Reovirus mutant jin-3 exhibits lytic and immune-stimulatory effects in preclinical human prostate cancer models.

Treatment of castration-resistant prostate cancer remains a challenging clinical problem. Despite the promising effects of immunotherapy in other solid cancers, prostate cancer has remained largely unresponsive. Oncolytic viruses represent a promising therapeutic avenue, as oncolytic virus treatment combines tumour cell lysis with activation of the immune system and mounting of effective anti-tumour responses. Mammalian Orthoreoviruses are non-pathogenic human viruses with a preference of lytic replication in human tumour cells. In this study, we evaluated the oncolytic efficacy of the bioselected oncolytic reovirus mutant jin-3 in multiple human prostate cancer models. The jin-3 reovirus displayed efficient infection, replication, and anti-cancer responses in 2D and 3D prostate cancer models, as well as in ex vivo cultured human tumour slices. In addition, the jin-3 reovirus markedly reduced the viability and growth of human cancer cell lines and patient-derived xenografts. The infection induced the expression of mediators of immunogenic cell death, interferon-stimulated genes, and inflammatory cytokines. Taken together, our data demonstrate that the reovirus mutant jin-3 displays tumour tropism, and induces potent oncolytic and immunomodulatory responses in human prostate cancer models. Therefore, jin-3 reovirus represents an attractive candidate for further development as oncolytic agent for treatment of patients with aggressive localised or advanced prostate cancer.

The Identification of Small Molecule Inhibitors That Reduce Invasion and Metastasis of Aggressive Cancers.

Transformed epithelial cells can activate programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype. This process is linked to the acquisition of an invasive phenotype and the formation of distant metastases. The development of compounds that block the acquisition of an invasive phenotype or revert the invasive mesenchymal phenotype into a more differentiated epithelial phenotype represent a promising anticancer strategy. In a high-throughput assay based on E-cadherin (re)induction and the inhibition of tumor cell invasion, 44,475 low molecular weight (LMW) compounds were screened. The screening resulted in the identification of candidate compounds from the PROAM02 class. Selected LMW compounds activated E-cadherin promoter activity and inhibited cancer cell invasion in multiple metastatic human cancer cell lines. The intraperitoneal administration of selected LMW compounds reduced the tumor burden in human prostate and breast cancer in vivo mouse models. Moreover, selected LMW compounds decreased the intra-bone growth of xenografted human prostate cancer cells. This study describes the identification of the PROAM02 class of small molecules that can be exploited to reduce cancer cell invasion and metastases. Further clinical evaluation of selected candidate inhibitors is warranted to address their safety, bioavailability and antitumor efficacy in the management of patients with aggressive cancers.

A multimodal molecular imaging approach targeting urokinase plasminogen activator receptor for the diagnosis, resection and surveillance of urothelial cell carcinoma.

With a 5-year recurrence rate of 30-78%, urothelial cell carcinoma (UCC) rates amongst the highest of all solid malignancies. Consequently, after transurethral resection, patients are subjugated to life-long endoscopic surveillance. A multimodal near-infrared (NIR) fluorescence-based imaging strategy can improve diagnosis, resection and surveillance, hence increasing quality of life. METHODS: Expression of urokinase plasminogen activator receptor (uPAR) and epithelial cell adhesion molecule (EpCAM) are determined on paraffin-embedded human UCC using immunohistochemistry and on UCC cell lines by flow cytometry. MNPR-101, a humanised monoclonal antibody targeting uPAR is conjugated to IRDye800CW and binding is validated in vitro using surface plasmon resonance and cell-based binding assays. In vivo NIR fluorescence and photoacoustic three-dimensional (3D) imaging are performed with subcutaneously growing human UM-UC-3luc2 cells in BALB/c-nude mice. The translational potential is confirmed in a metastasising UM-UC-3luc2 orthotopic mouse model. Infliximab-IRDye800CW and rituximab-IRDye800CW are used as controls. RESULTS: UCCs show prominent uPAR expression at the tumour-stroma interface and EpCAM on epithelial cells. uPAR and EpCAM are expressed by 6/7 and 4/7 UCC cell lines, respectively. In vitro, MNPR-101-IRDye800CW has a picomolar affinity for domain 2-3 of uPAR. In vivo fluorescence imaging with MNPR-101-IRDye800CW, specifically delineates both subcutaneous and orthotopic tumours with tumour-to-background ratios reaching as high as 6.8, differing significantly from controls (p < 0.0001). Photoacoustic 3D in depth imaging confirms the homogenous distribution of MNPR-101-IRDye800CW through the tumour. CONCLUSIONS: MNPR-101-IRDye800CW is suitable for multimodal imaging of UCC, awaiting clinical translation.

