A novel animal model of human breast cancer metastasis to the spine: a pilot study using intracardiac injection and luciferase-expressing cells.

J Neurosurg Spine. 2013 Mar;18(3):217-25.

Zadnik P, Sarabia-Estrada R, Groves ML, Molina C, Jackson C, McCarthy E, Gokaslan ZL, Bydon A, Wolinsky JP, Witham TF, Sciubba DM.

Departments of Neurosurgery, The Johns Hopkins University School of MedicineBaltimore, Maryland, USA.

Abstract

OBJECT: Metastatic spine disease is prevalent in cancer victims; 10%-30% of the 1.2 million new patients diagnosed with cancer in the US exhibit spinal metastases. Unfortunately, treatments are limited for these patients, as disseminated disease is often refractory to chemotherapy and is difficult to treat with surgical intervention alone. New animal models that accurately recapitulate the human disease process are needed to study the behavior of metastases in real time.

METHODS: In this study the authors report on a cell line that reliably generates bony metastases following intracardiac injection and can be tracked in real time using optical bioluminescence imaging. This line, RBC3, was derived from a metastatic breast adenocarcinoma lesion arising in the osseous spine of a rat following intracardiac injection of MDA-231 human breast cancer cells.

RESULTS: Upon culture and reinjection of RBC3, a statistically significantly increased systemic burden of metastatic tumor was noted. The resultant spine lesions were osteolytic, as demonstrated by small animal CT scanning.

CONCLUSIONS: This cell line generates spinal metastases that can be tracked in real time and may serve as a useful tool in the study of metastatic disease in the spine.

PMID: 23259542

 

Supplement:

Animal models of metastatic spine disease are needed to test novel therapeutic interventions for treating metastatic cancer in the spinal column. The goal of our experiment was to develop a model that more accurately reflected the physiology of human metastatic cancer, specifically a model that allowed the cancer cells to hematogenously spread and seed tumors in bone without direct implantation. In order to achieve this goal, we transfected MDA-231 human breast cancer cells with a Luciferase and GFP positive promoter, allowing us to track the cells’ movement within the body using a bioluminescence optical imaging system.

We then injected the cells into the left ventricle of immunocompromised rats and monitored cell movement throughout the body at day 10, 34 and 46. At day 10, diffuse signal was present throughout the body, however by day 34, three of six injected animals displayed distinct spinal metastases. On gross examination of the spinal column lesions, the tumor was confined to the vertebral body with no obvious extension into the abdominal cavity. On histological examination of the spinal lesion, there was evidence of osteolysis and invasion of the MDA-231 cells into the cancellous bone.

The second step of our experiment was to culture cells from a spinal metastatic lesion and reinject them into a second cohort of animals. The recovered cells were named RBC3. This method is known as serial culture and is illustrated below in the Figure.

Patricia Zadnik-1

Figure: Experimental work flow. Intracardiac injection of MDA-231 cells was followed by bioluminescence imaging and recovery of cells from a bioluminescence positive lesion. The identity of the cells was confirmed as GFP and Luciferase positive through imaging the cells in the bioluminescence optical imaging system in isolation in a 96-well plate (center). These cells were grown to confluence and renamed RBC3, and then were reinjected into the left ventricle of a second cohort of animals. These animals were subsequently subjected to bioluminescence imaging.

Bioluminescence was performed in the second cohort of RBC3 injected animals at day 11, 34 and 43. As in the first cohort, diffuse signal was noted at day 11. By day 34, six of 8 animal demonstrated focal lesions in the spinal column. By day 43, only half of the original cohort was alive and three of the four animals displayed metastatic spinal lesions. On quantification of the total bioluminescence positivity at day 34, a surrogate marker of tumor burden, animals injected with RBC3 had a significantly higher bioluminescence signal compared to the MDA-231 injected cohort (p=0.013).

This work demonstrates that bioluminescence is an effective tool for studying metastatic breast cancer in the spinal column, and further highlights the utility of serial culture in generating cell lines for specialized study.

Conflicts of Interest: The authors report that the RBC3 cell line is a tangible property of the Johns Hopkins Technology Transfer Office (Reference #C11928). The contents of this article were original published in the Journal of Neurosurgery: Spine 2013 Mar;18(3):217-25.

 

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