Experimental Breast Cancer Bone Metastasis Research Articles

Prediction of early metastatic disease in experimental breast cancer bone metastasis by combining PET/CT and MRI parameters to a Model-Averaged Neural Network.

Macrometastases in bone are preceded by bone marrow invasion of disseminated tumor cells. This study combined functional imaging parameters from FDG-PET/CT and MRI in a rat model of breast cancer bone metastases to a Model-averaged Neural Network (avNNet) for the detection of early metastatic disease and prediction of future macrometastases. Metastases were induced in 28 rats by injecting MDA-MB-231 breast cancer cells into the right superficial epigastric artery, resulting in the growth of osseous metastases in the right hind leg of the animals. All animals received FDG-PET/CT and MRI at days 0, 10, 20 and 30 after tumor cell injection. In total, 18/28 rats presented with metastases at days 20 or 30 (64.3%). None of the animals featured morphologic bone lesions during imaging at day 10, and the imaging parameters acquired at day 10 did not differ significantly between animals with metastases at or after day 20 and those without (all p > 0.3). The avNNet trained with the imaging parameters acquired at day 10, however, achieved an accuracy of 85.7% (95% CI 67.3-96.0%) in predicting future macrometastatic disease (ROCAUC 0.90; 95% CI 0.76-1.00), and significantly outperformed the predictive capacities of all single parameters (all p ≤ 0.02). The integration of functional FDG-PET/CT and MRI parameters into an avNNet can thus be used to predict macrometastatic disease with high accuracy, and their combination might serve as a surrogate marker for bone marrow invasion as an early metastatic process that is commonly missed during conventional staging examinations.

Retraction: Therapeutic Intervention of Experimental Breast Cancer Bone Metastasis by Indole-3-carbinol in SCID-human Mouse Model.

Retraction: Therapeutic Intervention of Experimental Breast Cancer Bone Metastasis by Indole-3-carbinol in SCID-human Mouse Model.

The C-Terminal Intact Forms of Periostin (iPTN) Are Surrogate Markers for Osteolytic Lesions in Experimental Breast Cancer Bone Metastasis

Periostin is an extracellular matrix protein that actively contributes to tumor progression and metastasis. Here, we hypothesized that it could be a marker of bone metastasis formation. To address this question, we used two polyclonal antibodies directed against the whole molecule or its C-terminal domain to explore the expression of intact and truncated forms of periostin in the serum and tissues (lung, heart, bone) of wild-type and periostin-deficient mice. In normal bones, periostin was expressed in the periosteum and specific periostin proteolytic fragments were found in bones, but not in soft tissues. In animals bearing osteolytic lesions caused by 4T1 cells, C-terminal intact periostin (iPTN) expression disappeared at the invasive front of skeletal tumors where bone-resorbing osteoclasts were present. In vitro, we found that periostin was a substrate for osteoclast-derived cathepsin K, generating proteolytic fragments that were not recognized by anti-periostin antibodies directed against iPTN. In vivo, using an in-house sandwich immunoassay aimed at detecting iPTN only, we observed a noticeable reduction of serum periostin levels (- 26%; P < 0.002) in animals bearing osteolytic lesions caused by 4T1 cells. On the contrary, this decrease was not observed in women with breast cancer and bone metastases when periostin was measured with a human assay detecting total periostin. Collectively, these data showed that mouse periostin was degraded at the bone metastatic sites, potentially by cathepsin K, and that the specific measurement of iPTN in serum should assist in detecting bone metastasis formation in breast cancer.

Tumor-Targeting Salmonella typhimurium A1-R: An Overview.

The present chapter reviews the development of the tumor-targeting amino-acid auxotrophic strain S. typhimurium A1 and the in vivo selection and characterization of the high-tumor-targeting strain S. typhimurium A1-R. Efficacy of S. typhimurium A1-R in nude-mouse models of prostate, breast, pancreatic, and ovarian cancer, as well as sarcoma and glioma in orthotopic mouse models is described. Also reviewed is efficacy of S. typhimurium A1-R targeting of primary bone tumor and lung metastasis of high-grade osteosarcoma, breast-cancer brain metastasis, and experimental breast-cancer bone metastasis in orthotopic mouse models. The efficacy of S. typhimurium A1-R on pancreatic cancer stem cells, on pancreatic cancer in combination with anti-angiogenic agents, as well as on cervical cancer, soft-tissue sarcoma, and pancreatic cancer patient-derived orthotopic xenograft (PDOX) mouse models, is also described.

