Craniofacial Phenomics: Three-Dimensional Assessment of the Size and Shape of Cranial and Dentofacial Structures.

Craniofacial phenomics has opened up numerous opportunities to correlate genetic and epigenetic factors to craniofacial phenotypes in order to improve our understanding of growth and development in health and disease. Three-dimensional (3D) imaging has played a key role in advancing craniofacial phenomics by facilitating highly sensitive and specific characterizations of craniofacial and dental morphology. Here we describe the use of micro-computed tomography (micro-CT) to image the murine craniofacial complex, followed by surface reconstruction for traditional morphometric analyses. We also describe the application of geometric morphometrics, based on Generalized Procrustes Analysis, for use in human premolars. These principles are interchangeable between various vertebrate species, and between various surface imaging techniques (including micro-CT and 3D surface scanners), offering a high level of versatility and precision for extensive phenotyping of the entire craniofacial complex.

Micro-CT-Based Bone Microarchitecture Analysis of the Murine Skull.

X-ray micro-computed tomography (micro-CT) imaging has important applications in microarchitecture analysis of cortical and trabecular bone structure. While standardized protocols exist for micro-CT-based microarchitecture assessment of long bones, specific protocols need to be developed for different types of skull bones taking into account differences in embryogenesis, organization, development, and growth compared to the rest of the body. This chapter describes the general principles of bone microarchitecture analysis of murine craniofacial skeleton to accommodate for morphological variations in different regions of interest.

Effect of Selenium and Iodine on Oxidative Stress in the First Trimester Human Placenta Explants.

Imbalanced maternal micronutrient status, poor placentation, and oxidative stress are associated with greater risk of pregnancy complications, which impact mother and offspring health. As selenium, iodine, and copper are essential micronutrients with key roles in antioxidant systems, this study investigated their potential protective effects on placenta against oxidative stress. First trimester human placenta explants were treated with different concentrations of selenium (sodium selenite), iodine (potassium iodide), their combination or copper (copper (II) sulfate). The concentrations represented deficient, physiological, or super physiological levels. Oxidative stress was induced by menadione or antimycin. Placenta explants were collected, fixed, processed, and embedded for laser ablation inductively coupled plasma-mass spectrometry (LA ICP-MS) element imaging or immunohistochemical labelling. LA ICP-MS showed that placenta could uptake selenium and copper from the media. Sodium selenite and potassium iodide reduced DNA damage and apoptosis (p < 0.05). Following oxidative stress induction, a higher concentration of sodium selenite (1.6 µM) was needed to reduce DNA damage and apoptosis while both concentrations of potassium iodide (0.5 and 1 µM) were protective (p < 0.05). A high concentration of copper (40 µM) increased apoptosis and DNA damage but this effect was no longer significant after induction of oxidative stress. Micronutrients supplementation can increase their content within the placenta and an optimal maternal micronutrient level is essential for placenta health.

Optimal Lateral Row Anchor Positioning in Posterior-Superior Transosseous Equivalent Rotator Cuff Repair: A Micro-Computed Tomography Study.

