Design and Manufacture of an Optimised Side-Shifted PPM EMAT Array for Use in Mobile Robotic Localisation.

Guided wave Electro Magnetic Acoustic Transducers (EMATs) offer an elegant method for structural inspection and localisation relative to geometric features, such as welds. This paper presents a Lorentz force EMAT construction framework, where a numerical model has been developed for optimising Printed Circuit Board (PCB) coil parameters as well as a methodology for optimising magnet array parameters to a user's needs. This framework was validated experimentally to show its effectiveness through comparison to an industry built EMAT. The framework was then used to design and manufacture a Side-Shifted Unidirectional Periodic Permanent Magnet (PPM) EMAT for use on a mobile robotic system, which uses guided waves for ranging to build internal maps of a given subject, identifying welded sections, defects and other structural elements. The unidirectional transducer setup was shown to operate in simulation and was then manufactured to compare to the bidirectional transmitter and two-receiver configurations on a localisation system. The unidirectional setup was shown to have clear benefits over the bidirectional setup for mapping an unknown environment using guided waves as there were no dead spots of mapping where signal direction could not be interpreted. Additionally, overall package size was significantly reduced, which in turn allows more measurements to be taken within confined spaces and increases robotic crawler mobility.

IQGAP1 Maintains Pancreatic Ductal Adenocarcinoma Clonogenic Growth and Metastasis.

OBJECTIVES: IQ motif containing GTPase-activating protein 1 (IQGAP1) acts as a scaffold for aberrant mitogen-activated protein kinase (MAPK) signaling driven by KRAS mutations in pancreatic ductal adenocarcinoma (PDAC). We determined the role of IQGAP1 in clonogenic growth and metastasis in PDAC. METHODS: We inhibited IQGAP1 expression using shRNA and assessed clonogenic growth, cell migration, and MAPK signaling in vitro and tumor initiation and metastasis in vivo. The efficacy of a peptide mimicking the IQGAP1 WW domain that binds and inhibits ERK1/2 was determined in vitro and in vivo. RESULTS: IQGAP1 loss inhibited clonogenic growth and migration of KRAS-dependent PDAC cells by disrupting MAPK signaling. In mice, IQGAP1 knockdown decreased tumor-initiating cell frequency and metastasis. WW peptide treatment inhibited clonogenic growth and in vivo tumor growth. CONCLUSIONS: Pancreatic ductal adenocarcinoma clonogenic growth, metastasis, and tumor initiation are dependent on MAPK signaling via IQGAP1. Treatment with a WW peptide disrupts IQGAP1 function and represents a novel targeting strategy for PDAC.

Direct Interactions With Cancer-Associated Fibroblasts Lead to Enhanced Pancreatic Cancer Stem Cell Function.

OBJECTIVE: Cancer-associated fibroblasts (CAFs) play an important role in the progression of pancreatic ductal adenocarcinoma (PDAC) by promoting tumor cell migration and drug resistance. We determined the impact of CAFs on PDAC cancer stem cells (CSCs). METHODS: Fibroblast cell lines from patients' tumors were cocultured with PDAC cells and examined for clonogenic growth and self-renewal using colony-forming assays and migration in vitro. Changes in the frequency of CSCs was determined by flow cytometry. The effect of integrin-focal adhesion kinase (FAK) signaling on CAF-mediated clonogenic growth was evaluated using short hairpin RNAs against ß1 integrin and FAK as well as a small-molecule FAK inhibitor. RESULTS: Cancer-associated fibroblasts enhanced PDAC clonogenic growth, self-renewal, and migration that was associated with an increase in the frequency of CSCs. These fibroblast cells were activated by PDAC cells and increased collagen synthesis resulting in FAK activation in PDAC cells. Knockdown of ß1-integrin and FAK or the inhibition of FAK kinase activity in PDAC cells abrogated the impact of CAFs on clonogenic growth. CONCLUSION: Therefore, CAFs enhance PDAC clonogenic growth, self-renewal, and the frequency of CSCs through type I collagen production that enhances integrin-FAK signaling in PDAC cells.

Ezrin Promotes Stem Cell Properties in Pancreatic Ductal Adenocarcinoma.

Self-renewal maintains the long-term clonogenic growth that is required for cancer relapse and progression, but the cellular processes regulating this property are not fully understood. In many diseases, self-renewal is enhanced in cancer stem cells (CSC), and in pancreatic ductal adenocarcinoma (PDAC), CSCs are characterized by the surface expression of CD44. In addition to cell adhesion, CD44 impacts cell shape and morphology by modulating the actin cytoskeleton via Ezrin, a member of the Ezrin/Radixin/Moesin (ERM) family of linker proteins. We examined the expression of Ezrin in PDAC cells and found higher levels of both total and activated Ezrin in CSCs compared with bulk tumor cells. We also found that the knockdown of Ezrin in PDAC cells decreased clonogenic growth, self-renewal, cell migration, and CSC frequency in vitro as well as tumor initiation in vivo. These effects were associated with cytoskeletal changes that are similar to those occurring during the differentiation of normal stem cells, and the inhibition of actin remodeling reversed the impact of Ezrin loss. Finally, targeting Ezrin using a small-molecule inhibitor limited the self-renewal of clinically derived low-passage PDAC xenografts. Our findings demonstrate that Ezrin modulates CSCs properties and may represent a novel target for the treatment of PDAC. IMPLICATIONS: Our findings demonstrate that Ezrin modulates CSCs' properties and may represent a novel target for the treatment of PDAC.

