A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

Summary

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

Abstract

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Introduction

Osteosarcoma (OS) is the most common primary bone tumor in children, adolescents, and young adults. The most recent advance in the treatment of OS occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone¹. OS develops during rapid bone growth, typically occurring in long tubular bones such as femur, tibia, and humerus. They are characterized by an osteolytic, osteoblastic, or mixed appearance with notable periosteal reaction². Chemotherapy and surgical resection can improve the outcome for patients with a 5-year survival for 65% of patients²

Protocol

All the procedures and experiments involving rats were performed according to protocols approved by Johns Hopkins Animal Care and Use Committee.

1. The SD rat OS cell line UMR-106 cell culture protocol

1. Grow cells in DMEM, supplemented with 10% (v/v) FBS, penicillin (10 U/mL)-streptomycin (10 U/mL) at 37 °C in humidified 5% CO₂ atmosphere. Perform experiments using cells with passages of 2-8¹².

2. Intratibial i.......

Discussion

Rats with OS tibial implants develop measurable tumors by 3 weeks post-implantation. If limbs with tumors are amputated 3 weeks post-implantation, the incidence of lung metastasis is reduced significantly. OSs are both osteolytic and osteoblastic. Rats without amputation develop lung metastases that are multiple and variably sized, observed by radiography or at necropsy by 7 weeks post-implantation.EGFR, ErbB2, and ErbB4 are expressed in rat UMR106 OS, similar to human OS^16,

Materials

Name	Company	Catalog Number	Comments
AKT	Cell Signaling TECHNOLOGY	4685S
absorbable suture	Ethicon	J214H
β-actin	SANTA CRUZ BIOTECHNOLOGY	sc-47778
β2-AR antibody	SANTA CRUZ BIOTECHNOLOGY	sc-569	replaced by β2-AR (E-3): sc-271322
Bis–Tris gels	Thermo Fisher	NP0321PK2
Buprenorphine SR Lab	ZooPharm	IZ-70000-201908
CD3 antibody	Dako	#A0452
CD68 antibody	eBioscience	#14-0688-82
Chemiluminescent substrate	cytiva	RPN2232
CL-Xposure film	Thermo Fisher	34089
Complete Anesthesia System	EVETEQUIP	922120
diaminobenzidine	VECTOR LABORATORIES	SK-4100
Doxorubicin	Actavis	NDC 45963-733-60
EGFR antibody	SANTA CRUZ BIOTECHNOLOGY	sc-03	replaced by EGFR (A-10): sc-373746
ERBB2 antibody	SANTA CRUZ BIOTECHNOLOGY	sc-284	replaced by Neu (3B5): sc-33684
ERBB4 antibody	SANTA CRUZ BIOTECHNOLOGY	sc-283	replaced by ErbB4 (C-7): sc-8050
ERK antibody	SANTA CRUZ BIOTECHNOLOGY	sc-514302
eye lubricant	PHARMADERM	NDC 0462-0211-38
Hamilton syringe (100 µL)	Hamilton	Model 1710 SN SYR
horseradish peroxidase-linked secondary antibody	cytiva	NA934
HRP polymer detection kit	VECTOR LABORATORIES	MP-7401
HRP polymer detection kit	VECTOR LABORATORIES	MP-7402
isoflurane	BUTLER SCHEIN	NDC 11695-6776-2
isoflurane vaporizer	EVETEQUIP	911103
UMR-106 cell	ATCC	CRL-1661
X-ray	Faxitron	UltraFocus
X-ray processor	Hope X-Ray Peoducts Inc	MicroMax X-ray Processor	Hope Processors are not available in USA anymore
wound clips	BECTON DICKINSON	427631