Lentivirus transduction of human osteoclast precursor cells and differentiation into functional osteoclasts.

Gene transfer into stem cells has been an ongoing priority as a treatment for genetic disease and cancer for more than two decades. Methods described herein, form the basis for providing the cell source to determine if osteoclast precursor cells (OcP) can be used as therapeutic gene delivery systems in vivo. Osteoclasts and tumor associated macrophages or OcP, support survival, tumor progression and osteolysis in bone cancers. Two sources of precursor cells are compared: CD14+ cells, the standard OcP, found abundantly in peripheral blood and CD34+ cells, hematopoietic stem cells that are rare, but which can be expanded into OcP. Our findings characterize cell yield at each step of the transduction process and thus provide essential data for planning future in vivo experiments. In addition we demonstrate that essential functions of OcP are preserved following lentiviral transduction. Specifically, neither the transduction method nor the lentiviral transduction influence the OcP's ability to form osteoclasts, express the marker gene, EGFP, or resorb bone. Finally, we conclude that CD34+ cells yield significantly more transduced cells and form functionally superior osteoclasts in vitro. This study represents a step towards considering human gene therapy for bone cancer by demonstrating successful transduction of human OcP for use as cellular delivery vehicles to sites of bone cancer.

Osteoclasts direct bystander killing of bone cancer.

Primary and metastatic bone cancers are difficult to eradicate and novel approaches are needed to improve treatment and extend life. As bone cancer grows, osteoclasts, the principal bone-resorbing cells of the body, are recruited to and activated at sites of cancer. In this investigation, we determined if osteoclast lineage cells could function as a cell-based gene delivery system to bone cancers. We used the cytosine deaminase (CD) 5-fluorocytosine (5-FC) enzyme/prodrug system and studied bone marrow and bones from transgenic mice expressing a novel CD gene regulated by the osteoclast tartrate-resistant acid phosphatase (TRAP) gene promoter (Tg/NCD). DsRed2-labeled 2472 sarcoma cells were placed in Tg/NCD osteoclastogenic cultures and treated with 5-FC. 5-FC treatment resulted in profound bystander killing (90%; P < 0.05). The effect of 5-FC treatment on osteoclast lineage cells was most dramatic when administered at the beginning of the 7-day cultures, suggesting that mature osteoclasts are less sensitive to 5-FC. Evaluation of osteoclast-directed bystander killing in vivo revealed dramatic killing of bone cancer with only a modest effect on osteoclast number. Specifically, 5-FC treatment of tumor-bearing Tg/NCD mice or Tg/NCD bone marrow transplanted C3H mice (Tg/NCD-C3H) resulted in 92% and 44% reductions in tumor area, respectively (P < 0.05). Eight of ten 5-FC-treated Tg/NCD mice had complete bone tumor killing and five of six 5-FC-treated Tg/NCD-C3H mice had reduced tumor compared with controls. In addition, Tg/NCD osteoclasts were resistant to 5-FC treatment in vivo, a very important feature, as it identifies osteoclasts as an ideal CD gene delivery system.

Novel cytosine deaminase fusion gene enhances the effect of radiation on breast cancer in bone by reducing tumor burden, osteolysis, and skeletal fracture.

BACKGROUND: Painful breast carcinoma metastases in bone are a common manifestation of malignant disease. Eradication of these tumors can be evasive, and as a result, skeletal morbidity increases with disease progression. EXPERIMENTAL DESIGN: The treatment potential of cytosine deaminase (CD) gene therapy combined with radiation treatment was evaluated in vitro and in vivo using a 4T1 murine breast carcinoma model. 4T1 carcinoma cells were transduced with a fusion gene encoding the extracellular and transmembrane domains of the human nerve growth factor receptor and the cytoplasmic portion of the yeast CD gene (NGFR-CD(y)). RESULTS AND CONCLUSIONS: CD-expressing tumor cells (4TCD(y)) were highly sensitive to treatment by 5-fluorocytosine prodrug (P < 0.0001). 5-Fluorocytosine treatment of 4TCD(y), but not 4T1 cells, enhanced the effects of radiation in vitro (P < 0.0001). 5-Fluorocytosine prodrug treatment also increased the therapeutic potential of radiation in vivo. Mice with 4TCD(y) intrafemoral tumors showed increased effectiveness of radiation based on improved reductions in tumor size, reductions in tumorigenic osteolysis, and a decrease in skeletal fractures (P < 0.01).

