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Cancer Research

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

Published: May 3rd, 2021

DOI:

10.3791/62139

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.

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.

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

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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.......

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Immunocompetent SD outbred rats are used for these OS studies, which offers an animal model with an intact immune system. We have used the UMR106 cell line from ATCC, developed from cells that were initially isolated from an OS from a SD rat. We implanted the cells into SD rats, thus providing a syngeneic model for OS. UMR106 cells are implanted into the tibia of 3-week-old male and female SD rats, simulating a pediatric OS model. Moreover, the orthotopic implantation of UMR106 cells directly into the tibia metaphysis/di.......

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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 OS16,

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NIH funding through National Cancer Institute, grant # CA228582. Shun Ishiyama is currently receiving a grant from Toray Medical Co., Ltd.

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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

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