Diffusion-weighted imaging and dynamic contrast-enhanced MRI of experimental breast cancer bone metastases – A correlation study with histology

PurposeTo validate imaging parameters from diffusion-weighted imaging and dynamic contrast-enhanced MRI with immunohistology and to non-invasively assess microstructure of experimental breast cancer bone metastases. Materials and methodsAnimals bearing breast cancer bone metastases were imaged in a clinical 1.5T MRI scanner. HASTE sequences were performed to calculate apparent diffusion coefficients. Saturation recovery turbo FLASH sequences were conducted while infusing 0.1mmol/l Gd–DTPA for dynamic contrast-enhanced MRI to quantify parameters amplitude A and exchange rate constant kep. After imaging, bone metastases were analyzed immunohistologically. ResultsWe found correlations of the apparent diffusion coefficients from diffusion-weighted imaging with tumor cellularity as assessed with cell nuclei staining. Histological vessel maturity was correlated negatively with parameters A and kep from dynamic contrast-enhanced MRI. Tumor size correlated inversely with cell density and vessel permeability as well as positively with mean vessel calibers. Parameters from the rim of bone metastases differed significantly from values of the center. ConclusionIn vivo diffusion-weighted imaging and dynamic contrast-enhanced MRI in experimental bone metastases provide information about tumor cellularity and vascularity and correlate well with immunohistology.

Combination of anti-angiogenic therapies reduces osteolysis and tumor burden in experimental breast cancer bone metastasis

The clinical efficacy of anti-angiogenic monotherapies in metastatic breast cancer is less than originally anticipated, and it is not clear what the response of bone metastasis to anti-angiogenic therapies is. Here, we examined the impact of neutralizing tumor-derived vascular endothelial growth factor (VEGF) in animal models of subcutaneous tumor growth and bone metastasis formation. Silencing of VEGF expression (Sh-VEGF) in osteotropic human MDA-MB-231/B02 breast cancer cells led to a substantial growth inhibition of subcutaneous Sh-VEGF B02 tumor xenografts, as a result of reduced angiogenesis, when compared to that observed with animals bearing mock-transfected (Sc-VEGF) B02 tumors. However, there was scant evidence that either the silencing of tumor-derived VEGF or the use of a VEGF-neutralizing antibody (bevacizumab) affected B02 breast cancer bone metastasis progression in animals. We also examined the effect of vatalanib (a VEGF receptor tyrosine kinase inhibitor) in this mouse model of bone metastasis. However, vatalanib failed to inhibit bone metastasis caused by B02 breast cancer cells. In sharp contrast, vatalanib in combination with bevacizumab reduced not only bone destruction but also skeletal tumor growth in animals bearing breast cancer bone metastases, when compared with either agent alone. Thus, our study highlights the importance of targeting both the tumor compartment and the host tissue (i.e., skeleton) to efficiently block the development of bone metastasis. We believe this is a crucially important observation as the clinical benefit of anti-angiogenic monotherapies in metastatic breast cancer is relatively modest.

