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The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

PDE9A is a cGMP-specific phosphodiesterase expressed in neurons throughout the brain that has attracted attention as a therapeutic target to treat cognitive disorders. Indeed, PDE9A inhibitors are under evaluation in clinical trials as a treatment for Alzheimer's disease and schizophrenia. However, little is known about the cGMP signaling cascades regulated by PDE9A. Canonical cGMP signaling in brain follows the activation of neuronal nitric oxide synthase (nNOS) and the generation of nitric oxide, which activates soluble guanylyl cyclase and cGMP synthesis. However, we show that in mice, PDE9A regulates a pool of cGMP that is independent of nNOS, specifically, and nitric oxide signaling in general. This PDE9A-regulated cGMP pool appears to be highly compartmentalized and independent of cGMP pools regulated by several PDEs. These findings provide a new foundation for study of the upstream and downstream signaling elements regulated by PDE9A and its potential as a therapeutic target for brain disease.

It is estimated that early detection of pancreatic ductal adenocarcinoma (PDAC) could increase long-term patient survival by as much as 30% to 40% (Seufferlein, T. et al., Nat. Rev. Gastroenterol. Hepatol. 2016 , 13 , 74⁻75). There is an unmet need for reagents that can reliably identify early cancerous or precancerous lesions through various imaging modalities or could be employed to deliver anticancer treatments specifically to tumor cells. However, to date, many PDAC tumor-targeting strategies lack selectivity and are unable to discriminate between tumor and nontumor cells, causing off-target effects or unclear diagnoses. Although a variety of approaches have been taken to identify tumor-targeting reagents that can effectively direct therapeutics or imaging agents to cancer cells (Liu, D. et al., J. Controlled Release 2015 , 219 , 632⁻643), translating these reagents into clinical practice has been limited, and it remains an area open to new methodologies and reagents (O'Connor, J.P. et al., Nat. Rev. Clin. Oncol . 2017 , 14 , 169⁻186). G protein⁻coupled receptors (GPCRs), which are key target proteins for drug discovery and comprise a large proportion of currently marketed therapeutics, hold significant promise for tumor imaging and targeted treatment, particularly for pancreatic cancer.

Computational modeling was used to direct the synthesis of analogs of previously reported phosphodiesterase 2A (PDE2A) inhibitor 1 with an imidazotriazine core to yield compounds of significantly enhanced potency. The analog PF-05180999 (30) was subsequently identified as a preclinical candidate targeting cognitive impairment associated with schizophrenia. Compound 30 demonstrated potent binding to PDE2A in brain tissue, dose responsive mouse brain cGMP increases, and reversal of N-methyl-d-aspartate (NMDA) antagonist-induced (MK-801, ketamine) effects in electrophysiology and working memory models in rats. Preclinical pharmacokinetics revealed unbound brain/unbound plasma levels approaching unity and good oral bioavailability resulting in an average concentration at steady state (C av,ss ) predicted human dose of 30 mg once daily (q.d.). Modeling of a modified release formulation suggested that 25 mg twice daily (b.i.d.) could maintain plasma levels of 30 at or above targeted efficacious plasma levels for 24 h, which became part of the human clinical plan.

Phosphodiesterase 2A (PDE2A) inhibitors have been reported to demonstrate in vivo activity in preclinical models of cognition. To more fully explore the biology of PDE2A inhibition, we sought to identify potent PDE2A inhibitors with improved brain penetration as compared to current literature compounds. Applying estimated human dose calculations while simultaneously leveraging synthetically enabled chemistry and structure-based drug design has resulted in a highly potent, selective, brain penetrant compound 71 (PF-05085727) that effects in vivo biochemical changes commensurate with PDE2A inhibition along with behavioral and electrophysiological reversal of the effects of NMDA antagonists in rodents. This data supports the ability of PDE2A inhibitors to potentiate NMDA signaling and their further development for clinical cognition indications.

