scholarly journals Monitoring of Tumor Growth and Vascularization with Repetitive Ultrasonography in the Chicken Chorioallantoic-Membrane-Assay.

2020 ◽  
Author(s):  
Jonas Eckrich ◽  
Philipp Kugler ◽  
Christoph Raphael Buhr ◽  
Benjamin Philipp Ernst ◽  
Simone Mendler ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tumor Angiogenesis ◽  
Imaging Techniques ◽  
Tumor Development ◽  
Three Dimensional ◽  
Chorioallantoic Membrane ◽  
Shielding Effect ◽  
Cam Assay ◽  
In Ovo ◽  
Cost Efficient

Abstract Background: The chorioallantoic-membrane (CAM)-assay is used for versatile experimentation and eligible for the analysis of tumor angiogenesis, development and metastasis. In contrast to rodent xenograft models, the CAM-assay does not require breeding of immunodeficient strains for tumor experimentation due to native immunodeficiency. This allows xenografts to grow on the non-innervated CAM without pain or impairment for the embyo.Taking into account the variability of multidirectional tumor growth, limited size monitoring capability is a major disadvantage of the CAM-assay as the enclosure of the tumor in ovo by the eggshell only allows for two-dimensional monitoring from above. The small size and the eggshell’s shielding effect further challenge established imaging techniques. We report the eligibility of ultrasonographic imaging for repetitive monitoring of tumor growth and vascularisation in the CAM-assay.Methods: Chicken eggs were placed in an incubator and cut open laterally on day three. On day seven a three-dimensional tumor was placed onto the CAM. Ultrasonographic imaging was then repetitively performed starting from day twelve. On day 14 the tumor was excised, fixed and histologically analyzed using light microscopy.Results: Tumor volume and vascularization were repetitively visualized using a commercial ultrasonographic scanner, allowing a longitudinal monitoring of tumor growth and tumor angiogenesis. Findings in ultrasonographic imaging significantly correlated with results obtained in histological analysis. Conclusion: Ultrasonography is cost efficient and widely available. It allows repetitive in ovo imaging and thereby enables visualization of tumor development. This increases the applicability of the CAM-assay as an alternative to xenograft rodent models in tumor research.

scholarly journals Monitoring of tumor growth and vascularization with repetitive ultrasonography in the chicken chorioallantoic-membrane-assay

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jonas Eckrich ◽  
Philipp Kugler ◽  
Christoph Raphael Buhr ◽  
Benjamin Philipp Ernst ◽  
Simone Mendler ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tumor Imaging ◽  
Imaging Techniques ◽  
Chorioallantoic Membrane ◽  
Cam Assay ◽  
In Ovo ◽  
Tumor Research ◽  
Xenograft Models ◽  
Cost Efficient

Abstract The chorioallantoic-membrane (CAM)-assay is an established model for in vivo tumor research. Contrary to rodent-xenograft-models, the CAM-assay does not require breeding of immunodeficient strains due to native immunodeficiency. This allows xenografts to grow on the non-innervated CAM without pain or impairment for the embryo. Considering multidirectional tumor growth, limited monitoring capability of tumor size is the main methodological limitation of the CAM-assay for tumor research. Enclosure of the tumor by the radiopaque eggshell and the small structural size only allows monitoring from above and challenges established imaging techniques. We report the eligibility of ultrasonography for repetitive visualization of tumor growth and vascularization in the CAM-assay. After tumor ingrowth, ultrasonography was repetitively performed in ovo using a commercial ultrasonographic scanner. Finally, the tumor was excised and histologically analyzed. Tumor growth and angiogenesis were successfully monitored and findings in ultrasonographic imaging significantly correlated with results obtained in histological analysis. Ultrasonography is cost efficient and widely available. Tumor imaging in ovo enables the longitudinal monitoring of tumoral development, yet allowing high quantitative output due to the CAM-assays simple and cheap methodology. Thus, this methodological novelty improves reproducibility in the field of in vivo tumor experimentation emphasizing the CAM-assay as an alternative to rodent-xenograft-models.

scholarly journals The Chorioallantoic Membrane Assay in Nanotoxicological Research—An Alternative for In Vivo Experimentation

