Monitoring AKT activity and targeting in live tissue and disease contexts using a real-time Akt-FRET biosensor mouse

Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications

Intravital imaging technology guides FAK-mediated priming in pancreatic cancer precision medicine according to Merlin status

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow–induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy

Intravital imaging technology guides FAK-mediated priming in pancreatic cancer precision medicine according to Merlin status

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Forster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micro-patterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flow-induced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy

Mortality of emergency abdominal surgery in high-, middle- and low-income countries

Background: Surgical mortality data are collected routinely in high-income countries, yet virtually no low- or middle-income countries have outcome surveillance in place. The aim was prospectively to collect worldwide mortality data following emergency abdominal surgery, comparing findings across countries with a low, middle or high Human Development Index (HDI). Methods: This was a prospective, multicentre, cohort study. Self-selected hospitals performing emergency surgery submitted prespecified data for consecutive patients from at least one 2-week interval during July to December 2014. Postoperative mortality was analysed by hierarchical multivariable logistic regression. Results: Data were obtained for 10 745 patients from 357 centres in 58 countries; 6538 were from high-, 2889 from middle- and 1318 from low-HDI settings. The overall mortality rate was 1⋅6 per cent at 24 h (high 1⋅1 per cent, middle 1⋅9 per cent, low 3⋅4 per cent; P < 0⋅001), increasing to 5⋅4 per cent by 30 days (high 4⋅5 per cent, middle 6⋅0 per cent, low 8⋅6 per cent; P < 0⋅001). Of the 578 patients who died, 404 (69⋅9 per cent) did so between 24 h and 30 days following surgery (high 74⋅2 per cent, middle 68⋅8 per cent, low 60⋅5 per cent). After adjustment, 30-day mortality remained higher in middle-income (odds ratio (OR) 2⋅78, 95 per cent c.i. 1⋅84 to 4⋅20) and low-income (OR 2⋅97, 1⋅84 to 4⋅81) countries. Surgical safety checklist use was less frequent in low- and middle-income countries, but when used was associated with reduced mortality at 30 days. Conclusion: Mortality is three times higher in low- compared with high-HDI countries even when adjusted for prognostic factors. Patient safety factors may have an important role. Registration number: NCT02179112 (http://www.clinicaltrials.gov)

Oseltamivir phosphate monotherapy ablates tumor neovascularization, growth, and metastasis in mouse model of human triple-negative breast adenocarcinoma.

BACKGROUND: Triple-negative breast cancers (TNBCs) lack the estrogen, progesterone, and epidermal growth factor (EGF) receptor-2 (HER2/neu) receptors. Patients with TNBC have typical high grading, more frequent relapses, and exhibit poorer outcomes or prognosis compared with the other subtypes of breast cancers. Currently, there are no targeted therapies that are effective for TNBC. Preclinical antitumor activity of oseltamivir phosphate (OP) therapy was investigated to identify its role in tumor neovascularization, growth, invasiveness, and long-term survival in a mouse model of human TNBC. METHODS: Live cell sialidase, water soluble tetrazolium, WST-1 cell viability, and immunohistochemistry assays were used to evaluate sialidase activity, cell survival, and the expression levels of tumor E-cadherin, N-cadherin, and host endothelial CD31+/PECAM-1 cells in archived paraffin-embedded TNBC MDA-MB-231 tumors grown in RAGxCγ double mutant mice. RESULTS: OP, anti-Neu1 antibodies, and matrix metalloproteinase-9-specific inhibitor blocked Neu1 activity associated with EGF-stimulated TNBC MDA-MB-231 cells. OP treatment of MDA-MB-231 and MCF-7 cells and their long-term tamoxifen-resistant clones reproducibly and dose-dependently reduced the sialidase activity associated with EGF-stimulated live cells and the cell viability after 72 hours of incubation. Combination of 1 μM cisplatin, 5-FU, paclitaxel, gemcitabine, or tamoxifen with OP dosages ≥300 μg/mL significantly reduced cell viability at 24, 48, and 72 hours when compared to the chemodrug alone. Heterotopic xenografts of MDA-MB-231 tumors developed robust and bloody tumor vascularization in RAG2xCγ double mutant mice. OP treatment at 30 mg/kg daily intraperitoneally reduced tumor vascularization and growth rate as well as significantly reduced tumor weight and spread to the lungs compared with the untreated cohorts. OP treatment at 50 mg/kg completely ablated tumor vascularization, tumor growth and spread to the lungs, with significant long-term survival at day 180 postimplantation, tumor shrinking, and no relapses after 56 days off-drug. OP 30 mg/kg cohort tumors expressed significantly reduced levels of human N-cadherins and host CD31+ endothelial cells with concomitant significant expression of E-cadherins compared to the untreated cohorts. CONCLUSION: OP monotherapy may be the effective treatment therapy for TNBC

Transcriptional factor snail controls tumor neovascularization, growth and metastasis in mouse model of human ovarian carcinoma.

