During avian development, the chorioallantoic membrane (CAM) is generated around 4 days after fertilization following the fusion of the allantois and the chorion. The CAM develops rapidly over the next several days and gets heavily vascularized and therefore has been explored widely as a tool for the study of angiogenesis. Additionally, being immunodeficient, the CAM can be used for tumor growth of human origin and its metastasis. Of note, the CAM assay is minimally invasive for the chicken embryo and lacks innervation, which gives this in vivo model a low ethical burden. Here, we describe the protocol for the generation of microtumors from human colorectal cancer cell lines on the CAM, incubated in a nutrient-deficient medium for the activation of autophagy. We show that pre-inoculation markers of autophagy induced through nutrient deficiency are retained in the microtumors generated on the CAM.

Olea europaea L. is the most valuable species of the Olea type, and its products offer a wide range of therapeutical uses. The olive tree has been extensively studied for its nourishing qualities, and the \"Mediterranean diet\", which includes virgin olive oil as a key dietary component, is strongly associated with a reduced risk of cardiovascular disease and various malignancies. Olive leaves, a by-product in the olive harvesting process, are valued as a resource for developing novel phytomedicines. For this purpose, two ethanolic extracts obtained from Olivae folium from Spain (OFS) and Greece (OFG) were investigated. Our findings contribute to a wider characterization of olive leaves. Both extracts displayed important amounts of phenolic compounds and pentacyclic triterpenes, OFG having higher concentrations of both polyphenols, such as oleuropein and lutein, as well as triterpenes, such as oleanolic acid and maslinic acid. The antioxidant capacity is similar for the two extracts, albeit slightly higher for OFG, possibly due to metal polyphenol complexes with antioxidant activity. The extracts elicited an antimicrobial effect at higher doses, especially against Gram-positive bacteria, such as Streptococcus pyogenes. The extract with lower inorganic content and higher content of polyphenols and triterpenic acids induced a strong anti-radical capacity, a selective cytotoxic effect, as well as antimigratory potential on A375 melanoma cells and antiangiogenic potential on the CAM. No irritability and a good tolerability were noted after evaluating the extracts on the in vivo Hen\'s Egg Test-Chorioallantoic Membrane (HET-CAM). Therefore, the present data are suggestive for the possible use of the two types of olive leaf products as high-antioxidant extracts, potentially impacting the healthcare system through their use as antimicrobial agents and as anticancer and anti-invasion treatments for melanoma.

Uveal melanoma (UM) is the most common intraocular tumor in adults, and nearly 50% of patients develop metastatic disease with a high mortality rate. Therefore, the development of relevant preclinical in vivo models that accurately recapitulate the metastatic cascade is crucial. We exploited the chick embryo chorioallantoic membrane (CAM) xenograft model to quantify both experimental and spontaneous metastasis by qPCR analysis. Our study found that the transplanted UM cells spread predominantly and early in the liver, reflecting the primary site of metastasis in patients. Visible signs of pigmented metastasis were observed in the eyes, liver, and distal CAM. Lung metastases occurred rarely and brain metastases progressed more slowly. However, UM cell types of different origins and genetic profiles caused an individual spectrum of organ metastases. Metastasis to multiple organs, including the liver, was often associated with risk factors such as high proliferation rate, hyperpigmentation, and epithelioid cell type. The severity of liver metastasis was related to the hepatic metastatic origin and chromosome 8 abnormalities rather than monosomy 3 and BAP1 deficiency. The presented CAM xenograft model may prove useful to study the metastatic potential of patients or to test individualized therapeutic options for metastasis in different organs.

Metastasis is a complex, multistep process. To study the molecular steps of the metastatic cascade, it is important to use an in vivo system that recapitulates the complex tumor microenvironment. The chicken embryo chorioallantoic membrane (CAM) is an in vivo system suitable for the implantation of xenograft tumor models. It allows the study of different aspects of the metastatic process, including the dormancy-awakening transition. The main advantages of this system are its high reproducibility, cost-effectiveness, and versatility. Here, by using two dormancy tumor models, one of head and neck squamous cell carcinoma and one of breast cancer, we described a detailed protocol for the use of the CAM model in metastasis assays and for the study of tumor growth and dormancy.