Huntington's disease (HD) symptoms are driven to a large extent by dysfunction of the basal ganglia circuitry. HD patients exhibit reduced striatal phoshodiesterase 10 (PDE10) levels. Using HD mouse models that exhibit reduced PDE10, we demonstrate the benefit of pharmacologic PDE10 inhibition to acutely correct basal ganglia circuitry deficits. PDE10 inhibition restored corticostriatal input and boosted cortically driven indirect pathway activity. Cyclic nucleotide signaling is impaired in HD models, and PDE10 loss may represent a homeostatic adaptation to maintain signaling. Elevation of both cAMP and cGMP by PDE10 inhibition was required for rescue. Phosphoproteomic profiling of striatum in response to PDE10 inhibition highlighted plausible neural substrates responsible for the improvement. Early chronic PDE10 inhibition in Q175 mice showed improvements beyond those seen with acute administration after symptom onset, including partial reversal of striatal deregulated transcripts and the prevention of the emergence of HD neurophysiological deficits. VIDEO ABSTRACT., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Duchenne muscular dystrophy is caused by mutations in the DYSTROPHIN gene. Although primarily associated with muscle wasting, a significant portion of patients (approximately 25%) are also diagnosed with autism spectrum disorder. We describe social behavioral deficits in dystrophin-deficient mice and present evidence of cerebellar deficits in cGMP production. We demonstrate therapeutic potential for selective inhibitors of the cGMP-specific PDE5A and PDE9A enzymes to restore social behaviors in dystrophin-deficient mice., Competing Interests: LMK is a consultant for Pfizer, Summit Corporation PLC and Sarepta Therapeutics for muscle disease drug therapies. MTP, CAM, all hold equity in Pfizer. MTP is a current employee of Autism Speaks. RJK is a consultant for Ironwood Pharmaceuticals. CJS, JFH and ELPC are employees of Pfizer. MS is on the Scientific Advisory Board of Sage Therapeutics and has received research support from Novartis and Shire. The remaining authors declare no conflict of interest.

The striatum contains a rich variety of cyclic nucleotide phosphodiesterases (PDEs), which play a critical role in the regulation of cAMP and cGMP signaling. The dual-substrate enzyme PDE10A is the most highly expressed PDE in striatal medium-sized spiny neurons (MSNs) with low micromolar affinity for both cyclic nucleotides. Previously, we have shown that systemic and local administration of the selective PDE10A inhibitor TP-10 potently increased the responsiveness of MSNs to cortical stimulation. However, the signaling mechanisms underlying PDE10A inhibitor-induced changes in corticostriatal transmission are only partially understood. The current studies assessed the respective roles of cAMP and cGMP in the above effects using soluble guanylyl cyclase (sGC) or adenylate cyclase (AC) specific inhibitors. Cortically evoked spike activity was monitored in urethane-anesthetized rats using in vivo extracellular recordings performed proximal to a microdialysis probe during local infusion of vehicle, the selective sGC inhibitor ODQ, or the selective AC inhibitor SQ 22536. Systemic administration of TP-10 (3.2 mg/kg) robustly increased cortically evoked spike activity in a manner that was blocked following intrastriatal infusion of ODQ (50 μm). The effects of TP-10 on evoked activity were due to accumulation of cGMP, rather than cAMP, as the AC inhibitor SQ was without effect. Consistent with these observations, studies in neuronal NO synthase (nNOS) knock-out (KO) mice confirmed that PDE10A operates downstream of nNOS to limit cGMP production and excitatory corticostriatal transmission. Thus, stimulation of PDE10A acts to attenuate corticostriatal transmission in a manner largely dependent on effects directed at the NO-sGC-cGMP signaling cascade., (Copyright © 2015 the authors 0270-6474/15/355781-11$15.00/0.)

Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.

There currently are no tests available for early diagnosis or for the identification of patients at risk for development of pancreatic cancer. We report the discovery of single nucleotide polymorphism (SNP) in the cholecystokinin B receptor (CCKBR) gene predicts survival and risk of pancreatic cancer. Growth of human pancreatic cancer is stimulated by gastrin through the CCKBR and an alternatively spliced isoform of the CCKBR gene called CCKCR. One hundred and ten surgically resected benign and malignant pancreatic tissues as well as normal pancreas were prospectively evaluated for CCKBR genotype and protein expression. Analysis demonstrated the expression of the spliced isoform, CCKCR, was associated with a (SNP) (C > A) at position 32 of the intron 4 (IVS 4) of the CCKBR gene. Since the SNP is within an intron, it has not previously been identified in the GWAS studies. Only patients with the A/A or A/C genotypes, exhibited immunoreactivity to a selective CCKCR antibody. Survival among pancreatic cancer patients with the A-SNP was significantly shorter (p = 0.0001, hazard ratio = 3.63) compared with individuals with C/C genotype. Other variables such as surgical margins, lymph node status, histologic grade or adjuvant chemotherapy were not associated with survival. Furthermore, having one or two of the A-alleles was found to increase the risk of pancreatic adenocarcinoma by 174% (p = 0.0192) compared with the C/C wild type. Cancer cells transfected to overexpress the CCKCR demonstrated increased proliferation over controls. Genetic screening for this SNP may aid in early detection of pancreatic cancer in high risk subjects.

