The chick chorioallantoic membrane as an in vivo xenograft model for Burkitt lymphoma

Abstract

BackgroundBurkitt lymphoma (BL) is an aggressive malignancy that arises from B-cells and belongs to the group of Non-Hodgkin lymphomas (NHL). Due to the lack of appropriate in vivo models NHL research is mainly performed in vitro. Here, we studied the use of the chick chorioallantoic membrane (CAM) for the generation of human BL xenograft tumors, which we compared with known characteristics of the human disease.MethodsIn order to generate experimental BL tumors, we inoculated human BL2B95 and BL2-GFP cells on the CAM. BL2B95 xenograft-tumors were grown for seven days and subsequently analyzed with transmission electron and immunofluorescence microscopy, as well as histological staining approaches. BL2-GFP cells were studied at regular intervals up to seven days, and their metastatic behavior was visualized with intravital immunofluorescence techniques.ResultsXenografted BL2B95 cells formed solid tumors in the CAM model with a Ki67-index greater than 90%, preservation of typical tumor markers (CD10, CD19, CD20), a ‘starry sky’ morphology, production of agyrophilic fibers in the stroma, formation of blood and lymphatic vessels and lymphogenic dissemination of BL2B95 to distant sites. We identified macrophages, lymphocytes and heterophilic granulocytes (chick homolog of neutrophils) as the most abundant immune cells in the experimental tumors. BL2-GFP cells could be traced in real-time during their distribution in the CAM, and the first signs for their dissemination were visible after 2-3 days.ConclusionsWe show that xenografted BL2B95 cells generate tumors in the CAM with a high degree of cellular, molecular and proliferative concord with the human disease, supporting the application of the CAM model for NHL research with a focus on tumor-stroma interactions. Additionally we report that BL2-GFP cells, grafted on the CAM of ex ovo cultured chick embryos, provide a powerful tool to study lymphogenic dissemination in real-time.

Multi-Modal PET and MR Imaging in the Hen’s Egg Test-Chorioallantoic Membrane (HET-CAM) Model for Initial In Vivo Testing of Target-Specific Radioligands

“…Since no approval for animal testing is required in most countries, a study based on HET-CAM can start much faster than a comparable study in mice. The HET-CAM model allows the easy introduction of various tumor models, not only based on human cell lines but also on rodent cell lines or patient-derived material [15,[29][30][31]39,[83][84][85]. This facilitates the transfer from an established mouse model to the HET-CAM model for at least initial evaluation.…”

Proteomic Differences in Feline Fibrosarcomas Grown Using Doxorubicin-Sensitive and -Resistant Cell Lines in the Chick Embryo Model

“…The chick embryo chorioallantoic membrane (CAM) model is a preclinical in vivo model that follows the “3R” (reduction, replacement, refinement) guidelines, which need to be strictly followed after the implementation of directive 2010/63/EU on the protection of animals used for scientific purposes. The CAM model is widely used for angiogenesis assays, acute toxicological studies, and studies of neoplastic cell extravasation, bone regeneration, and molecular cancer biology [ 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. In veterinary medicine, it has been applied in canine osteosarcoma, canine soft-tissue sarcoma, canine mammary gland tumors, feline mammary carcinoma, and feline fibrosarcoma research [ 35 , 36 , 37 , 38 , 39 ].…”

High CD206 levels in Hodgkin lymphoma‐educated macrophages are linked to matrix‐remodeling and lymphoma dissemination

“…To test the interaction of lymphoma cells with monocytes or Mφ in an in vivo setting, we used the CAM assay as it recapitulates important aspects of human lymphoma as shown recently (Klingenberg et al , , ). One million Mφ derived by incubation of monocytes with HL‐CM were mixed with 2 × 10 6 lymphoma cells (L428, L1236) in Matrigel and applied on the CAM.…”

“…Chicken eggs (Valo BioMedia GmbH, Osterholz‐Scharmbeck, Germany) were bred and regularly turned for 3 days at 37.8 °C and 80% humidity as described recently (Klingenberg et al , ; Klingenberg et al , ).On day 3, a window was sawed into the egg and sealed with sellotape. On day 10, 2 × 10 6 L428 cells with or without 1 × 10 6 CD14 + PBMCs or corresponding 1 × 10 6 Mφ embedded in 20 µL Matrigel (BD Biosciences) were inoculated onto the CAM.…”