BACKGROUND: Snail, a transcriptional factor and repressor of E-cadherin is well known for its role in cellular invasion. It can regulate epithelial to mesenchymal transition (EMT) during embryonic development and in epithelial cells. Snail also mediates tumor progression and metastases. Silencing of Snail and its associate member Slug in human A2780 ovarian epithelial carcinoma cell line was investigated to identify its role in tumor neovascularization. METHODS: Live cell sialidase, WST-1 cell viability and immunohistochemistry assays were used to evaluate sialidase activity, cell survival and the expression levels of tumor E-cadherin, N-cadherin, VE-cadherin, and host endothelial CD31+(PECAM-1) cells in archived paraffin-embedded ovarian A2780, A2780 Snail shRNA GIPZ lentiviral knockdown (KD) and A2780 Slug shRNA GIPZ lentiviral KD tumors grown in RAGxCγ double mutant mice. RESULTS: Oseltamivir phosphate (OP), anti-Neu1 antibodies and MMP-9 specific inhibitor blocked Neu1 activity associated with epidermal growth factor (EGF) stimulated A2780 ovarian epithelial carcinoma cells. Silencing Snail in A2780 cells abrogated the Neu1 activity following EGF stimulation of the cells compared to A2780 and A2780 Slug KD cells. OP treatment of A2780 and cisplatin-resistant A2780cis cells reproducibly and dose-dependently abated the cell viability with a LD50 of 7 and 4 μm, respectively, after 48 h of incubation. Heterotopic xenografts of A2780 and A2780 Slug KD tumors developed robust and bloody tumor vascularization in RAG2xCγ double mutant mice. OP treatment at 50 mg/kg daily intraperitoneally did not significantly impede A2780 tumor growth rate but did cause a significant reduction of lung metastases compared with the untreated and OP 30mg/kg cohorts. Silencing Snail in A2780 tumor cells completely abrogated tumor vascularization, tumor growth and spread to the lungs in RAGxCγ double mutant mice. A2780 and A2780 Slug KD tumors expressed high levels of human N- and VE-cadherins, and host CD31+ endothelial cells, while A2780 Snail KD tumors expressed E-cadherin and reduced host CD31+ cells. OP 50mg/kg cohort tumors had reduced numbers of host CD31+ cells compared to a higher expression levels of CD31+ cells in tumors from the untreated control and OP 30mg/kg cohorts. CONCLUSION: Snail transcriptional factor is an important intermediate player in human ovarian tumor neovascularization

Therapeutic targeting of Neul sialidase with oseltamivir phosphate (Tamiflu (R)) disables cancer cell survival in human pancreatic cancer with acquired chemoresistance

BACKGROUND: Resistance to drug therapy, along with high rates of metastasis, contributes to the low survival rate in patients diagnosed with pancreatic cancer. An alternate treatment for human pancreatic cancer involving targeting of Neu1 sialidase with oseltamivir phosphate (Tamiflu®) was investigated in human pancreatic cancer (PANC1) cells with acquired resistance to cisplatin and gemcitabine. Its efficacy in overcoming the intrinsic resistance of the cell to chemotherapeutics and metastasis was evaluated. METHODS: Microscopic imaging, immunocytochemistry, immunohistochemistry, and WST-1 cell viability assays were used to evaluate cell survival, morphologic changes, and expression levels of E-cadherin, N-cadherin, and VE-cadherin before and after treatment with oseltamivir phosphate in PANC1 cells with established resistance to cisplatin, gemcitabine, or a combination of the two agents, and in archived paraffin-embedded PANC1 tumors grown in RAGxCγ double mutant mice. RESULTS: Oseltamivir phosphate overcame the chemoresistance of PANC1 to cisplatin and gemcitabine alone or in combination in a dose-dependent manner, and disabled the cancer cell survival mechanism(s). Oseltamivir phosphate also reversed the epithelial-mesenchymal transition characteristic of the phenotypic E-cadherin to N-cadherin changes associated with resistance to drug therapy. Low-dose oseltamivir phosphate alone or in combination with gemcitabine in heterotopic xenografts of PANC1 tumors growing in RAGxCγ double mutant mice did not prevent metastatic spread to the liver and lung. CONCLUSION: Therapeutic targeting of Neu1 sialidase with oseltamivir phosphate at the growth factor receptor level disables the intrinsic signaling platform for cancer cell survival in human pancreatic cancer with acquired chemoresistance. These findings provide evidence for oseltamivir phosphate (Tamiflu) as a potential therapeutic agent for pancreatic cancer resistant to drug therapy