Cholecystokinin (CCK) and gastrin stimulate growth of pancreatic cancer. Although down-regulation of gastrin inhibits growth of pancreatic cancer, the contribution of endogenous CCK to tumor growth is unknown. The purpose of this study was to evaluate the role of endogenous CCK on autocrine growth of pancreatic cancer. Pancreatic cancer cell lines were analyzed for CCK mRNA and peptide expression by real-time RT-PCR and radioimmunoassay, respectively. The effect of endogenous CCK on growth was evaluated by treating cancer cells with CCK neutralizing antibodies and by down-regulating CCK mRNA by RNAi. Wild-type pancreatic cancer cells expressed significantly lower CCK mRNA and peptide levels than gastrin. Neither treatment of pancreatic cancer cells with CCK antibodies nor the down-regulation of CCK mRNA and peptide by shRNAs altered growth in vitro or in vivo. Conversely, when gastrin mRNA expression was down-regulated, the same cells failed to produce tumors in spite of having sustained levels of endogenous CCK. Pancreatic cancer cells produce CCK and gastrin; however, the autocrine production of gastrin is more important for stimulating tumor growth.

Inhibition of phosphodiesterase 10A (PDE10A) promotes cyclic nucleotide signaling, increases striatal activation, and decreases behavioral activity. Enhanced cyclic nucleotide signaling is a well established route to producing changes in gene expression. We hypothesized that chronic suppression of PDE10A activity would have significant effects on gene expression in the striatum. A comparison of the expression profile of PDE10A knockout (KO) mice and wild-type mice after chronic PDE10A inhibition revealed altered expression of 19 overlapping genes with few significant changes outside the striatum or after administration of a PDE10A inhibitor to KO animals. Chronic inhibition of PDE10A produced up-regulation of mRNAs encoding genes that included prodynorphin, synaptotagmin10, phosphodiesterase 1C, glutamate decarboxylase 1, and diacylglycerol O-acyltransferase and a down-regulation of mRNAs encoding choline acetyltransferase and Kv1.6, suggesting long-term suppression of the PDE10A enzyme is consistent with altered striatal excitability and potential utility as a antipsychotic therapy. In addition, up-regulation of mRNAs encoding histone 3 (H3) and down-regulation of histone deacetylase 4, follistatin, and claspin mRNAs suggests activation of molecular cascades capable of neuroprotection. We used lentiviral delivery of cAMP response element (CRE)-luciferase reporter constructs into the striatum and live animal imaging of 2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid (TP-10)-induced luciferase activity to further demonstrate PDE10 inhibition results in CRE-mediated transcription. Consistent with potential neuroprotective cascades, we also demonstrate phosphorylation of mitogen- and stress-activated kinase 1 and H3 in vivo after TP-10 treatment. The observed changes in signaling and gene expression are predicted to provide neuroprotective effects in models of Huntington's disease.

By utilizing structure-based drug design (SBDD) knowledge, a novel class of phosphodiesterase (PDE) 10A inhibitors was identified. The structure-based drug design efforts identified a unique "selectivity pocket" for PDE10A inhibitors, and interactions within this pocket allowed the design of highly selective and potent PDE10A inhibitors. Further optimization of brain penetration and drug-like properties led to the discovery of 2-[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)-phenoxymethyl]-quinoline (PF-2545920). This PDE10A inhibitor is the first reported clinical entry for this mechanism in the treatment of schizophrenia.

