Chick Chorioallantoic Membrane Research Articles

The Chick Chorioallantoic Membrane (CAM) Model as a Tool to Study Ovarian Tissue Transplantation

Ovarian tissue cryopreservation and transplantation is an effective strategy for preserving fertility but has one major drawback, namely massive follicle loss occurring shortly after reimplantation due to abnormal follicle activation and death. Rodents are benchmark models for investigating follicle activation, but the cost, time, and ethical considerations are becoming increasingly prohibitive, thus driving the development of alternatives. The chick chorioallantoic membrane (CAM) model is particularly attractive, being inexpensive and maintaining natural immunodeficiency up to day 17 postfertilization, making it ideal to study short-term xenografting of human ovarian tissue. The CAM is also highly vascularized and has been widely used as a model to explore angiogenesis. This gives it a remarkable advantage over in vitro models and allows the investigation of mechanisms affecting the early post-grafting follicle loss process. The protocol outlined herein aims to describe the development of a CAM xenografting model for human ovarian tissue, with specific insights into the effectiveness of the technique, the graft revascularization time frame, and the tissue viability across a 6 day grafting period.

AATF inhibition exerts antiangiogenic effects against human hepatocellular carcinoma.

Angiogenesis is a key factor in the growth and metastasis of hepatic tumors and thus a potential therapeutic target in hepatocellular carcinoma (HCC). In this study, we aim to identify the key role of apoptosis antagonizing transcription factor (AATF) in tumor angiogenesis and its underlying mechanisms in HCC. HCC tissues were analyzed for AATF expression by qRT-PCR and immunohistochemistry. Stable clones of control and AATF knockdown (KD) were established in human HCC cells. The effect of AATF inhibition on the angiogenic processes was determined by proliferation, invasion, migration, chick chorioallantoic membrane (CAM) assay, zymography, and immunoblotting techniques. We identified high levels of AATF in human HCC tissues compared to adjacent normal liver tissues, and the expression was found to be correlated with the stages and tumor grades of HCC. Inhibiting AATF in QGY-7703 cells resulted in higher levels of pigment epithelium-derived factor (PEDF) than controls due to decreased matric metalloproteinase activity. Conditioned media from AATF KD cells inhibited the proliferation, migration, and invasion of human umbilical vein endothelial cells as well as the vascularization of the chick chorioallantoic membrane. Furthermore, the VEGF-mediated downstream signaling pathway responsible for endothelial cell survival and vascular permeability, cell proliferation, and migration favoring angiogenesis was suppressed by AATF inhibition. Notably, PEDF inhibition effectively reversed the anti-angiogenic effect of AATF KD. Our study reports the first evidence that the therapeutic strategy based on the inhibition of AATF to disrupt tumor angiogenesis may serve as a promising approach for HCC treatment.

ATF2 loss promotes 5-FU resistance in colon cancer cells via activation of the ATR-Chk1 damage response pathway

BackgroundThe role of ATF2 in colon cancer (CC) is controversial. Recently, we reported that low ATF2 expression is characteristic of highly invasive tumors, suggesting that ATF2 might also be involved in therapy resistance. 5-Fluorouracil (5-FU) is the best-known chemotherapeutic drug for CC, but drug resistance affects its curative effect. To date, the role of ATF2 in the 5-FU response remains elusive.Methods/ResultsFor our study, we had available HCT116 cells (wild-type p53) and HT29 colon tumor cells (mutant p53) and their corresponding CRISPR‒Cas9-generated ATF2-KO clones. We observed that loss of ATF2 triggered dose- and time-dependent 5-FU resistance in HCT116 cells by activating the DNA damage response (DDR) pathway with high p-ATRThr1989 and p-Chk1Ser317 levels accompanied by an increase in the DNA damage marker γ-H2AX in vitro and in vivo using the chicken chorioallantoic membrane (CAM) model. Chk1 inhibitor studies causally displayed the link between DDR and drug resistance. There were contradictory findings in HT29 ATF2-KO cells upon 5-FU exposure with low p-Chk1Ser317 levels, strong apoptosis induction, but no effects on DNA damage. In ATF2-silenced HCT116 p53−/− cells, 5-FU did not activate the DDR pathway. Co-immunoprecipitation and proximity ligation assays revealed that upon 5-FU treatment, ATF2 binds to ATR to prevent Chk1 phosphorylation. Indeed, in silico modelling showed reduced ATR-Chk1 binding when ATF2 was docked into the complex.ConclusionsWe demonstrated a novel ATF2 scaffold function involved in the DDR pathway. ATF2-negative cells are highly resistant due to effective ATR/Chk1 DNA damage repair. Mutant p53 seems to overwrite the tumor suppressor function of ATF2.

