Chelsea K. Martin

Dickkopf-1 (DKK-1) stimulated prostate cancer growth and metastasis and inhibited bone formation in osteoblastic bone metastases

Osteoblastic bone metastasis is the predominant phenotype observed in prostate cancer patients and is associated with high patient mortality and morbidity. However, the mechanisms determining the development of this phenotype are not well understood. Prostate cancer cells secrete several osteogenic factors including Wnt proteins, which are not only osteoinductive but also oncogenic. Therefore, the purpose of the study was to investigate the contribution of the Wnt signaling pathway in prostate cancer growth, incidence of bone metastases, and osteoblastic phenotype of bone metastases. The strategy involved overexpressing the Wnt antagonist, DKK‐1, in the mixed osteoblastic and osteolytic Ace‐1 prostate cancer cells.

Evidence for a stepwise program of extrathymic T cell development within the human tonsil

The development of a broad repertoire of T cells, which is essential for effective immune function, occurs in the thymus. Although some data suggest that T cell development can occur extrathymically, many researchers remain skeptical that extrathymic T cell development has an important role in generating the T cell repertoire in healthy individuals. However, it may be important in the setting of poor thymic function or congenital deficit and in the context of autoimmunity, cancer, or regenerative medicine. Here, we report evidence that a stepwise program of T cell development occurs within the human tonsil. We identified 5 tonsillar T cell developmental intermediates: (a) CD34⁺CD38dimLin⁻ cells, which resemble multipotent progenitors in the bone marrow and thymus; (b) more mature CD34⁺CD38brightLin⁻ cells; (c) CD34⁺CD1a⁺CD11c⁻ cells, which resemble committed T cell lineage precursors in the thymus; (d) CD34⁻CD1a⁺CD3⁻CD11c⁻ cells, which resemble CD4⁺CD8⁺ double-positive T cells in the thymus; and (e) CD34⁻CD1a⁺CD3⁺CD11c⁻ cells. The phenotype of each subset closely resembled that of its thymic counterpart. The last 4 populations expressed RAG1 and PTCRA, genes required for TCR rearrangement, and all 5 subsets were capable of ex vivo T cell differentiation. TdT⁺ cells found within the tonsillar fibrous scaffold expressed CD34 and/or CD1a, indicating that this distinct anatomic region contributes to pre-T cell development, as does the subcapsular region of the thymus. Thus, we provide evidence of a role for the human tonsil in a comprehensive program of extrathymic T cell development.

Zoledronic Acid Decreased Osteolysis But Not Bone Metastasis in a Nude Mouse Model of Canine Prostate Cancer With Mixed Bone Lesions

Bone metastasis is the most common cause of morbidity and mortality in patients with advanced prostate cancer and is manifested primarily as mixed osteoblastic and osteolytic lesions. However, the mechanisms responsible for bone metastases in prostate cancer are not clearly understood, in part due to the lack of relevant in vivo models that mimic the clinical presentation of the disease in humans. We previously established a nude mouse model with mixed bone metastases using intracardiac injection of canine prostate cancer cells (Ace‐1). In this study, we hypothesized that tumor‐induced osteolysis promoted the incidence of bone metastases and osteoblastic activity.

Bone-Invasive Oral Squamous Cell Carcinoma in Cats Pathology and Expression of Parathyroid Hormone-Related Protein