Abstract P5-04-13: Antibody-based therapy targeting integrin a5 is an effective strategy to treat experimental breast cancer bone metastasis

Abstract Background: Integrin a5β1 is a specific fibronectin receptor that is often upregulated in breast cancer cells undergoing epithelial-to-mesenchymal transition. Preclinical evidence showed that α5β1-fibronectin interaction promotes in vitro the survival of growth-arrested breast cancer cells in the bone marrow. This is in line with the observation that disseminated tumor cells (DTCs) in the bone marrow, that are the earliest sign of development of metastatic disease in patients, express α5β1 integrin. However, the role of α5β1 in bone marrow micrometastasis formation remains unknown. Here, we investigated its role in experimental breast cancer bone metastasis, using an antibody-based strategy targeting the α5 subunit. Methods: Integrin α5 mRNA expression levels were quantified by qRT-PCR, using radically resected primary tumors of 427 breast cancer patients. Comparison between Kaplan-Meier curves was performed using the log-rank test. The Cox model was used for multivariate analysis. For experimental metastasis, female BALB/c immunodeficient mice were treated with a chimeric IgG4 monoclonal antibody that specifically binds to human integrin subunit α5 (M200;15mg/kg) or the vehicle. Treatment was performed 3 times per week starting the day before intra-arterial inoculation with luciferase-expressing human MDA-MB-231/B02 breast cancer cells, which selectively metastasize to bone. Vehicle and M200-treated mice were analyzed by radiography and bioluminescence. On day 28 after tumor cell inoculation, animals were sacrificed and histomorphometric analysis of metastatic legs was performed. Alternatively, animals were culled on day 7 after tumor cell inoculation, and the bone marrow was flushed for DTC colony assay. In-vitro cell-based assays were conducted to explore the functions of α5β1 on adhesion, invasion and survival. Statistical analysis was carried out by performing a Mann-Whitney U test. Results: Median durations of metastasis-free survival were 6.2 (high α5 expression) and 9.5 years (low α5 expression) (P = 0.0008). Additionally, compared with low expression, a high α5 expression was associated with shorter bone metastasis-free survival, both in univariate (P = 0.024) and multivariate analysis (HR = 1.65,95%CI = 1.02 to 2.67; P = 0.04).The treatment of tumor-bearing animals with M200 antibody statistically significantly delayed the onset of skeletal lesions (P = 0.02) and caused a 50% reduction in the extent of osteolytic lesions (P = 0.038), compared with vehicle. This difference was accompanied with a sharp reduction of tumor burden (P = 0.02), as determined by bioluminescence. Histomorphometric analysis of metastatic legs showed that M200 treatment decreased skeletal tumor burden (P = 0.027) and increased the bone volume (P = 0.02), compared with vehicle. Additionally, the number of DTC colonies in the bone marrow from M200-treated mice was dramatically decreased compared with vehicle (P&amp;lt;0.001). In vitro, M200 antibody did not affect tumor cell survival. By contrast, it specifically inhibited fibronectin-mediated tumor cell adhesion and invasion. Conclusion: Our results suggest that α5β1 integrin expression in breast cancer cells facilitates bone marrow micrometastasis formation and the subsequent development of osteolytic lesions. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-13.

Quantitative Contrast-Enhanced Ultrasound for Imaging Antiangiogenic Treatment Response in Experimental Osteolytic Breast Cancer Bone Metastases

The aim of this study was to investigate the feasibility of using contrast-enhanced ultrasound (CEUS) in experimental breast cancer bone metastases and its utilization for assessment of early antiangiogenic treatment response in these lesions. Nude rats bearing bone metastases (n = 20) were treated with the antiangiogenic tyrosine kinase inhibitor sunitinib daily from days 30 to 35 after MDA-MB-231 tumor cell inoculation (n = 10) and compared with sham-treated controls (n = 10). Imaging with ultrasound (US) and magnetic resonance imaging (MRI) was performed on days 30, 32, and 35 after tumor cell inoculation to determine tumor volume and parameters of vascularization in bone metastases. Contrast-enhanced US images were used to calculate wash-in and wash-out values, peak enhancement, and area under the curve from time intensity curves. In addition, a quantitative analysis software was used to determine regional blood volume and flow as well as filling times within bone metastases. For comparison, dynamic contrast-enhanced MRI provided amplitude A and exchange rate constant kep, respectively. Immunohistological analysis of the vasculature in bone metastases followed in vivo imaging experiments. Although no changes in tumor volume were assessed in the observation time, significantly decreased values for peak enhancement, area under the curve, and wash-out were determined by CEUS in animals treated with sunitinib at day 35 after tumor cell inoculation. Analysis of CEUS images with quantitative analysis software showed significantly lower values for regional blood volume and regional blood flow as well as higher values for filling time in treated animals as early as 2 days after therapy onset. Dynamic contrast-enhanced MRI revealed significantly decreased values for parameter A at day 35 and kep at days 32 and 35 after tumor cell inoculation for treated animals. Immunohistology of bone metastases revealed significantly larger vessels and decreased positive area fraction for von Willebrand Factor in animals treated with sunitinib. Contrast-enhanced US is feasible in experimental breast cancer bone metastases and depicts early antiangiogenic treatment response in advanced osteolytic lesions.

