Lung Tumor Cell Recruitment Assay

Summary

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

Abstract

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

Introduction

Tumor metastasis accounts for high mortality in cancer-related diseases. From the viewpoint of investigations at the molecular level, metastasis can be divided into multiple steps: tumor initiation at the primary tumor site, primary tumor growth and invasion into surrounding tissues, intravasation, circulation through blood vessels, extravasation at distant organs, and tumor re-growth. Each step involves different sets of molecules/signals¹.

Studies to date have been concerned with events occurring in distant organs before the tumor cells start circulating in the blood, which is also known as the pre-metastatic phase^2,3,4,5,6. Our mouse model studies revealed that the pre-metastatic phase facilitates lung metastasis by creating long-lasting and low-level inflammatory responses in the lungs. The pre-metastatic phase is characterized by hyperpermeability of themicrovasculature, recruitment of bone marrow derived cells (BMDC), and upregulation of cytokine/chemokine-like molecules including S100A8 and SAA3^3,4. It has been reported that lysyl oxidase modifies the extracellular matrix in the lungs to recruit BMDCs⁷. Another report has revealed the importance of Angpt2, MMP3, and MMP10 in the pre-metastatic phase⁸. In other words, the intricate interactions between primary tumors, bone marrow, and remote organs need to be understood and properly modulated (for instance, by using drugs that target proteins involved in these interactions) to prevent future metastasis⁹. To regulate the pre-metastatic phase, it should first be visualized and evaluated for diagnostic or therapeutic interventions. Here, we report a lung tumor cell recruitment assay that enables the study of the pre-metastatic phase in the lungs.

Tumor cells directly injected into the blood vessel of the mouse are similar to circulating tumor cells in cancer patients, although there may be differences between cultured cells and circulating tumor cells. Circulating tumor cells themselves undergo some characteristic changes when they enter circulation from the primary tumor. Nevertheless, cultured cells can form tumors in immunodeficient mice when they are injected in the tail vein. Additionally, in the mouse model system, fluorescence labeled tumor cells can be used that allow tracking of these cells in the body. The behavior of circulating tumor cells is critical as they determine the ultimate consequences in cancer patients¹⁰. Blood is not a good place for survival of tumor cells². They need to escape from attack by the host immune system and need anti-apoptotic signals to prevent apoptosis due to a lack of physical support. Tumor cells with higher abilities of tumor initiation at metastatic sites generate more tumor nodules. If the tumor cells show specific organ tropism, metastasis should be observed in those particular organs. In this assay, the initial steps of the metastatic cascade (primary tumor growth and invasion) are not included, and so the focus is on the interaction between circulating tumor cells and the stromal cells (endothelial cells, epithelial cells, and blood cells). In conventional tumor cell injection assays, researchers have to wait until tumor nodules grow to a tangible size to detect the metastasis. In this case, the exact number of tumor cells in the blood vessel cannot be quantified. Additionally, this process includes a tumor regrowth phase, indicating that other factors in the tumor cells may affect the outcome.

Counting labeled tumor cells under a fluorescence stereomicroscope is a simple process. The stereomicroscope provides a wide field of view so that the whole lung can be scanned. Flow cytometry is also a useful tool that instantly gives an accurate number of tumor cells in the tissue. Counting fluorescent cells in the tissue under a confocal microscope is also possible and displays the most accurate pictures of metastatic tumor cells, but it is time-consuming and may give biased results if only a selected area is counted. The ultimate goal of this assay is to observe how easily tumor cells accumulate in the lungs. As reported previously^3,4, if the lungs are in pre-metastatic phase due to inflammatory signaling from the primary tumor, the injected tumor cells are prone to accumulate in the lungs, thus implying higher chances of lung metastasis. Other conditions (rheumatoid arthritis, asthma, etc.), and their treatment with anti-inflammatory agents (such as anti-TNFα) may attenuate lung metastasis. Thus, we conclude that this assay is a powerful tool to assess the possibility of lung metastasis.

Protocol

All procedures performed with mice were approved by the Animal Research Committee of Tokyo Women's Medical University.