Feline oral squamous cell carcinoma (OSCC) is the most common oral tumor in cats. There is no effective treatment, and the average duration of survival after diagnosis is only 2 months. Feline OSCC is frequently associated with osteolysis; however, the mechanisms responsible are unknown. The objective of this study was to characterize the epidemiology and pathology of bone-invasive OSCC in cats and to determine the expression of select bone resorption agonists. In sum, 451 cases of feline OSCC were evaluated. There was no sex or breed predisposition, although there were more intact cats in the OSCC group compared to the control group. Gingiva was the most common site, followed by the sublingual region and tongue. Cats with lingual OSCC were younger (mean, 11.9 years) compared to cats with gingival OSCC (mean, 13.6 years). In addition to osteolysis, there was periosteal new bone formation, osseous metaplasia of tumor stroma, and direct apposition of OSCC to fragments of bone, suggestive of bone-binding behavior. Eighty-two cases were selected for immunohistochemical detection of parathyroid hormone-related protein (PTHrP). Specimens with osteolysis had increased PTHrP expression and nuclear localization, compared to OSCC without osteolysis. Thirty-eight biopsies of OSCC with osteolysis were evaluated for tumor necrosis factor α expression, and only 4 biopsies had such expression in a small proportion of tumor cells. Increased tumor expression of PTHrP and increased localization of PTHrP to the nucleus were associated with osteolysis and may play an important role in bone resorption and tumor invasion in cats with OSCC.

Zoledronic acid reduces bone loss and tumor growth in an orthotopic xenograft model of osteolytic oral squamous cell carcinoma.

Squamous cell carcinoma (SCC) is the most common form of oral cancer. Destruction and invasion of mandibular and maxillary bone frequently occurs and contributes to morbidity and mortality. We hypothesized that the bisphosphonate drug zoledronic acid (ZOL) would inhibit tumor-induced osteolysis and reduce tumor growth and invasion in a murine xenograft model of bone-invasive oral SCC (OSCC) derived from an osteolytic feline OSCC. Luciferase-expressing OSCC cells (SCCF2Luc) were injected into the perimaxillary subgingiva of nude mice, which were then treated with 100 μg/kg ZOL or vehicle. ZOL treatment reduced tumor growth and prevented loss of bone volume and surface area but had no effect on tumor invasion. Effects on bone were associated with reduced osteolysis and increased periosteal new bone formation. ZOL-mediated inhibition of tumor-induced osteolysis was characterized by reduced numbers of tartrate-resistant acid phosphatase-positive osteoclasts at the tumor-bone interface, where it was associated with osteoclast vacuolar degeneration. The ratio of eroded to total bone surface was not affected by treatment, arguing that ZOL-mediated inhibition of osteolysis was independent of effects on osteoclast activation or initiation of bone resorption. In summary, our results establish that ZOL can reduce OSCC-induced osteolysis and may be valuable as an adjuvant therapy in OSCC to preserve mandibular and maxillary bone volume and function.

Osteolytic bone resorption in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is caused by human T lymphotropic virus type 1 (HTLV-1). Patients with ATLL frequently develop humoral hypercalcemia of malignancy (HHM) resulting from increased osteoclastic bone resorption. Our goal was to investigate the mechanisms of ATLL-induced osteoclastic bone resorption. Murine calvaria co-cultured with HTLV-1-infected cells directly or conditioned media from cell cultures had increased osteoclast activity that was dependent on RANKL, indicating that factors secreted from ATLL cells had a stimulatory effect on bone resorption. Factors released from resorbing bone stimulated proliferation of HTLV-1-infected T-cells. Parathyroid hormone-related protein (PTHrP) and macrophage inflammatory protein-1α (MIP-1α), both osteoclast stimulators, were expressed in HTLV-1-infected T-cell lines. Interestingly, when HTLV-1-infected T-cells were co-cultured with pre-osteoblasts, the expression of osteoprotegerin (OPG), an osteoclast inhibitory factor, was significantly down-regulated in the pre-osteoblasts. When OPG was added into the ex vivo osteoclastogenesis assay induced by HTLV-1-infected T-cells, osteoclastogenesis was strongly inhibited. In addition, HTLV-1-infected T-cells inhibited expression of early osteoblast genes and induced late genes. These regulators will serve as future therapeutic targets for the treatments of HHM in ATLL.