Patient-derived tumour models for personalized therapeutics in urological cancers.

Preclinical knowledge of dysregulated pathways and potential biomarkers for urological cancers has undergone limited translation into the clinic. Moreover, the low approval rate of new anticancer drugs and the heterogeneous drug responses in patients indicate that current preclinical models do not always reflect the complexity of malignant disease. Patient-derived tumour models used in preclinical uro-oncology research include 3D culture systems, organotypic tissue slices and patient-derived xenograft models. Technological innovations have enabled major improvements in the capacity of these tumour models to reproduce the clinical complexity of urological cancers. Each type of patient-derived model has inherent advantages and limitations that can be exploited, either alone or in combination, to gather specific knowledge on clinical challenges and address unmet clinical needs. Nevertheless, few opportunities exist for patients with urological cancers to benefit from personalized therapeutic approaches. Clinical validation of experimental data is needed to facilitate the translation and implementation of preclinical knowledge into treatment decision making.

Cationic amphiphilic drugs as potential anticancer therapy for bladder cancer.

More effective therapy for patients with either muscle-invasive or high-risk non-muscle-invasive urothelial carcinoma of the bladder (UCB) is an unmet clinical need. For this, drug repositioning of clinically approved drugs represents an interesting approach. By repurposing existing drugs, alternative anticancer therapies can be introduced in the clinic relatively fast, because the safety and dosing of these clinically approved pharmacological agents are generally well known. Cationic amphiphilic drugs (CADs) dose-dependently decreased the viability of a panel of human UCB lines in vitro. CADs induced lysosomal puncta formation, a hallmark of lysosomal leakage. Intravesical instillation of the CAD penfluridol in an orthotopic mouse xenograft model of human UCB resulted in significantly reduced intravesical tumor growth and metastatic progression. Furthermore, treatment of patient-derived ex vivo cultured human UCB tissue caused significant partial or complete antitumor responses in 97% of the explanted tumor tissues. In conclusion, penfluridol represents a promising treatment option for bladder cancer patients and warrants further clinical evaluation.

An ex vivo Tissue Culture Model for the Assessment of Individualized Drug Responses in Prostate and Bladder Cancer.

Urological malignancies, including prostate and bladder carcinoma, represent a major clinical problem due to the frequent occurrence of therapy resistance and the formation of incurable distant metastases. As a result, there is an urgent need for versatile and predictive disease models for the assessment of the individualized drug response in urological malignancies. Compound testing on ex vivo cultured patient-derived tumor tissues could represent a promising approach. In this study, we have optimized an ex vivo culture system of explanted human prostate and bladder tumors derived from clinical specimens and human cancer cell lines xenografted in mice. The explanted and cultured tumor slices remained viable and tissue architecture could be maintained for up to 10 days of culture. Treatment of ex vivo cultured human prostate and bladder cancer tissues with docetaxel and gemcitabine, respectively, resulted in a dose-dependent anti-tumor response. The dose-dependent decrease in tumor cells upon administration of the chemotherapeutic agents was preceded by an induction of apoptosis. The implementation and optimization of the tissue slice technology may facilitate the assessment of anti-tumor efficacies of existing and candidate pharmacological agents in the complex multicellular neoplastic tissues from prostate and bladder cancer patients. Our model represents a versatile "near-patient" tool to determine tumor-targeted and/or stroma-mediated anti-neoplastic responses, thus contributing to the field of personalized therapeutics.

Protocols for Migration and Invasion Studies in Prostate Cancer.

