Anti-nicastrin monoclonal antibodies elicit pleiotropic anti-tumour pharmacological effects in invasive breast cancer cells.

The goal of targeted cancer therapies is to specifically block oncogenic signalling, thus maximising efficacy, while reducing side-effects to patients. The gamma-secretase (GS) complex is an attractive therapeutic target in haematological malignancies and solid tumours with major pharmaceutical activity to identify optimal inhibitors. Within GS, nicastrin (NCSTN) offers an opportunity for therapeutic intervention using blocking monoclonal antibodies (mAbs). Here we explore the role of anti-nicastrin monoclonal antibodies, which we have developed as specific, multi-faceted inhibitors of proliferation and invasive traits of triple-negative breast cancer cells. We use 3D in vitro proliferation and invasion assays as well as an orthotopic and tail vail injection triple-negative breast cancer in vivo xenograft model systems. RNAScope assessed nicastrin in patient samples. Anti-NCSTN mAb clone-2H6 demonstrated a superior anti-tumour efficacy than clone-10C11 and the RO4929097 small molecule GS inhibitor, acting by inhibiting GS enzymatic activity and Notch signalling in vitro and in vivo. Confirming clinical relevance of nicastrin as a target, we report evidence of increased NCSTN mRNA levels by RNA in situ hybridization (RNAScope) in a large cohort of oestrogen receptor negative breast cancers, conferring independent prognostic significance for disease-free survival, in multivariate analysis. We demonstrate here that targeting NCSTN using specific mAbs may represent a novel mode of treatment for invasive triple-negative breast cancer, for which there are few targeted therapeutic options. Furthermore, we propose that measuring NCSTN in patient samples using RNAScope technology may serve as companion diagnostic for anti-NCSTN therapy in the clinic.

Inhibition of thrombin receptor signaling on α-smooth muscle actin(+) CD34(+) progenitors leads to repair after murine immune vascular injury.

OBJECTIVE: The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). METHODS AND RESULTS: BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. CONCLUSIONS: Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage.

Evaluation of [18F]AlF-EMP-105 for Molecular Imaging of C-Met.

C-Met is a receptor tyrosine kinase that is overexpressed in a range of different cancer types, and has been identified as a potential biomarker for cancer imaging and therapy. Previously, a 68Ga-labelled peptide, [68Ga]Ga-EMP-100, has shown promise for imaging c-Met in renal cell carcinoma in humans. Herein, we report the synthesis and preliminary biological evaluation of an [18F]AlF-labelled analogue, [18F]AlF-EMP-105, for c-Met imaging by positron emission tomography. EMP-105 was radiolabelled using the aluminium-[18F]fluoride method with 46 ± 2% RCY and >95% RCP in 35-40 min. In vitro evaluation showed that [18F]AlF-EMP-105 has a high specificity for c-Met-expressing cells. Radioactive metabolite analysis at 5 and 30 min post-injection revealed that [18F]AlF-EMP-105 has good blood stability, but undergoes transformation-transchelation, defluorination or demetallation-in the liver and kidneys. PET imaging in non-tumour-bearing mice showed high radioactive accumulation in the kidneys, bladder and urine, demonstrating that the tracer is cleared predominantly as [18F]fluoride by the renal system. With its high specificity for c-Met expressing cells, [18F]AlF-EMP-105 shows promise as a potential diagnostic tool for imaging cancer.

Effective Detection and Monitoring of Glioma Using [18F]FPIA PET Imaging.

BACKGROUND: Reprogrammed cellular metabolism is a cancer hallmark. In addition to increased glycolysis, the oxidation of acetate in the citric acid cycle is another common metabolic phenotype. We have recently developed a novel fluorine-18-labelled trimethylacetate-based radiotracer, [18F]fluoro-pivalic acid ([18F]FPIA), for imaging the transcellular flux of short-chain fatty acids, and investigated whether this radiotracer can be used for the detection of glioma growth. METHODS: We evaluated the potential of [18F]FPIA PET to monitor tumor growth in orthotopic patient-derived (HSJD-GBM-001) and cell line-derived (U87, LN229) glioma xenografts, and also included [18F]FDG PET for comparison. We assessed proliferation (Ki-67) and the expression of lipid metabolism and transport proteins (CPT1, SLC22A2, SLC22A5, SLC25A20) by immunohistochemistry, along with etomoxir treatment to provide insights into [18F]FPIA uptake. RESULTS: Longitudinal PET imaging showed gradual increase in [18F]FPIA uptake in orthotopic glioma models with disease progression (p < 0.0001), and high tumor-to-brain contrast compared to [18F]FDG (p < 0.0001). [18F]FPIA uptake correlated positively with Ki-67 (p < 0.01), SLC22A5 (p < 0.001) and SLC25A20 (p = 0.001), and negatively with CPT1 (p < 0.01) and SLC22A2 (p < 0.01). Etomoxir reduced [18F]FPIA uptake, which correlated with decreased Ki-67 (p < 0.05). CONCLUSIONS: Our findings support the use of [18F]FPIA PET for the detection and longitudinal monitoring of glioma, showing a positive correlation with tumor proliferation, and suggest transcellular flux-mediated radiotracer uptake.