Saikosaponin b2 inhibits tumor angiogenesis in liver cancer via down‑regulation of VEGF/ERK/HIF‑1α signaling.

Saikosaponin b2 (SSb2) is an active component of Radix Bupleuri, which is commonly used in traditional Chinese medicine for defervescence and liver protection. In the present study, it was demonstrated that SSb2 exhibited potent antitumor activity by inhibiting tumor angiogenesis invivo and invitro. As measured by tumor weight and measures of immune function such as thymus index, spleen index and white blood cell count, SSb2 inhibited tumor growth, with low immunotoxicity, in H22 tumor‑bearing mice. Furthermore, proliferation and migration of HepG2 liver cancer cells was inhibited following SSb2 treatment, which demonstrated SSb2's antitumor effect. The angiogenesis marker CD34 was downregulated in the SSb2‑treated tumor samples, which suggested the antiangiogenic activity of SSb2. Furthermore, the chick chorioallantoic membrane assay demonstrated the potent inhibitory effect of SSb2 on basic fibroblast growth factor‑induced angiogenesis. Invitro, SSb2 significantly inhibited numerous stages of angiogenesis, including the proliferation, migration and invasion of human umbilical vein endothelial cells. Further mechanistic studies demonstrated that SSb2 treatment reduced the levels of key proteins involved in angiogenesis, including vascular endothelial growth factor (VEGF), phosphorylated ERK1/2, hypoxia‑inducible factor (HIF)‑1α, MMP2 and MMP9 in H22 tumor‑bearing mice, which supported the HepG2 liver cancer cell results. Overall, SSb2 effectively inhibited angiogenesis via the VEGF/ERK/HIF‑1α signal pathway and may serve as a promising natural agent for liver cancer treatment.

A new 3D-printed polylactic acid-bioglass composite for bone tissue engineering induces angiogenesis in vitro and in ovo.

Large bone defects such as those that occur after trauma or resections due to cancer still are a challenge for surgeons. Main challenge in this area is to find a suitable alternative to the gold-standard therapy, which is highly risky, and a promising option is to use biomaterials manufactured by 3D printing. In former studies, we demonstrated that the combination of polylactic acid (PLA) and bioglass (BG) resulted in a stable 3D-printable material, and porous and finely structured scaffolds were printed. These scaffolds exhibited osteogenic and anti-inflammatory properties. This 3D-printed material fulfills most of the requirements described in the diamond concept of bone healing. However, the question remains as to whether it also meets the requirements concerning angiogenesis. Therefore, the aim of this study was to analyze the effects of the 3D-printed PLA-BG composite material on angiogenesis. In vitro analyses with human umbilical vein endothelial cells (HUVECs) showed a positive effect of increasing BG content on viability and gene expression of endothelial markers. This positive effect was confirmed by an enhanced vascular formation analyzed by Matrigel assay and chicken chorioallantoic membrane (CAM) assay. In this work, we demonstrated the angiogenic efficiency of a 3D-printed PLA-BG composite material. Recalling the osteogenic potential of this material demonstrated in former work, we manufactured a mechanically stable, 3D-printable, osteogenic and angiogenic material, which could be used for bone tissue engineering.

Bioinspired gold coated phage nanosomes for anti-microbial and anti-cancer theranostics

We report a unique bioinspired phage nanosome as a biological template for tuning the NIR absorbance of gold nanoparticles (ФNSAu) coated on its surface. Spectroscopical analysis revealed that the presence of phage significantly enhances the optical properties of encapsulated dyes, rendering them excellent imaging agents. In vitro, studies showed the excellent photothermal transduction efficacy of the developed ФNSAu in combination with a chemotherapeutic drug, Mitoxantrone (ФNSAuM). ФNSAuM mediated photothermal therapy (PTT) using a 690 nm laser exhibited excellent anti-microbial activity against E. coli and C. albicans. In vitro and in ovo studies using zebrafish embryos revealed excellent biocompatibility, no/minimal cardiotoxicity, and enhanced internalization of phage-based nanosomes when compared with respective liposomal controls. When tested against 4T1 cell lines, the synthesized ФNSAuM exhibited significant synergistic anti-cancer activity with enhanced ROS generation and subsequent mitochondrial damage. The blood vessel rupturing potential of ФNSAuM mediated PTT has been demonstrated using an in ovo chick chorioallantoic membrane (CAM) assay, thus proving it as a potential photothermal agent. Overall, the developed phage-based nanosomes facilitated enhanced PTT efficacy, thereby showing an encouraging potential for anti-microbial and anti-cancer theranostics.

