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Cancer Research

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

Published: February 3rd, 2021

DOI:

10.3791/62077

1Department of Oncology, University of Alberta, Edmonton, Alberta, Canada, 2Department of Cell Biology & Anatomy, Cumming School of Medicine, University of Calgary

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.

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.

Metastasis is the main cause of cancer patient death1,2,3. 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.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

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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 regulatio.......

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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.......

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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.......

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This work was supported by Canadian Cancer Society Research Institute Grant #702849 to JDL and KS. Dr. Lewis holds the Frank and Carla Sojonky Chair in Prostate Cancer Research supported by the Alberta Cancer Foundation.

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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

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