Repurposed floxacins targeting RSK4 prevent chemoresistance and metastasis in lung and bladder cancer.

Lung and bladder cancers are mostly incurable because of the early development of drug resistance and metastatic dissemination. Hence, improved therapies that tackle these two processes are urgently needed to improve clinical outcome. We have identified RSK4 as a promoter of drug resistance and metastasis in lung and bladder cancer cells. Silencing this kinase, through either RNA interference or CRISPR, sensitized tumor cells to chemotherapy and hindered metastasis in vitro and in vivo in a tail vein injection model. Drug screening revealed several floxacin antibiotics as potent RSK4 activation inhibitors, and trovafloxacin reproduced all effects of RSK4 silencing in vitro and in/ex vivo using lung cancer xenograft and genetically engineered mouse models and bladder tumor explants. Through x-ray structure determination and Markov transient and Deuterium exchange analyses, we identified the allosteric binding site and revealed how this compound blocks RSK4 kinase activation through binding to an allosteric site and mimicking a kinase autoinhibitory mechanism involving the RSK4's hydrophobic motif. Last, we show that patients undergoing chemotherapy and adhering to prophylactic levofloxacin in the large placebo-controlled randomized phase 3 SIGNIFICANT trial had significantly increased (P = 0.048) long-term overall survival times. Hence, we suggest that RSK4 inhibition may represent an effective therapeutic strategy for treating lung and bladder cancer.

Evaluation of apoptosis imaging biomarkers in a genetic model of cell death.

PURPOSE: We have previously developed the caspase-based radiotracer, 18F-ICMT-11, for PET imaging to monitor treatment response. We further validated 18F-ICMT-11 specificity in a murine melanoma death-switch tumour model with conditional activation of caspase-3 induced by doxycycline. METHODS: Caspase-3/7 activity and cellular uptake of 18F-ICMT-11, 18F-ML-10 and 18F-FDG were assessed in B16ova and B16ovaRevC3 cells after death-switch induction. Death-switch induction was confirmed in vivo in xenograft tumours, and 18F-ICMT-11 and 18F-ML-10 biodistribution was assessed by ex vivo gamma counting of select tissues. PET imaging was performed with 18F-ICMT-11, 18F-ML-10 and 18F-FDG. Caspase-3 activation was confirmed by immunohistochemistry. RESULTS: Significantly increased caspase-3/7 activity was observed only in B16ovaRevC3 cells after death-switch induction, accompanied by significantly increased 18F-ICMT-11 (p < 0.001) and 18F-ML-10 (p < 0.05) and decreased 18F-FDG (p < 0.001) uptake compared with controls. B16ova and B16ovaRevC3 tumours had similar growth in vivo; however, B16ovaRevC3 growth was significantly reduced with death-switch induction (p < 0.01). Biodistribution studies showed significantly increased 18F-ICMT-11 tumour uptake following death-switch induction (p < 0.01), but not for 18F-ML-10. Tumour uptake of 18F-ICMT-11 was higher than that of 18F-ML-10 after death-switch induction. PET imaging studies showed that 18F-ICMT-11 can be used to detect apoptosis after death-switch induction, which was accompanied by significantly increased expression of cleaved caspase-3. 18F-FDG signal decreased in tumours after death-switch induction. CONCLUSIONS: We demonstrate that 18F-ICMT-11 can be used to detect caspase-3 activation in a death-switch tumour model, independent of the confounding effects of cancer therapeutics, thus confirming its specificity and supporting the development of this radiotracer for clinical use to monitor tumour apoptosis and therapy response.

Malingering on the Social Security disability consultative exam: a new rating scale.

In disability examinations, benefits may depend on the findings of a psychological consultative examination (PCE), which in Louisiana usually involves a mental status examination and a Wechsler Scale. The disability determinations service (DDS) requires a warning that failure to do one's best may result in an unfavorable decision on the claim, but psychologists are officially discouraged from determining effort by the use of formal effort tests. Consequently, there is a need for internal indicators of effort. Formal testing of effort was undertaken in order to identify indicators of effort within the PCE in WAIS-age and WISC-age claimants. Our findings indicated that the total score of indicators was more predictive of effort than any single indicator. Regression equations yielded information on how much effort contributes to IQ. Classification accuracy for the new rating scale was described for a "dose-response" of effort. Disincentives for malingering detection in the PCE were identified.

