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

Abstract

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.

Biosynthesized Silver Nanoparticles for Cancer Therapy and In Vivo Bioimaging

“…The chicken egg-based CAM assay model is a well-established study for evaluating anticancer activity. Here, only the nanoparticles are incubated to visualize the effect of blood vessel inhibition by the nanoparticles for prevention of tumor angiogenesis [ 43 , 44 ]. Based on the in vitro anticancer activity of AgZE; the anticancer activity of AgZE is further validated by CAM assay, an in vivo approach.…”

Combination of Resminostat with Ruxolitinib Exerts Antitumor Effects in the Chick Embryo Chorioallantoic Membrane Model for Cutaneous T Cell Lymphoma

“…The chick embryo CAM model was developed as previously described [ 21 , 22 ], by first preparing the eggs for xenografting tumor cells, then preparing tumor cells for grafting, grafting the tumor cells onto the CAM, and, finally, harvesting tumors and chick embryo tissues. More specifically, 10 6 MyLa or SeAx cells were injected onto CAM embryos and induced tumor formation.…”

“…In the present study, we have initially investigated the preclinical experimental model by implanting MF/SS cells in the Chick Embryo Chorioallantoic Membrane (CAM) [ 21 , 22 ]. This experimental model has become an appealing tool for in vivo assays for drug testing, tumor growth, and metastasis [ 23 ].…”

Replacement in angiogenesis research: Studying mechanisms of blood vessel development by animal-free in vitro, in vivo and in silico approaches

“…In the classic in ovo assay, a circular, sealable observation window is prepared into the eggshell for the repeated analysis of the CAM ( Schmitd et al, 2019 ). Alternatively, ex ovo assays with shell-less embryo cultures have been introduced, which enable an easier access to larger areas of the CAM ( Vargas et al, 2009 ; Merlos Rodrigo et al, 2021 ). On the other hand, these assays require a more demanding preparation and complex incubation environment and, thus, also bear a higher risk of infection and embryo mortality.…”