Anti-angiogenic effect of nano-formulated water soluble kaempferol and combretastatin in an in vivo chick chorioallantoic membrane model and HUVEC cells

The present study evaluated the efficacy of nano-formulated water-soluble kaempferol and combretastatin alone and combined against the native kaempferol and combretastatin on angiogenesis. The solvent evaporation method was used to synthesize the nano-formulated water-soluble kaempferol and combretastatin and characterized using various analyses such as dynamic light scattering (DLS) and Fourier-transform infrared (FT-IR) spectroscopy.The anti-angiogenic activity of native, nano-formulated water-soluble kaempferol and combretastatin was investigated by cell viability on HUVEC and A498 cell lines, while chick chorioallantoic membrane (CAM) assay was utilized to assess morphometric and histopathological changes, and mRNA expressions of VEGF-A and FGF2 using qRT-PCR. MTT assay results revealed that the combination of nano-formulated water-soluble kaempferol and combretastatin significantly reduced the cell viability compared to control, individual treatments of native, nano-formulated water-soluble kaempferol, and combretastatin. Morphometric analysis of CAM showed that treatment with nano-formulated water-soluble kaempferol and combretastatin caused a substantial decrease in density, vessel network, branch points, and nets of CAM blood vessels. The histopathological results of CAM showed the irregular shape of blood vessels at the thin stratum of chronic endoderm, and blood capillaries were diminished compared to the control. In addition, the mRNA expression levels of VEGF-A and FGF2 were significantly decreased compared with native forms. Therefore, the findings of this study indicate that nano-formulated water-soluble combretastatin and kaempferol suppress angiogenesis by preventing the activation of endothelial cells and suppressing factors of angiogenesis. Moreover, a combination of nano-formulated water-soluble kaempferol and combretastatin worked much better than individual treatments.

The angiogenic potential of pH-neutral borophosphate bioactive glasses.

Borate bioactive glasses have gained attention in recent years due to their therapeutic and regenerative effects in vivo. However, borate bioactive glasses release alkaline ions, increasing the local pH and creating a toxic environment for cell culture studies. A partial compositional substitution of phosphate for borate can create a pH-neutral glass that does not significantly affect the local pH while still releasing therapeutic ions. In the present study, a series of Na-Ca-borophosphate bioactive glasses with different borate-to-phosphate ratios was evaluated in vitro and in vivo for cytotoxicity and angiogenic effects. Compared to more basic borate glasses, the pH-neutral glasses supported endothelial cell migration and stimulated greater blood vessel formation in a chick chorioallantoic membrane model. The results from this study indicate that these pH-neutral glasses are promising angiogenic biomaterials for use in tissue engineering and regenerative medicine.

The Antiangiogenic Effect and Ocular Pharmacology of Novel Modified Nonsteroidal Anti-Inflammatory Drugs in the Treatment of Oxygen-Induced Retinopathy.

Purpose: To evaluate the hypothesis that 3 novel compounds, OXT-328, Q-922, and CL-717 show efficacy in the treatment of oxygen-induced retinopathy (OIR) and whether or not their route of administration is intravitreal, topical, or systemic. Methods: The OIR mouse model, characterized by an avascular area (AVA) and a neovascular area (NVA) of the retina, was used to study retinopathy of prematurity and other retinal diseases characterized by abnormal vessel growth. We measured the effect of our compounds on both the AVA and NVA in whole mounts of mouse retinal tissue. We also evaluated their ability to prevent new vessel formation in chicken chorioallantoic membranes (CAMs). Finally, we measured the in vitro uptake and biodistribution of topically applied CL-717 in human eye explants. Results: In mice with OIR, compared to controls, a single intravitreal administration of Q-922 or OXT-328 significantly reduced both AVA and NVA. CL-717 administered as eye drops over 5 days also reduced AVA and NVA, whereas OXT-328 eye drops had no effect. Q-922 given intraperitoneal (150 mg/kg/day × 5 days) reduced AVA and NVA. Remarkably, explanted human eyes bathed in CL-717 show rapid uptake and biodistribution in ocular tissues. In the chicken CAM model, all 3 compounds reduced the formation of new blood vessels by about one-third. No side effect in mice was observed, except for mild ocular surface irritation with Q-922. Conclusions: Systemic administration of Q-922 or topical administration of CL-717 holds particular promise for a simplified treatment of proliferative retinopathies without the necessity of intravitreal injections.