Abstract 379: Monitoring adjuvant treatment effects of MMP 2/9 inhibition in experimental breast cancer bone metastases using VCT and ultra high-field MRI in a longitudinal study

Abstract In the microenvironment of bone metastases, matrix metalloproteinases (MMPs) 2/9 are expressed by osteoblasts, osteoclasts and tumor cells, and in particular MMP 9 has been associated with osteoclast-mediated migration, invasion and angiogenesis. Using non-invasive imaging techniques including magnetic resonance imaging (MRI) and volumetric computed tomography (VCT) monitoring various aspects of treatment response including osteolysis, invasion and angiogenesis can be assessed longitudinally. The purpose of the study was to image effects of adjuvant MMP 2/9 inhibition in experimental breast cancer bone metastases using VCT and ultra high-field MRI. Human MDA-MB-231 breast cancer cells were inoculated into the femoral artery of nude rats, resulting in localized osteolytic bone metastases of the hind leg. Animals were either treated with a MMP 2/9 inhibitor (n=14) beginning immediately after tumor cell inoculation (p.i.) for 30 days (200 mg/kg orally) or sham-treated (n=17). Animals were imaged at days 10, 20 and 30 p.i. using VCT (Volume CT, Siemens) and MRI (7 Tesla, Siemens). Dynamic contrast-enhanced MRI (DCE-MRI) was performed to determine amplitude A (associated with blood volume) and exchange rate constant Kep (associated with perfusion and vessel permeability) in skeletal metastases. Volumetry of osteolysis and of the corresponding soft tissue tumor was obtained by VCT and MRI, respectively. All values were expressed as treatment over control ratios in percent (T/C%). Beginning from day 20 p.i., osteolyses and corresponding soft tissue tumors of bone metastases could be assessed by VCT and MRI at high isotropic resolution (200 µm for VCT, 110 µm for MRI). Strong uptake of contrast media within the soft tissue tumor enabled detailed assessment of volume and invasive behavior of the soft tissue tumor using ultra high-field MRI. Bone metastasis formation could not be prevented by MMP inhibition as the tumor take rate was not significantly different between the two groups within the observation time. However, the mean volumes of osteolysis and soft tissue tumor were significantly smaller upon MMP inhibition as compared to controls at days 20 (MRI, 28 T/C%; VCT, 46 T/C%) and 30 (MRI, 24 T/C%; VCT, 38 T/C%) p.i. As determined by DCE-MRI in skeletal metastases, a significant reduction of the amplitude A was assessed after MMP inhibition (day 30 p.i., 63 T/C%), whereas no significant differences for the exchange rate constant Kep were found between the groups. In conclusion, inhibition of MMPs 2/9 in experimental breast cancer bone metastases resulted in anti-resorptive, anti-invasive and anti-angiogenic effects as imaged longitudinally and at high resolution by VCT and ultra high-field MRI after adjuvant treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 379. doi:1538-7445.AM2012-379

A novel PET tracer for the imaging of αvβ3 and αvβ5 integrins in experimental breast cancer bone metastases

