Exploring novel roles of tumor pericytes

Tumor biology has been extensively studied over the last few decades, with a principal focus on how neoplastic cells obtain cellular immortality. A number of oncogenes and tumor suppressor genes have been uncovered that regulate cancerous transformation. However, tumors are now believed to be complex tissues consisting of various kinds of tumor stromal cells as well as transformed cancer cells. The tumor stroma is mainly comprised of tumorinfiltrating leukocytes, cancer-associated fibroblasts, vascular endothelial cells, lymphatic endothelial cells, tumor pericytes, and extracellular matrix. These cells have an extensive interplay with one another or with cancer cells, from the initiation stage of tumor development to its metastatic dissemination. The aim of this thesis was to investigate the tumor pericytes—one of the tumor stroma constituents that has not been widely explored—in order to determine their novel roles in tumor malignancy. The use of a pericyte-deficient mouse model (pdgfbret/ret), in paper I, confirmed that the myeloid-derived suppressor cells (MDSCs), one of the most aggressive types oftumor-infiltrating leukocytes, are significantly increased both at the tumor site and in the peripheral blood in B16 melanoma and Lewis lung carcinoma (LLC) subcutaneous mouse models ofpdgfbret/ret, compared to their littermate controls. The increase in the MDSC number was dependent on expression oftumor-derived IL-6, induced by the hypoxic tumor microenvironment in pericyte-deficient B16 and LLC tumors. Analysis of gene expression in human samples (253 breast cancer patients of an Uppsala dataset) showed an inverse correlation between human pericyte-related genes and human MDSC markers and a subsequent relevance to the survival rate ofbreast cancer patients. The relevance of the tumor pericytes to other tumor stroma cells was studied in paper II, which revealed a comparable abundance of PDGFRα- expressing perivascular cells in pericyte-deficient B16 melanomas. A further investigation identified the PDGFRα-expressing perivascular cells as “specialized myofibroblasts” with gene signature features of both fibroblast-related (Fap, Pdgfra, Hgf) and pericyte-related (Cspg4, Pdgfrb, Asma) gene sets. Moreover, pericyte-deficient, B16-melanoma-bearing mice showed an elevated level of the serum S100B protein, which is widely considered to be a distinctive prognostic marker for malignant melanoma patients. The B16 melanoma tumor cell-derived S100B was then confirmed to pass into the peripheral blood. Presumably “activated endothelial cells” in pdgfbret/ret mice would facilitate more rapid transport of S100B protein via endothelial caveola-mediated transcytosis. In conclusion, tumor pericytes directly interact with adjacent endothelial cells, thereby controlling the tumor vasculature and further changing the tumor microenvironment. Tumor pericytes favor tumor immunogenicity by blocking systemic MDSC bursts in experimental mouse models (B16, LLC) and are also negatively involved in recruitment of the perivascular myofibroblasts. However, the biological relevance ofthe perivascular myofibroblasts should be further investigated

Molecular biology on a microfluidic chip

We have developed microfluidic chips for automating molecular biology processes such as gene ligation and gene transformation from nanolitre sample volumes with parallel architecture. Unlike conventional tube methods with cumbersome pipetting procedures, all processes, including metering of samples, ligation and transformation, were carried out in the microfluidic chips through pneumatic control of the nanofluid. The microfluidic devices presented here offer an illustration of some of the basic physics that arises when trying to miniaturize and automate biological techniques

Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria

Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA–based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment

Evaluation of peristaltic micromixers for highly integrated microfluidic systems

Microfluidic devices based on the multilayer soft lithography allow accurate manipulation of liquids, handling reagents at the sub-nanoliter level, and performing multiple reactions in parallel processors by adapting micromixers. Here, we have experimentally evaluated and compared several designs of micromixers and operating conditions to find design guidelines for the micromixers. We tested circular, triangular, and rectangular mixing loops and measured mixing performance according to the position and the width of the valves that drive nanoliters of fluids in the micrometer scale mixing loop. We found that the rectangular mixer is best for the applications of highly integrated microfluidic platforms in terms of the mixing performance and the space utilization. This study provides an improved understanding of the flow behaviors inside micromixers and design guidelines for micromixers that are critical to build higher order fluidic systems for the complicated parallel bio/chemical processes on a chip

Eco-friendly Technologies for Physical and Chemical Recycling of PVC-Related Wasteful Resources