BACKGROUND: The optimal placement of suture anchors in transosseous-equivalent (TOE) double-row rotator cuff repair remains controversial. PURPOSE: A 3-dimensional (3D) high-resolution micro-computed tomography (micro-CT) histomorphometric analysis of cadaveric proximal humeral greater tuberosities (GTs) was performed to guide optimal positioning of lateral row anchors in posterior-superior (infraspinatus and supraspinatus) TOE rotator cuff repair. STUDY DESIGN: Descriptive laboratory study. METHODS: Thirteen fresh-frozen human cadaveric proximal humeri underwent micro-CT analysis. The histomorphometric parameters analyzed in the standardized volumes of interest included cortical thickness, bone volume, and trabecular properties. RESULTS: Analysis of the cortical thickness of the lateral rows demonstrated that the entire inferior-most lateral row, 15 to 21 mm from the summit of the GT, had the thickest cortical bone (mean, 0.79 mm; P = .0001), with the anterior-most part of the GT, 15 to 21 mm below its summit, having the greatest cortical thickness of 1.02 mm (P = .008). There was a significantly greater bone volume (BV; posterior, 74.5 ± 27.4 mm3; middle, 55.8 ± 24.9 mm3; anterior, 56.9 ± 20.7 mm3; P = .001) and BV as a percentage of total tissue volume (BV/TV; posterior, 7.3% ± 2.7%, middle, 5.5% ± 2.4%; anterior, 5.6% ± 2.0%; P = .001) in the posterior third of the GT than in intermediate or anterior thirds. In terms of both BV and BV/TV, the juxta-articular medial row had the greatest value (BV, 87.3 ± 25.1 mm3; BV/TV, 8.6% ± 2.5%; P = .0001 for both) followed by the inferior-most lateral row 15 to 21 mm from the summit of the GT (BV, 62.0 ± 22.7 mm3; BV/TV, 6.1% ± 2.2%; P = .0001 for both). The juxta-articular medial row had the greatest value for both trabecular number (0.3 ± 0.06 mm-1; P = .0001) and thickness (0.3 ± 0.08 µm; P = .0001) with the lowest degree of trabecular separation (1.3 ± 0.4 µm; P = .0001). The structure model index (SMI) has been shown to strongly correlate with bone strength, and this was greatest at the inferior-most lateral row 15 to 21 mm from the summit of the GT (2.9 ± 0.9; P = .0001). CONCLUSION: The inferior-most lateral row, 15 to 21 mm from the tip of the GT, has good bone stock, the greatest cortical thickness, and the best SMI for lateral row anchor placement. The anterior-most part of the GT 15 to 21 mm below its summit had the greatest cortical thickness of all zones. The posterior third of the GT also has good bone stock parameters, second only to the medial row. The best site for lateral row cortical anchor placement is 15 to 21 mm below the summit of the GT. CLINICAL RELEVANCE: Optimal lateral anchor positioning is 15 to 21 mm below the summit of the greater tuberosity in TOE.

Bone marrow lesions detected by specific combination of MRI sequences are associated with severity of osteochondral degeneration.

BACKGROUND: Bone marrow lesions (BMLs) are useful diagnostic and prognostic markers in knee osteoarthritis (OA), but what they represent at the tissue level remains unclear. The aim of this study was to provide comprehensive tissue characterization of BMLs detected using two specific MRI sequences. METHODS: Tibial plateaus were obtained from 60 patients (29 females, 31 males), undergoing knee arthroplasty for OA. To identify BMLs, MRI was performed ex vivo using T1 and PDFS-weighted sequences. Multi-modal tissue level analyses of the osteochondral unit (OCU) were performed, including cartilage volume measurement, OARSI grading, micro-CT analysis of bone microstructure, routine histopathological assessment and quantitation of bone turnover indices. RESULTS: BMLs were detected in 74 % of tibial plateaus, the remainder comprising a No BML group. Of all BMLs, 59 % were designated BML 1 (detected only by PDFS) and 41 % were designated BML 2 (detected by both PDFS + T1). The presence of a BML was related to degeneration of the OCU, particularly within BML 2. When compared to No BML, BML 2 showed reduced cartilage volume (p = 0.008), higher OARSI scores (p = 0.004), thicker subchondral plate (p = 0.002), increased trabecular bone volume and plate-like structure (p = 0.0004), increased osteoid volume (p = 0.002) and thickness (p = 0.003), more bone marrow oedema (p = 0.03), fibrosis (p = 0.002), necrosis (p = 0.01) and fibrovascular cysts (p = 0.04). For most measures, BML 1 was intermediate between No BML and BML 2. CONCLUSIONS: BMLs detected by specific MRI sequences identify different degrees of degeneration in the OCU. This suggests that MRI characteristics of BMLs may enable identification of different BML phenotypes and help target novel approaches to treatment and prevention of OA.

