Elsevier

Bone

Volume 38, Issue 1, January 2006, Pages 4-12
Bone

Osteoclasts direct bystander killing of cancer cells in vitro

https://doi.org/10.1016/j.bone.2005.07.016Get rights and content

Abstract

Cytosine deaminase (CD) catalyzes the deamination of 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic agent 5-fluorouracil (5FU). A unique feature of the CD/5FC enzyme/prodrug system is its ability to kill adjacent cells via bystander killing. Bystander killing of cancer cells can be mediated by non-cancerous accessory cells transduced with the CD gene; one type of non-cancerous accessory cell found in primary bone cancer and breast cancer metastases to bone is the osteoclast. This manuscript determines if osteoclast precursor cells, transduced with the CD gene, can function as a gene delivery system capable of killing cancer cells. An osteoclast precursor cell line (RAW 264.7, RAW) and authentic bone marrow-derived osteoclast precursor cells were transduced with a retroviral vector containing the cytosine deaminase fusion gene (NCD) composed of the human nerve growth factor receptor and CD genes. RAW cells and bone marrow-derived osteoclast precursor cells transduced with NCD expressed NCD protein and converted 5FC to 5FU. Treatment of NCD-transduced osteoclast precursor cells with the 5FC prodrug resulted in significant killing in vitro. NCD-transduced osteoclasts were co-cultured with either DsRed2-labeled sarcoma cells (2472-DSR) or green fluorescent protein (GFP)-labeled breast cancer cells (GFP-4T1). Treatment of the NCD osteoclast/tumor cell co-cultures with 5FC resulted in bystander killing of 2472-DSR cells (P < 0.006) and GFP-4T1 cells (P < 0.004). These findings demonstrate that NCD-transduced osteoclasts can promote killing of cancer cells and introduce the exciting possibility for developing osteoclast-mediated, CD-based treatment of primary bone cancers and breast cancer metastases to bone.

Introduction

One approach for cancer gene therapy involves using the cytosine deaminase gene (CD) to kill tumor cells [1], [2], [3], [4], [5], [6], [7]. The CD suicide gene catalyzes the deamination of non-toxic 5-fluorocytosine (5FC) to produce the highly toxic chemotherapeutic drug 5-fluorouracil (5FU). An interesting property of the CD enzyme/prodrug system is its bystander effect, which causes the death of unmodified tumor cells adjacent to genetically modified cells [8], [9]. Therapeutic efficacy of this approach relies on the bystander effect, where 5FU released from CD-expressing cells diffuses across tumor cell membranes. Recent reports indicate that CD gene delivery to tumors by non-cancerous accessory cells is a promising treatment for gliomas and lung cancer. The non-cancerous accessory cell chosen as a gene delivery system for gliomas is the neural progenitor cell [1], [10], and the cellular gene delivery system for lung cancers is the endothelial precursor cell [7], [11].

Recent advances in understanding the pathophysiology of bone cancer have provided keen insight into the pathologic role of osteoclasts [12], [13], [14]. It has been shown that these bone-resorbing cells form in high numbers at sites of bone cancer, destroy bone at sites of tumor, and stimulate tumor progression. As a first step toward developing a novel gene therapy for treating bone metastases, we hypothesized that CD-expressing osteoclast precursor cells can serve as a cellular gene delivery system capable of killing cancer cells.

To examine the possibility that osteoclasts can direct killing of cancer cells, we have transduced an osteoclast precursor cell line and authentic osteoclasts with a customized yeast CD gene. Findings indicate that CD-transduced osteoclast precursors form osteoclasts, express active CD, convert the prodrug 5FC to 5FU, and direct bystander killing when co-cultured with murine sarcoma or breast cancer cells. Findings represent a crucial first step toward proving the exciting possibility that CD-expressing osteoclast precursor cells can be used to develop new cancer gene therapy treatments for primary bone cancer and bone cancer metastases.

Section snippets

Cell culture

RAW 264.7, a monocyte/macrophage cell line which can be differentiated into osteoclasts [16], [17], 2472 cell line, originally derived from a malignant tissue tumor (sarcoma) in a C3H mouse, NCTC clone L929 (L929), a macrophage colony-stimulating factor (MCSF) producing tumor line, NIH 3T3 normal mouse fibroblasts, and PA317 packaging cell line were obtained from the American Type Culture Collection (Rockville, MD). The 4T1 murine breast cancer cell line was a gift from Dr. Fred Miller [18] and

Characterization of transduced osteoclast precursor (RAW 264.7) cell line

RAW cells transduced with LNGFR-CDSN vector expressed high levels of the NCD fusion protein. As expected, the parent RAW cells and transduced cells (RAW/NCD) were osteoclast precursors in that they expressed surface membrane CD11b (Fig. 1). In contrast to RAW cells, RAW/NCD cells expressed the NGFR and CD portions of the NCD fusion protein. FACS analysis revealed abundant cell surface NGFR in RAW/NCD cells and no NGFR expression in RAW cells (Fig. 1). Western analysis of RAW/NCD cell lysates,

Discussion

Findings from this investigation demonstrate that CD-transduced osteoclasts can direct killing of sarcoma and breast cancer cells. Authentic bone marrow-derived osteoclast precursors and the osteoclast precursor RAW cell line were both transduced with the LNGFR-CDSN retroviral vector. Under osteoclastogenic conditions, each population of transduced cells formed osteoclasts and each population expressed the therapeutic NCD fusion protein. Measures of CD enzyme activity determined that both

Acknowledgments

We would like to acknowledge the assistance of the Flow Cytometry Core Facility of the University of Minnesota Cancer Center, a comprehensive cancer center designated by the National Cancer Institute, supported in part by P30 CA77598. Support was also provided by the National Cancer Institute; CA90434 and the National Institute of Arthritis, Musculoskeletal and Skin Diseases; and the Roby C. Thompson Endowment.

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