Prostate cancer is the most common malignancy diagnosed in men in the western world. The development of distant metastases and therapy resistance are major clinical problems in the management of prostate cancer patients. In order for prostate cancer to metastasize to distant sites in the human body, prostate cancer cells have to migrate and invade neighboring tissue. Cancer cells can acquire a migratory and invasive phenotype in several ways, including single cell and collective migration. As a requisite for migration, epithelial prostate cancer cells often need to acquire a motile, mesenchymal-like phenotype. This way prostate cancer cells often lose polarity and epithelial characteristics (e.g., expression of E-cadherin homotypic adhesion receptor), and acquire mesenchymal phenotype (for example, cytoskeletal rearrangements, enhanced expression of proteolytic enzymes and other repertory of integrins). This process is referred to as epithelial-to-mesenchymal transition (EMT). Cellular invasion, one of the hallmarks of cancer, is characterized by the movement of cells through a three-dimensional matrix, resulting in remodeling of the cellular environment. Cellular invasion requires adhesion, proteolysis of the extracellular matrix, and migration of cells. Studying the migratory and invasive ability of cells in vitro represents a useful tool to assess the aggressiveness of solid cancers, including those of the prostate.This chapter provides a comprehensive description of the Transwell migration assay, a commonly used technique to investigate the migratory behavior of prostate cancer cells in vitro. Furthermore, we will provide an overview of the adaptations to the Transwell migration protocol to study the invasive capacity of prostate cancer cells, i.e., the Transwell invasion assay. Finally, we will present a detailed description of the procedures required to stain the Transwell filter inserts and quantify the migration and/or invasion.

Transplantable Animal Studies and Whole-Body Optical Imaging in Prostate Carcinoma.

Current treatments of advanced prostate cancer only marginally increase overall survival and can be regarded as predominantly palliative. Hence, there is an urgent need for novel therapeutic strategies for the treatment of primary tumors and, more importantly perhaps, for the prevention of tumor progression and metastasis formation. Clinically relevant preclinical models are therefore urgently needed. An ideal, clinically relevant preclinical model would mimic the genetic and phenotypic changes that occur at the different stages of human prostate cancer progression and subsequent metastasis. In this chapter, transplantable xenograft prostate cancer models are described, in which human prostate cancer cells are transplanted into host animals (e.g., immune-deficient mice). Cancer cells can be administered to the small laboratory animals in various ways, including inoculation of the prostate tumor cells subcutaneously, at the anatomical site of origin (orthotopically), or at the metastatic site. In addition, we describe imaging methods suitable for small laboratory animals with emphasis on optical imaging (bioluminescence and fluorescence).

Spontaneous development of Epstein-Barr Virus associated human lymphomas in a prostate cancer xenograft program.

Prostate cancer research is hampered by the lack of in vivo preclinical models that accurately reflect patient tumour biology and the clinical heterogeneity of human prostate cancer. To overcome these limitations we propagated and characterised a new collection of patient-derived prostate cancer xenografts. Tumour fragments from 147 unsupervised, surgical prostate samples were implanted subcutaneously into immunodeficient Rag2-/-γC-/- mice within 24 hours of surgery. Histologic and molecular characterisation of xenografts was compared with patient characteristics, including androgen-deprivation therapy, and exome sequencing. Xenografts were established from 47 of 147 (32%) implanted primary prostate cancers. Only 14% passaged successfully resulting in 20 stable lines; derived from 20 independent patient samples. Surprisingly, only three of the 20 lines (15%) were confirmed as prostate cancer; one line comprised of mouse stroma, and 16 were verified as human donor-derived lymphoid neoplasms. PCR for Epstein-Barr Virus (EBV) nuclear antigen, together with exome sequencing revealed that the lymphomas were exclusively EBV-associated. Genomic analysis determined that 14 of the 16 EBV+ lines had unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements, confirming their B-cell origin. We conclude that the generation of xenografts from tumour fragments can commonly result in B-cell lymphoma from patients carrying latent EBV. We recommend routine screening, of primary outgrowths, for latent EBV to avoid this phenomenon.

Glucocorticoid receptor antagonism reverts docetaxel resistance in human prostate cancer.

Resistance to docetaxel is a major clinical problem in advanced prostate cancer (PCa). Although glucocorticoids (GCs) are frequently used in combination with docetaxel, it is unclear to what extent GCs and their receptor, the glucocorticoid receptor (GR), contribute to the chemotherapy resistance. In this study, we aim to elucidate the role of the GR in docetaxel-resistant PCa in order to improve the current PCa therapies. GR expression was analyzed in a tissue microarray of primary PCa specimens from chemonaive and docetaxel-treated patients, and in cultured PCa cell lines with an acquired docetaxel resistance (PC3-DR, DU145-DR, and 22Rv1-DR). We found a robust overexpression of the GR in primary PCa from docetaxel-treated patients and enhanced GR levels in cultured docetaxel-resistant human PCa cells, indicating a key role of the GR in docetaxel resistance. The capability of the GR antagonists (RU-486 and cyproterone acetate) to revert docetaxel resistance was investigated and revealed significant resensitization of docetaxel-resistant PCa cells for docetaxel treatment in a dose- and time-dependent manner, in which a complete restoration of docetaxel sensitivity was achieved in both androgen receptor (AR)-negative and AR-positive cell lines. Mechanistically, we demonstrated down-regulation of Bcl-xL and Bcl-2 upon GR antagonism, thereby defining potential treatment targets. In conclusion, we describe the involvement of the GR in the acquisition of docetaxel resistance in human PCa. Therapeutic targeting of the GR effectively resensitizes docetaxel-resistant PCa cells. These findings warrant further investigation of the clinical utility of the GR antagonists in the management of patients with advanced and docetaxel-resistant PCa.