Nanomaterials ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 2328
Author(s):  
Christoph R. Buhr ◽  
Nadine Wiesmann ◽  
Rachel C. Tanner ◽  
Jürgen Brieger ◽  
Jonas Eckrich
Keyword(s):  
Imaging Techniques ◽  
Chorioallantoic Membrane ◽  
Drug Carriers ◽  
Human Organism ◽  
Cam Assay ◽  
In Ovo ◽  
In Vivo Models ◽  
Toxicological Profile ◽  
Application Fields

Nanomaterials unveil many applicational possibilities for technical and medical purposes, which range from imaging techniques to the use as drug carriers. Prior to any human application, analysis of undesired effects and characterization of their toxicological profile is mandatory. To address this topic, animal models, and rodent models in particular, are most frequently used. However, as the reproducibility and transferability to the human organism of animal experimental data is increasingly questioned and the awareness of animal welfare in society increases at the same time, methodological alternatives are urgently required. The chorioallantoic membrane (CAM) assay is an increasingly popular in ovo experimental organism suitable for replacement of rodent experimentation. In this review, we outline several application fields for the CAM assay in the field of nanotoxicology. Furthermore, analytical methods applicable with this model were evaluated in detail. We further discuss ethical, financial, and bureaucratic aspects and benchmark the assay with other established in vivo models such as rodents.

scholarly journals The Role of Pre-Clinical 3-Dimensional Models of Osteosarcoma

2020 ◽  
Vol 21 (15) ◽  
pp. 5499
Author(s):  
Hannah L. Smith ◽  
Stephen A. Beers ◽  
Juliet C. Gray ◽  
Janos M. Kanczler
Keyword(s):  
Three Dimensional ◽  
Chorioallantoic Membrane ◽  
3D Models ◽  
In Ovo ◽  
Future Directions ◽  
3 Dimensional ◽  
In Vivo Animal Models

Treatment for osteosarcoma (OS) has been largely unchanged for several decades, with typical therapies being a mixture of chemotherapy and surgery. Although therapeutic targets and products against cancer are being continually developed, only a limited number have proved therapeutically active in OS. Thus, the understanding of the OS microenvironment and its interactions are becoming more important in developing new therapies. Three-dimensional (3D) models are important tools in increasing our understanding of complex mechanisms and interactions, such as in OS. In this review, in vivo animal models, in vitro 3D models and in ovo chorioallantoic membrane (CAM) models, are evaluated and discussed as to their contribution in understanding the progressive nature of OS, and cancer research. We aim to provide insight and prospective future directions into the potential translation of 3D models in OS.

scholarly journals Extending the Applicability of In Ovo and Ex Ovo Chicken Chorioallantoic Membrane Assays to Study Cytostatic Activity in Neuroblastoma Cells

2021 ◽  
Vol 11 ◽  
Author(s):  
Miguel Angel Merlos Rodrigo ◽  
Berta Casar ◽  
Hana Michalkova ◽  
Ana Maria Jimenez Jimenez ◽  
Zbynek Heger ◽  
...  
Keyword(s):  
High Throughput Screening ◽  
Neuroblastoma Cells ◽  
Chorioallantoic Membrane ◽  
Stage Iv ◽  
Metastatic Potential ◽  
Significant Inhibition ◽  
Cam Assay ◽  
Anticancer Activities ◽  
In Ovo

PurposeThe chick chorioallantoic membrane (CAM) assay can provide an alternative versatile, cost-effective, and ethically less controversial in vivo model for reliable screening of drugs. In the presented work, we demonstrate that CAM assay (in ovo and ex ovo) can be simply employed to delineate the effects of cisplatin (CDDP) and ellipticine (Elli) on neuroblastoma (Nbl) cells in terms of their growth and metastatic potential.MethodsThe Nbl UKF-NB-4 cell line was established from recurrent bone marrow metastases of high-risk Nbl (stage IV, MYCN amplification, 7q21 gain). Ex ovo and in ovo CAM assays were optimized to evaluate the antimetastatic activity of CDDP and Elli. Immunohistochemistry, qRT-PCR, and DNA isolation were performed.ResultsEx ovo CAM assay was employed to study whether CDDP and Elli exhibit any inhibitory effects on growth of Nbl xenograft in ex ovo CAM assay. Under the optimal conditions, Elli and CDDP exhibited significant inhibition of the size of the primary tumor. To study the efficiency of CDDP and Elli to inhibit primary Nbl tumor growth, intravasation, and extravasation in the organs, we adapted the in ovo CAM assay protocol. In in ovo CAM assay, both studied compounds (CDDP and Elli) exhibited significant (p < 0.001) inhibitory activity against extravasation to all investigated organs including distal CAM.ConclusionsTaken together, CAM assay could be a helpful and highly efficient in vivo approach for high-throughput screening of libraries of compounds with expected anticancer activities.