The aim of this study was the evaluation of (68)Ga-DOTA-E-[c(RGDfK)](2) as a novel PET tracer to image αvβ3 and αvβ5 integrins. For this purpose, DOTA-E-[c(RGDfK)](2) was labeled with (68)Ga, which was obtained from a (68)Ge/(68)Ga generator, purified by solid-phase extraction and the radiochemical purity analyzed by radio-RP-HPLC. (68) Ga-DOTA-E-[c(RGDfK)](2) was obtained reproducibly in radiochemical yields of 60 ± 6% and with an excellent radiochemical purity of >99%. In nude rats bearing bone metastases after injection of MDA-MB-231 human breast cancer cells, biodistribution studies were performed to evaluate the accumulation of the radiotracer in selected organs, blood and bone metastases 0.5, 1, 2 and 3 h post injection. A rapid uptake into the bone metastases and rapid blood clearance was observed, resulting in tumor-blood ratios of up to 26.6 (3 h post injection) and tumor-muscle ratios of up to 7.9 (3 h post injection). A blocking experiment with coinjected αvβ3/αvβ5 antagonist showed the tumor uptake to be receptor-specific. In an initial in vivo micro PET evaluation of the tracer using the same animal model, the bone metastasis was clearly visualized. These results suggest that (68)Ga-DOTA-E-[c(RGDfK)](2) is a promising PET tracer suitable for the imaging of αvβ3 and αvβ5 integrins in bone metastases. This novel PET tracer should be further evaluated concerning its usefulness for early detection of bone metastases and monitoring treatment response of these lesions.

Sorafenib tosylate and paclitaxel induce anti-angiogenic, anti-tumour and anti-resorptive effects in experimental breast cancer bone metastases

Purpose In this study we investigated sorafenib tosylate and paclitaxel as single and combination therapies regarding their effects on tumour growth and vasculature as well as their potency to inhibit osteolysis in experimental breast cancer bone metastases. Experimental design Nude rats bearing breast cancer bone metastases were treated with sorafenib tosylate (7 mg/kg, n = 11), paclitaxel (5 mg/kg, n = 11) or the combination of both ( n = 10) and were compared to untreated controls ( n = 11). In a longitudinal study, volumes of osteolyses and respective soft tissue tumours were measured in these groups by MRI and volume CT, while changes in cellularity within bone metastases were assessed by diffusion-weighted imaging. Dynamic contrast-enhanced MRI and vessel size imaging was performed to determine changes of tumour vasculature within osseous lesions non-invasively. Results Animals treated with sorafenib tosylate or paclitaxel showed significantly reduced growth of both, the osteolytic lesions and the soft tissue tumours as well as a decreased cellularity in bone metastases compared to control rats. Effects on the tumour vasculature of these drugs included significantly reduced blood volume as well as significant changes of the vessel permeability and the mean vessel calibers. When combining sorafenib tosylate with paclitaxel for the treatment of bone metastases positive combination effects were observed, particularly on reducing vessel permeability in these lesions. Conclusion The application of sorafenib tosylate monotherapy or in combination with paclitaxel is effective against experimental breast cancer bone metastases resulting in anti-angiogenic, anti-tumour and anti-resorptive effects.

Cilengitide inhibits progression of experimental breast cancer bone metastases as imaged noninvasively using VCT, MRI and DCE‐MRI in a longitudinal in vivo study

The aim of this study was to investigate the effect of inhibiting αvβ(3)/α(v) β(5) integrins by cilengitide in experimentally induced breast cancer bone metastases using noninvasive imaging techniques. For this purpose, nude rats bearing established breast cancer bone metastases were treated with cilengitide, a small molecule inhibitor of αvβ(3) and αvβ(5) integrins (75 mg/kg, five days per week; n = 12 rats) and compared to vehicle-treated control rats (n = 12). In a longitudinal study, conventional magnetic resonance imaging (MRI) and flat panel volumetric computed tomography were used to assess the volume of the soft tissue tumor and osteolysis, respectively, and dynamic contrast-enhanced (DCE-) MRI was performed to determine functional parameters of the tumor vasculature reflecting blood volume and blood vessel permeability. In rats treated with cilengitide, VCT and MRI showed that osteolytic lesions and the respective bone metastatic soft tissue tumors progressed more slowly than in vehicle-treated controls. DCE-MRI indicated a decrease in blood volume and an increase in vessel permeability and immunohistology revealed increased numbers of immature vessels in cilengitide-treated rats compared to vehicle controls. In conclusion, treatment of experimental breast cancer bone metastases with cilengitide resulted in pronounced antiresorptive and antitumor effects, suggesting that αvβ(3)/αvβ(5) inhibition may be a promising therapeutic approach for bone metastases.

