Experimental Metastasis Assay
August 24th, 2010
This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.
The overall goal of the following experiment is to test the metastatic abilities of human cancer cells by tail vein injections. In immunodeficient mice, the cells are prepared for injection. The cells are then injected into immunodeficient mice, which will generate lung metastases after one to two months.
The mice are dissected and lung metastases are quantified and cultured. Results of this type of assay indicate the extent of metastases of a particular type of cancer cell. Hi, I am Sonali Mohanty from Dr.LE's Lab in the Department of Biomedical Genetics at the University of Rochester.
Today we will show you a procedure to perform experimental metastasis assay. We use this procedure in our lab to study cancer metastasis. So let's get started.
Begin with cells grown to about 70%Co fluency aspirate media from the plate and gently wash once with one XPBS. Next, add two milliliters of 0.05%Trypsin inversing. Gently rock the plate to facilitate cell detachment.
Observe the cells under a microscope as they detach. Add an ample volume of media containing FBS to quench the trips in activity. Then transfer to a 50 milliliter tube.
Count the cells using a hemo cytometer. Once the concentration has been determined, centrifuge at about 200 times gravity for three minutes without disturbing the cell pellet. Carefully remove the media Resus suspending an appropriate volume of Hank's balance buffer solution or HBSS to reach a final concentration of about five times 10 to the six cells per milliliter.
Next, filter through a Falcon's 70 micrometer cell strainer. To exclude large cell aggregates, place the tip of the pipette directly onto the filter, placed over a five milliliter culture tube. Quickly eject the suspension through the filter into the tube.
Count the cells again using trian blue to determine viability. The viability should be greater than 90%prior to injection. Then dilute to a final concentration of 2.5 times 10 to the six cells per milliliter.
Keep on ice. Gently grab the tail of an immunodeficient mouse and pull it into the mouse restrainer with its back against the slit and its tail sticking outta the small opening in the back. Slowly slide the ring inward along the slit.
Once the ring catches the mouth of the mouse, lock it into place. The mouse should not be able to move freely, but should have a normal rate of breathing. Find the major tail veins.
Four major blood vessels are present in a mouse tail. The arteries are located on the dorsal and ventral sides of the tail. The veins are located on the lateral sides.
Draw at least 200 microliters of the prepared cells into a one milliliter syringe. Attach a 30 gauge half needle and push out any air bubbles. The final volume in the syringe should be 200 microliters.
Prepare to inject into the distal end of the tail. So if the first trial fails, a more proximal region of the tail could be used for a second try. After wiping the injection site with 70%ethanol, pull the tail straight.
Hold the tip with the thumb and support the point for injection with the index finger. Insert the needle into the vein and inject the cells. Make sure the needle and syringe are parallel to the vein during injection.
Otherwise, the needle will poke through the vessel wall and cells will be injected into the adjacent tail tissues. Withdraw the needle if the procedure went successfully. Blood should perfuse from the injection site.
Press a clean cotton ball to facilitate flossing and palpate the tail upwards to push any residual sample into circulation. Release the mouse from the restrainer and return it to the cage. Record the number of injection trials and the number of cells injected in a laboratory notebook.
Determine the viability of cells after injection. As before this step provides reassurance that cells stay alive throughout the injection process. At the end of an injection experiment, the viability of cells will decline, but should remain above 80%As an optional step, spin down the leftover cells and rinse the pellets with PBS once.
Then freeze the pellets at minus 80 degrees Celsius for future analyses such as western blotting. To confirm gene expression on knockdowns After one to two months, dissect the mice to determine the locations of metastases. Begin by placing the euthanized mouse on a dissection station.
Spray the mouse generously with ethanol. Cut open the rib cage to expose the lungs and heart. Then carefully excise them and place them in PBS.
The lung is the primary site for metastasis since it contains the first capillary bed that cells encounter. After they enter the circulation, remove the heart and rinse the lungs in PBS then observe each lobe of the lung under a dissecting microscope. Count the number of detectable metastases on both sides of the lung, then at the numbers of lung metastases on all four lobes together to determine the total number of lung metastases.
Next, isolate individual lung metastases by excision Using scissors. Transfer each metastasis to a 70 micrometer cell strainer and mince it using the end of a sterile needle cap. Rinse the cell strainer with several milliliters of medium and collect the cells that pass through the filter.
In a culture dish, each dish contain the cells from one metastasis. Incubate the cells at 37 degrees Celsius for at least four days without disturbance. Initially, both cancer cells and fibroblast cells will grow on the plates, but gradually the fibroblast cells will die out and be replaced by cancer cells.
If the cancer cells carry any drug resistant genes, select the cells with corresponding antibiotics After three days, wash away any blood or tissue debris, debris on the dish with PBS and add fresh medium. Assess the purity of the derived cells by immuno staining with antibodies against human specific proteins here and anti-human menting antibody is used even in the absence of drug selection. Greater than 99%of the cells should be human.
The newly derived cells can now be injected again into immunodeficient mice to test their metastatic abilities at the end of this assay. A highly metastatic cancer cell line typically gives rise to many lung metastases, while very few lung metastases will come from a poorly metastatic cancer cell line. These images show lung metastases two months after injection of the human melanoma cell line.