Preparation and characterization of the farnesiferol C-loaded solid lipid nanoparticles decorated with folic acid-bound chitosan and evaluation of its in vitro anti-cancer and anti-angiogenic activities

Farnesiferol C (FC) has long been used as a cancer prevention agent. In the present study, FC was loaded into solid lipid nanoparticles (SLN) coated with folic acid (FA) bound chitosan (CS), FC-SLN-CS-FA, by homogenization followed by ultrasonication. Then its toxicity effect on cancer cells compared to normal cells was investigated by the MTT method. In the following, the pro-apoptotic effects of synthesized nanoparticles were evaluated by flow cytometry and acridine orange, propidium iodide, and DAPI staining, and the expression of genes involved in apoptosis was evaluated. The anti-oxidant effects of formulations have been evaluated using DPPH and ABTS assay. To investigate the anti-angiogenic effects of nanoparticles, the chicken chorioallantoic membrane (CAM) assay was used and the expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) genes was used by real-time quantitative PCR (qPCR). The formed nanoparticles with a size of 150 nm and a dispersion index of 0.30 had a surface charge of +17 mV. These nanoparticles exhibited significant growth and proliferation inhibitory effects in PANC, AGS, HT-29, MCF7, and A549 human cancer cells from the lowest concentration (15.6 µg/mL, *** p < 0.001). The flow cytometry analysis shows 10.1%, 48.8%, and 74.4% subG1 arrest at the concentrations of 6, 36, and 66 µg/mL of the FC-SLN-CS-FA, respectively. The formulation caused apoptotic cell death by 3-fold overregulating BAX at concentrations of 36 and 66 µg/mL, and 2- and 2.5-fold overexpression of caspase-9 at concentrations of 36 and 66 µg/mL respectively. The treatment with FC-SLN-CS-FA resulted in decreasing BCL2 and NF-κB gene expression. The ABTS and DPPH assays showed the anti-oxidant properties of formulation in a concentration-dependent manner. FC-SLN-CS-FA significantly inhibited angiogenesis in CAM by reducing the length and number of blood vessels. Considering the pro-apoptotic and anti-angiogenic effects of FC-SLN-CS-FA, it can be said that these nanoparticles can effectively suppress the carcinogenesis of multipotent cancer stem cells. Therefore, they have the potential to be studied as an efficient anticancer agent.

Development of PCL PolyHIPE Substrates for 3D Breast Cancer Cell Culture.

Cancer is a becoming a huge social and economic burden on society, becoming one of the most significant barriers to life expectancy in the 21st century. In particular, breast cancer is one of the leading causes of death for women. One of the most significant difficulties to finding efficient therapies for specific cancers, such as breast cancer, is the efficiency and ease of drug development and testing. Tissue-engineered (TE) in vitro models are rapidly developing as an alternative to animal testing for pharmaceuticals. Additionally, porosity included within these structures overcomes the diffusional mass transfer limit whilst enabling cell infiltration and integration with surrounding tissue. Within this study, we investigated the use of high-molecular-weight polycaprolactone methacrylate (PCL-M) polymerised high-internal-phase emulsions (polyHIPEs) as a scaffold to support 3D breast cancer (MDA-MB-231) cell culture. We assessed the porosity, interconnectivity, and morphology of the polyHIPEs when varying mixing speed during formation of the emulsion, successfully demonstrating the tunability of these polyHIPEs. An ex ovo chick chorioallantoic membrane assay identified the scaffolds as bioinert, with biocompatible properties within a vascularised tissue. Furthermore, in vitro assessment of cell attachment and proliferation showed promising potential for the use of PCL polyHIPEs to support cell growth. Our results demonstrate that PCL polyHIPEs are a promising material to support cancer cell growth with tuneable porosity and interconnectivity for the fabrication of perfusable 3D cancer models.

Targeting exosomes enveloped EBV-miR-BART1-5p-antagomiRs for NPC therapy through both anti-vasculogenic mimicry and anti-angiogenesis.