The aim of this study is to enhance the recycled PVC related material property by formulation technology and develop the recycling product processing technology furthermore develop the chemical recycling technology for last stage of PVC wastes. The formulation technology is composed of pre-treatment (crushing, separation etc.) and post-treatment (material ratio, additives, stabilizer etc.) to enhance the recyclate property. The formulation for recycled PVC by application basis and processing technology is applied to produce the structural product for civil and construction application such as pipe fittings and water drainage cap for environmental waterway. Also chemical recycling technology for end life PVC scrap which causes environmental pollution by incineration or landfill is studied for producing hydrocarbon and hydrogen chloride for VCM

Is poststroke complex regional pain syndrome the combination of shoulder pain and soft tissue injury of the wrist?: A prospective observational study STROBE of ultrasonographic findings in complex regional pain syndrome

Patients with poststroke complex regional pain syndrome (CRPS) show different symptoms compared to other types of CRPS, as they usually complain of shoulder and wrist pain with the elbow relatively spared. It is thus also known by the term “shoulder-hand syndrome.” The aim of this study is to present a possible pathophysiology of poststroke CRPS through ultrasonographic observation of the affected wrist before and after steroid injection at the extensor digitorum communis (EDC) tendon in patients suspected with poststroke CRPS. Prospective evaluation and observation, the STROBE guideline checklist was used. Twenty-three patients diagnosed as poststroke CRPS in accordance to clinical criteria were enrolled. They had a Three Phase Bone Scan (TPBS) done and the cross-sectional area (CSA) of EDC tendon was measured by using ultrasonography. They were then injected with steroid at the EDC tendon. The CSA of EDC tendon, visual analogue scale (VAS), and degree of swelling of the wrist were followed up 1 week after the injection. TPBS was interpreted as normal for 4 patients, suspected CRPS for 10 patients, and CRPS for 9 patients. Ultrasonographic findings of the affected wrist included swelling of the EDC tendon. After the injection of steroid to the wrist, CSA and swelling of the affected wrist compared to that before the treatment was significantly decreased (P < 0.001). The VAS score declined significantly after the injection (P < 0.001). Our results suggest that the pathophysiology of poststroke CRPS might be the combination of frozen shoulder or rotator cuff tear of shoulder and soft tissue injury of the wrist caused by the hemiplegic nature of patients with stroke

Subtle cytotoxicity and genotoxicity differences in superparamagnetic iron oxide nanoparticles coated with various functional groups

Superparamagnetic iron oxide nanoparticles (SPIONs) have been widely utilized for the diagnosis and therapy of specific diseases, as magnetic resonance imaging (MRI) contrast agents and drug-delivery carriers, due to their easy transportation to targeted areas by an external magnetic field. For such biomedical applications, SPIONs must have multifunctional characteristics, including optimized size and modified surface. However, the biofunctionality and biocompatibility of SPIONs with various surface functional groups of different sizes have yet to be elucidated clearly. Therefore, it is important to carefully monitor the cytotoxicity and genotoxicity of SPIONs that are surfaced-modified with various functional groups of different sizes. In this study, we evaluated SPIONs with diameters of approximately 10 nm and 100~150 nm, containing different surface functional groups. SPIONs were covered with −O− groups, so-called bare SPIONs. Following this, they were modified with three different functional groups – hydroxyl (−OH), carboxylic (−COOH), and amine (−NH2) groups – by coating their surfaces with tetraethyl orthosilicate (TEOS), (3-aminopropyl)trimethoxysilane (APTMS), TEOS-APTMS, or citrate, which imparted different surface charges and sizes to the particles. The effects of SPIONs coated with these functional groups on mitochondrial activity, intracellular accumulation of reactive oxygen species, membrane integrity, and DNA stability in L-929 fibroblasts were determined by water-soluble tetrazolium, 2′,7′-dichlorodihydrofluorescein, lactate dehydrogenase, and comet assays, respectively. Our toxicological observations suggest that the functional groups and sizes of SPIONs are critical determinants of cellular responses, degrees of cytotoxicity and genotoxicity, and potential mechanisms of toxicity. Nanoparticles with various surface modifications and of different sizes induced slight, but possibly meaningful, changes in cell cytotoxicity and genotoxicity, which would be significantly valuable in further studies of bioconjugation and cell interaction for drug delivery, cell culture, and cancer-targeting applications