Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

Summary

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

Abstract

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Protocol

1. Preparing the eggs for xenografting (spontaneous metastasis)

1. Freshly laid fertilized chicken eggs are incubated in a rotating incubator for 10 days at 100°F and 60% humidity. The eggs are rotated four times each hour.
2. On developmental day 10 the eggs are placed on their side in an egg rack. A goose-neck lamp or other suitable light source is used to candle the eggs by shining the light into the eggshell at the blunt end of the egg where the airsac is located.
3. The chorioallantoic vein is located and marked. This vein is located at the top of the eggshell where it is the junction of several large blood vessels. At this branch point the b....

Discussion

The chick CAM has been used for decades as a model system to study various aspects of cancer biology and metastatic progression. Principle evaluation of metastatic abilities have been performed in this model by a variety of groups including the analysis of dormancy (Aguirre Ghiso et al., 1999; Ossowski and Reich, 1983), tissue remodeling (Deryugina et al., 2005; Hahn-Dantona et al., 1999), and metastasis (Zijlstra et al., 2008; Zijlstra et al., 2002). It continues to be a very.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1X Dulbecco’s Modified Eagle Medium (DMEM)	Invitrogen	11995073
Dulbecco’s Phosphate-Buffered Saline (D-PBS) (1X), liquid	Invitrogen	14190250	pH 7.4
Trypsin, 0.05% (1X) with EDTA 4Na, liquid	Invitrogen	25300054
Sportsman hatcher	Berry Hill	1550HA	These incubators are some of the most readily available. However, the video will show different equipment.
Sportsman incubator	Berry Hill	1502EA	These incubators are some of the most readily available. However, the video will show different equipment.
Dremel rotary tool	Dremel
Dremel cutoff wheels no. 36	Dremel	409
Portable Pipette Aid	Fisher Scientific	1368115E
Cotton Tipped Applicators	Fisher Scientific	23-400-001)
Fertilized Eggs	Various		Many different vendor can be used. The eggs should be approximately 35-55 grams. The hens that lays them should be between 36 and 55 weeks of age.
Hemocytometer	Hausser Scientific	15170-090
Fiber-optic microscope illuminator	Amscope	HL250-AY	150W
25 gauge needle
Sybr Green Extract—N-Amp Tissue PCR Kit	Sigma-Aldrich	XNATRG