Analysis of distinct tartrate-resistant acid phosphatase promoter regions in transgenic mice.

The tartrate-resistant acid phosphatase (TRAP) is present in multiple tissues, including kidney, liver, lung, spleen, and bone. Recent study of (TRAP) gene expression has provided evidence for distinct promoters within the (TRAP) gene, suggesting that the gene has alternative, tissue-preferred mRNA transcripts. Examination of endogenous (TRAP) exon 1B and 1C mRNA transcripts revealed tissue-preferred transcript abundance with increased exon 1B transcripts detected in liver and kidney and increased exon 1C transcripts detected in bone and spleen. In this investigation, we have made transgenic mice that express a marker gene driven by two candidate promoters, designated BC and C, within the (TRAP) gene. The BC and C promoters are 2.2 and 1.6 kb, respectively, measured from the translation initiation site. Evaluation of BC transgenic lines demonstrated robust expression in multiple tissues. In contrast, significant transgene expression was not detected in C transgenic lines. Evaluation of transgene mRNAs in BC transgenic lines revealed that virtually all expression was in the form of B transcripts, suggesting that the tissue-preferred pattern of endogenous (TRAP) was not replicated in the BC transgenic line. Likewise, osteoclastogenic cultures from BC, but not C, transgenic bone marrow cells expressed the transgene following receptor activator of NFkappaB ligand/macrophage colony-stimulating factor stimulation. In conclusion, when compared with the 2.2-kb BC portion of the (TRAP) promoter region, the 1.6-kb C portion does not account for significant gene expression in vivo or in vitro; production of the bone- and spleen-preferred (TRAP) C transcript must depend on regulatory elements outside of the 2.2-kb promoter. As the majority of currently investigated transcription factors that influence transcriptional regulation of osteoclast gene expression bind within the 1.6-kb C portion of the (TRAP) promoter, it is likely that transcription binding sites outside of the 2.2-kb region will have profound effects on regulation of the gene in vivo and in vitro.

Tumor implantation in mouse humerus evokes movement-related hyperalgesia exceeding that evoked by intramuscular carrageenan.

In this paper we compare two innovative models of movement-related pain: tumor-induced nociception following implantation of fibrosarcoma cells into bone and muscle inflammation-induced nociception following injection of the irritant carrageenan into muscle. Importantly, using the grip force test, an assay of movement-related hyperalgesia, both non-malignant and malignant pain are examined in parallel. Movement-related hyperalgesia, known clinically as a specific type of 'breakthrough pain', is a common feature of bone cancer and is thought to be a predictor of poor response to conventional analgesic pharmacotherapy (Bruera et al., 1995, J. Pain Symptom. Manage. 10 (1995) 348; Mercadaute et al., 1992, Pain 50 (1992) 151; Pain 81 (1999) 129). Implantation of NCTC 2472 sarcoma cells in both humeri or injection of carrageenan (4%) in both triceps of C3H/He mice produced apparent forelimb hyperalgesia that was not associated with mechanical hyperalgesia in the forepaw, whereas carrageenan at 6 and 8% did evoke significant cutaneous hyperalgesia of the forepaw as well. Control groups receiving implants of vehicle or no treatment at all did not manifest this forelimb hyperalgesia. B6C3/F1 mice implanted with non-lysis-inducing G3.26 melanoma cells or vehicle did not manifest significant hyperalgesia when compared to B6C3/F1 mice receiving fibrosarcoma cells, indicating a dependence on bone involvement for induction of hyperalgesia in this model. Histological examination at days 3, 7, and 10 post-implantation showed a clear correlation of tumor growth-induced bone destruction with behavioral hyperalgesia. Morphine was more potent in decreasing the maximal hyperalgesia induced by carrageenan than that induced by tumor implantation. Acutely administered morphine (3-100mg/kg, i.p.) attenuated peak hyperalgesia of carrageenan-injected mice (ED(50) 6.9 mg/kg) and tumor-bearing mice (ED(50) 23.9 mg/kg) in a dose-related manner with a difference in potency of 3.5. Tumor-implanted mice with a level of hyperalgesia comparable to that induced by carrageenan required almost three times more morphine (ED(50) 18.5mg/kg) for equivalent attenuation of forelimb hyperalgesia. These animal models of movement-related hyperalgesia may aid in discerning the peripheral and central mechanisms underlying pain that accompanies bone metastases and distinguishing it from the pain associated with muscular inflammation. Importantly, they may also aid in predicting differences in analgesic efficacy in different types of musculoskeletal pain.