Objectives: This study evaluated the effects of gastrin messenger RNA (mRNA) down-regulation on growth of human pancreatic cancer., Methods: Gastrin expression was examined in human pancreatic cancer cell lines by reverse transcriptase-polymerase chain reaction, and peptide expression was assessed by immunocytochemistry. Gastrin was down-regulated using either stable transfection of an antisense gastrin cDNA or 1 of 3 shRNA (short hairpin RNA) constructs. Tumor formation was evaluated after either subcutaneous or orthotopic injections into nude mice. The effect of nanoliposomes loaded with gastrin siRNA (small interfering RNA) was tested in mice bearing pancreatic tumors., Results: Stable transfection of gastrin antisense or shRNAs into BxPC-3 cells resulted in clones with more than 90% reduction in gastrin mRNA. Tumor growth rate and incidence of metastases in both wild-type and transfected pancreatic cancer cells were directly proportional to the degrees of gastrin mRNA expression. Immunofluorescence analysis confirmed that gastrin peptide levels were decreased in antisense and shRNA tumors. Gastrin knockdown clones had lower Ki-67 and increased cleaved caspase-3 staining, consistent with known effects of gastrin on proliferation and apoptosis. Tumors in mice treated with gastrin siRNA were smaller than controls., Conclusions: These results suggest that RNAi targeting of gastrin could serve as an effective treatment for pancreatic cancer.

We have previously demonstrated that inactivation of capsaicin-sensitive sensory neurons enhances lung and heart metastases of breast carcinoma. Because a significant part of sensory innervation of lung tissue is supplied by the vagus nerve, we here examined the effects of unilateral mid-cervical vagotomy in the metastases of 4THMpc breast carcinoma and tissue Substance P (SP) levels. Balb-c mice were injected orthotopically with 4THMpc cells 1 week after vagotomy. Animals were sacrificed 27-30 days after injection of 4THMpc cells and the extent of metastases was determined. Unilateral vagotomy, right or left significantly increased the lung, liver and kidney metastases without altering the growth rate of the primary tumor. Heart metastases were increased only following left vagotomy. The changes in SP levels were somewhat surprising such that vagotomy actually increased while sham-operation decreased SP levels in lung. The effect of sham-operation was reversed by unilateral vagotomy demonstrating that vagal activity decreases total SP levels in the lung. Increased SP levels might be due to decreased degradation of the peptide. Presence of the tumor markedly increased SP level in the lung, which was more prominent in vagotomized animals. These results provide evidence that vagal activity may protect against metastatic disease.

We have recently proposed the hypothesis that inhibition of the cyclic nucleotide phosphodiesterase (PDE) 10A may represent a new pharmacological approach to the treatment of schizophrenia (Curr Opin Invest Drug 8:54-59, 2007). PDE10A is highly expressed in the medium spiny neurons of the mammalian striatum (Brain Res 985:113-126, 2003; J Histochem Cytochem 54:1205-1213, 2006; Neuroscience 139:597-607, 2006), where the enzyme is hypothesized to regulate both cAMP and cGMP signaling cascades to impact early signal processing in the corticostriatothalamic circuit (Neuropharmacology 51:374-385, 2006; Neuropharmacology 51:386-396, 2006). Our current understanding of the physiological role of PDE10A and the therapeutic utility of PDE10A inhibitors derives in part from studies with papaverine, the only pharmacological tool for this target extensively profiled to date. However, this agent has significant limitations in this regard, namely, relatively poor potency and selectivity and a very short exposure half-life after systemic administration. In the present report, we describe the discovery of a new class of PDE10A inhibitors exemplified by TP-10 (2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid), an agent with greatly improved potency, selectivity, and pharmaceutical properties. These new pharmacological tools enabled studies that provide further evidence that inhibition of PDE10A represents an important new target for the treatment of schizophrenia and related disorders of basal ganglia function.

Inflammatory bowel disease (IBD) is a condition of the intestine with significant morbidity. Although hereditary, environmental, immunologic, and bacterial factors have been implicated, the etiology of IBD remains unknown. Since opioid peptides modulate inflammatory cytokine production and opioid antagonists promote tissue growth and repair, we hypothesized the opioid antagonist naltrexone could reduce inflammation of the bowel. Using a chemically-induced mouse model of IBD, C57BL/6J mice received either untreated drinking water or water containing 2% dextran sulfate sodium (DSS) in two parallel regimens modeling moderate and severe colitis. After colitis was established, animals in the moderate colitis study were administered either saline (control) or naltrexone (NTX; 8 or 400 microg/kg) daily, while those in the severe colitis study received 0.1 or 10 mg/kg NTX. DSS-treated animals had significant weight loss (p = 0.006) and higher disease activity index (DAI) scores (p < 0.001) compared to water controls. However, NTX treatment of mice with moderate colitis resulted in less weight loss, lower DAI scores, and less histologic evidence of inflammation compared to controls. Significantly, elevated levels of colonic RNA for pro-inflammatory cytokines interleukin (IL)-6 and IL-12 were also decreased toward normal with NTX. Similar to patients with severe and unresponsive disease, animals in the severe colitis study did not significantly respond to treatment. Thus, NTX therapy reverses physical symptoms, histologic evidence, and molecular markers of inflammation in moderate colitis. The mechanism by which NTX acts to reverse colitis is related in part to the decreased expression of pro-inflammatory cytokines.