Anticancer efficacy of the hypoxia-activated prodrug evofosfamide (TH-302) in osteolytic breast cancer murine models.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, hypoxia offers treatment opportunities, exemplified by the development of compounds that target hypoxic regions within tumors. Evofosfamide (TH-302) is a prodrug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide mustard (Br-IPM). When evofosfamide is delivered to hypoxic regions, the DNA cross-linking effector, Br-IPM, is released. This study assessed the cytotoxic activity of evofosfamide in vitro and its antitumor activity against osteolytic breast cancer either alone or in combination with paclitaxel in vivo. A panel of human breast cancer cell lines were treated with evofosfamide under hypoxia and assessed for cell viability. Osteolytic MDA-MB-231-TXSA cells were transplanted into the mammary fat pad, or into tibiae of mice, allowed to establish and treated with evofosfamide, paclitaxel, or both. Tumor burden was monitored using bioluminescence, and cancer-induced bone destruction was measured using micro-CT. In vitro, evofosfamide was selectively cytotoxic under hypoxic conditions. In vivo evofosfamide was tumor suppressive as a single agent and cooperated with paclitaxel to reduce mammary tumor growth. Breast cancer cells transplanted into the tibiae of mice developed osteolytic lesions. In contrast, treatment with evofosfamide or paclitaxel resulted in a significant delay in tumor growth and an overall reduction in tumor burden in bone, whereas combined treatment resulted in a significantly greater reduction in tumor burden in the tibia of mice. Evofosfamide cooperates with paclitaxel and exhibits potent tumor suppressive activity against breast cancer growth in the mammary gland and in bone.

Hypoxia-activated pro-drug TH-302 exhibits potent tumor suppressive activity and cooperates with chemotherapy against osteosarcoma.

Tumor hypoxia is a major cause of treatment failure for a variety of malignancies. However, tumor hypoxia also offers treatment opportunities, exemplified by the development compounds that target hypoxic regions within tumors. TH-302 is a pro-drug created by the conjugation of 2-nitroimidazole to bromo-isophosphoramide (Br-IPM). When TH-302 is delivered to regions of hypoxia, Br-IPM, the DNA cross linking toxin, is released. In this study we assessed the cytotoxic activity of TH-302 against osteosarcoma cells in vitro and evaluated its anticancer efficacy as a single agent, and in combination with doxorubicin, in an orthotopic mouse model of human osteosarcoma (OS). In vitro, TH-302 was potently cytotoxic to osteosarcoma cells selectively under hypoxic conditions, whereas primary normal human osteoblasts were protected. Animals transplanted with OS cells directly into their tibiae and left untreated developed mixed osteolytic/osteosclerotic bone lesions and subsequently developed lung metastases. TH-302 reduced tumor burden in bone and cooperated with doxorubicin to protect bone from osteosarcoma induced bone destruction, while it also reduced lung metastases. TH-302 may therefore be an attractive therapeutic agent with strong activity as a single agent and in combination with chemotherapy against OS.

Doxorubicin overcomes resistance to drozitumab by antagonizing Inhibitor of Apoptosis Proteins (IAPs).

BACKGROUND/AIM: Drozitumab is a fully human agonistic monoclonal antibody that binds to death receptor DR5 and induces apoptosis. However, drozitumab resistance is a major obstacle limiting anticancer efficacy. MATERIALS AND METHODS: We examined the potential for the chemotherapeutic agent doxorubicin to overcome resistance against drozitumab-resistant breast cancer cells both in vitro and in vivo. RESULTS: Treatment with doxorubicin increased cell surface expression of DR5, reduced levels of Inhibitors of Apoptosis Proteins (cIAPs) and re-sensitised cells to drozitumab-induced apoptosis. Animals implanted with resistant breast cancer cells into the mammary fat pad and treated with a combination of drozitumab and doxorubicin showed inhibition of tumor growth and a substantial delay in tumor progression compared to untreated controls and mice treated with each agent alone. CONCLUSION: These results suggest that combination of drozitumab with chemotherapy and agents that modulate IAP levels could potentially be a useful strategy in the treatment of breast cancer.

Pharmacologic inhibition of bone resorption prevents cancer-induced osteolysis but enhances soft tissue metastasis in a mouse model of osteolytic breast cancer.

