CRISPR-Mediated VHL Knockout Generates an Improved Model for Metastatic Renal Cell Carcinoma.

Metastatic renal cell carcinoma (mRCC) is nearly incurable and accounts for most of the mortality associated with RCC. Von Hippel Lindau (VHL) is a tumour suppressor that is lost in the majority of clear cell RCC (ccRCC) cases. Its role in regulating hypoxia-inducible factors-1α (HIF-1α) and -2α (HIF-2α) is well-studied. Recent work has demonstrated that VHL knock down induces an epithelial-mesenchymal transition (EMT) phenotype. In this study we showed that a CRISPR/Cas9-mediated knock out of VHL in the RENCA model leads to morphologic and molecular changes indicative of EMT, which in turn drives increased metastasis to the lungs. RENCA cells deficient in HIF-1α failed to undergo EMT changes upon VHL knockout. RNA-seq revealed several HIF-1α-regulated genes that are upregulated in our VHL knockout cells and whose overexpression signifies an aggressive form of ccRCC in the cancer genome atlas (TCGA) database. Independent validation in a new clinical dataset confirms the upregulation of these genes in ccRCC samples compared to adjacent normal tissue. Our findings indicate that loss of VHL could be driving tumour cell dissemination through stabilization of HIF-1α in RCC. A better understanding of the mechanisms involved in this phenomenon can guide the search for more effective treatments to combat mRCC

Low-Engine-Friction Technology for Advanced Natural-Gas Reciprocating Engines

This program aims at improving the efficiency of advanced natural-gas reciprocating engines (ANGRE) by reducing piston and piston ring assembly friction without major adverse effects on engine performance, such as increased oil consumption and wear. An iterative process of simulation, experimentation and analysis is being followed towards achieving the goal of demonstrating a complete optimized low-friction engine system. To date, a detailed set of piston and piston-ring dynamic and friction models have been developed and applied that illustrate the fundamental relationships between design parameters and friction losses. Low friction ring designs have already been recommended in a previous phase, with full-scale engine validation partially completed. Current accomplishments include the addition of several additional power cylinder design areas to the overall system analysis. These include analyses of lubricant and cylinder surface finish and a parametric study of piston design. The Waukesha engine was found to be already well optimized in the areas of lubricant, surface skewness and honing cross-hatch angle, where friction reductions of 12% for lubricant, and 5% for surface characteristics, are projected. For the piston, a friction reduction of up to 50% may be possible by controlling waviness alone, while additional friction reductions are expected when other parameters are optimized. A total power cylinder friction reduction of 30-50% is expected, translating to an engine efficiency increase of two percentage points from its current baseline towards the goal of 50% efficiency. Key elements of the continuing work include further analysis and optimization of the engine piston design, in-engine testing of recommended lubricant and surface designs, design iteration and optimization of previously recommended technologies, and full-engine testing of a complete, optimized, low-friction power cylinder system

Rapamycin enhances adenovirus-mediated cancer imaging and therapy in pre-immunized murine hosts.

Tumor-specific adenoviral vectors comprise a fruitful gene-based diagnostic imaging and therapy research area for advanced stage of cancer, including metastatic disease. However, clinical translation of viral vectors has encountered considerable obstacles, largely due to host immune responses against the virus. Here, we explored the utilization of an immunosuppressant, rapamycin, to circumvent the anti-adenovirus immunity in immunocompetent murine prostate cancer models. Rapamycin diminished adenoviral-induced acute immune response by inhibiting NF-κB activation; it also reduced the scale and delayed the onset of inflammatory cytokine secretion. Further, we found that rapamycin abrogated anti-adenovirus antibody production and retarded the function of myeloid cells and lymphocytes that were activated upon viral administration in pre-immunized hosts. Thus, the co-administration of rapamycin prolonged and enhanced adenovirus-delivered transgene expression in vivo, and thereby augmented the imaging capability of adenoviral vectors in both bioluminescent and positron emission tomography modalities. Furthermore, we showed that despite an excellent response of cancer cells to a cytotoxic gene therapeutic vector in vitro, only minimal therapeutic effects were observed in vivo in pre-immunized mice. However, when we combined gene therapy with transient immunosuppression, complete tumor growth arrest was achieved. Overall, transient immunosuppression by rapamycin was able to boost the diagnostic utility and therapeutic potentials of adenoviral vectors

