstract 2670: Natural compound tetrocarcin-A downregulates junctional adhesion molecule-A in conjunction with erα, her2 and inhibitor of apoptosis proteins and inhibits tumor cell growth

Abstract Overexpression of the tight junction protein Junctional Adhesion Molecule-A (JAM-A) has been linked to aggressive disease in breast and several other cancers, but JAM-targeting drugs remain elusive. Screening of a natural compound library identified the cytotoxic antibiotic Tetrocarcin-A as a novel downregulator of JAM-A protein expression in breast cancer cells. Specifically, Tetrocarcin-A-induced cytotoxicity was accompanied by downregulation of JAM-A protein expression in conjunction with that of estrogen receptor alpha (ERα) and human epidermal growth factor receptor 2 (HER2) in MCF7-HER2-overexpressing breast cells. Lysosomal inhibition partially rescued the downregulation of JAM-A, HER2 and ERα caused by Tetrocarcin-A, and attenuated its cytotoxicity. Interestingly, Tetrocarcin-A repressed the transcriptional activity of c-FOS by inhibiting ERK1/2-mediated phosphorylation at Threonine-232, and downregulated protein expression of the inhibitor of apoptosis proteins (IAP). Investigation of cell death pathways revealed that Tetrocarcin-A induced caspase-dependent apoptosis. To begin testing the potential clinical relevance of our findings, we extended our studies into various other models. Encouragingly, Tetrocarcin-A downregulated JAM-A expression and caused cytotoxicity in primary breast cancer and lung cancer stem cells, and inhibited the growth of model human breast tumors in an in vivo model involving invasion across the chicken egg chorioallantoic membrane. Taken together, our data suggest that Tetrocarcin-A merits future evaluation as a novel cancer therapeutic by virtue of its ability to downregulate JAM-A expression, induce apoptosis and reduce tumorigenic signalling. This work was financially supported by Science Foundation Ireland (grant 13/IA/1994 to AMH). Tetrocarcin-A was provided by the NCI/DTP Open Chemical Repository https://dtp.cancer.gov. (NSC 333856) Citation Format: Sri Harikrishna Vellanki, Lance Hudson, Hanne Jahns, Rodrigo G.B. Cruz, Giovanni Sette, Adriana Eramo, Ann M. Hopkins. Natural compound tetrocarcin-A downregulates junctional adhesion molecule-A in conjunction with erα, her2 and inhibitor of apoptosis proteins and inhibits tumor cell growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2670.

Preparation and Evaluation of Tibia- and Calvarium-Derived Decellularized Periosteum Scaffolds.

The periosteum plays a key role in bone regeneration and an artificial bionic material is urgently required. The periostea on the tibia and skull differ with respect to the types of cells, microstructure, and components, leading to different biological functions and biomechanical properties. We aimed to prepare decellularized periosteum scaffolds derived from different origins and evaluate their angiogenic and osteogenic activities. Histological assessment of α-smooth muscle actin, bone morphogenetic protein-2, and alkaline phosphatase in tibial and calvarial periosteum tissues provided preliminary information on their differing angiogenic and osteogenic properties. We developed decellularization protocols to completely remove the periosteum cellular components and for good maintenance of the hierarchical multilayer structures and components of the extracellular matrix (ECM) with no cytotoxicity. Moreover, using a chicken egg chorioallantoic membrane assay and a nude mouse implantation model, we found that tibia-derived periosteum ECM had superior osteogenic activity and calvarium-derived ECM had good angiogenic activity. The preliminary mechanisms of differing activities were then evaluated by osteogenesis- and angiogenesis-related gene expression in human umbilical vein endothelial cell- and MC-3T3 cell-seeded ECM scaffolds. Thus, this study provides periosteum biomaterials that are derived from specific tissues and have different functional properties and structures, for use in bone regeneration.