Osteoprotegerin (OPG) is a secreted member of the TNF receptor superfamily, which binds to the receptor activator of nuclear factor κB ligand (RANKL) and inhibits osteoclast activity and bone resorption. Systemic administration of recombinant OPG was previously shown to inhibit tumor growth in bone and to prevent cancer-induced osteolysis. In this study, we examined the effect of OPG, when produced locally by breast cancer cells located within bone, using a mouse model of osteolytic breast cancer. MDA-MB-231-TXSA breast cancer cells, tagged with a luciferase reporter gene construct and engineered to overexpress full-length human OPG, were transplanted directly into the tibial marrow cavity of nude mice. Overexpression of OPG by breast cancer cells protected the bone from breast cancer-induced osteolysis and diminished intra-osseous tumor growth but had no effect on extra-skeletal tumor growth. This effect was associated with a significant reduction in the number of osteoclasts that lined the bone surface, resulting in a net increase in bone volume. Despite limiting breast cancer-mediated bone loss, OPG overexpression resulted in a significant increase in the incidence of pulmonary metastasis. Our results demonstrate that inhibition of osteoclastic bone resorption by OPG when secreted locally by tumors in bone may affect the behaviour of cancer cells within the bone microenvironment and their likelihood of spreading and establishing metastasis elsewhere in the body.

RECK in osteosarcoma: a novel role in tumour vasculature and inhibition of tumorigenesis in an orthotopic model.

BACKGROUND: Targeted therapy in osteosarcoma (OS) is needed to improve patient outcomes. Human RECK may have a role because it inhibits cancer invasion and regulates angiogenesis. This study aimed to characterize RECK expression in human OS, to examine in vitro effects of RECK on vascular endothelium and OS cell behavior, and to analyze the effect of RECK on OS grown orthotopically in nude mice. METHODS: RECK was examined in human OS samples. Interactions between RECK and VEGF were studied in tissue and cells. RECK transfection was used to study its effects on vascular endothelial (HMEC-1) and OS (SaOS-2) cell behavior in vitro and in vivo. SaOS-2 co-culture with RAW 246.7-derived osteoclasts on osteoslides was used to assess effects on osteoclast activity. RESULTS: RECK was absent from OS cells but was expressed in tumor vessel endothelium. Via microarray analysis, RECK mRNA was elevated in samples with low proliferative activity, a trend most evident in poorly differentiated samples. VEGF induced RECK expression in HMEC-1. RECK transfection inhibited HMEC-1 invasion and induced thicker, although more numerous, tube formation. RECK inhibited SaOS-2 invasion, proliferation, colony formation, and osteoclast activity but supported SaOS-2 adhesion to collagen I. In vivo, RECK inhibited SaOS-2 tumor growth, bone destruction, and consequent metastasis. CONCLUSIONS: RECK expression is downregulated in highly proliferative OS but is present in tumor vessels and upregulated in endothelium by VEGF. RECK inhibits invasion and tumorigenic properties in SaOS-2, as confirmed in vivo. Further testing of RECK delivery in OS is warranted.

Anticancer efficacy of Apo2L/TRAIL is retained in the presence of high and biologically active concentrations of osteoprotegerin in vivo.