scholarly journals The Anti-Tumorigenic Activity of Sema3C in the Chick Embryo Chorioallantoic Membrane Model

2019 ◽  
Vol 20 (22) ◽  
pp. 5672 ◽  
Author(s):  
Valiulytė ◽  
Curkūnavičiūtė ◽  
Ribokaitė ◽  
Kazlauskas ◽  
Vaitkevičiūtė ◽  
...  
Keyword(s):  
Protein Function ◽  
Protein A ◽  
Tumor Development ◽  
Chorioallantoic Membrane ◽  
In Ovo ◽  
Promising Target ◽  
Anti Epileptic Drug ◽  
Chick Embryo Chorioallantoic Membrane ◽  
Angiogenesis Process ◽  
Tumorigenic Activity

Sema3C protein, a member of the class 3 family of secreted semaphorins, play an important role in tumor development by regulating cell proliferation, migration, invasion, and angiogenesis processes. Depending on the type and malignancy grade of the tumor, Sema3C function remains controversial. In this study, we constructed a stably overexpressing Sema3C glioblastoma cell line U87 MG and tested it on the chicken embryo chorioallantoic membrane (CAM) model with the aim to reveal Sema3C protein function on angiogenesis process in ovo. Our experiments showed that Sema3C not only affects angiogenesis of CAM by inhibiting neovascularization but also acts as an anti-tumorigenic molecule by hampering U87 MG cell invasion into mesenchyme. The effects of Sema3C on CAM were similar to the effects of anti-epileptic drug sodium valproate (NaVP). Both, anti-angiogenic and anti-tumorigenic activities of Sema3C were enhanced by the treatment of NaVP and, importantly, were not attributed to the cytotoxic effects. Our studies suggest that Sema3C could be a promising target for glioblastoma treatment.

scholarly journals The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

10.3791/52411 ◽  
2015 ◽  
Author(s):  
Michael Li ◽  
Ravi R. Pathak ◽  
Esther Lopez-Rivera ◽  
Scott L. Friedman ◽  
Julio A. Aguirre-Ghiso ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Chorioallantoic Membrane ◽  
Xenograft Model ◽  
Cam Assay ◽  
In Ovo ◽  
Chick Chorioallantoic Membrane

scholarly journals Determination of the Antiangiogenic Activity of Telescopium telescopium (horn snail) Extract using in ovo Chorioallantoic Membrane (CAM) Assay

2018 ◽  
Vol 52 (4) ◽  
Author(s):  
Milany Anne M. Luay ◽  
Mary France R. Gonzaga ◽  
Sharmaine Kae D. Po ◽  
Erna C. Arollado
Keyword(s):  
Crude Extract ◽  
Chorioallantoic Membrane ◽  
Vessel Diameter ◽  
Biochemical Screening ◽  
Cam Assay ◽  
In Ovo ◽  
Percent Inhibition ◽  
Butanol Fraction ◽  
Antiangiogenic Activity ◽  
Significant Difference

Objective. To determine the antiangiogenic activity of Telescopium telescopium (Horn snail) extract using in ovo chorioallantoic membrane (CAM) assay. Methods. Methanolic extract of Telescopium telescopium was subjected to modified Kupchan partitioning. Four treatment groups – negative control, positive control (quercetin), test samples, and blanks – were used for the in ovo chorioallantoic membrane (CAM) assay. ImageJ software was used to measure average vessel diameter (DV) and total length (LT) to determine the degree of vascularization, percent inhibition, and antiangiogenic activity. Biochemical screening was done for the crude extract and the fraction with the highest percent inhibition. Results. Butanol fraction showed the highest percent inhibition in both average vessel diameter (DV) (417.30% ± 300.83) and total length (L T) (44.21% ± 8.11). There was no significant difference in the antiangiogenic activity of both crude and butanol fraction compared with quercetin. Biochemical screening confirmed the presence of sterols and carbohydrates in both fractions. Conclusion. Crude extract and butanol fraction resulted in positive percent inhibition values, indicating inhibition of angiogenesis. They were found to have no significant difference with quercetin in regard to their antiangiogenic activity. Sterols were assumed as the biochemical class of the antiangiogenic compound of interest.