Biphenotypic Differentiation of Pancreatic Cancer in 3-Dimensional Culture.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the third most common cause of cancer death in the United States. Improved characterized models of PDAC are needed for drug screening. METHODS: We grew 4 established pancreatic cancer cell lines in hanging drop cultures to produce spheroids. We also grew organoids from explanted xenografted PDAC and surgically resected primary PDAC. We performed transmission and scanning electron microscopy and compared findings with those of the normal pancreatic duct. We also performed single-cell cloning to determine the potential options for differentiation. RESULTS: Spheroids contained tight junctions and desmosomes but lacked zymogen granules, as expected. The former features were present in normal pancreatic duct but absent from PDAC cell lines grown in standard 2-dimensional culture. Spheroids functionally excluded macromolecules in whole mounts. Cells on the surface of PDAC spheroids were carpeted by microvilli except for rare cells with prominent stereocilia. Carpets of microvilli were also seen in low passage organoids produced from xenografts and surgically resected human PDAC, in addition to normal human pancreatic duct. We performed single-cell cloning and resulting spheroids produced both cell phenotypes at the same approximate ratios as those from bulk cultures. CONCLUSIONS: Pancreatic cancer spheroids/organoids are capable of biphenotypic differentiation.

The extracellular matrix and focal adhesion kinase signaling regulate cancer stem cell function in pancreatic ductal adenocarcinoma.

Cancer stem cells (CSCs) play an important role in the clonogenic growth and metastasis of pancreatic ductal adenocarcinoma (PDAC). A hallmark of PDAC is the desmoplastic reaction, but the impact of the tumor microenvironment (TME) on CSCs is unknown. In order to better understand the mechanisms, we examined the impact of extracellular matrix (ECM) proteins on PDAC CSCs. We quantified the effect of ECM proteins, ß1-integrin, and focal adhesion kinase (FAK) on clonogenic PDAC growth and migration in vitro and tumor initiation, growth, and metastasis in vivo in nude mice using shRNA and overexpression constructs as well as small molecule FAK inhibitors. Type I collagen increased PDAC tumor initiating potential, self-renewal, and the frequency of CSCs through the activation of FAK. FAK overexpression increased tumor initiation, whereas a dominant negative FAK mutant or FAK kinase inhibitors reduced clonogenic PDAC growth in vitro and in vivo. Moreover, the FAK inhibitor VS-4718 extended the anti-tumor response to gemcitabine and nab-paclitaxel in patient-derived PDAC xenografts, and the loss of FAK expression limited metastatic dissemination of orthotopic xenografts. Type I collagen enhances PDAC CSCs, and both kinase-dependent and independent activities of FAK impact PDAC tumor initiation, self-renewal, and metastasis. The anti-tumor impact of FAK inhibitors in combination with standard chemotherapy support the clinical testing of this combination.

IQGAP1 Scaffold-MAP Kinase Interactions Enhance Multiple Myeloma Clonogenic Growth and Self-Renewal.

Despite improved outcomes in newly diagnosed multiple myeloma, virtually all patients relapse and ultimately develop drug-resistant disease. Aberrant RAS/MAPK signaling is activated in the majority of relapsed/refractory multiple myeloma patients, but its biological consequences are not fully understood. Self-renewal, as defined by the long-term maintenance of clonogenic growth, is essential for disease relapse, and we examined the role of RAS/MAPK activation on multiple myeloma self-renewal by targeting IQ motif-containing GTPase-activating protein 1 (IQGAP1), an intracellular scaffold protein required for mutant RAS signaling. We found that loss of IQGAP1 expression decreased MAPK signaling, cell-cycle progression, and tumor colony formation. Similarly, a peptide mimicking the WW domain of IQGAP1 that interacts with ERK inhibited the clonogenic growth and self-renewal of multiple myeloma cell lines and primary clinical specimens in vitro as well as tumor-initiating cell frequency in immunodeficient mice. During multiple myeloma progression, self-renewal may be enhanced by aberrant RAS/MAPK signaling and inhibited by targeting IQGAP1. Mol Cancer Ther; 15(11); 2733-9. ©2016 AACR.

Molecular pathways: the hedgehog signaling pathway in cancer.

The Hedgehog (Hh) signaling pathway regulates embryonic development and may be aberrantly activated in a wide variety of human cancers. Efforts to target pathogenic Hh signaling have steadily progressed from the laboratory to the clinic, and the recent approval of the Hh pathway inhibitor vismodegib for patients with advanced basal cell carcinoma represents an important milestone. On the other hand, Hh pathway antagonists have failed to show significant clinical activity in other solid tumors. The reasons for these negative results are not precisely understood, but it is possible that the impact of Hh pathway inhibition has not been adequately measured by the clinical endpoints used thus far or that aberrancies in Hh signal transduction limits the activity of currently available pathway antagonists. Further basic and correlative studies to better understand Hh signaling in human tumors and validate putative antitumor mechanisms in the clinical setting may ultimately improve the success of Hh pathway inhibition to other tumor types.