MiR-127 and miR-376a act as tumor suppressors by in vivo targeting of COA1 and PDIA6 in giant cell tumor of bone

Giant cell tumors of bone (GCTB) are generally benign bone tumors associated with expansive osteolytic defects, a high rate of recurrence and potential malignant transformation. We recently observed silencing of miR-127-3p and miR-376a-3p in GCTB and identified COA1 and PDIA6 as their target genes. Here, we investigate the impact of these microRNAs and their target genes on tumor engraftment and progression of giant cell tumor stromal cells (GCTSC) in vivo by xenotransplantation on the chorioallantoic membrane of chicken eggs. Prior to transplantation, the neoplastic GCTSCs were transfected with miRNA mimics or siRNAs directed against their target genes. Restoration of miR-127-3p and miR-376a-3p reduced the tumor take rate to 17% and 47% compared to 95% in the controls. The tumor volumes were significantly reduced to 29% by both miRNAs. Silencing of COA1 and PDIA6 significantly decreased the tumor volumes to 37.7% and 42.7%, while the tumor take rates remained stable. Our results indicate that re-expression of miR-127-3p and miR-376a-3p induces a strong tumor suppressor effect in GCTSC, which is partially mediated via COA1 and PDIA6.

Evaluation of Angiogenic Potential of 1-Monocaproin Using Chick Chorioallantoic Membrane Assay

1-Monocaproin, a 1-monoacylglycerol, was synthesized with the reaction of glycidol by chromium-caproic fatty acid complexes and was found to be amphiphilic in nature. Synthesized 1-monocaproin was characterized by thin-layer chromatography, and the residual chromium was analyzed using inductively coupled plasma mass spectroscopy. Vascular endothelium growth factor (VEGF)-mediated angiogenesis pathways are being targeted for the development of novel drugs suitable for inhibition or stimulation of angiogenesis in various pathologies. In the chicken egg Chorioallantoic membrane (CAM) assay, 1-monocaproin exhibited VEGF-induced neovascularization in a dose-dependent manner. Synthesized 1-monocaproin was found to show the angiogenic effect at a dose concentration equivalent to VEGF at 1,000 ppm. The results thus suggest that 1-monocaproin should be considered as a useful drug candidature targeting angiogenesis in coronary artery disease, stroke, and postsurgical therapy.

Ferrocenyl-Coupled N-Heterocyclic Carbene Complexes of Gold(I): A Successful Approach to Multinuclear Anticancer Drugs.

Four gold(I) carbene complexes featuring 4-ferrocenyl-substituted imidazol-2-ylidene ligands were investigated for antiproliferative and antivascular properties. They were active against a panel of seven cancer cell lines, including multidrug-resistant ones, with low micromolar or nanomolar IC50 (72 h) values, according to their lipophilicity and cellular uptake. The delocalized lipophilic cationic complexes 8 and 10 acted by increasing the reactive oxygen species in two ways: through a genuine ferrocene effect and by inhibiting the thioredoxin reductase. Both complexes gave rise to a reorganization of the F-actin cytoskeleton in endothelial and melanoma cells, associated with a G1 phase cell cycle arrest and a retarded cell migration. They proved antiangiogenic in tube formation assays with endothelial cells and vascular-disruptive on real blood vessels in the chorioallantoic membrane of chicken eggs. Biscarbene complex 10 was also tolerated well by mice where it led to a volume reduction of xenograft tumors by up to 80 %.

Solanum nigrum Suppress Angiogenesis-Mediated Tumor Growth Through Inhibition of the AKT/mTOR Pathway.

Solanum nigrum L., an edible plant and local dish, has been assigned anticancer activities. However, the anticancer mechanisms of S. nigrum are poorly understood. Here, we investigated whether the water or polyphenol extracts of S. nigrum (SNWE or SNPE) could inhibit angiogenesis-mediated tumor growth. In nude mice bearing tumor xenografts, SNWE or SNPE significantly reduced the volume and weight of the tumors, and decreased the expression of CD31, a marker for angiogenesis. SNWE or SNPE was found to inhibit the VEGF-induced capillary structure formation of endothelial cells. The chicken egg chorioallantoic membrane (CAM) and matrigel plug assays showed further that SNWE or SNPE inhibited tumor angiogenesis. In human umbilical vascular endothelial cells (HUVECs), SNWE or SNPE suppressed the VEGF-induced activation of AKT and mTOR. Moreover, SNWE or SNPE inhibited the viability of human hepatoma HepG2 cells, and these effects were correlated with the extent of inhibition of the AKT/mTOR pathway. Taken together, our data imply that SNWE or SNPE downregulated the AKT/mTOR pathway in HUVECs and HepG2 cells, which lead to reduced tumor growth and angiogenesis.