Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Summary

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Abstract

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

Introduction

Osteosarcoma is the most common primary bone cancer in children and adolescents^1,2, which mainly infiltrates the surrounding tissue, and even metastasizes to the lungs when the patients are diagnosed. Pulmonary metastasis is the main challenge for osteosarcoma therapy, and the five-year survival rate of osteosarcoma patients with pulmonary metastasis remains as low as 20%-30%^3,4,5. However, the five-year survival rate of primary osteosarcoma has been increased to about 70% since the 1970s due to the introduction of ch....

Protocol

All animal experiments were approved by the animal welfare committee of Shanghai University of Traditional Chinese Medicine. Four-week-old male BALB/c athymic mice were acclimated for a week before the surgery for orthotopic injection of osteosarcoma cells. Mice were housed in individually ventilated mice cages with five mice per cage in a 12-hour light/dark cycle with ad libitum access to SPF feed and sterile water.

1. Preparation of cells

1. On the day of osteo.......

Discussion

Orthotopic injection of osteosarcoma cells is an ideal model to study the function and mechanism of specific factors in osteosarcoma carcinogenesis and development to evaluate the therapeutic efficacy. To avoid differences in tumor growth, most active osteosarcoma cells at 80%-90% confluent with the same number are carefully injected into the tibia of each mouse, and the cell trypsinization time is strictly controlled without affecting the cell viability. As cell clumps affect cell counting leading to inaccurate cell num.......

Materials

Name	Company	Catalog Number	Comments
Automatic cell counter	Shanghai Simo Biological Technology Co., Ltd	IC1000	Counting cells
Anesthesia machine	Shenzhen RWD Life Technology Co., Ltd	R500IP	The Equipment of Anesthesia mice
BALB/c athymic mice	Shanghai SLAC Laboratory Animal Co, Ltd.	/	animal
Basement Membrane Matrix	Shanghai Uning Bioscience Technology Co., Ltd	356234, BD, Matrigel	re-suspende cells
Bioluminescence imaging system	Shanghai Baitai Technology Co., Ltd	Vieworks	tracking the tumor growth and pulmonary metastasis, if the injection cell is labeled by luciferase
Centrifuge tube (15 mL)	Shanghai YueNian Biotechnology Co., Ltd	430790, Corning	Centrifuge the cells
isoflurane	Shenzhen RWD Life Technology Co., Ltd	VETEASY	Anesthesia mice
MEM media	Shanghai YueNian Biotechnology Co., Ltd	LM-E1141	Cell culture medium
Micro-volume syringe	Shanghai high pigeon industry and trade Co., Ltd	0-50 μL	Inject precise cells into the tibia
Phosphate-buffered saline	Beyotime Biotechnology	ST447	wash the human osteosarcoma cells
1ml syringes	Shandong Weigao Group Medical Polymer Co., Ltd	20200411	drilling
143B cell line	ATCC	CRL-8303	osteosarcoma cell line
Trypsin (0.25%)	Shanghai YueNian Biotechnology Co., Ltd	25200056, Gibco	trypsin treatment of cells
Trypan blue	Beyotime Biotechnology	ST798	Staining cells to assess activity
vector (pLV-luciferase)	Shanghai YueNian Biotechnology Co., Ltd	VL3613	Plasmid
Lipofectamine 2000	Shanghai YueNian Biotechnology Co., Ltd	11668027,Thermo fisher	Plasmid transfection reagent
X-ray imaging system	Brook (Beijing) Technology Co., Ltd	FX PRO	X-ray images were obtained to detect tumor growth