The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma
Summary
The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.
Abstract
The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.
Introduction
Hepatocellular carcinoma (HCC) is the 3rd leading cause of cancer mortality in the world1. Currently only 30% of HCC patients are eligible for potentially curative surgical treatments2, and systemic chemotherapy is not efficacious3. Therefore, there is a pressing unmet clinical need for novel HCC therapies, and the development of model systems suitable for efficient testing of new agents. The chick chorioallantoic membrane (CAM) assay provides a reproducible, cost-effective, and fast medium-throughput method of testing potential anti-tumor drugs in vivo.
The CAM assay has been used extensively to study angiogenesis4. It has also been successfully developed into a tumor xenograft model of cancers, including glioblastoma5, pancreatic cancer6, melanoma7-9, and osteosarcoma10-11. Both in ovo12 and ex ovo13 techniques have been utilized in the literature, with details varying from protocol to protocol. One major challenge to the CAM xenograft model is the relatively high incidence of embryonic death after manipulation of the egg, with published chick embryo mortality rates ranging from 25 – 50 percent11-14.
In this article, we describe the development of an in ovo xenograft model of HCC that reliably produces growth of three-dimensional, vascularized tumors that histologically resemble undifferentiated HCC. We have adapted a protocol first described by Ossowski et al.14 and have achieved chick embryonic survival rates of up to 93% with extremely high tumor engraftment.
Protocol
Representative Results
Discussion
Several key steps in this protocol most likely account for the improved embryonic survival as well as increased reliability of tumor growth. Dropping the CAM away from the shell by applying suction to the air sac is less invasive than other existing methods (using a needle to remove albumin from the egg, blunt dissection, etc.). Using a sterile push pin to create the two small holes required for this method is the most challenging part of the protocol. Using too much force can result in damage to the CAM and its vasculat…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Premium incubated eggs | Charles River | N/A | http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx |
| Egg incubators | GQF | Hova-Bator 2362N | |
| Rotating egg trays | GQF | 1611 Automatic Egg Turner | |
| Egg candler | Lyon | Hi-Power 950-070 | |
| Dremel 100 rotary tool with 15/16 cut-off wheel | Dremel | 100-N/7 | |
| Sterile forceps, push pin, dissection scissors, Scotch tape | |||
| Matrigel | BD Biosciences | 356234 | |
| Cryogenic vials, external thread with silicone washer | Corning | 430659 | |
| Collagenase from Clostridium histolyticum | Sigma | C9891 |
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