A 3 7 5 sm. The cells derived using this method usually give rise to more metastasis than the parental line when they're tested. Again using this assay, We have just shown you the procedure to perform experimental metastasis assay.
When doing this procedure, it is very important to remember to ensure that the cells are injected into the tail vein, but not the its and tail tissue. So that's it. Thank you for watching and good luck with your experiments.
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Overview
This article describes an experimental metastasis assay used to evaluate the metastatic potential of human cancer cell lines through tail vein injections in immunodeficient mice.
Key Study Components
Area of Science
- Oncology
- Metastasis Research
- Experimental Biology
Background
- Understanding cancer metastasis is crucial for developing effective treatments.
- Experimental assays help in quantifying the metastatic potential of cancer cells.
- Immunodeficient mice provide a suitable model for studying human cancer cell behavior.
- Tail vein injections are a common method for assessing lung metastases.
Purpose of Study
- To test the metastatic abilities of human cancer cells.
- To quantify and culture lung metastases generated from injected cells.
- To provide insights into the metastatic potential of different cancer cell lines.
Methods Used
- Preparation of human cancer cells for injection.
- Injection of cells into the tail vein of immunodeficient mice.
- Monitoring the development of lung metastases over one to two months.
- Dissection of mice to quantify and culture lung metastases.
Main Results
- Successful generation of lung metastases from injected cancer cells.
- Quantification of metastases provides data on the metastatic potential.
- Cultured metastases can be used for further analysis.
- Results indicate variability in metastatic abilities among different cancer cell lines.
Conclusions
- The experimental metastasis assay is effective for evaluating cancer cell behavior.
- Insights gained can inform therapeutic strategies against metastatic cancer.
- Further studies can build on these findings to explore underlying mechanisms.
Frequently Asked Questions
Executive Industry Relevance
The experimental metastasis assay provides a quantitative in vivo model to assess the metastatic potential of human cancer cell lines, supporting target validation and mechanistic de-risking in oncology drug discovery. By enabling the isolation and propagation of highly metastatic derivatives, the assay facilitates the identification of genes and pathways regulating metastatic progression, thereby informing lead identification and preclinical prioritization. This approach enhances predictive confidence in downstream therapeutic development by linking in vitro findings to functional metastatic outcomes in a disease-relevant system.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses by quantifying metastatic ability of cancer cells following genetic or pharmacological perturbation.
- Operational Value: Provides a reproducible in vivo readout for functional target validation in metastasis-relevant pathways.
- Scientific Value: Supports mechanistic de-risking by linking molecular alterations to metastatic phenotypes in immunocompromised mice.
Screening & Assay Development
- Scientific Value: Generates quantitative metastasis counts that serve as dependent variables for screening modulators of metastatic dissemination.
- Operational Value: Standardizes cell preparation, injection, and tissue harvesting procedures to ensure reproducibility across experiments.
- Scientific Value: Enables derivation of metastatic cell lines with enhanced invasive properties for use in secondary screening and target deconvolution.
Translational & Preclinical Research
- Scientific Value: Uses a disease-relevant system where lung metastasis formation mirrors clinical patterns of hematogenous spread.
- Operational Value: Supports longitudinal studies by allowing re-injection of cultured metastases to assess stability of the metastatic phenotype.
- Scientific Value: Facilitates biomarker discovery by enabling molecular profiling of isolated metastases compared to parental lines.
Pipeline & Workflow Integration
The assay functions as a discovery-stage tool that bridges target hypothesis testing with phenotypic validation, informing decisions on compound progression and mechanistic follow-up in oncology pipelines.
- Discovery Biology: Supports hypothesis-driven interrogation of metastasis regulators through quantitative lung colonization assays.
- Screening: Delivers standardized, quantifiable outputs (metastasis number per lung) enabling comparison across genetic or chemical conditions.
- Analytics: Generates count-based data suitable for statistical analysis to determine significant differences in metastatic potential.
- Translational Research: Connects early discovery to preclinical validation by providing a model that reflects organ-specific metastatic tropism.
- Enterprise Reuse: Establishes a reusable platform for evaluating metastatic capacity across multiple cancer types and therapeutic modalities.
Operational & Enterprise Impact
- Scientific Value: Increases predictive confidence in target selection by demonstrating causal links between gene modulation and metastatic suppression or enhancement.
- Operational Value: Ensures reproducibility through standardized cell viability thresholds (>90% pre-injection, >80% post-injection) and standardized injection volumes.
- Strategic Value: Improves go/no-go decisions by providing functional metastasis data that complements in vitro invasion and proliferation assays.
- Portfolio Impact: Enables risk-adjusted prioritization of targets based on their demonstrated effect on metastatic burden in vivo.
Implementation Considerations
- Requires expertise in animal handling, intravenous injection techniques, and aseptic tissue dissection.
- Dependent on access to immunodeficient mouse models and facilities for long-term animal housing (one to two months).
- Necessitates standardized protocols for cell preparation, viability assessment, and metastasis quantification to minimize variability.
- Involves technical considerations for metastasis isolation, including enzymatic or mechanical dissociation and filtration to obtain single-cell cultures.
- Limited to modeling early dissemination and lung colonization; does not capture later stages of metastasis such as extravasation or distant organ growth beyond the lung.