Rapamycin Enhances Adenovirus-Mediated Cancer Imaging and Therapy in Pre-Immunized Murine Hosts

<div><p>Tumor-specific adenoviral vectors comprise a fruitful gene-based diagnostic imaging and therapy research area for advanced stage of cancer, including metastatic disease. However, clinical translation of viral vectors has encountered considerable obstacles, largely due to host immune responses against the virus. Here, we explored the utilization of an immunosuppressant, rapamycin, to circumvent the anti-adenovirus immunity in immunocompetent murine prostate cancer models. Rapamycin diminished adenoviral-induced acute immune response by inhibiting NF-κB activation; it also reduced the scale and delayed the onset of inflammatory cytokine secretion. Further, we found that rapamycin abrogated anti-adenovirus antibody production and retarded the function of myeloid cells and lymphocytes that were activated upon viral administration in pre-immunized hosts. Thus, the co-administration of rapamycin prolonged and enhanced adenovirus-delivered transgene expression <i>in vivo</i>, and thereby augmented the imaging capability of adenoviral vectors in both bioluminescent and positron emission tomography modalities. Furthermore, we showed that despite an excellent response of cancer cells to a cytotoxic gene therapeutic vector <i>in vitro</i>, only minimal therapeutic effects were observed <i>in vivo</i> in pre-immunized mice. However, when we combined gene therapy with transient immunosuppression, complete tumor growth arrest was achieved. Overall, transient immunosuppression by rapamycin was able to boost the diagnostic utility and therapeutic potentials of adenoviral vectors.</p> </div

Rapamycin enhanced Ad-mediated transgene expression in pre-immunized mice.

<p>(A) The timeline for the pre-immunity models. Animals were primed with an intraperitoneal dose of 1×10⁸ PFU empty Ad and, 3 weeks later, subcutaneous tumors were inoculated. RM9 tumors became palpable 4-5 days post implantation; MycCaP tumors became palpable 5-7 days post implantation. At this point, rapamycin or control treatment initiated and intratumoral Ad imaging vectors were administered 4 days later. Daily rapamycin treatment was continued till the end of the study. FL bioluminescent imaging was conducted at time points indicated in B and C. (B) FL bioluminescent imaging of RM-9 bearing C57BL/6 mice on day 4 and 7. High: 5 mg/kg/day rapamycin; Medium: 1.5 mg/kg/day; Low: 0.5 mg/kg/day. n=3. (C) FL bioluminescent imaging of MycCaP bearing FVB mice (n=3 or 4) at indicated time points. (D) Quantification of imaging signal from C. Color bar of bioluminescent imaging: photon count. Error bars: mean ± SEM (n=4 for Ad only; n=3 for Ad + RAPA). * P<0.05, ** P<0.01 by two-tailed <i>t</i> test.</p

Macrophage Blockade Using CSF1R Inhibitors Reverses the Vascular Leakage Underlying Malignant Ascites in Late-Stage Epithelial Ovarian Cancer.

Malignant ascites is a common complication in the late stages of epithelial ovarian cancer (EOC) that greatly diminishes the quality of life of patients. Malignant ascites is a known consequence of vascular dysfunction, but current approved treatments are not effective in preventing fluid accumulation. In this study, we investigated an alternative strategy of targeting macrophage functions to reverse the vascular pathology of malignant ascites using fluid from human patients and an immunocompetent murine model (ID8) of EOC that mirrors human disease by developing progressive vascular disorganization and leakiness culminating in massive ascites. We demonstrate that the macrophage content in ascites fluid from human patients and the ID8 model directly correlates with vascular permeability. To further substantiate macrophages' role in the pathogenesis of malignant ascites, we blocked macrophage function in ID8 mice using a colony-stimulating factor 1 receptor kinase inhibitor (GW2580). Administration of GW2580 in the late stages of disease resulted in reduced infiltration of protumorigenic (M2) macrophages and dramatically decreased ascites volume. Moreover, the disorganized peritoneal vasculature became normalized and sera from GW2580-treated ascites protected against endothelial permeability. Therefore, our findings suggest that macrophage-targeted treatment may be a promising strategy toward a safe and effective means to control malignant ascites of EOC