Nasopharyngeal carcinoma (NPC) is a type of head and neck cancer with high incidence in China. The molecular mechanisms of vasculogenic mimicry (VM) and angiogenesis are not fully elucidated in NPC. More specially, it has seldomly been reported that Epstein-Barr virus-encoded miRNA can regulate VM and angiogenesis in NPC. The aim of this study was to investigate the function and molecular mechanism of a targeting exosome system (iRGD-exo-antagomiR) against VM and angiogenesis in NPC, and to provide new approaches for improving the comprehensive treatment of NPC. Exosomes were isolated by differential ultracentrifugation. Dynamic light scattering, transmission electron microscopy and western blotting were performed to characterize the exosomes. The 3D-Culture assay, tube formation assay, chicken chorioallantoic membrane assay, Matrigel plug assay, mouse xenograft tumor modeling and immunohistochemical staining were applied to evaluate the anti-VM and anti-angiogenic effects of the targeting exosome system in vitro and in vivo. Western blot was performed to detect the changes of downstream regulated networks following interference and recovery of the target gene. In vitro or in vivo treatment with iRGD-tagged exosome containing antagomiR-BART1-5p specifically suppressed VM and angiogenesis in NPC. EBV-miR-BART1-5p promoted VM and angiogenesis in vitro and in vivo by regulating VEGF, PI3K, Akt, mTOR and HIF1-α in a Spry2-dependent manner. Our findings demonstrated that targeting exosomes enveloped EBV-miR-BART1-5p-antagomiRs in a Spry2-dependent manner for NPC therapy through both anti-VM and anti-angiogenesis in vitro and in vivo.

Deciphering the Precise Target for Saroglitazar Associated Antiangiogenic Effect: A Computational Synergistic Approach.

Antidiabetic drugs that have a secondary pharmacological effect on angiogenesis inhibition may help diabetic patients delay or avoid comorbidities caused by angiogenesis including malignancies. In recent studies, saroglitazar has exhibited antiangiogenic effects in diabetic retinopathy. The current study investigates the antiangiogenic effects of saroglitazar utilizing the chicken chorioallantoic membrane (CAM) assay and then identifies its precise mode of action on system-level protein networks. To determine the regulatory effect of saroglitazar on the protein-protein interaction network (PIN), 104 target genes were retrieved and tested using an acid server and Swiss target prediction tools. A string-based interactome was created and analyzed using Cytoscape. It was determined that the constructed network was scale-free, making it biologically relevant. Upon topological analysis of the network, 37 targets were screened on the basis of centrality values. Submodularization of the interactome resulted in the formation of four clusters. A total of 20 common targets identified in topological analysis and modular analysis were filtered. A total of 20 targets were compiled and were integrated into the pathway enrichment analysis using ShinyGO. The majority of hub genes were associated with cancer and PI3-AKT signaling pathways. Molecular docking was utilized to reveal the most potent target, which was validated by using molecular dynamic simulations and immunohistochemical staining on the chicken CAM. The comprehensive study offers an alternate research paradigm for the investigation of antiangiogenic effects using CAM assays. This was followed by the identification of the precise off-target use of saroglitazar using system biology and network pharmacology to inhibit angiogenesis.

Quercetin activates vitamin D receptor and ameliorates breast cancer induced hepatic inflammation and fibrosis.

To explore the hepatoprotective role of quercetin and its novel molecular mechanism of action on breast cancer associated hepatic inflammation and fibrosis via Vitamin D receptor (VDR). We used Ehrlich Ascites Carcinoma (mouse mammary carcinoma) model for our in-vivo experiments and human breast cancer cell lines for in-vitro assays. We inoculated 1.5 × 106 Ehrlich ascites carcinoma cells into female Swiss albino mice. Quercetin (50 mg/kg) was administered intraperitoneally for 15 days. Liver enzymes activity was determined using a spectrophotometric assay. The hallmarks of inflammation and fibrosis were determined using Immunohistochemistry. The effect of quercetin on tumor formation was elucidated using human breast cancer cell lines and chick chorioallantoic membrane assay. Docking study was performed to explore the binding mode of quercetin with VDR. In EAC tumor-bearing mice, cell numbers, tumor volume, body weight and liver weight were dramatically increased, while they significantly decreased in mice treated with quercetin. Additionally, the peritoneal neo-angiogenesis was also significantly suppressed in the quercetin-treated mice, compared to the control. In addition, quercetin treated EAC tumor bearing mice had lower levels of liver enzymes, decreased hepatic inflammation and fibrosis compared with EAC tumor bearing mice. Docking study confirmed VDR-quercetin interaction. Furthermore, in-vitro assays and chick chorioallantoic membrane assay revealed the Vitamin D mimicking effect of quercetin. Dietary flavonoid, quercetin could act as a promising therapeutic drug to suppress the breast cancer induced tumor angiogenesis, hepatic inflammation, and fibrosis possibly via activation of VDR.