Therapeutic targeting of Neu1 sialidase with oseltamivir phosphate (Tamiflu&reg;) disables cancer cell survival in human pancreatic cancer with acquired chemoresistance

Leah K O&#39;Shea,1 Samar Abdulkhalek,1 Stephanie Allison,2 Ronald J Neufeld,2 Myron R Szewczuk11Department of Biomedical and Molecular Sciences, 2Department of Chemical Engineering, Queen&#39;s University, Kingston, ON, CanadaBackground: Resistance to drug therapy, along with high rates of metastasis, contributes to the low survival rate in patients diagnosed with pancreatic cancer. An alternate treatment for human pancreatic cancer involving targeting of Neu1 sialidase with oseltamivir phosphate (Tamiflu&reg;) was investigated in human pancreatic cancer (PANC1) cells with acquired resistance to cisplatin and gemcitabine. Its efficacy in overcoming the intrinsic resistance of the cell to chemotherapeutics and metastasis was evaluated.Methods: Microscopic imaging, immunocytochemistry, immunohistochemistry, and WST-1 cell viability assays were used to evaluate cell survival, morphologic changes, and expression levels of E-cadherin, N-cadherin, and VE-cadherin before and after treatment with oseltamivir phosphate in PANC1 cells with established resistance to cisplatin, gemcitabine, or a combination of the two agents, and in archived paraffin-embedded PANC1 tumors grown in RAGxC&gamma; double mutant mice.Results: Oseltamivir phosphate overcame the chemoresistance of PANC1 to cisplatin and gemcitabine alone or in combination in a dose-dependent manner, and disabled the cancer cell survival mechanism(s). Oseltamivir phosphate also reversed the epithelial-mesenchymal transition characteristic of the phenotypic E-cadherin to N-cadherin changes associated with resistance to drug therapy. Low-dose oseltamivir phosphate alone or in combination with gemcitabine in heterotopic xenografts of PANC1 tumors growing in RAGxC&gamma; double mutant mice did not prevent metastatic spread to the liver and lung.Conclusion: Therapeutic targeting of Neu1 sialidase with oseltamivir phosphate at the growth factor receptor level disables the intrinsic signaling platform for cancer cell survival in human pancreatic cancer with acquired chemoresistance. These findings provide evidence for oseltamivir phosphate (Tamiflu) as a potential therapeutic agent for pancreatic cancer resistant to drug therapy.Keywords: human pancreatic cancer, cancer cell survival, chemoresistance, PANC1 cell line

Tropoelastin Promotes the Formation of Dense, Interconnected Endothelial Networks

Tropoelastin, the soluble precursor of elastin, has been used for regenerative and wound healing purposes and noted for its ability to accelerate wound repair by enhancing vascularization at the site of implantation. However, it is not clear whether these effects are directly due to the interaction of tropoelastin with endothelial cells or communicated to endothelial cells following interactions between tropoelastin and neighboring cells, such as mesenchymal stem cells (MSCs). We adapted an endothelial tube formation assay to model in vivo vascularization with the goal of exploring the stimulatory mechanism of tropoelastin. In the presence of tropoelastin, endothelial cells formed less tubes, with reduced spreading into capillary-like networks. In contrast, conditioned media from MSCs that had been cultured on tropoelastin enhanced the formation of more dense, complex, and interconnected endothelial tube networks. This pro-angiogenic effect of tropoelastin is mediated indirectly through the action of tropoelastin on co-cultured cells. We conclude that tropoelastin inhibits endothelial tube formation, and that this effect is reversed by pro-angiogenic crosstalk from tropoelastin-treated MSCs. Furthermore, we find that the known in vivo pro-angiogenic effects of tropoelastin can be modeled in vitro, highlighting the value of tropoelastin as an indirect mediator of angiogenesis