Regenerative repair after endoluminal injury in mice with specific antagonism of protease activated receptors on CD34+ vascular progenitors.

Tissue factor (TF) and thrombin are involved in intimal hyperplasia (IH) and remodelling following vascular injury. Because many neointimal smooth muscle cells (VSMCs) derive from circulating vascular progenitors (VPs), we investigated how thrombin influences VP phenotype and function. Following wire-induced carotid artery injury in mice, the majority of circulating VPs expressed TF, were capable of initiating clotting in vitro, and had protease-activated receptors (PAR)-1, -2, and -4. Thrombin, through PAR-1, inhibited apoptosis and caused proliferation, resulting in the outgrowth of VP coexpressing markers of activated endothelial cells and VSMCs, even in the presence of growth factors. These mixed-phenotype VPs circulated as a minority population after injury and shared a similar phenotype with many neointimal cells. Labeled CD34(+) cells, injected up to 2 weeks after injury, could be detected in the injured vessel wall, suggesting that continued recruitment may contribute to progressive IH. Finally, CD34(+) cells incubated with thrombin prior to injection promoted florid neointimal lesions, whereas those incubated with PAR antagonists inhibited IH and promoted regenerative repair characterized by the development of a quiescent endothelium. We conclude that IH after vascular injury is due to the direct actions of thrombin on mobilized VPs.

Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1-dependent leukocyte recruitment in vivo.

Thrombin, acting through a family of protease-activated receptors (PARs), is known to amplify inflammatory responses, but the in vivo importance of PARs in inflammation is not fully appreciated. In a mouse heart-to-rat transplant model, where it is possible to distinguish graft (mouse) from systemic (rat) chemokines, we show that donor PAR-1 is required to generate the local monocyte chemoattractant protein (MCP)-1 needed to recruit rat natural killer cells and macrophages into the hearts. We have confirmed the importance of this mechanism in a second model of thioglycollate-induced peritonitis and also show that PAR-1 is important for the production of MCP-3 and MCP-5. Despite the presence of multiple other mediators capable of stimulating chemokine production in these models, these data provide the first evidence that thrombin and PAR activation are required in vivo to initiate inflammatory cell recruitment.

Disaster reanimatology potentials : A structural interview study in Armenia

The study of a disaster which has accurred cannot using the traditional techniques as it is not possible to conceive or conduct controlled, randomized experiments for such an event. Paper I of this series described non-experimental, scientific methodologies which were applied to study the detection-extrication-resucitatioj activities which occurred following the devastating 1988 earthquake in the Republic of Armenia, USSR. This paper critically evaluates the methodologies used for the Armenia study and proposes modification in these methods for application to the study of future disasters.(AU)

Disaster reanimatology potentials : A structural interview study in Armenia : I. Methodology and preliminary results

In general, preparations for disasters which results in mass casualties do not incorporate a modern resuscitation approach. We explored the life-saving potential of, and time limits for life-supporting first aid (LSFA), advanced trauma life support (ATLS), resuscitative surgey, and prolonged life support (PLS: intensive care) following the earthquake in Armenia on 7 December 1988. We used a structured, retrospective interview method applied previously to evaluation of emergency medical services (EMS) in the United States. A total of 120 survivors of, and participants in the earthquake in Armenia were interviewed on site (49 lay eyewitnesses, 20 search-rescue personnel, 39 medical personnel and records, and 12 administrators). Answers were verified by crosschecks.Preliminary results permit the following generalizations: 1)a significant nomber of victims died slowly as the result of iinjuries such as external hemorrhage, head injury with coma,shock, or crush sundrome; 2) early search and rescue was perfomed primarily by uninjured covictims using hand tools; 3) many livespotentially could have been saved by the use of LSFA and ATLS started during extrication of crushed victims. 4) medical teams from neighboring EMS systems started to arrive at the site at 2-3 hours and therefore, ATLS could have been provided in time to save lives and limbs; 5) some amputations had to be perfomed in the field to enable extrication; 6) the usefulness of other resuscitative surgery in the fieldneeds to be clarified; 7) evacuations were rapid; 8) air evacuation proved essential; 9) hospital intensive care was well organized; and 10)international medical aid, which arrived after 48 hours, was too late top impact on resuscitation. definitive analysis of data in the near future will lead to recommendations for local, regional, and National Disaster Medical Systems (NDMS).(AU)