Cannabidiol (CBD) is the main non-psychotropic cannabinoid. It has attracted a great deal of interest in the treatment of several diseases such as inflammatory disorders and cancer. Despite its promising clinical interest, its administration is very challenging. In situ forming implants (ISFIs) could be a simple and cheap strategy to administer CBD while obtaining a prolonged effect with a single administration. This work aims to design, develop, and characterize for the first time ISFIs for the parenteral administration of CBD with potential application in cancer disease. Formulations made of PLGA-502, PLGA-502H, and PLA-202 in NMP or DMSO and PLA-203 in DMSO at a polymer concentration of 0.25 mg/µL and loaded with CBD at a drug: polymer ratio of 2.5:100 and 5:100 (w/w) were developed. The formulations prepared with NMP exhibited a faster drug release. CBD implants elaborated with PLGA-502 and DMSO with the highest CBD: polymer ratio showed the most suitable drug release for one month. This formulation was successfully formed in ovo onto the chorioallantoic chick membrane without exhibiting signs of toxicity and exhibited a superior antiangiogenic activity than CBD in solution administered at the same doses. Consequently, implants made of PLGA-502 and DMSO represent a promising strategy to effectively administer CBD subcutaneously as combination therapy in cancer disease.

The complement system, composed of complement components and complement control proteins, plays an essential role in innate immunity. Complement system molecules are expressed at the maternal-conceptus interface, and inappropriate activation of the complement system is associated with various adverse pregnancy outcomes in humans and rodents. However, the expression, regulation, and function of the complement system at the maternal-conceptus interface in pigs have not been studied. In this study, we investigated the expression, localization, and regulation of complement system molecules at the maternal-conceptus interface in pigs. Complement components and complement control proteins were expressed in the endometrium, early-stage conceptus, and chorioallantoic tissues during pregnancy. The expression of complement components acting on the early stage of complement activation increased in the endometrium on Day 15 of pregnancy, with greater levels on that day compared with the estrous cycle. Localization of several complement components and complement control proteins was cell-type specific in the endometrium. The expression of C1QC, C2, C3, C4A, CFI, ITGB2, MASP1, and SERPING1 was increased by IFNG in endometrial explant tissues. Furthermore, cleaved C3 fragments were detected in endometrial tissues and uterine flushings on Day 15 of the estrous cycle and Day 15 of pregnancy, with greater levels on Day 15 of pregnancy. These results suggest that complement system molecules in pigs expressed at the maternal-conceptus interface play important roles in the establishment and maintenance of pregnancy by regulating innate immunity and modulating the maternal immune environment during pregnancy.

Clinical therapies, including dermatology and oncology, require safe application. In vitro experiments allow only limited conclusions about in vivo effects, while animal studies in, e.g., rodents have ethical constraints at a large scale. Chicken embryos lack pain reception until day 15 postfertilization, making the in ovo model a suitable alternative to in vivo safety assessment. In addition, the hen\'s egg test on chorioallantoic membrane assay allows irritation potential analysis for topical treatments, but standardized analysis has been limited so far. Medical gas plasma is a topical, routine, approved dermatology treatment. Recent work suggests the potential of this technology in oncology. Its main mode of action is the release of various reactive species simultaneously. Intriguingly, varying plasma feed gas compositions generates customized reactive species profiles previously shown to be optimized for specific applications, such as skin cancer treatment. To support clinical implications, we developed a novel chicken embryo CAM scoring and study scheme and employed the model to analyze 16 different plasma feed gas settings generated by the atmospheric pressure plasmajet kINPen, along with common anticancer drugs (e.g., cisplatin) and physiological mediators (e.g., VEGF). Extensive gas- and liquid-phase plasma reactive species profiling was done and was found to have a surprisingly low correlation with irritation potential parameters. Despite markedly different reactive species patterns, feed gas-modulated kINPen plasma was equally tolerated compared to standard argon plasma. CAM irritation with gas plasmas but not anticancer agents was reversed 48 h after treatment, underlining the only temporary tissue effects of medical gas plasma. Our results indicate a safe therapeutic application of reactive species.

