Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Summary

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Abstract

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Introduction

Mouse models of human and animal disease are becoming increasingly popular in biomedical research. The utility of using mice in this context is that their anatomy and physiology are very similar to humans. They have a relatively short gestation period and time in post-natal life to achieve maturity, and are largely associated with a relatively low cost and ease of housing, albeit increasing costs of development or purchase are associated with greater degrees of genetic modification, immunodeficiency, and/or humanization¹. Use of inbred strains results in a largely uniform animal population prior to study inclusion. A complete knowledge of their....

Protocol

All described animal experiments were approved by the institutional animal care and use committee of University of Cambridge, Cambridge, UK.

1. Preparation of cell lines

1. Grow cell lines in accordance with the laboratory’s standard cell culture protocols for traditional cell culture or injection into mice. Standard protocols used here are growth in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS), L-glutamine, and penicillin/streptomycin (hereafter known as complete growth medium).
   NOTE: In this experiment, Abrams osteosarcoma cells are used in Balb/c Foxn1 nu/nu mice. F....

Results

A positive result would be associated with tumor engraftment and progressive tumor growth over time. Depending on the tumor type, intraosseous tumor growth may be associated with progressive hind limb lameness, but many tumors do not cause lameness despite signs of attendant bone disease. Successful engraftment was documented with advanced imaging, whereby there would be progressive radiographic, µCT, or µMRI changes in the proximal tibia associated with the bone phenotype of the cell line of interest (osteolytic, osteob.......

Discussion

This report documents our model to create primary bone tumors or bone metastasis following the intratibial implantation of a tumor allograft. We believe that there are several critical steps in this process. A safe anesthetic plane should be established for both subcutaneous injection of the tumor cell suspension and intratibial placement of the resultant tumor fragments. There should be sterile preparation of the surgical site for both removal of the subcutaneous allograft and intratibial placement of the allograft. Tum.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#15 scalpel blade	Henry Schein Ltd.	75614	None
6-well tissue culture plates	Thermo Fisher Scientific	10578911	Used for mincing tumor pieces. Can also be used for cell culture
Abrams osteosarcoma cell line	Not applicable	Not applicable	None
Anesthesia machine with isoflurane vaporiser and oxygen tank(s)	VetEquip	901805	None
Animal weighing scale	Kent Scientific	SCL- 1015	None
BALB/c nude mouse (nu/nu)	Charles River Ltd.	NA	6-8 weeks of age. Male or female mice
Bone cement	Depuy Synthes	160504	Optional use instead of bone wax
Bone wax	Ethicon	W31G	Optional
Buprenorphine	Animalcare Ltd.	N/A	Buprecare 0.3 mg/ml Solution for Injection for Dogs and Cats
Carbon dioxide euthanasia station	N/A	N/A	Should be provided within animal facility
Cell culture incubator set at 37 °C and 5% carbon dioxide	Heraeus	Various	None
Chlorhexidine surgical scrub	Vetoquinol	411412	None
Cryovials (2 ml)	Thermo Scientific Nalgene	5000-0020	Optional if cryopreserving tumor fragments
D-luciferin (Firefly), potassium salt	Perkin Elmer	122799	Optional if cell line of interest has a bioluminescent reporter gene
Digital caliper	Mitutoyo	500-181-30	Can be manual
Digital microradiography cabinet	Faxitron Bioptics, LLC	MX-20	Optional to evaluate bone response to tumor growth
Dimethyl sulfoxide (DMSO)	Sigma Aldrich	1371171000	Optional if cryopreserving tumor fragments
Dulbecco’s modified Eagle’s medium	Thermo Fisher Scientific	11965092	None
Ethanol (70%)	Sigma Aldrich	2483	None
Fetal bovine serum	Thermo Fisher Scientific	26140079	None
Forceps, Dumont	Fine Science Tools, Inc.	11200-33	None
Freezer (– 80 °C)	Sanyo	MDF-794C	Optional if cryopreserving or snap freezing tumor fragments
Hemocytometer	Thermo Fisher Scientific	11704939	Can also use automated cell counter, if available
Hypodermic needles (27 gauge)	Henry Schein Ltd.	DIS55510	May also use 25G (DIS55509) and 30G (Catalog DIS599) needles
Ice	N/A	N/A	Ideally small pieces in a container for syringe and cell suspension storage
Iris scissors	Fine Science Tools, Inc.	14084-08	None
Isoflurane	Henry Schein Ltd.	1182098	None
IVIS Lumina III bioluminescence/fluorescence imaging system	Perkin Elmer	CLS136334	Optional if cell line of interest has bioluminescent or fluorescent reporter genes
L-glutamine	Thermofisher scientifc	25030081	None
Liquid nitrogen	British Oxygen Corporation	NA	Optional if cryopreserving or snap freezing tumor fragments
Liquid nitrogen dewar, 5 litres	Thermo Fisher Scientific	TY509X1	Optional if cryopreserving tumor fragments
Matrigel® Matrix GFR, LDEV-Free, 5 ml	Corning Life Sciences	356230	Optional. Also available in 10 ml size (354230)
Microcentrifuge	Thermo Fisher Scientific	75002549	Pellet cells at 1200 rpm for 5-6 minutes
Mr. Frosty freezing containiner	Fisher Scientific	10110051	Optional if cryopreserving tumor fragments
NAIR Hair remover lotion/oil	Thermo Fisher Scientific	NC0132811	Can alternatively use an electric clipper with fine blade
Penicillin/streptomycin	Sigma-Aldrich	P4333	None
Scalpel handle, #7 Short	Fine Science Tools, Inc.	10007-12	User preference as long as it accepts #15 scalpel blade
Small animal heated pad	VetTech	HE006	None
Stereomicroscope	GT Vision Ltd.	H600BV1	None
Sterile phosphate-buffered saline (PBS)	Thermo Fisher Scientific	10010023	Use for injections and also as part of the surgical scrub, alternating with chlorhexidine
Tissue adhesive (sterile)	3M Corporation	84-1469SB	Can alternatively use non-absorbable skin suture (6-0 size)
Trypan blue	Thermo Fisher Scientific	5250061	None
Trypsin-EDTA	Thermo Fisher Scientific	25300054	Use 0.05%-0.25%
Tuberculin syringe (1 ml with 0.1 ml gradations)	Becton Dickinson	309659	Slip tip preferred over Luer
Vented tissue culture flasks, T-75	Corning Life Sciences	CLS3290	Can also use smaller or larger flasks, as needed