The phenotype of genetically modified animals is strongly influenced by both the genetic background of the animal as well as environmental factors. We have previously reported the behavioral and neurochemical characterization of PDE10A knockout mice maintained on a DBA1LacJ (PDE10A(DBA)) genetic background. The aim of the present studies was to assess the behavioral and neurochemical phenotype of PDE10A knockout mice on an alternative congenic C57BL/6N (PDE10A(C57)) genetic background. Consistent with our previous results, PDE10A(C57) knockout mice showed a decrease in exploratory locomotor activity and a delay in the acquisition of conditioned avoidance responding. Also consistent with previous studies, the elimination of PDE10A did not alter basal levels of striatal cGMP or cAMP or affect behavior in several other well-characterized behavioral assays. PDE10A(C57) knockout mice showed a blunted response to MK-801, although to a lesser degree than previously observed in the PDE10A(DBA) knockout mice, and no differences were observed following a PCP challenge. PDE10A(C57) knockout mice showed a significant change in striatal dopamine turnover, which was accompanied by an enhanced locomotor response to AMPH, These studies demonstrate that while many of the behavioral effects of the PDE10A gene deletion appear to be independent of genetic background, the impact of the deletion on behavior can vary in magnitude. Furthermore, the effects on the dopaminergic system appear to be background-dependent, with significant effects observed only in knockout mice on the C57BL6N genetic background.

Phosphodiesterase 10A (PDE10A) is a recently identified cyclic nucleotide phosphodiesterase expressed primarily in dopaminoreceptive medium spiny neurons of the striatum. We report that papaverine is a potent, specific inhibitor of PDE10A and use this compound to explore the role of PDE10A in regulating striatal function. Papaverine administration produces an increase in striatal tissue levels of cGMP and an increase in extracellular cAMP measured by microdialysis. These cyclic nucleotide changes are accompanied by increases in the phosphorylation of CREB and ERK, downstream markers of neuronal activation. In rats, papaverine potentiates haloperidol-induced catalepsy, consistent with the hypothesis that inhibition of PDE10A can increase striatal output and prompting a further evaluation of papaverine in models predictive of antipsychotic activity. Papaverine is found to inhibit conditioned avoidance responding in rats and mice and to inhibit PCP- and amphetamine-stimulated locomotor activity in rats. The effects of papaverine on striatal cGMP and CREB and ERK phosphorylation, as well as on conditioned avoidance responding, were absent in PDE10A knockout mice, indicating that the effects of the compound are the result of PDE10A inhibition. These results indicate that PDE10A regulates the activation of striatal medium spiny neurons through effects on cAMP- and cGMP-dependent signaling cascades. Furthermore, the present results demonstrate that papaverine has efficacy in behavioral models predictive of antipsychotic activity. Thus, inhibition of PDE10A may represent a novel approach to the treatment of psychosis.

Purpose: In vivo studies have focused on the latter stages of the bone metastatic process (osteolysis), whereas little is known about earlier events, e.g., arrival, localization, and initial colonization. Defining these initial steps may potentially identify the critical points susceptible to therapeutic intervention., Experimental Design: MDA-MB-435 human breast cancer cells engineered with green fluorescent protein were injected into the cardiac left ventricle of athymic mice. Femurs were analyzed by fluorescence microscopy, immunohistochemistry, real-time PCR, flow cytometry, and histomorphometry at times ranging from 1 hour to 6 weeks., Results: Single cells were found in distal metaphyses at 1 hour postinjection and remained as single cells up to 72 hours. Diaphyseal arrest occurred rarely and few cells remained there after 24 hours. At 1 week, numerous foci (2-10 cells) were observed, mostly adjacent to osteoblast-like cells. By 2 weeks, fewer but larger foci (> or =50 cells) were seen. Most bones had a single large mass at 4 weeks (originating from a colony or coalescing foci) which extended into the diaphysis by 4 to 6 weeks. Little change (<20%) in osteoblast or osteoclast numbers was observed at 2 weeks, but at 4 to 6 weeks, osteoblasts were dramatically reduced (8% of control), whereas osteoclasts were reduced modestly (to approximately 60% of control)., Conclusions: Early arrest in metaphysis and minimal retention in diaphysis highlight the importance of the local milieu in determining metastatic potential. These results extend the Seed and Soil hypothesis by demonstrating both intertissue and intratissue differences governing metastatic location. Ours is the first in vivo evidence that tumor cells influence not only osteoclasts, as widely believed, but also eliminate functional osteoblasts, thereby restructuring the bone microenvironment to favor osteolysis. The data may also explain why patients receiving bisphosphonates fail to heal bone despite inhibiting resorption, implying that concurrent strategies that restore osteoblast function are needed to effectively treat osteolytic bone metastases.

