The Chick Chorioallantoic Membrane (CAM) Assay as a Three-dimensional Model to Study Autophagy in Cancer Cells

Background

The chick chorioallantoic membrane (CAM) is a highly vascularized extra-embryonic membrane that has multiple functions during embryonic development, such as gas exchange (Romanoff, 1960). It is a well-known model to study blood vessels and angiogenesis in the context of tumor biology (Kain et al., 2014; Nowak-Sliwinska et al., 2014). The chick development in the egg lasts 21 days (Romanoff, 1960). Three days after starting incubating the eggs, we cut a small round window into the eggshell for easy access to the CAM later in the procedure (Figure 1). At this time point the membrane is not yet formed and thus there is no risk to damage it. On Day 7 of embryo development the tumor cells are seeded on the CAM and seven days later (Day 14) the microtumors that developed on the membrane are harvested and analyzed. To this end, the tumors are excised, fixed, dehydrated, embedded and cut to apply immunohistochemical stainings for the autophagy-related markers: microtubule associated protein 1 light chain 3 beta (MAP1LC3B, short LC3B) and sequestosome 1 (SQSTM1, aka p62).

Experimentally assessing autophagy is challenging, since it is a dynamic process, and steady state expression levels of autophagy related genes do not give a complete picture of the active autophagic process (Barth et al., 2010). In tissue, dot like staining of LC3B and p62 is considered a better indication of autophagic flux, defined as the degradation rate of cargo via autophagy (Martinet et al., 2013). Our group previously established an automated LC3B and p62 staining and scoring protocol for tumor tissue (Schläfli et al., 2015), which we apply here on microtumors generated in the CAM model. Using a 3D CAM xenograft model to broaden our knowledge on autophagy in tumor biology has several advantages. It is easy accessible for manipulation (addition of an autophagy blocking agent), for microscopy (for instance to analyze the growth and vascularization of the tumors) and it provides small tumors that are suitable for immunohistochemical analyses. Thus, the expression of different markers can be visualized in the tissue.


Figure 1. Overview of the workflow. The development of the chicken in the egg takes in total 21 days. At Day 3 of incubation, when the CAM is not yet formed a window is cut into the eggshell. At Day 7 of incubation when the membrane is visible but not yet completely developed the cancer cells are seeded in a collagen scaffold and then transferred to the membrane. After another 7 days in the incubator, at Day 14 of development the tumor is harvested, fixed and embedded.