Gas-modulating microcapsules for spatiotemporal control of hypoxia

Oxygen is a vital molecule involved in regulating development, homeostasis, and disease. The oxygen levels in tissue vary from 1 to 14% with deviations from homeostasis impacting regulation of various physiological processes. In this work, we developed an approach to encapsulate enzymes at high loading capacity, which precisely controls the oxygen content in cell culture. Here, a single microcapsule is able to locally perturb the oxygen balance, and varying the concentration and distribution of matrix-embedded microcapsules provides spatiotemporal control. We demonstrate attenuation of hypoxia signaling in populations of stem cells, cancer cells, endothelial cells, cancer spheroids, and intestinal organoids. Varying capsule placement, media formulation, and timing of replenishment yields tunable oxygen gradients, with concurrent spatial growth and morphogenesis in a single well. Capsule containing hydrogel films applied to chick chorioallantoic membranes encourages neovascularization, providing scope for topical treatments or hydrogel wound dressings. This platform can be used in a variety of formats, including deposition in hydrogels, as granular solids for 3D bioprinting, and as injectable biomaterials. Overall, this platform's simplicity and flexibility will prove useful for fundamental studies of oxygen-mediated processes in virtually any invitro or invivo format, with scope for inclusion in biomedical materials for treating injury or disease.

Structural, vibrational, optical, and anticancer properties of Hyphaene thebaica‐reduced nano‐lanthanum oxide (La2O3)

Phytoextracts modulated synthesis of nano‐scaled spherical lanthanum oxide nanoparticles (La2O3 NPs) are reported using the aqueous extracts of Hyphaene thebaica as a bioreducing and chelating agent. X‐ray diffraction (XRD) pattern revealed sharp peaks and single‐phase purity. The Debye–Scherrer approximation indicated an average crystallite size of 37 nm, whereas W‐H plot revealed a crystallite size of 59 nm, complemented by the high‐resolution transmission electron microscopy (HR‐TEM). The infrared spectroscopy revealed characteristic La‐O band at 426 and 648 cm−1. K‐M function revealed the bandgap of 5.6 eV, whereas broad intense emission peaks between 345 and 750 nm. The Raman spectra revealed peaks at 182, 325, 384, and 491 cm−1. Elemental composition was confirmed using energy‐dispersive X‐ray spectroscopy (EDS) spectrum, and the spotty rings from selected area electron diffraction (SAED) indicated the crystalline nature of the HT‐La2O3 NPs. In addition, the HT‐La2O3 NPs were investigated for in vitro antioxidant, anticancer, antiangiogenic, and cytotoxic potentials. DPPH free radical scavenging activity was highest, that is, 49.54 ± 0.88% at 1000 μg/mL. MTT assay on MCF‐7 and 3T3 cell lines revealed a median lethal concentration of 114 and 43 μg/mL, respectively. The antiangiogenic effect examined by chick chorioallantoic membrane (CAM) assay indicated inhibition of 57.14 ± 0.96% at 1000‐μg/mL concentration with an IC50 of 551 μg/mL. IC50 for the brine shrimp lethality assay was 314.2 μg/mL. Our study indicates cytotoxicity of HT‐La2O3 NPs, and it further concluded that HT‐La2O3 NPs were more potent against 3T3 cells as compared with MCF‐7 cells. Further research on the biological synthesis and anticancer studies are recommended for the large‐scale applications of the La2O3 NPs.