Drug-Induced Vessel Remodeling in Bone Metastases as Assessed by Dynamic Contrast Enhanced Magnetic Resonance Imaging and Vessel Size Imaging: A LongitudinalIn vivoStudy

The aim of this study was to assess the antiangiogenic treatment effects of zoledronic acid (ZA) and sunitinib malate (SM) noninvasively in experimental breast cancer bone metastases by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and vessel size imaging. Nude rats bearing bone metastases after inoculation of MDA-MB-231 breast cancer cells were treated with ZA (40 microg/kg weekly; n = 8 rats), SM (20 mg/kg daily; n = 8 rats), or their combination (ZA and SM; n = 8 rats) and compared with sham-treated controls (n = 10 rats). Vascular changes in bone metastases were longitudinally imaged in vivo using DCE-MRI [amplitude (A) and exchange rate coefficient (k(ep))] and vessel size imaging [blood volume (BV) and vessel size index (VI)]. In addition, antiresorptive and antitumor changes were assessed in these lesions by flat-panel volumetric computed tomography as well as morphologic MRI and diffusion-weighted imaging. In bone metastases, significant changes in A, k(ep), BV, and VI in accordance with decreased blood volume and vessel permeability as well as with increased mean vessel diameters were observed after application of ZA and SM as compared with controls. In this longitudinal study, antiangiogenic changes preceded the inhibition of osteolysis and antitumor effects after treatment. These results indicate vessel remodeling in breast cancer bone metastases on ZA and SM treatment and implicate substantial effects on imaging and treatment of malignant bone lesions.

Highlights of This Issue

Angiogenesis plays a crucial role in the development of bone metastases. To examine vascular changes in experimental breast cancer bone metastases upon treatment with zoledronic acid and sunitinib malate, Bäuerle and colleagues used dynamic contrast enhanced MRI and vessel size imaging in a longitudinal in vivo study. Resulting parameters from noninvasive imaging indicated vessel remodeling in these osseous lesions after application of zoledronic acid and sunitinib malate, which could be assessed before antiresorptive and antitumor effects of these treatments were detected. These results suggest that imaging could be used for early assessment of treatment response in malignant bone lesions.In recent years, the study of cancer stem cells (CSCs) has garnered much attention. In this issue of CCR Focus, guest editor Percy Ivy provides an overview of research surrounding CSCs and addresses some of the controversies in the field. Topics in CSC biology are discussed, including CSC identification and characterization, the CSC self-renewal pathway, and interaction of CSCs with the microenvironment. A particular emphasis is placed on translating our underlying knowledge of CSC biology into the development of anticancer therapeutics, and efforts to target several pathways involved in CSCs, including the Wnt, Notch, and Hedgehog signaling pathways, are described.Histone deacetylases (HDACs) 1–11 are differentially expressed in cancer tissues; however, more research is needed on the role of individual HDACs in tumor biology. In this study, Milde and colleagues found that class II HDACs 5 and 9 were significantly upregulated in medulloblastoma tumors with poor prognosis, and high HDAC5 and HDAC9 mRNA expression was an independent risk factor for poor survival in multifactorial analysis. Furthermore, siRNA-mediated knockdown of HDAC5 or HDAC9 resulted in decreased tumor cell growth. These findings implicate HDAC5 and HDAC9 as potential therapeutic targets in medulloblastoma.Local CD40-directed therapies, which aim to tilt tumor-induced immunosuppression in favor of antitumor responses, have shown promise in experimental models. Malmström and colleagues used adenovector-based gene therapy to introduce the human CD40 ligand (CD40L) gene into the tumor milieu. In a Phase I/IIa trial of patients with urinary bladder carcinoma, they found that AdCD40L therapy was safe and that it stimulated a massive immune cell infiltration into the bladder wall. Further, several patients had few, or no, remaining high-grade malignant cells following treatment. These results suggest that AdCD40L therapy has potential for local application on patients with solid tumors.