Osteoprotegerin (OPG) is a secreted member of the tumor necrosis factor (TNF) receptor superfamily that binds to the ligand for receptor activator of nuclear factor κB (RANKL) and inhibits bone resorption. OPG can also bind and inhibit the activity of the TNF-related apoptosis-inducing ligand (Apo2L/TRAIL), raising the possibility that the anticancer efficacy of soluble Apo2L/TRAIL may be abrogated in the bone microenvironment where OPG expression is high. In this study we used a murine model of breast cancer growth in bone to evaluate the efficacy of recombinant soluble Apo2L/TRAIL against intratibial tumors that were engineered to overexpress native full-length human OPG. In vitro, OPG-overexpressing breast cancer cells were protected from Apo2L/TRAIL-induced apoptosis, an effect that was reversed with the addition of soluble RANKL or neutralizing antibodies to OPG. In vivo, mice injected intratibially with cells containing the empty vector developed large osteolytic lesions. In contrast, OPG overexpression preserved the integrity of bone and prevented breast cancer-induced bone destruction. This effect was due primarily to the complete absence of osteoclasts in the tibias of mice inoculated with OPG-transfected cells, confirming the biologic activity of the transfected OPG in vivo. Despite the secretion of supraphysiologic levels of OPG, treatment with Apo2L/TRAIL resulted in strong growth inhibition of both empty vector and OPG-overexpressing intratibial tumors. While Apo2L/TRAIL-induced apoptosis may be abrogated in vitro by OPG overexpression, the in vivo anticancer efficacy of recombinant soluble Apo2L/TRAIL is retained in the bone microenvironment even in the presence of biologically active OPG at supraphysiologic concentrations.

Zoledronic acid protects against osteosarcoma-induced bone destruction but lacks efficacy against pulmonary metastases in a syngeneic rat model.

Osteosarcoma (OS) is the most common primary malignant tumor of bone in children and adolescents. In spite of successful control of the primary tumor, death from lung metastasis occurs in more than a third of patients. To investigate the efficacy of zoledronic acid (ZOL) on the development, progression and metastatic spread of OS, we used a rat model of OS, with features of the disease similar to human patients, including spontaneous metastasis to lungs. Rat OS cells were inoculated into the tibial marrow cavity of syngeneic rats. OS development was associated with osteolysis mixed with new bone formation, adjacent to the periosteum and extended into the surrounding soft tissue. Metastatic foci in the lungs formed 3-4 weeks postcancer cell transplantation. Treatment with a clinically relevant dose of ZOL was initiated 1 week after tumors were established and continued once weekly or as a single dose. ZOL preserved the integrity of both trabecular and cortical bone and reduced tumor-induced bone formation. However, the overall tumor burden at the primary site was not reduced because of the persistent growth of cancer cells in the extramedullary space, which was not affected by ZOL treatment. ZOL treatment failed to prevent the metastatic spread of OS to the lungs. These findings suggest that ZOL as a single agent protects against OS-induced bone destruction but lacks efficacy against pulmonary metastases in this rat model. ZOL may have potential value as an adjuvant therapy in patients with established OS.

Apomab, a fully human agonistic antibody to DR5, exhibits potent antitumor activity against primary and metastatic breast cancer.

Apomab, a fully human agonistic DR5 monoclonal antibody, triggers apoptosis through activation of the extrinsic apoptotic signaling pathway. In this study, we assessed the cytotoxic effect of Apomab in vitro and evaluated its antitumor activity in murine models of breast cancer development and progression. MDA-MB-231-TXSA breast cancer cells were transplanted into the mammary fat pad or directly into the tibial marrow cavity of nude mice. Apomab was administered early, postcancer cell transplantation, or after tumors progressed to an advanced stage. Tumor burden was monitored progressively using bioluminescence imaging, and the development of breast cancer-induced osteolysis was measured using microcomputed tomography. In vitro, Apomab treatment induced apoptosis in a panel of breast cancer cell lines but was without effect on normal human primary osteoblasts, fibroblasts, or mammary epithelial cells. In vivo, Apomab exerted remarkable tumor suppressive activity leading to complete regression of well-advanced mammary tumors. All animals transplanted with breast cancer cells directly into their tibiae developed large osteolytic lesions that eroded the cortical bone. In contrast, treatment with Apomab following an early treatment protocol inhibited both intraosseous and extraosseous tumor growth and prevented breast cancer-induced osteolysis. In the delayed treatment protocol, Apomab treatment resulted in the complete regression of advanced tibial tumors with progressive restoration of both trabecular and cortical bone leading to full resolution of osteolytic lesions. Apomab represents a potent immunotherapeutic agent with strong activity against the development and progression of breast cancer and should be evaluated in patients with primary and metastatic disease.

Targeted disruption of the CXCL12/CXCR4 axis inhibits osteolysis in a murine model of myeloma-associated bone loss.