Abstract 4866: Pharmacological mechanisms of osimertinib in advanced non-small lung cancer bearing the deletion exon 19 of EGFR In an original chicken chorioallantoic membrane (CAM) Model

Abstract Introduction: NSCLC accounts for about 80-85% of all lung cancers. Approximately 10-50% of patients with NSCLC harbor EGFR activating mutations, such as in-frame deletions in exon 19 (Ex19del). Currently, for patients with advanced NSCLC, testing for sensitizing mutations in EGFR is mandatory prior to the administration of anti-EGFR inhibitors such as Osimertinib. The CAM xenograft offers a rich environment in nutrients and embryonic growth factors, favorable for aggressive tumor development, with its highly vascularized membrane and natural immunodeficiency at engraftment (at EDD9). Methods: HCC827 Ex19del cells have been engrafted in ovo, and treated with various doses of osimertinib for seven days (4 treatments). The xenografts were collected and immunohistopathological, transcriptional and genetic analyses were carried out. Based on human transcriptional data, supervised multivariate discrimination between conditions and ontology analysis was performed through GSEA. Results: The xenograft growth was inversely dependent on the exposure dose of osimertinib, with a reduction in tumor weight of 58% for 10 µM (corresponding to 8.3 µg/Kg, T1/2 =4hr). The histological analysis showed epithelial tumor cells composed of solid sheets. The quantity of tumor cells was significantly lower in the treated group, with an area of tumor necrosis. The immunohistochemistry of tumor cells confirmed the human pulmonary epithelial origin, with TTF1 positive in treated and control groups. As expected, the ontology analysis of transcriptomic findings highlighted a downregulation of the EGFR pathway and its downstream effectors. Furthermore, the transcriptomic responses associated with chemotaxis, immune cell recruitment, and angiogenesis were dampened in the presence of osimertinib. This suggests that therapeutic efficacy was not uniquely guided through cell-autonomous mechanisms, but also takes place at the tissue level. We confirmed this in an independent experiment of in ovo HCC827 engraftment, showing a statically significant reduction of 15% in the number of vessels surrounding the xenograft in the osimertinib-treated condition. This demonstrates the advantage of using the in ovo model to decipher the regulations associated with therapeutics in a complex tissue context. Conclusions: For the first time, we have observed that osimertinib modulates angiogenesis and chemotaxis, which apparently reduces immune system recruitment and immune checkpoint responses. The ontologic analysis of transcriptomic findings in the CAM model strongly support the clinical observations of the pharmacological tumoral response to targeted therapy. The fact that immune infiltration was reduced in osimertinib-treated tumors may partially explain the lower response to immunotherapy regimens in osimertinib resistance contexts. Citation Format: David Barthelemy, Arnaud Vigneron, Xavier Rousset, Jerome Guitton, Emmanuel Grolleau, Margaux Raffin, Julie Balandier, Gaelle Lescuyer, Florence Geiguer, Sebastien Couraud, Jean Vaillet, Nazim Benzerdjeb, Lea Payen-Gay. Pharmacological mechanisms of osimertinib in advanced non-small lung cancer bearing the deletion exon 19 of EGFR In an original chicken chorioallantoic membrane (CAM) Model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4866.

Abstract 3620: N-myristoylation inhibition reduces angiogenesis and cancer cell migration

Abstract Myristoylation is the modification of proteins by the 14-carbon fatty acid myristate. In humans, two N-myristoyltransferases (NMT1 and NMT2) catalyze myristate transfer onto &amp;gt;200 proteins to regulate membrane binding and signal transduction. The pan-NMT inhibitor PCLX-001 has been in phase I clinical trials over a year for the treatment of lymphoma and advanced solid malignancies. Lack of myristoylation promotes the degradation of numerous normally myristoylated proteins like the proto-oncogenic Src family kinases (SFKs). SFKs like Src and Lyn are essential for downstream signaling of Vascular Endothelial Growth Factor (VEGF) and Epidermal Growth Factor (EGF) receptor tyrosine kinases. Thus, SFKs are critical for angiogenesis and cell motility. Angiogenesis is essential for tumor growth and dissemination of cancer cells to distal locations. VEGF-induced angiogenesis requires Src activation, suggesting that PCLX-001 may have antiangiogenic properties. SFKs also promote cell migration/metastasis, the leading cause of cancer-associated mortality. Lyn SFK knockdown was shown to reduce breast cancer migration and neighbouring tissue invasion, suggesting that PCLX-001 may also inhibit cell migration/metastasis. We investigated the antiangiogenic properties of PCLX-001 in vitro using HUVEC umbilical cord cells. PCLX-001 treatment significantly reduced HUVEC sprouting and tube-formation to levels lower than two FDA-approved angiogenesis inhibitors Sorafenib and Sunitinib. PCLX-001 also significantly reduced neo-vascularisation in vivo at lower concentrations than these two drugs using a chicken chorioallantoic membrane model, demonstrating the high potential of myristoylation inhibitors to reduce angiogenesis. We then validated our observations in vivo using a HT-1376 bladder mouse xenograft model. Tumors from mice treated for 28 days at increasing PCLX-001 concentrations showed significant reduction of Src, Lyn, and VEGF receptor protein levels as measured by immunohistochemistry, further validating PCLX-001 effect in vivo. We hypothesize that PCLX-001 acts downstream of VEGF/EGF receptors by inducing degradation of un-myristoylated SFKs. We confirmed this by knocking down NMT1 or treating breast cancer cells with PCLX-001 and found that both significantly reduced SFK protein levels. In a wound-healing assay using PCLX-001, we observed significant dose-dependent cell migration inhibition. PCLX-001 also dose-dependently reduced migration and invasion in transwell migration/invasion assays, indicating that myristoylation is required for optimal migration and potentially cancer cell metastasis. Overall, by lowering SFK levels, PCLX-001 reduces angiogenesis, cell migration/invasion, and thus potentially cancer metastasis. Reducing these classical cancer hallmarks in malignant cells may benefit a wide array of cancer patients and ultimately improve cancer treatment outcomes. Citation Format: Rony Pain, Erwan Beauchamp, Katia Carmine-Simmen, Jay Gamma, Rebecca Reif, Abul Azad, Allan Murray, John Lewis, Luc Berthiaume. N-myristoylation inhibition reduces angiogenesis and cancer cell migration. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3620.