Tumor αvβ3 Integrin Is a Therapeutic Target for Breast Cancer Bone Metastases

In breast cancer bone metastasis, tumor cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone stimulate tumor growth. The alphavbeta3 integrin is an adhesion receptor expressed by breast cancer cells and osteoclasts. It is implicated in tumor cell invasion and osteoclast-mediated bone resorption. Here, we hypothesized that the therapeutic targeting of tumor alphavbeta3 integrin would prevent bone metastasis formation. We first showed that, compared with mock-transfected cells, the i.v. inoculation of alphavbeta3-overexpressing MDA-MB-231 breast cancer cells in animals increased bone metastasis incidence and promoted both skeletal tumor burden and bone destruction. The direct inoculation of alphavbeta3-overexpressing transfectants into the tibial bone marrow cavity did not however enhance skeletal tumor burden and bone destruction, suggesting that alphavbeta3 controls earlier events during bone metastasis formation. We next examined whether a nonpeptide antagonist of alphavbeta3 (PSK1404) exhibits meaningful antitumor effects in experimental breast and ovarian cancer bone metastasis. A continuous PSK1404 treatment, which inhibited osteoclast-mediated bone resorption in an animal model of bone loss, substantially reduced bone destruction and decreased skeletal tumor burden. Importantly, a short-term PSK1404 treatment that did not inhibit osteoclast activity also decreased skeletal tumor burden and bone destruction. This dosing regimen caused a profound and specific inhibition of bone marrow colonization by green fluorescent protein, alphavbeta3-expressing tumor cells in vivo and blocked tumor cell invasion in vitro. Overall, our data show that tumor alphavbeta3 integrin stands as a therapeutic target for the prevention of skeletal metastases.

Antitumor Effects of Clinical Dosing Regimens of Bisphosphonates in Experimental Breast Cancer Bone Metastasis

Bisphosphonates exhibit direct antitumor activity in animal models, but only at high doses that are incompatible with the clinical dosing regimens approved for the treatment of cancer patients with skeletal metastases. We compared the antitumor effects of clinical dosing regimens of the bisphosphonates zoledronic acid and clodronate in a mouse model of bone metastasis. Mice (n = 6-10 per group) were treated with zoledronic acid, clodronate, or vehicle starting before (preventive protocols) or after (treatment protocols) intravenous injection with human B02/GFP.2 breast cancer cells, which express green fluorescent protein (GFP) and luciferase and metastasize to bone. Zoledronic acid was given as daily, weekly, or single doses at a cumulative dose of 98-100 microg/kg body weight, equivalent to the 4-mg intravenous dose given to patients. Clodronate was given as a daily dose (530 microg/kg/day), equivalent to the daily 1600-mg oral clinical dose given to patients. Bone destruction was measured by radiography, x-ray absorptiometry or tomography, and histomorphometry (as the ratio of bone volume to tissue volume). Skeletal tumor burden was measured by histomorphometry (as the ratio of tumor burden to soft tissue volume [TB/STV]) and luciferase activity. All statistical tests were two-sided. In treatment protocols, daily clodronate was less effective at decreasing the TB/STV ratio than daily (53% versus 87%, difference = 34%, 95% confidence interval [CI] = 16% to 44%, P < .001) or weekly (53% versus 90%, difference = 37%, 95% CI = 19% to 46%, P < .001) zoledronic acid-dosing regimens. Compared with vehicle, a single dose of zoledronic acid decreased tumor burden by only 16% (95% CI = 9% to 22%, P < .001). In preventive protocols, daily clodronate and daily or weekly zoledronic acid decreased the TB/STV ratio by 49% (95% CI = 40% to 57%, P = .006), 83% (95% CI = 68% to 98%, P < .001), and 66% (95% CI = 47% to 84%, P < .001), respectively, compared with vehicle, whereas a single dose of zoledronic acid decreased tumor burden by only 13% (95% CI = -2% to 28%, P = .84). Mice treated with a daily preventive regimen of clodronate or with a daily or weekly preventive regimen of zoledronic acid showed a decreased B02/GFP.2 cell tumor burden compared with vehicle, whereas a single preventive dose of zoledronic acid had no effect. Daily or repeated intermittent therapy with clinical doses of bisphosphonates inhibits skeletal tumor growth in a mouse model.