Crotalus snakebites induce various toxicological effects, encompassing neurological, myotoxic, and cytotoxic symptoms, with potentially fatal outcomes. Investigating venom toxicity is essential for public health, and developing new tools allows for these effects to be studied more comprehensively. The research goals include the elucidation of the physiological consequences of venom exposure and the assessment of toxicity using animal models. Chicken embryos serve as valuable models for assessing venom toxicity through the chick embryotoxicity screening test (CHEST) and the chick chorioallantoic membrane (CAM) assay, particularly useful for evaluating vascular impacts. C. adamanteus venom application resulted in higher embryotoxicity and morphological abnormalities, such as Siamese twins. The CAM assay demonstrated the hemorrhagic effects of venom, varying with venom type and concentration. The irritant potential of both venom types was classified as slight or moderate depending on their concentration. Additionally, acetylcholinesterase (AChE) activity was performed to receive information about organ toxicity. The results show that both venoms induced changes in the whole embryo, heart, and liver weights, but the C. adamanteus venom was identified as more toxic. Specific venom concentrations affected AChE activity in embryonic tissues. These findings underscore the embryotoxic and vasoactive properties of Crotalus venoms, providing valuable insights into their mechanisms of toxicity and potential applications in biomedicine.

Herein, we synthesized hydrogel films from crosslinked polyethylene oxide (PEO) and guar gum (GG) which can offer hydrophilicity, antibacterial efficacy, and neovascularization. This study focuses on synthesis and material/biological characterization of rosemary (RM) and citric acid (CA) loaded PEO/GG hydrogel films. Scanning Electron Microscopy images confirmed the porous structure of the developed hydrogel film matrix (PEO/GG) and the dispersion of RM and CA within it. This porous structure promotes moisture adsorption, cell attachment, proliferation, and tissue layer formation. Fourier Transform Infrared Spectroscopy (FTIR) further validated the crosslinking of the PEO/GG matrix, as confirmed by the appearance of C-O-C linkage in the FTIR spectrum. PEO/GG and PEO/GG/RM/CA revealed similar degradation and release kinetics in Dulbecco\'s Modified Eagle Medium, Simulated Body Fluid, and Phosphate Buffer Saline (degradation of ∼55 % and release of ∼60 % RM in 168 h.). The developed hydrogel film exhibited a zone of inhibition against Escherichia. coli (2 mm) and Staphylococcus. aureus (9 mm), which can be attributed to the presence of RM in the hydrogel film. Furthermore, incorporating CA in the hydrogel film promoted neovascularization, as confirmed by the Chorioallantoic Membrane Assay. The developed RM and CA-loaded PEO/GG-based hydrogel films offered suitable in-vitro properties that may aid in potential wound healing applications.

Uveal melanoma is one of the most common and aggressive intraocular malignancies, and, due to its great capability of metastasize, it constitutes the most incident intraocular tumor in adults. However, to date there is no effective treatment since achieving the inner ocular tissues still constitutes one of the greatest challenges in actual medicine, because of the complex structure and barriers. Uncoated and PEGylated nanostructured lipid carriers were developed to achieve physico-chemical properties (mean particle size, homogeneity, zeta potential, pH and osmolality) compatible for the ophthalmic administration of (S)-(-)-MRJF22, a new custom-synthetized prodrug for the potential treatment of uveal melanoma. The colloidal physical stability was investigated at different temperatures by Turbiscan® Ageing Station. Morphology analysis and mucoadhesive studies highlighted the presence of small particles suitable to be topically administered on the ocular surface. In vitro release studies performed using Franz diffusion cells demonstrated that the systems were able to provide a slow and prolonged prodrug release. In vitro cytotoxicity test on Human Corneal Epithelium and Human Uveal Melanoma cell lines and Hen\'s egg-chorioallantoic membrane test showed a dose-dependent cytotoxic effect of the free prodrug on corneal cells, whose cytocompatibility improved when encapsulated into nanoparticles, as also confirmed by in vivo studies on New Zealand albino rabbits. Antiangiogenic capability and preventive anti-inflammatory properties were also investigated on embryonated eggs and rabbits, respectively. Furthermore, preliminary in vivo biodistribution images of fluorescent nanoparticles after topical instillation in rabbits\' eyes, suggested their ability to reach the posterior segment of the eye, as a promising strategy for the treatment of choroidal uveal melanoma.