1.Tail Vein Injection of Lewis Lung Carcinoma (LLC) Cells

1. Maintain LLC cells in DMEM supplemented with 10% FCS, in a humidified 5% CO₂ incubator at 37 °C. Cells should contain a fluorescent protein expression system (e.g., GFP).
2. Remove cells from culture dish by using a non-enzymatic cell dissociation reagent. Follow manufacturer's protocol.
3. Collect cells in a 15 mL tube, and centrifuge at 400 x g for 3 min. Resuspend the cells in 5 mL of PBS by pipetting, and centrifuge the tube (400 x g, 3 min). Then, remove the supernatant.
   1. Repeat this washing step one more time.
4. After the second wash, pass the cells through a cell strainer (40 µm pore size), and resuspend in PBS to give a final cell density of 1 x 10⁶ cells/mL.
5. Inject 0.1 mL of cell suspension (1 x 10⁵ cells, 29G needle) into each C57BL/6 mouse (8 - 10 week old males) via the tail vein.

2. Isolation of the Lungs and Counting the Cells Under a Fluorescence Stereomicroscope

1. Euthanize the mice by CO₂ inhalation.
2. Remove the skin on the ventral surface of the chest with scissors. Then, cut the ribs and the diaphragm to expose the thoracic cavity. Flush PBS (100 cmH₂O, total 15 mL) through the right ventricle with a winged infusion 25G needle and tubing.
3. Cut out the heart and the thymus. Grip the trachea with forceps, and pull up, dissecting the connective tissues around trachea with scissors. Dissect out the lungs and wash them in PBS.
4. Detach each lobe and observe it under a fluorescence stereomicroscope.

3. Lung Digestion

1. Dissolve 10 mg of collagenase, 10 mg of dispase, and 10 µg of DNase in 10 mL of serum-free DMEM. Filter through a 0.22 µm syringe filter to prepare the enzymatic digestion solution.
2. Dice the isolated caudal lobe with scissors. Place the lung pieces in a 10 mL syringe, followed by the plunger.
3. Aspirate the digestion solution (5 mL) into the syringe by pulling back on the plunger. Operate the plunger up and down until the lung sinks into the solution. Then, place the lung and digestion buffer in a 50 mL tube.
4. Shake the tube for 15 min at 37 °C on a reciprocal shaker (150 rpm). Lungs may become tattered during this incubation.
5. Pipette up and down until lung cells are completely dispersed. Shake the tube for additional 30 min at 37 °C on a reciprocal shaker (150 rpm).
6. Pass the lung cells through a 40 µm cell strainer. Spin down the cells at 400 x g for 3 min.

4. Flow Cytometry of Lung Cells

1. Dissolve 100 mg of BSA in 10 mL of PBS and filter through a 0.22 µm syringe filter to prepare PBS-1% BSA.
2. Remove supernatant of the sample obtained at step 3.6 using a pipette and add 2 mL of red blood cell lysis solution. Then, spin down the cells at 400 x g for 3 min. Discard the supernatant.
3. Resuspend the cells in 5 mL of PBS-1% BSA by pipetting, and centrifuge the tube (400 x g, 3 min). Discard the supernatant.
   1. Repeat this washing step one more time.
4. Suspend cells in 1 mL of PBS-1% BSA and pass through a new 40 µm cell strainer.
5. Analyze the cells with a flow cytometer to count tumor cells (using filter setup FL1 for GFP-LLC).

Results

The lungs present many GFP-LLC cells 2 h after injection (Figure 1A). It should be noted that the fluorescent spots also detected in the red filter should be excluded from the cell number count. The vast majority of LLC cells disappear from the lungs 24 h after injection (Figure 1C).

To confirm the number of the GFP-LLC in the lungs, one of the lobes can be used for flo...

Discussion

Pre-treatment of mice with antibodies, drugs, a high-fat diet or tumor-conditioned medium prior to tumor cell injection is possible. The most difficult step in this assay is the tail vein injection. Incomplete injection results in inconclusive data. The tail vein in C57BL/6 is particularly difficult to identify, resulting in failed injections. Placing the mice on a heating pad (37 °C) helps dilate the tail vein so that injection becomes easier.

This assay uses a large number of (1 x 10

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Collagenase	Sigma	C6885
Dispase	Gibco	17105-041
Bovine serum albumin	Sigma	A7030
DPBS	Gibco	14190-144	no calcium, no magnesium
Deoxyribonuclease I	Sigma	DN-25	trace amount
RBC lysis buffer	Sigma	R7757
Cell strainer	BD	352340	40 micrometer mesh
Fluorescence labeling kit	Sigma	MIN26-KIT
DMEM	Gibco	11965-092
0.22 μm syringe filter	sartorius	17597K
O.C.T. compound	Sakura Finetek	4583
4% Paraformaldehyde Phosphate Buffer Solution	Wako	163-20145