Therapeutic intervention of experimental breast cancer bone metastasis by indole-3-carbinol in SCID-human mouse model

Several lines of experimental evidence have suggested that chemokine receptor CXCR4, a metastasis-promoting molecule, may play important roles in breast cancer bone metastasis. There is emerging evidence linking CXCR4 to matrix metalloproteinases (MMP) as well as their regulator nuclear factor-kappaB (NF-kappaB), a key transcription factor, which is known to activate metastasis-promoting molecules for many types of malignancies, including breast cancer. A recent study also showed that promoter region of CXCR4 has several NF-kappaB-binding sites, suggesting that there may be a cross-talk between CXCR4 and NF-kappaB. We have shown previously that indole-3-carbinol (I3C), a natural compound present in vegetables of the genus Brassica, can inhibit NF-kappaB in breast cancer cells. However, there are no reports in the literature showing any effect of I3C on CXCR4 expression in vitro and in vivo. We therefore examined whether I3C could inhibit bone metastasis of breast cancer by inhibiting CXCR4 and MMP-9 expression mediated via the inhibition of the NF-kappaB signaling pathway. Here, we have modified the severe combined immunodeficient (SCID)-human mouse model of experimental bone metastasis for use with the MDA-MB-231 breast cancer cell line. In this animal model, we found that I3C significantly inhibited MDA-MB-231 bone tumor growth, and our results were correlated with the down-regulation of NF-kappaB. Moreover, we found that I3C significantly inhibited the expression of multiple genes involved in the control of metastasis and invasion in vitro and in vivo, especially the expression of CXCR4 and MMP-9 along with pro-MMP-9, with concomitant decrease in Bcl-2 and increase in the proapoptotic protein Bax. From these results, we conclude that the CXCR4/NF-kappaB pathway is critical during I3C-induced inhibition of experimental breast cancer bone metastasis. These results also suggest that I3C could be a promising agent for the prevention and/or treatment of breast cancer bone metastasis in the future.

SU11248 inhibits tumor growth and CSF-1R-dependent osteolysis in an experimental breast cancer bone metastasis model.

The aim of the study was to investigate inhibitory effects of the receptor tyrosine kinase (RTK) inhibitor SU11248 against CSF-1R and osteoclast (OC) formation. We developed an in vivo model of breast cancer metastasis to evaluate efficacy of SU11248 against tumor growth and tumor-induced osteolysis in bone. The in vitro effects of SU11248 on CSF-1R phosphorylation, OC formation and function were evaluated. Effects on 435/HAL-Luc tumor growth in bone were monitored by in vivo bioluminescence imaging (BLI), and inhibition of osteolysis was evaluated by measurement of serum pyridinoline (PYD) concentration and histology. Phosphorylation of the receptor for M-CSF (CSF-1R) expressed by NIH3T3 cells was inhibited by SU11248 with an IC50 of 50-100 nM, consistent with CSF-1R belonging to the class III split kinase domain RTK family. The early M-CSF-dependent phase of in vitro murine OC development and function were inhibited by SU11248 at 10-100 nM. In vivo inhibition of osteolysis was confirmed by significant lowering of serum PYD levels following SU11248 treatment of tumor-bearing mice (P = 0.047). Using BLI, SU11248 treatment at 40 mg/kg/day for 21 days showed 64% inhibition of tumor growth in bone (P = 0.006), and at 80 mg/kg/day showed 89% inhibition (P = 0.001). Collectively, these data suggest that SU11248 may be an effective and tolerated therapy to inhibit growth of breast cancer bone metastases, with the additional advantage of inhibiting tumor-associated osteolysis.