Skeletal metastases occur with high incidence in patients with breast cancer and cause long-term skeletal morbidity. Osteonectin (SPARC, BM-40) is a bone matrix factor that is an in vitro chemoattractant for breast and prostate cancer cells. Increased expression of osteonectin is found in malignant breast tumors. We infected MDA-231 breast cancer cells with an adenovirus expressing osteonectin to examine the role of osteonectin expression in breast cancer cells and its effect on metastasis, in particular to bone. Expression of osteonectin did not affect MDA-231 cell proliferation, apoptosis, migration, cell aggregation, or protease cleavage of collagen IV. However, in vitro invasion of these osteonectin-infected cells through Matrigel and colony formation on Matrigel was decreased. Interestingly, high osteonectin expression in MDA-231 cells inhibited metastasis in a dose-dependent manner to many different organs including bone. The reduction in metastasis may be due to decreased platelet-tumor cell aggregation, because exogenous osteonectin inhibited platelet aggregation in vitro and the high osteonectin expression in MDA-231 cells reduced tumor cell-induced thrombocytopenia in vivo compared with control-infected cells. These studies suggest that high endogenous expression of osteonectin in breast cancer cells may reduce metastasis via reduced invasive activity and reduced tumor cell-platelet aggregation.

Previous studies have demonstrated that some tumor cells occupy a pericyte-like location in melanoma, forming angio-tumoral complexes. We hypothesized that these tumor cells are migrating along the abluminal surface of the endothelium, a mechanism termed "extravascular migratory metastasis." In the present study, we have used human and murine melanoma cells that stably express enhanced green fluorescence protein (GFP) to examine, in an ex vivo co-culture model, melanoma cell interactions with vessels that have sprouted from rat aortic rings. We also used in vivo tumor growth on the chick chorioallantoic membrane (CAM) to observe the dissemination pathway of melanoma cells. In the ex vivo rat aorta system, we observed a pericyte-like location of tumor cells that were spreading along the vascular channels. For examination of the CAM in vivo, we have used the Lugassy preparation, allowing one to obtain striking images of the relationship between fluorescent GFP cells and microvessels. Melanoma cells were found cuffing the outside of vessels around the tumor. Tumor cells were observed along the vessels several centimeters from the tumor. Confocal microscopy and histopathology confirmed the pericyte-like location of tumor cells, without any observable intravasation. The results indicate that melanoma cells can migrate along the abluminal surface of vessels. This study also demonstrates that these models can provide quantitation analysis that may prove useful in elucidating the molecular interactions involved in extravascular migratory metastasis.

Introduction: Breast cancer is one of the most common malignancies affecting women in the United States and Europe. Approximately three out of every four women with breast cancer develop metastases in bone which, in turn, diminishes quality of life. The alpha(v)beta3 integrin has previously been implicated in multiple aspects of tumor progression, metastasis and osteoclast bone resorption. Therefore, we hypothesized that the alpha(v)beta3-selective inhibitor, S247, would decrease the development of osteolytic breast cancer metastases., Materials and Methods: Cells were treated in vitro with S247 and assessed for viability and adhesion to matrix components. Athymic mice received intracardiac (left ventricle) injections of human MDA-MB-435 breast carcinoma cells expressing enhanced green-fluorescent protein. Mice were treated with vehicle (saline) or S247 (1, 10, or 100 mg/kg/d) using osmotic pumps beginning either one week before or one week after tumor cell inoculation. Bones were removed and examined by fluorescence microscopy and histology. The location and size of metastases were recorded., Results and Conclusions: IC50 for S247 adhesion to alpha(v)beta3 or alpha(IIB)beta3a substrates was 0.2 nM vs. 244 nM, respectively. Likewise, S247 was not toxic at doses up to 1000 microM. However, osteoclast cultures treated with S247 exhibited marked morphological changes and impaired formation of the actin sealing zone. When S247 was administered prior to tumor cells, there was a significant, dose-dependent reduction (25-50% of vehicle-only-treated mice; P = 0.002) in osseous metastasis. Mice receiving S247 after tumor cell inoculation also developed fewer bone metastases, but the difference was not statistically significant. These data suggest that, in the MDA-MB-435 model, the alpha(v)beta3 integrin plays an important role in early events (e.g., arrest of tumor cells) in bone metastasis. Furthermore, the data suggest that alpha(v)beta3 inhibitors may be useful in the treatment and/or prevention of breast cancer metastases in bone.

