Feasibility of the chick chorioallantoic membrane model for preclinical studies on tumor radiofrequency ablation

Abstract

Background: We evaluated the feasibility of a chick chorioallantoic membrane (CAM) tumor model for preclinical research on tumor radiofrequency ablation (RFA).

Methods: Fertilized chicken eggs were incubated and divided into five cohorts: RFA for 30 s (n = 5), RFA for 60 s (n = 5), RFA for 120 s (n = 4), sham (n = 8), and controls (n = 6). Xenografting using pancreatic neuroendocrine tumor cells of the BON-1 cell line was performed on embryonic day (ED) 8. The RFA was performed on ED 12. Survival, stereomicroscopic observations, and histological observations using hematoxylin-eosin (H&E) and Ki67 staining were evaluated.

Results: The survival rates in the 30-s, 60-s, and 120-s, sham and control cohort were 60%, 60%, 0%, 100%, and 50%, respectively. Signs of bleeding and heat damage were common findings in the evaluation of stereomicroscopic observations. Histological examination could be performed in all but one embryo. Heat damage, bleeding, thrombosis, and leukocyte infiltration and hyperemia were regular findings in H&E-stained cuts. A complete absence of Ki67 staining was recorded in 33.3% and 50% of embryos in the 30-s and 60-s cohorts that survived until ED 14, respectively.

Conclusions: The CAM model is a feasible and suiting research model for tumor RFA with many advantages over other animal models. It offers the opportunity to conduct in vivo research under standardized conditions. Further studies are needed to optimize this model for tumor ablations in order to explore promising but unrefined strategies like the combination of RFA and immunotherapy.

Relevance statement: The chick chorioallantoic membrane model allows in vivo research on tumor radiofrequency ablation under standardized conditions that may enable enhanced understanding on combined therapies while ensuring animal welfare in concordance with the "Three Rs."

Key points: • The chorioallantoic membrane model is feasible and suiting for tumor radiofrequency ablation. • Radiofrequency ablation regularly achieved reduction but not eradication of Ki67 staining. • Histological evaluation showed findings comparable to changes in humans after RFA. • The chorioallantoic membrane model can enable studies on combined therapies after optimization.

Keywords: Chickens; Chorioallantoic membrane; Neuroendocrine tumors; Radiofrequency ablation; Radiology (interventional).

Fig. 1

Overview of methods. Parts of the figure were drawn using pictures from Servier Medical Art (smart.servier.com), provided by Servier, licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/). ED Embryonic day, RFA Radiofrequency ablation

Fig. 2

Stereomicroscopic observations. a Tumor cell formation (arrow) is visible inside the silicone ring on the CAM on ED 12 before RFA. b Signs of heat damage (*) and bleeding (arrow, bordered by dashed line) can be seen 2 h after ablation on ED 12. No tumor cells are visible on a chick embryo of the control cohort before the RFA on ED 12 (c), while signs of bleeding (arrow, bordered by dashed line) and heat damage (*) are displayed 2 h after ablation (d). CAM Chorioallantoic membrane, ED Embryonic day, RFA Radiofrequency ablation

Fig. 3

(Immune-)histological observations. a and b display a cluster of BON-1 cells (arrow) in 40 × magnification H&E staining (a) and Ki67 IHC (b) in a chick embryo on which no ablation was performed on. Vital BON-1 cells appear larger and more basophilic in H&E staining (a) than surrounding tissue and Matrigel (*) and also show a high nuclear to cytoplasmatic ratio. Proliferating tumor cells show brown nuclei in Ki67 IHC, while surrounding chicken cells are negative because the antibody does not bind to chicken Ki67 (b). c depicts the overview of an ablated chorioallantoic membrane (CAM) in H&E (10 × magnification). In the corresponding Ki67 staining, a single Ki67-positive cell (arrow) can be seen within the Matrigel (d, 10 × magnification). Common findings after RFA were thrombosis (e) and the presence of leukocytes and erythrocytes indicating inflammatory response with leukocyte infiltration (f). Black scale bars in a, b, e, and f (40 × magnification) indicate 50 μm, while black scale bars in c and d (10 × magnification) indicate 100 μm. CAM Chorioallantoic membrane, ED Embryonic day, H&E Hematoxylin–eosin, IHC Immunohistochemistry, RFA Radiofrequency ablation