Characterization of bone resorption in novel in vitro and in vivo models of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most commonly diagnosed oral malignancy in humans and cats and frequently invades bone. The objective of this study was to determine if feline OSCC serves as a relevant model of human OSCC in terms of osteolytic behavior and expression of bone resorption agonists. Novel feline OSCC cell lines (SCCF2 and SCCF3) were derived from spontaneous carcinomas. Gene expression and osteolytic behavior were compared to an established feline OSCC cell line (SCCF1) and three human OSCC cell lines (UMSCC-12, A253 and SCC25). Interaction of OSCC with bone and murine pre-osteoblasts (MC3T3) was investigated using in vitro co-culture techniques. In vivo bioluminescent imaging, Faxitron radiography and microscopy were used to measure xenograft growth and bone invasion in nude mice. Human and feline OSCC expressing the highest levels of parathyroid hormone-related protein (PTHrP) were associated with in vitro and in vivo bone resorption and osteoclastogenesis. MC3T3 cells had increased receptor activator of nuclear factor κB ligand (RANKL) expression and reduced osteoprotegerin (OPG) expression in conditioned medium from bone-invasive SCCF2 cells compared to minimally bone invasive SCCF3 cells, which was partially reversed with a neutralizing anti-PTHrP antibody. Human and feline OSCC cells cultured in bone-conditioned medium had increased PTHrP secretion and proliferation. Feline OSCC-induced bone resorption was associated with tumor cell secretion of PTHrP and with increased RANKL:OPG expression ratio in mouse preosteoblasts. Bone-CM increased OSCC proliferation and secretion of PTHrP. The preclinical models of feline OSCC recapitulated the bone-invasive phenotype characteristic of spontaneous OSCC and will be useful to future preclinical and mechanistic studies of bone invasive behavior.

Development of a brain metastatic canine prostate cancer cell line

Prostate cancer in men has a high mortality and morbidity due to metastatic disease. The pathobiology of prostate cancer metastasis is not well understood and cell lines and animal models that recapitulate the complex nature of the disease are needed. Therefore, the goal of the study was to establish and characterize a new prostate cancer line derived from a dog with spontaneous prostate cancer.

Animal models of head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is the most common oral cancer worldwide. Local bone invasion into the maxilla or mandible and metastasis to regional lymph nodes often result in a poor prognosis, decreased quality of life and shortened survival time for HNSCC patients. Poor response to treatment and clinical outcomes are the major concerns in this aggressive cancer. Multiple animal models have been developed to replicate spontaneous HNSCC and investigate genetic alterations and novel therapeutic targets. This review provides an overview of HNSCC as well as the traditional animal models used in HNSCC preclinical research. The value and challenges of each in vivo model are discussed. Similarity between HNSCC in humans and cats and the possibility of using spontaneous feline oral squamous cell carcinoma (FOSCC) as a model for HNSCC in translational research are highlighted.

Effect of zoledronic acid and amputation on bone invasion and lung metastasis of canine osteosarcoma in nude mice

Osteosarcoma (OSA) is an aggressive, highly metastatic and lytic primary bone neoplasm commonly affecting the appendicular skeleton of dogs and children. Current treatment options include amputation of the afflicted limb, limb-sparing procedures, or palliative radiation with or without adjunct chemotherapy. Therapies that inhibit bone resorption, such as the bisphosphonates, may be an effective palliative therapy by limiting the local progression of OSA in those patients that are not viable candidates for amputation. We have developed a mouse model of canine skeletal OSA following intratibial inoculation of OSCA40 cells that spontaneously metastasized to the lungs. We demonstrated that therapy with a nitrogen-containing bisphosphonate, zoledronic acid (Zol), reduced OSA-induced bone lysis; however, Zol monotherapy or in combination with amputation was not effective at inhibiting pulmonary metastasis. While not reaching statistical significance, amputation of the tumor-bearing limb reduced the average incidence of lung metastases; however, this effect was nullified when Zol was added to the treatment protocol. In untreated mice, the magnitude of proximal tibial lysis was significantly correlated with the incidence of metastasis. The data support amputation alone for the management of appendicular OSA rather than combining amputation with Zol. However, in patients that are not viable candidates for amputation, Zol may be a useful palliative therapy for OSA by reducing the magnitude of lysis and therefore bone pain, despite the risk of increased pulmonary metastasis.