Breast cancer metastasizes to bone with high frequency and incidence. However, studies of breast cancer metastasis to bone have been limited by two factors. First, the number of models that colonize bone are limited. Second, detection of bone metastases is too insensitive or too laborious for routine, large-scale studies or for studying the earliest steps in bone colonization. To partially alleviate these problems, the highly metastatic MDA-MB-435 (435) human breast carcinoma cell line was engineered to constitutively express enhanced green fluorescent protein (GFP). While 435GFP cells did not form femoral metastases following orthotopic or intravenous injections, they produced widespread osteolytic skeletal metastases following injection into the left ventricle of the heart. All mice developed at least one femur metastasis as well as a mandibular metastasis. As in humans, osseous metastases localized predominantly to trabecular regions, especially proximal and distal femur, proximal tibia, proximal humerus and lumbar vertebrae. 435GFP cells also developed metastases in adrenal glands, brain and ovary following intracardiac injection, suggesting that this model may also be useful for studying organotropism to other tissues as well. Additionally, GFP-tagging permitted detection of single cells and microscopic metastases in bone at early time points following arrival and at stages of proliferation prior to coalescence of individual metastases.

Metastatic disease is the most critical impediment to cancer patient survival. However, comparatively little is known concerning the intricate pathways which govern the complex phenotypes associated with metastasis. The KISS1 metastasis suppressor gene inhibits metastasis in both in vivo melanoma and breast carcinoma models. Despite its clear physiological activity, the mechanism of KISS1 remains unclear. Recent identification of a 54 amino acid peptide of KISS1, termed metastin or kisspeptin-54, and its cognate G-protein coupled receptor (hOT7T175, AXOR12, GPR54) have provided additional clues and avenues of research. While studies have attributed KISS1 with modulation of NFkappaB regulation, experiments with metastin and its receptor implicate MAP kinase pathways and also suggest the potential of autocrine, paracrine and endocrine roles. Impacts on motility, chemotaxis, adhesion and invasion have each been documented in disparate cell lines and conflicting observations require resolution. Nevertheless, mounting clinical evidence, particularly the loss of KISS1 in metastases, correlates KISS1 and metastin receptor expression with human tumor progression. Together, the data substantiate roles for these molecules in metastasis regulation.

We have recently shown that administration of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine (PA) biosynthesis reduces pulmonary metastasis from MDA-MB-435 breast cancer xenografts in nude mice. The present experiments were designed to further explore PA involvement in breast cancer metastasis, using GFP-tagged MDA-MB-435 cells that can be tracked at the single cell level. Administration of DFMO significantly reduced the number of mice with pulmonary metastasis as well as the number of metastases per mouse. Both single-cell and multicellular metastatic deposits were similarly suppressed, thus suggesting that DFMO was inhibiting lung colonization by tumor cells rather than preventing progression of single-cell deposits to overt metastasis. DFMO administration also significantly reduced local recurrences following removal of the primary tumor. Prolongation of DFMO treatment to 14 weeks did not yield a superior antimetastatic effect beyond that provided by a 10-week course of therapy. Discontinuation of DFMO, on the other hand, was associated with local regrowth of the tumors and, possibly, recurrence of pulmonary metastasis. These data provide a rationale for testing the efficacy of anti-PA treatment within the context of adjuvant therapy of breast cancer.

