Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Summary

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Abstract

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.

The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature.

Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

Introduction

Metastasis is the main cause of cancer patient death^1,2,3. Metastatic cancer cells utilize distinct signaling pathways, dependent on the type of cancer, throughout the five steps of the metastatic cascade: local invasion, intravasation, survival in the circulation, extravasation, and colony expansion at distant metastatic sites. Current understanding of this metastatic process suggests that there are two bottleneck steps, one is directional invasion of the cancer cell from the primary tumor, and the second is the establishment of the distant site metastatic lesion

Protocol

All experiments were performed in accordance with the regulations and guidelines of the Institutional Animal Care and Use Committee at the University of Alberta. Avian embryos are not considered to be live animals by many research institutes and no animal protocols are required. It is, however, an accepted view that avian embryos can feel pain and, therefore, must be treated as humanely as possible. The local animal research authority must be contacted prior commencing of any research work to ensure that proper regulations are followed.

1. Shell-less egg culture

1. Acquire fertilized chicken eggs from the local provider. This exper....

Results

Cancer cell injection is considered to be successful if the majority of the cells that are lodged in the capillaries are single and located at a significant difference from each other (~0.05-0.1 cm) so the colonies will not overlap after 5-6 days of incubation period (Figure 3A). The injection was not successful if a buildup of cancer cells can be seen in most of capillaries, embryos displaying this should be discarded (Figure 2E.......

Discussion

Here we describe a rapid fluorescence microscopy based intravital screening protocol that can be utilized for important applications such as genetic or drug candidate screens. Cancer cells that have been transduced with a genetic library of interest or transfected with individual expression constructs can be rapidly screened and quantified as to phenotype of interest using this chick CAM model. Since transduction or transfection protocols vary significantly, depending on the library type, they are not included in this pr.......

Materials

Name	Company	Catalog Number	Comments
1 mL disposable syringes	BD	BD309659
15 mL conical centrifuge tubes.	Corning	CLS430791-500EA
18 gauge x 1 1/2  BD precision needle	BD	BD305196	we use 1.2mm x 40mm, it is possible to use shorter needles if preferred
2.5% Trypsin solution	many sources are available
4T1 mouse breast cancer	ATCC	CRL-2539
B16F10 mouse melanoma cell line	ATCC	CRL-6475
Benchtop centrifuge.	many sources are available		Any TC compatible centrifuge that can be used to spin down the cells is suitable
Circular coverslips, 22 mm.	Fisher Scientific	12-545-101
Collagenase	Sigma	C0130-100MG
Confocal microscope			We use Nikon A1r
cotton swabs	many sources are available		must be sterilized before use
Culture media appropriate for the cell lines used	many sources are available		We grow HT1080, HEp3 and b16 cell lines in DMEM, 10% FBS media
Egg incubator	many sources are available		An exact model that is necessary depends on the scale of the screen. Available sources are MGF Company Inc., Savannah, GA, or  Lyon Electric Company Inc., Chula Vista, CA
eppendor tubes , 1.5ml	Sigma	T4816-250EA
Fertilized White Leghorn eggs	any local supplyer
fine forceps	many sources are available		must be sterilized begfore use
Hemocytometer	Millipore-Sigma	MDH-4N1-50PK
HT1080 human fibrosarcoma cell line	ATCC	CCL-121
Image analysis software			We use Nikon Elements
Lectin Lens Culinary Agglutinin (LCA) conjugated with Fluorescein or Rhodamine	Vector Laboratories	RL-1042, FL-1041	Dilute stock (5mg/ml) 50-100x depending on the microscope sensetivity. Must be a different color from the color of cell line used for screening
MDA-MB-468 human breast cancer	ATCC	HTB-132
PBS (1x)	many sources are available
Plastic weighting dishes	Simport	CA11006-614	dimensions are 78x78x25mm; many other sources are available
small surgical scissors	many sources are available		must be sterilized before use
Sodium borosilicate glass capillary tubes, outer diameter 1.0 mm, inner diameter 0.58 mm, 10 cm length	Sutter Instrument	BF100-58-10
Square petri dishes (used as lids for the weighting dishes).	VWR	CA25378-115	dimensions are 100x100x15mm; many other sources are available
Stereo fluorescent microscope			We use Zeiss Lumar v12
Tygon R-3603 laboratory tubing	Cole-Parmer	AAC00001	1/32 in inner diameter, 3/32 in. outer diameter, 1/32 in. wall thickness
U-118 MG human glioblastoma	ATCC	HTB-15
U-87 MG human glioblastoma	ATCC	HTB-14
Vertical pipette puller	many sources are available		we use David Kopf Instruments, Tujunga, CA; Model 720