Experimental Metastasis Assay

Summary

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Abstract

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

Protocol

1. Sample Preparation

1. Grow cells to ~70% confluent in their specific media with growth factors or FBS (e.g., DMEM with 10% FBS for MC-1 cells). Aspirate media from plate and gently wash several times with 1 x PBS (8 g/L of NaCl, 0.2 g/L of KCl, 1.15 g/L of Na₂HPO₄.7H₂O, 0.2 g/L of KH₂PO₄, pH 7.3).
2. Aspirate PBS and add 2 mL of 0.05% Trypsin in versene (0.014 g/L of phenol red and 0.2 g/L of EDTA-Na in 1 x PBS, pH 7.2). Gently rock plate to facilitate cell detachment from plate. Observe the cells under a microscope. It usually takes 2-5 minutes for the cells to detach.
3. Add an ample volume of media containing FBS (or soybean trypsin inhibitor) to quench the trypsin activity and collect cells in a 50mL falcon tube. Count cells using a hemacytometer. Load 10μL of cell suspension onto a clean hemacytometer. The number of cells per mL is equal to the average # of cells in each of the five squares multiplied by 10⁴.
4. Centrifuge the cells at 1000 RPM (or ~200 xg) in a benchtop centrifuge for 3 minutes.
5. Carefully remove the media without disturbing the cell pellet. Resuspend cells in an appropriate volume of Hanks Balanced Buffer Solution (HBSS) to reach a final concentration of ~5 x 10⁶ cells/mL.
6. Filter cells through a Falcon 70 μm cell strainer to exclude large cell aggregates. Place the tip of the pipette directly onto the filter over a labeled 5mL Falcon culture tube. Quickly eject the suspension through the filter into the culture tube.
7. Count cells again using a hemacytometer and dilute them to a final concentration of 2.5 x 10⁶/mL. Keep cells on ice.
8. Determine the viability of the cells. Mix some cells with trypan blue and measure the percentage of dead (blue) cells over the total cells using a hemacytometer. The viability of cells should be ≥ 90% prior to injection.

2. Intravenous Injection

1. Change gloves. Gently grab the tail of an immunodeficient mouse (nude, NOD-SCID, or NSG, Jackson's Laboratory) and pull it into the mouse restrainer, with its back against the slit and its tail sticking out of the small opening in the back of the restrainer.
2. Slowly slide the ring inward along the slit and lock it in place once the ring catches the mouth of the mouse. The mouse should not be able to move freely, but should have normal rate of breathing.
3. Find the major tail veins. Four major blood vessels are present in a mouse tail. Blood vessels on the dorsal and ventral sides of the tail are arteries. Veins are on the lateral sides of the tail.
4. Draw more than 200 μL of the prepared cells into a 1mL syringe. Attach the 30 G1/2 inch needle and push out any air bubbles that may exist. The final volume of cells in the syringe should be 200 μL (i.e., 5 x 10⁵ cells in total).
5. Inject cells into the tail vein.
   1. Start from 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.
   2. Wipe the tail with 70% ethanol. Pull the tail straight. Hold the tip of tail with thumb and support the point for injection with the index finger.
   3. Insert the needle to the vein and inject cells. Make sure the needle and syringe are parallel to the vein during injection, otherwise the needle will poke through the vessel wall and inject the cells into the adjacent tail tissues.
   4. Withdraw the needle after injection. Blood should profuse from the injection site if the injection went successfully. Press a clean piece of paper towel or cotton swab on the injection site to facilitate clotting, and palpate the tail upwards to push any residual sample in the vein into circulation.
   5. Release the mouse from the restrainer and return it to the cage. Record the injection process (e.g., how many trials it took to inject the cells and how much cells were injected) in a lab notebook.
   6. Determine the viability of cells after injection, as described in the Section of Sample Preparation, Step 9. 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 be above 80%.
   7. (optional) Spin down the leftover cells and rinse the pellets with PBS once. Freeze the pellets at -80 oC for future analyses (e.g., western blots to confirm gene expression or knockdowns).
6. Typically after one or two months, the mice will be dissected and the locations of metastases are grossly determined. Lung is the primary site for metastasis, since it contains the first capillary bed that the cells encounter after they enter the circulation.
7. After rinsing with PBS, each lobe of the lung (or other tissues containing metastases) is observed under a dissecting microscope (Figure 1). The number of detectable metastases on both sides of the lung is counted and the numbers of lung metastases on all the four lobes are added together as the total number of lung metastases.¹ Lung metastases are more easily detected if the lungs are fixed in formalin overnight, since the metastases will apppear as whitish spots in contrast to the adjacent dark brown lung tissues.