Introduction of normal, neomycin-tagged human chromosome 11 (neo11) reduces the metastatic capacity of MDA-MB-435 human breast carcinoma cells by 70-90% without affecting tumorigenicity. Differential display comparing MDA-MB-435 and neo11/435 led to the discovery of a human breast carcinoma metastasis suppressor gene, BRMS1, which maps to chromosome 11q13.1-q13.2. Stable transfectants of MDA-MB-435 and MDA-MB-231 breast carcinoma cells with BRMS1 cDNA still form progressively growing, locally invasive tumors when injected in mammary fat pads of athymic mice but exhibit significantly lower metastatic potential (50-90% inhibition) to lungs and regional lymph nodes. To begin elucidating the mechanism(s) of action, we measured the ability of BRMS1 to perturb individual steps of the metastatic cascade modeled in vitro. Consistent differences were not observed for adhesion to extracellular matrix components (laminin, fibronectin, type IV collagen, type I collagen, Matrigel); growth rates in vitro or in vivo; expression of matrix metalloproteinases, heparanase, or invasion. Likewise. BRMS1 expression did not up regulate expression of other metastasis suppressors, such as NM23, Kai1, KiSS1 or E-cadherin. Motility of BRMS1 transfectants was modestly inhibited (30-60%) compared to parental and vector-only transfectants. Ability to grow in soft agar was also decreased in MDA-MB-435 cells by 80-89%, but the decrease for MDA-MB-231 was less (13-15% reduction). Also, transfection and re-expression of BRMS1 restored the ability of human breast carcinoma cells to form functional homotypic gap junctions. Collectively, these data suggest that BRMS1 suppresses metastasis of human breast carcinoma by complex, atypical mechanisms.

The incidence of melanoma continues to increase at a rapid rate. As for most cancers, it is melanoma metastases, rather than the primary malignancy, that is the principal cause of death. We previously showed that the introduction of a normal copy of chromosome 6 into the metastatic human melanoma cell line C8161 suppresses metastasis at a step subsequent to tumor cells entering the bloodstream. To better define the step(s) in metastasis blocked by the addition of chromosome 6 we engineered cells that constitutively express green fluorescent protein (GFP). When these tagged, chromosome 6 hybrid cells were injected intravenously into athymic mice, grossly detectable metastases did not form. However, fluorescence microscopy revealed micro-metastases (single cells or clusters of <10 cells) in the lungs, suggesting that these cells lodged in the lungs but failed to proliferate. Cells isolated from lung up to 60 days post-injection grew in culture and/or formed tumors when injected into the skin, indicating that they were still viable, but dormant. This result implies that the gene(s) on chromosome 6 interfere specifically with growth regulatory response in the lung, but not in the skin. Thus, the gene(s) responsible for metastasis suppression represents a new class of metastasis inhibitors acting at the final stages of the metastatic cascade--that is, affecting the ability of the cells to survive and proliferate at a specific secondary site.

Acute subdural hematoma kills or disables more severely head injured patients than any other complication of cranial trauma. The main pathological factor involved is ischemic neuronal damage, which is caused by raised intracranial pressure and local effect. The authors have evaluated the hypothesis that a novel use-dependent sodium channel antagonist, 619C89, could reduce ischemic brain damage in the rat subdural hematoma model. Because previous studies have shown that focal neuronal damage may be mediated by "excitotoxic" mechanisms, and because excitatory amino acid levels have been shown to be markedly elevated after brain trauma in humans, the authors have measured levels of glutamate, aspartate, and threonine within the cortex underneath the hematoma, using in vivo microdialysis before and after induction of hematoma, in both vehicle- and drug-treated rats. Postinjury treatment with 619C89 (30 mg/kg) significantly reduced the volume of hemispheric ischemic damage produced by subdural hematoma, from 99.77 +/- 7.51 mm3 in vehicle-treated control rats to 46.07 +/- 11.06 mm3 (p = 0.0007) in drug-treated animals. In the vehicle-treated animals, induction of subdural hematoma led to a fourfold increase in glutamate in the first 30 minutes after subdural hematoma occurred. The mean extracellular glutamate concentration in these animals remained 2- to 2.6-fold increased over the following 2.5 hours. In the 619C89-treated animals, the release of glutamate from the cortex underneath the hematoma was significantly attenuated. In these rats, induction of subdural hematoma led to a 2.7-fold increase in the first 30-minute sample, but extracellular glutamate concentration returned to near-basal levels thereafter, compared with vehicle-treated animals (p < 0.05). These results show that 619C89 is highly neuroprotective in this model and that its effects may, in part, be mediated by the inhibition of glutamate release from the ischemic cortex underneath the hematoma.