Liposomal delivery of dexamethasone attenuates prostate cancer bone metastatic tumor growth in vivo.

BACKGROUND: The inflammatory tumor microenvironment, and more specifically the tumor-associated macrophages, plays an essential role in the development and progression of prostate cancer towards metastatic bone disease. Tumors are often characterized by a leaky vasculature, which - combined with the prolonged circulation kinetics of liposomes - leads to efficient tumor localization of these drug carriers, via the so-called enhanced permeability and retention (EPR) -effect. In this study, we evaluated the utility of targeted, liposomal drug delivery of the glucocorticoid dexamethasone in a model of prostate cancer bone metastases. METHODS: Tumor-bearing Balb-c nu/nu mice were treated intravenously with 0.2-1.0-5.0 mg/kg/week free- and liposomal DEX for 3-4 weeks and tumor growth was monitored by bioluminescent imaging. RESULTS: Intravenously administered liposomes localize efficiently to bone metastases in vivo and treatment of established bone metastases with (liposomal) dexamethasone resulted in a significant inhibition of tumor growth up to 26 days after initiation of treatment. Furthermore, 1.0 mg/kg liposomal dexamethasone significantly outperformed 1.0 mg/kg free dexamethasone, and was found to be well-tolerated at clinically-relevant dosages that display potent anti-tumor efficacy. CONCLUSIONS: Liposomal delivery of the glucocorticoid dexamethasone inhibits the growth of malignant bone lesions. We believe that liposomal encapsulation of dexamethasone offers a promising new treatment option for advanced, metastatic prostate cancer which supports further clinical evaluation.

SYK is a candidate kinase target for the treatment of advanced prostate cancer.

Improved targeted therapies are needed to combat metastatic prostate cancer. Here, we report the identification of the spleen kinase SYK as a mediator of metastatic dissemination in zebrafish and mouse xenograft models of human prostate cancer. Although SYK has not been implicated previously in this disease, we found that its expression is upregulated in human prostate cancers and associated with malignant progression. RNAi-mediated silencing prevented invasive outgrowth in vitro and bone colonization in vivo, effects that were reversed by wild-type but not kinase-dead SYK expression. In the absence of SYK expression, cell surface levels of the progression-associated adhesion receptors integrin α2ß1 and CD44 were diminished. RNAi-mediated silencing of α2ß1 phenocopied SYK depletion in vitro and in vivo, suggesting an effector role for α2ß1 in this setting. Notably, pharmacologic inhibitors of SYK kinase currently in phase I-II trials for other indications interfered similarly with the invasive growth and dissemination of prostate cancer cells. Our findings offer a mechanistic rationale to reposition SYK kinase inhibitors for evaluation in patients with metastatic prostate cancer.

Glycogen synthase kinase-3ß inhibition depletes the population of prostate cancer stem/progenitor-like cells and attenuates metastatic growth.

Cancer cells with stem or progenitor properties play a pivotal role in the initiation, recurrence and metastatic potential of solid tumors, including those of the human prostate. Cancer stem cells are generally more resistant to conventional therapies thus requiring the characterization of key pathways involved in the formation and/or maintenance of this malignant cellular subpopulation. To this end, we identified Glycogen Synthase Kinase-3ß (GSK-3ß) as a crucial kinase for the maintenance of prostate cancer stem/progenitor-like cells and pharmacologic inhibition of GSK-3ß dramatically decreased the size of this cellular subpopulation. This was paralleled by impaired clonogenicity, decreased migratory potential and dramatic morphological changes. In line with our in vitro observations, treatment with a GSK-3ß inhibitor leads to a complete loss of tumorigenicity and a decrease in metastatic potential in preclinical in vivo models. These observed anti-tumor effects appear to be largely Wnt-independent as simultaneous Wnt inhibition does not reverse the observed antitumor effects of GSK-3ß blockage. We found that GSK-3ß activity is linked to cytoskeletal protein F-actin and inhibition of GSK-3ß leads to disturbance of F-actin polymerization. This may underlie the dramatic effects of GSK-3ß inhibition on prostate cancer migration. Furthermore, GSK-3ß inhibition led to strongly decreased expression of several integrin types including the cancer stem cell-associated α2ß1 integrin. Taken together, our mechanistic observations highlight the importance of GSK-3ß activity in prostate cancer stemness and may facilitate the development of novel therapy for advanced prostate cancer.

