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....

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....

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