Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Summary

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Abstract

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Introduction

Despite being a highly complex and inefficient process¹, metastasis is a significant contributor to the morbidity and mortality of cancer patients². In fact, most cancer-related deaths are attributed to metastatic spread of disease^3,4. In order for tumor cells to successfully metastasize, they must detach from the primary site, invade through adjoining stroma, intravasate into blood circulation or lymphatics, travel to the capillary bed of a secondary site, extravasate into the secondary tissue, and proliferate or grow to form metastatic lesions^....

Protocol

Animal use followed University Laboratory Animal Resources (ULAR) regulations under the OSU Institutional Animal Care and Use Committee (IACUC)–approved protocol 2007A0120-R4 (PI: Dr. Gina Sizemore).

1. Tail-vein injection of breast cancer cells

1. Preparation of cells and syringe for injection
   1. Plate an appropriate number of cells based on the number of mice and cell concentration to be used.
      NOTE: The number of cells injected and time to the development of metastases will depend on the cell line used and will need to be optimized. In this demonstration, 1 x 10⁶ MDA-MB-231 cells are injected int....

Results

If using unlabeled cells for tail-vein injection, it may be difficult to confirm lung colonization until (1) the time of necropsy if macrometastases can be observed or (2) following histological analysis if microscopic metastases exist. With extensive metastatic lung tumor burden, mice will have labored breathing. As with any tumor study, mice should be carefully monitored throughout the study duration. The use of labeled cells is an easy way to confirm successful tail-vein injection; hence the use of luciferase-tagged M.......

Discussion

As researchers continue to use intravenous injection of tumor cells as an experimental model for metastasis, standard practices to analyze the resulting metastatic tumor burden are lacking. In some cases, significant differences in metastatic tumor burden upon manipulation of particular cell lines and/or use of chemical compounds can be observed macroscopically. However, in other instances, subtle differences in metastatic seeding and growth may be overlooked or misinterpreted without thorough pathological analysis. This.......

Materials

Name	Company	Catalog Number	Comments
alcohol prep pads	Fisher Scientific	22-363-750	for cleaning tail prior to injection
dissection scissors	Fisher Scientific	08-951-5	for mouse dissection and lung tissue inflation
DMEM with L-Glutamine, 4.5g/L Glucose and Sodium Pyruvate	Fisher Scientific	MT10013CV	cell culture media base for MDA-MB-231 and MVT1 cell lines
Dulbecco's Phosphate-Buffered Salt Solution 1x	Fisher Scientific	MT21030CV	used for resuspending tumor cells for injection
ethanol (70 % solution)	OSU		used to minimize mouse's fur during dissection; use caution - flammable
Evan's blue dye	Millipore Sigma	E2129	used at 1 % in sterile PBS for practice with tail-vein injection method; use caution - dangerous reagent
Fetal Bovine Serum	Millipore Sigma	F4135	cell culture media additive; used at 10% in DMEM
forceps	Fisher Scientific	10-270	for dissection and lung tissue inflation
FVB/NJ mice	The Jackson Laboratory	001800	syngeneic mouse strain for MVT1 cells
hemacytometer (Bright-Line)	Millipore Sigma	Z359629	for use in cell culture to obtain cell counts
insulin syringe (28 G)	Fisher Scientific	14-829-1B	for tail-vein injections (BD 329424)
MDA-MB-231 cells	ATCC		human breast cancer cell line
MVT1 cells			mouse mammary tumor cells
needles (26 G)	Fisher Scientific	14-826-15	used to inflate the mouse's lungs
neutral buffered formalin (10%)	Fisher Scientific	245685	used as a tissue fixative and to inflate lung tissue; use caution - dangerous reagent
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice	The Jackson Laboratory	005557	maintained by OSUCCC Target Validation Shared Resource
Penicillin Streptomycin 100x	ThermoFisher	15140163	cell culture media additive
sterile gauze	Fisher Scientific	NC9379092	for applying pressue to mouse's tail if bleeding occurs
syringe (5 mL)	Fisher Scientific	14-955-458	used to inflate mouse lung tissue
tail-vein restrainer	Braintree Scientific, Inc.	TV-150 STD	used to restrain mouse for tail-vein injections
Trypan blue (0.4 %)	ThermoFisher	15250061	used in cell culture to assess viability
Trypsin-EDTA 0.25 %	ThermoFisher	25200-114	used in cell culture to detach tumor cells from plate