Targeting of alpha-v integrins reduces malignancy of bladder carcinoma.

Low survival rates of metastatic cancers emphasize the need for a drug that can prevent and/or treat metastatic cancer. αv integrins are involved in essential processes for tumor growth and metastasis and targeting of αv integrins has been shown to decrease angiogenesis, tumor growth and metastasis. In this study, the role of αv integrin and its potential as a drug target in bladder cancer was investigated. Treatment with an αv integrin antagonist as well as knockdown of αv integrin in the bladder carcinoma cell lines, resulted in reduced malignancy in vitro, as illustrated by decreased proliferative, migratory and clonogenic capacity. The CDH1/CDH2 ratio increased, indicating a shift towards a more epithelial phenotype. This shift appeared to be associated with downregulation of EMT-inducing transcription factors including SNAI2. The expression levels of the self-renewal genes NANOG and BMI1 decreased as well as the number of cells with high Aldehyde Dehydrogenase activity. In addition, self-renewal ability decreased as measured with the urosphere assay. In line with these observations, knockdown or treatment of αv integrins resulted in decreased metastatic growth in preclinical in vivo models as assessed by bioluminescence imaging. In conclusion, we show that αv integrins are involved in migration, EMT and maintenance of Aldehyde Dehydrogenase activity in bladder cancer cells. Targeting of αv integrins might be a promising approach for treatment and/or prevention of metastatic bladder cancer.

Metastasis of prostate cancer and melanoma cells in a preclinical in vivo mouse model is enhanced by L-plastin expression and phosphorylation.

BACKGROUND: Tumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking. METHODS: We investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells. RESULTS: Knock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed. CONCLUSIONS: Our data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable.

BMP7 activates brown adipose tissue and reduces diet-induced obesity only at subthermoneutrality.

BACKGROUND/AIMS: Brown adipose tissue (BAT) dissipates energy stored in triglycerides as heat via the uncoupling protein UCP-1 and is a promising target to combat hyperlipidemia and obesity. BAT is densely innervated by the sympathetic nervous system, which increases BAT differentiation and activity upon cold exposure. Recently, Bone Morphogenetic Protein 7 (BMP7) was identified as an inducer of BAT differentiation. We aimed to elucidate the role of sympathetic activation in the effect of BMP7 on BAT by treating mice with BMP7 at varying ambient temperature, and assessed the therapeutic potential of BMP7 in combating obesity. METHODS AND RESULTS: High-fat diet fed lean C57Bl6/J mice were treated with BMP7 via subcutaneous osmotic minipumps for 4 weeks at 21 °C or 28 °C, the latter being a thermoneutral temperature in which sympathetic activation of BAT is largely diminished. At 21 °C, BMP7 increased BAT weight, increased the expression of Ucp1, Cd36 and hormone-sensitive lipase in BAT, and increased total energy expenditure. BMP7 treatment markedly increased food intake without affecting physical activity. Despite that, BMP7 diminished white adipose tissue (WAT) mass, accompanied by increased expression of genes related to intracellular lipolysis in WAT. All these effects were blunted at 28 °C. Additionally, BMP7 resulted in extensive 'browning' of WAT, as evidenced by increased expression of BAT markers and the appearance of whole clusters of brown adipocytes via immunohistochemistry, independent of environmental temperature. Treatment of diet-induced obese C57Bl6/J mice with BMP7 led to an improved metabolic phenotype, consisting of a decreased fat mass and liver lipids as well as attenuated dyslipidemia and hyperglycemia. CONCLUSION: Together, these data show that BMP7-mediated recruitment and activation of BAT only occurs at subthermoneutral temperature, and is thus likely dependent on sympathetic activation of BAT, and that BMP7 may be a promising tool to combat obesity and associated disorders.

Epithelial plasticity, cancer stem cells, and the tumor-supportive stroma in bladder carcinoma.