3. Culturing Cells from Lung Metastases

1. Lung metastases are isolated from injected mice. Each should be cultured separately.
2. Each metastasis is minced by the end of a needle cap (sterile) on a 70 μm cell strainer.
3. Rinse the cell strainer with several mLs of medium and collected cells that pass through the filter in a culture dish.
4. Incubate the cells at 37 °C for at least four days without disturbance.
5. Wash away blood or tissue debris on the dish with PBS.
6. Add fresh medium. At the beginning, both cancer cells and fibroblast cells 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.
7. The purity of the derived cells is assessed by immunostaining using antibodies against human-specific proteins. We use an anti-human vimentin antibody (NCL-VIM-V9, Novocastra). The cells we derived contain typically over 99% of human cells, even in the absence of drug selection. The newly derived cells could be injected again into immunodeficient mice to test their metastatic abilities, as described above.

4. Representative Results

1. At the end of this assay, a highly metastatic cancer cell line typically gives rise to many lung metastases (Figure 1),¹ while very few lung metastases will come from a poorly metastatic cancer cell line.
2. The cells derived using this method usually give rise to more metastases than the parental line, when they are tested again using this assay.^1-3

Figure 1. Representative images of mouse lungs after tail vein injections of cancer cells. 5 x 10⁵ of the metastatic human melanoma cell line, SM cells,⁴ were injected into the tail vein of immunodeficient mice. Two months later, the lungs were isolated and metastases were found dispersed among the normal lung tissue.

Discussion

Metastasis is the leading cause of death in cancer patients. It involves four major steps: detachment of cancer cells from their primary loci, their entry into circulation (intravasation), their exit from circulation (extravasation), and survival and growth in a distant organ. Metastasis in human is considered a slow process and often manifested after years of latency. To study its progression in a timely manner, the above relatively quick experimental assay was established in immunodeficient mice.⁴ Since its...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM	Invitrogen	10313-021
FBS	Hyclone	SH30088.03
HBSS	Invitrogen	14175-095
NaCl	Sigma-Aldrich	S9888
KCl	Mallinckrodt Baker Inc.	6838-04
Na2 HPO4.7H2O	Mallinckrodt Baker Inc.	7896-04
KH2PO4	Mallinckrodt Baker Inc.	7100-12
Phenol red	Sigma-Aldrich	P3532
EDTA-Na	EMD Millipore	4010
Trypan Blue	Invitrogen	15250-061
Trypsin 2.5%	Invitrogen	15090-046
Ethanol	Ultrapure	200-CSGP
50 ml Falcon Tube	VWR international	89039-656
Microcentrifuge Tubes	Axygen Scientific	MCT-175-C
Falcon 70μm Cell Strainer	BD Biosciences	352350
5ml Falcon Culture Tube	VWR international	60818-576
Hemacytometer	Hausser Scientific	15170-208
1 ml syringe	BD Biosciences	329650
30 ½ gauge needle	BD Biosciences	305106
Mouse restrainer	Plas Labs Inc.	551-BSRR
Surgical scissors	Fisher Scientific	08-940
Forceps	Fisher Scientific	08-902
Dissecting Microscope	Nikon Instruments	SMZ1500
Benchtop Centrifuge	Thermo Fisher Scientific, Inc.	75003491