The plasma cell (PC) malignancy, multiple myeloma (MM), is unique among hematological malignancies in its capacity to cause osteoclast (OC)-mediated skeletal destruction. We have previously shown that elevated plasma levels of PC-derived CXCL12 are associated with presence of X-ray detectable osteolytic lesions in MM patients. To further investigate this relationship, plasma levels of CXCL12 and betaCrossLaps, a marker of bone loss, were measured. A strong correlation between levels of CXCL12 and OC-mediated bone resorption was identified. To confirm the OC-activating potential of MM PC-derived CXCL12 in vivo, we established a model of MM-mediated focal osteolysis, wherein MM PC lines, such as RPMI-8226, were injected into the tibias of nude mice. Implanting RPMI-8226 gave rise to osteolytic lesions proximal to the tumor, resulting in a 5% decrease in bone volume (BV) compared with vehicle control. Importantly, bone loss was significantly inhibited with systemic administration of the CXCL12/CXCR4 antagonist T140. Furthermore, implanting CXCL12-overexpressing RPMI-8226 cells resulted in a 13% decrease in BV and was associated with increased OC recruitment proximal to the tumor, increased serum matrix metalloproteinase activity, and increased levels of collagen I degradation products. These findings confirm our hypothesis that MM PC-derived CXCL12 stimulates the recruitment and activity of OC, thereby contributing to the formation of MM osteolytic lesions.

Zoledronic acid inhibits both the osteolytic and osteoblastic components of osteosarcoma lesions in a mouse model.

PURPOSE: To evaluate the efficacy of zoledronic acid (ZOL) against osteosarcoma (OS) growth, progression, and metastatic spread using an animal model of human OS that closely resembles the human disease. EXPERIMENTAL DESIGN: Human K-HOS or KRIB OS cells, tagged or untagged with a luciferase reporter construct, were transplanted directly into the tibial cavity of nude mice. ZOL was given as weekly, or a single dose of 100 microg/kg body weight, equivalent to the 4 mg i.v. dose used clinically. Tumor growth at the primary site and as pulmonary metastases was monitored by bioluminescence imaging and histology, and OS-induced bone destruction was measured using high-resolution micro-computed tomography. RESULTS: Mice transplanted with OS cells exhibited aberrant bone remodeling in the area of cancer cell transplantation, with areas of osteolysis mixed with extensive new bone formation extending from the cortex. ZOL administration prevented osteolysis and significantly reduced the amount of OS-induced bone formation. However, ZOL had no effect on tumor burden at the primary site. Importantly, ZOL failed to reduce lung metastasis and in some cases was associated with larger and more numerous metastatic lesions. CONCLUSIONS: Our data show that clinically relevant doses of ZOL, while protecting the bone from OS-induced bone destruction, do not inhibit primary tumor growth. Moreover, lung metastases were not reduced and may even have been promoted by this treatment, indicating that caution is required when the clinical application of the bisphosphonate class of antiresorptives is considered in OS.

Apo2L/TRAIL inhibits tumor growth and bone destruction in a murine model of multiple myeloma.

PURPOSE: Multiple myeloma is an incurable disease, for which the development of new therapeutic approaches is required. Here, we report on the efficacy of recombinant soluble Apo2L/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to inhibit tumor progression and bone destruction in a xenogeneic model of human multiple myeloma. EXPERIMENTAL DESIGN: We established a mouse model of myeloma, in which Apo2L/TRAIL-sensitive RPMI-8226 or KMS-11 cells, tagged with a triple reporter gene construct (NES-HSV-TK/GFP/Luc), were transplanted directly into the tibial marrow cavity of nude mice. Tumor burden was monitored progressively by bioluminescence imaging and the development of myeloma-induced osteolysis was measured using high resolution in vivo micro-computed tomography. RESULTS: Tumor burden increased progressively in the tibial marrow cavity of mice transplanted with Apo2L/TRAIL-sensitive RPMI-8226 or KMS-11 cells associated with extensive osteolysis directly in the area of cancer cell transplantation. Treatment of mice with recombinant soluble Apo2L/TRAIL reduced myeloma burden in the bone marrow cavity and significantly protected against myeloma-induced osteolysis. The protective effects of Apo2L/TRAIL treatment on bone were mediated by the direct apoptotic actions of Apo2L/TRAIL on myeloma cells within the bone microenvironment. CONCLUSIONS: This is the first in vivo study that investigates the efficacy of recombinant Apo2L/TRAIL on myeloma burden within the bone microenvironment and associated myeloma-induced bone destruction. Our findings that recombinant soluble Apo2L/TRAIL reduces myeloma burden within the bone microenvironment and protects the bone from myeloma-induced bone destruction argue against an inhibitory role of osteoprotegerin in Apo2L/TRAIL-induced apoptosis in vivo and highlight the need to clinically evaluate Apo2L/TRAIL in patients with multiple myeloma.