High recurrence rates and poor survival rates of metastatic bladder cancer emphasize the need for a drug that can prevent and/or treat bladder cancer progression and metastasis formation. Accumulating evidence suggests that cancer stem/progenitor cells are involved in tumor relapse and therapy resistance in urothelial carcinoma. These cells seem less affected by the antiproliferative therapies, as they are largely quiescent, have an increased DNA damage response, reside in difficult-to-reach, protective cancer stem cell niches and express ABC transporters that can efflux drugs from the cells. Recent studies have shown that epithelial-to-mesenchymal transition (EMT), a process in which sessile, epithelial cells switch to a motile, mesenchymal phenotype may render cancer cells with cancer stem cells properties and/or stimulate the expansion of this malignant cellular subpopulation. As cancer cells undergo EMT, invasiveness, drug resistance, angiogenesis, and metastatic ability seem to increase in parallel, thus giving rise to a more aggressive tumor type. Furthermore, the tumor microenvironment (tumor-associated stromal cells, extracellular matrix) plays a key role in tumorigenesis, tumor progression, and metastasis formation. Taken together, the secret for more effective cancer therapies might lie in developing and combining therapeutic strategies that also target cancer stem/progenitor cells and create an inhospitable microenvironment for highly malignant bladder cancer cells. This review will focus on the current concepts about the role of cancer stem cells, epithelial plasticity, and the supportive stroma in bladder carcinoma. The potential implications for the development of novel bladder cancer therapy will be discussed.

Preclinical imaging of the cellular and molecular events in the multistep process of bone metastasis.

Bone metastasis is a complex process that ultimately leads to devastating metastatic bone disease. It is therefore of key interest to unravel the mechanisms underlying the multistep process of skeletal metastasis and cancer-induced bone disease, and to develop better treatment and management of patients with this devastating disease. Fortunately, novel technologies are rapidly emerging that allow real-time imaging of molecules, pathogenic processes, drug delivery and drug response in preclinical in vivo models. The outcome of these experimental studies will facilitate clinical cancer research by improving the detection of cancer cell invasion, metastasis and therapy response.

Preclinical models that illuminate the bone metastasis cascade.

In this chapter currently available preclinical models of tumor progression and bone metastasis, including genetically engineered mice that develop primary and metastatic carcinomas and transplantable animal models, will be described. Understanding the multistep process of incurable bone metastasis is pivotal to the development of new therapeutic strategies. Novel technologies for imaging molecules or pathologic processes in cancers and their surrounding stroma have emerged rapidly and have greatly facilitated cancer research, in particular the cellular behavior of osteotropic tumors and their response to new and existing therapeutic agents. Optical imaging, in particular, has become an important tool in preclinical bone metastasis models, clinical trials and medical practice. Advances in experimental and clinical imaging will-in the long run-result in significant improvements in diagnosis, tumor localization, enhanced drug delivery and treatment.

BMP-7 inhibits TGF-ß-induced invasion of breast cancer cells through inhibition of integrin ß(3) expression.

BACKGROUND: The transforming growth factor (TGF)-ß superfamily comprises cytokines such as TGF-ß and Bone Morphogenetic Proteins (BMPs), which have a critical role in a multitude of biological processes. In breast cancer, high levels of TGF-ß are associated with poor outcome, whereas inhibition of TGF-ß-signaling reduces metastasis. In contrast, BMP-7 inhibits bone metastasis of breast cancer cells. METHODS: In this study, we investigated the effect of BMP-7 on TGF-ß-induced invasion in a 3 dimensional invasion assay. RESULTS: BMP-7 inhibited TGF-ß-induced invasion of the metastatic breast cancer cell line MCF10CA1a, but not of its premalignant precursor MCF10AT in a spheroid invasion model. The inhibitory effect appears to be specific for BMP-7, as its closest homolog, BMP-6, did not alter the invasion of MCF10CA1a spheroids. To elucidate the mechanism by which BMP-7 inhibits TGF-ß-induced invasion, we analyzed invasion-related genes. BMP-7 inhibited TGF-ß-induced expression of integrin α(v)ß(3) in the spheroids. Moreover, targeting of integrins by a chemical inhibitor or knockdown of integrin ß(3) negatively affected TGF-ß-induced invasion. On the other hand, overexpression of integrin ß(3) counteracted the inhibitory effect of BMP7 on TGF-ß-induced invasion. CONCLUSION: Thus, BMP-7 may exert anti-invasive actions by inhibiting TGF-ß-induced expression of integrin ß(3).