scholarly journals Novel multimodal MRI and MicroCT imaging approach to quantify angiogenesis and 3D vascular architecture of biomaterials

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Anna Woloszyk ◽  
Petra Wolint ◽  
Anton S. Becker ◽  
Andreas Boss ◽  
Weston Fath ◽  
...  
Keyword(s):  
Comprehensive Evaluation ◽  
Imaging Techniques ◽  
Three Dimensional ◽  
Chorioallantoic Membrane ◽  
High Tech ◽  
3D Analysis ◽  
Analysis Strategy ◽  
Vessel Volume ◽  
Magnetic Resonance Imaging Mri ◽  
Microct Imaging

AbstractQuantitative assessment of functional perfusion capacity and vessel architecture is critical when validating biomaterials for regenerative medicine purposes and requires high-tech analytical methods. Here, combining two clinically relevant imaging techniques, (magnetic resonance imaging; MRI and microcomputed tomography; MicroCT) and using the chorioallantoic membrane (CAM) assay, we present and validate a novel functional and morphological three-dimensional (3D) analysis strategy to study neovascularization in biomaterials relevant for bone regeneration. Using our new pump-assisted approach, the two scaffolds, Optimaix (laminar structure mimicking entities of the diaphysis) and DegraPol (highly porous resembling spongy bone), were shown to directly affect the architecture of the ingrowing neovasculature. Perfusion capacity (MRI) and total vessel volume (MicroCT) strongly correlated for both biomaterials, suggesting that our approach allows for a comprehensive evaluation of the vascularization pattern and efficiency of biomaterials. Being compliant with the 3R-principles (replacement, reduction and refinement), the well-established and easy-to-handle CAM model offers many advantages such as low costs, immune-incompetence and short experimental times with high-grade read-outs when compared to conventional animal models. Therefore, combined with our imaging-guided approach it represents a powerful tool to study angiogenesis in biomaterials.

scholarly journals The Effect of Sodium Valproate on the Glioblastoma U87 Cell Line Tumor Development on the Chicken Embryo Chorioallantoic Membrane and on EZH2 and p53 Expression

2017 ◽  
Vol 2017 ◽  
pp. 1-12 ◽  
Author(s):  
Dovilė Kavaliauskaitė ◽  
Donatas Stakišaitis ◽  
Justė Martinkutė ◽  
Lina Šlekienė ◽  
Arūnas Kazlauskas ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Sodium Valproate ◽  
Chicken Embryo ◽  
Tumor Development ◽  
Chorioallantoic Membrane ◽  
Tumor Formation ◽  
P53 Expression ◽  
Human Gbm ◽  
Patients Experience ◽  
Cam Model

Literature data support evidences that glioblastoma (GBM) patients experience prolonged survival due to sodium valproate (NaVP) treatment. The study assessed the human GBM cell U87 xenograft studied in the chicken embryo chorioallantoic membrane (CAM) model evaluating NaVP effect on tumor. Three groups of tumors (eachn= 10) were studied: nontreated, treated with 4 mM, and treated with 8 mM of NaVP. The majority of tumors without NaVP treatment during tumor growth destroyed the chorionic epithelium, invaded the mesenchyme, and induced angiogenesis. Incidence of tumor formation on CAM without invasion into the mesenchyme was higher when U87 cells were treated with NaVP; the effect significantly increased with NaVP concentration. Treatment with 8 mM of NaVP did not show clear dynamics of tumor growth during 5 days; at the same time, the angiogenesis failed. With a strong staining of EZH2, p53 in tumors without NaVP treatment was found, and NaVP significantly decreased the expression of EZH2- and p53-positive cells; the effect was significantly higher at its 8 mM concentration. NaVP has a function in blocking the growth, invasion, and angiogenesis of tumor in the CAM model; tumor growth interferes with EZH2 and p53 molecular pathways, supporting the NaVP potential in GBM therapy.

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