Apo2l/Tumor necrosis factor-related apoptosis-inducing ligand prevents breast cancer-induced bone destruction in a mouse model.

Breast cancer is the most common carcinoma that metastasizes to bone. To examine the efficacy of recombinant soluble Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against breast cancer growth in bone, we established a mouse model in which MDA-MB-231 human breast cancer cells were transplanted directly into the marrow cavity of the tibiae of athymic nude mice producing osteolytic lesions in the area of injection. All vehicle-treated control animals developed large lesions that established in the marrow cavity, eroded the cortical bone, and invaded the surrounding soft tissue, as assessed by radiography, micro-computed tomography, and histology. In contrast, animals treated with recombinant soluble Apo2L/TRAIL showed significant conservation of the tibiae, with 85% reduction in osteolysis, 90% reduction in tumor burden, and no detectable soft tissue invasion. Tumor cells explanted from Apo2L/TRAIL-treated animals were significantly more resistant to the effects of Apo2L/TRAIL when compared with the cells explanted from the vehicle-treated control animals, suggesting that prolonged treatment with Apo2/TRAIL in vivo selects for a resistant phenotype. However, such resistance was readily reversed when Apo2L/TRAIL was used in combination with clinically relevant chemotherapeutic drugs, including taxol, etoposide, doxorubicin, cisplatin, or the histone deacetylase inhibitor suberoylanilide hydroxamic acid. These studies show for the first time that Apo2L/TRAIL can prevent breast cancer-induced bone destruction and highlight the potential of this ligand for the treatment of metastatic breast cancer in bone.

The histone deacetylase inhibitor, suberoylanilide hydroxamic acid, overcomes resistance of human breast cancer cells to Apo2L/TRAIL.

While the apoptosis-inducing ligand Apo2L/TRAIL is a promising new agent for the treatment of cancer, the sensitivity of cancer cells for induction of apoptosis by Apo2L/TRAIL varies considerably. Identification of agents that can be used in combination with Apo2L/TRAIL to enhance apoptosis in breast cancer cells would increase the potential utility of this agent as a breast cancer therapeutic. Here, we show that the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize Apo2L/TRAIL-resistant breast cancer cells to Apo2L/TRAIL-induced apoptosis. Importantly, neither Apo2L/TRAIL alone, nor in combination with SAHA, affected the viability of normal human cells in culture. Apo2L/TRAIL-resistant MDA-MB-231 breast cancer cells, generated by long-term culture in the continuous presence of Apo2L/TRAIL, were resensitized to Apo2L/TRAIL-induced apoptosis by SAHA. The sensitization of these cells by SAHA was accompanied by activation of caspase 8, caspase 9 and caspase 3 and was concomitant with Bid and PARP cleavage. The expression of the proapoptotic protein, Bax, increased significantly with SAHA treatment and high levels of Bax were maintained in the combined treatment with Apo2L/TRAIL. Treatment with SAHA increased cell surface expression of DR5 but not DR4. Interestingly, SAHA treatment also resulted in a significant increase in cell surface expression of DcR1. Taken together, our findings indicate that the use of these 2 agents in combination may be effective for the treatment of breast cancer.