Chick chorioallantoic membrane (CAM) assay for the evaluation of the antitumor and antimetastatic activity of platinum-based drugs in association with the impact on the amino acid metabolism.

The combination of in ovo and ex ovo chorioallantoic membrane (CAM) assay provides an excellent platform which extends its relevance in studying carcinogenesis to the field of screening of anticancer activity of platinum nanoparticles (PtNPs) and further study of the amino acids' fluctuations in liver and brain. PtNPs are promising candidates for replacing cisplatin (CDDP); however, insufficient data of their antitumor efficiency and activity on the cancer-related amino acid metabolism are available, and the assessment of the in vivo performance has barely scratched the surface. Herein, we used CAM assay as in vivo model for screening of novel therapeutic modalities, and we conducted a comparative study of the effects of CDDP and polyvinylpyrrolidone coated PtNPs on MDA-MB-231 breast cancer xenograft. PtNPs showed a higher efficiency to inhibit the tumor growth and metastasis compared to CDDP. The amino acids profiling in the MDA-MB-231​cells revealed that the PtNPs had an overall depleting effect on the amino acids content. Noteworthy, more side effects to amino acid metabolism were deduced from the depletion of the amino acids in tumor, brain, and liver upon CDDP treatment. Different sets of enzymes of the tricarboxylic acid (TCA) cycle were targeted by PtNPs and CDDP, and while mRNA encoding multiple enzymes was downregulated by PtNPs, the treatment with CDDP affected only two TCA enzymes, indicating a different mechanism of action. Taken together, CAM assay represents and invaluable model, demonstrating the PtNPs capability of repressing angiogenesis, decrease amino acid contents and disrupt the TCA cycle.

Antiangiogenic Activity of Aerva lanata Flowers: In Vitro and In Vivo Evaluation

Background: A well-known traditional herb Aerva lanata is widely used in India for the management of different ailments including urolithiasis. Objective: In the current research, flowers of Aerva lanata were chosen for isolation of active constituents and screened for antiangiogenic and anticancer potentials, as literature supports those flowers have highest quantity of natural constituents when compared with the other parts of the plant. Materials and methods: Hydroalcoholic (80%-water, 20%-alcohol) extract of Aerva Lanata flowers was prepared. Antiangiogenic activity was screened by chick chorioallantoic membrane assay (CAM) (in vivo) and sponge implantation method (SIM) (in vivo). Anti-cancer potency was screened by cell migration (in vitro), tube formation assay (in vitro) and cell proliferation assay (in vitro). Results: The results of test groups were matched up to control group by applying one-way ANOVA, followed by a post hoc and Dunnett’s test. In CAM assay method significant branching points and angiogenic score was obtained at 10-5 M and 10-4 M. Considerable decline was observed in sponge weight, formation of blood vessels and hemoglobin content (Hb)at various concentrations of hydroalcoholic extract of Aerva Lanata flower (HAL) in SIM. The study on human umbilical vein endothelial cells displayed major inhibition of proliferation along with reduction in network length of cord-alike tubes in a dosage-dependent pattern. Conclusions: Aerva lanata has shown potent antiangiogenic and anticancer effect in a dose dependent manner.