The initiator methionine tRNA drives secretion of type II collagen from stromal fibroblasts to promote tumor growth and angiogenesis

Summary: Expression of the initiator methionine tRNA (tRNAi Met) is deregulated in cancer. Despite this fact, it is not currently known how tRNAi Met expression levels influence tumor progression. We have found that tRNAi Met expression is increased in carcinoma-associated fibroblasts, implicating deregulated expression of tRNAi Met in the tumor stroma as a possible contributor to tumor progression. To investigate how elevated stromal tRNAi Met contributes to tumor progression, we generated a mouse expressing additional copies of the tRNAi Met gene (2+tRNAi Met mouse). Growth and vascularization of subcutaneous tumor allografts was enhanced in 2+tRNAi Met mice compared with wild-type littermate controls. Extracellular matrix (ECM) deposited by fibroblasts from 2+tRNAi Met mice supported enhanced endothelial cell and fibroblast migration. SILAC mass spectrometry indicated that elevated expression of tRNAi Met significantly increased synthesis and secretion of certain types of collagen, in particular type II collagen. Suppression of type II collagen opposed the ability of tRNAi Metoverexpressing fibroblasts to deposit pro-migratory ECM. We used the prolyl hydroxylase inhibitor ethyl- 3,4-dihydroxybenzoate (DHB) to determine whether collagen synthesis contributes to the tRNAi Met-driven pro-tumorigenic stroma in vivo. DHB had no effect on the growth of syngeneic allografts in wild-type mice but opposed the ability of 2+tRNAi Met mice to support increased angiogenesis and tumor growth. Finally, collagen II expression predicts poor prognosis in high-grade serous ovarian carcinoma. Taken together, these data indicate that increased tRNAi Met levels contribute to tumor progression by enhancing the ability of stromal fibroblasts to synthesize and secrete a type II collagen-rich ECM that supports endothelial cell migration and angiogenesis

Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion

Developing cell identification methods using atomic force microscopy

This body of work describes the development of a non-invasive and label-free method for characterization of cell surface markers. The motivation for such a method is the ability to measure cells whilst maintaining function, minimizing contamination and disturbance but enabling downstream applications. The technique would impact on life sciences applications including; phenotype identification of both individual and populations of cells, dynamic measurement of cellular response and monitoring cell-microenvironment interactions. The method described centers on molecular recognition interactions which are associated with specific binding forces. These specific forces can be measured in a highly sensitive manner using force instruments. In this study atomic force microscopy (AFM) was employed because of its powerful capability of highly sensitive force measurement at a nanoscale spatial resolution. The objective to develop a force based method for characterization of cell surface molecules may be considered in more specific aims; the development of a functional AFM probe for identification of specific molecules and establishment of quantitative measurement of surface markers. The probe developed has a colloidal geometry which encourages multivalent binding due to greater contact areas, which can reveal presence on cells in just few measurements. On non-deformable surfaces few interactions occur and regular force increments and probability of unbinding indicate presence of target molecules. With multivalent interactions on deformable samples other variables of adhesion indicate identification of interactions; namely distance of total separation, total peaks of unbinding and energy for total separation. With these variables, the identity of HeLa and HFF1 cells was indicated by cluster of differentiation markers 24, 44 and 98 in a semi-quantitative manner. Additionally individual mesenchymal stems cells are identified by the presence of cluster of differentiation marker 90 and dynamic measurement of Human Leukocyte Antigen. Single-cell force spectroscopy was employed to investigate cellular binding to cancerous matrices to gain greater understanding of tumour angiogenesis. Total internal reflection fluorescence microscopy was employed to inform the experimental setting of contact area and sampling density. The method developed illustrates the potential of force based measurement for label-free, non-invasive measurements on cells. Further development and automation may allow the dynamic measurement of multiple markers. This would allow for a number of applications; the identification of true stem cell clones which is of great importance for stem cells therapies, for monitoring of differentiation, where both short and long term activations could be investigated

Bericht der Mobil Oil AG ueber das Geschaeftsjahr vom 1.1.1989 bis 31.12.1989

Available from Mobil Oil A.G., Hamburg (DE) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Creating “Living” Polymer Surfaces to Pattern Biomolecules and Cells on Common Plastics

Creating patterns of biomolecules and cells has been applied widely in many fields associated with the life sciences, including diagnostics. In these applications it has become increasingly apparent that the spatiotemporal arrangement of biological molecules in vitro is important for the investigation of the cellular functions found in vivo. However, the cell patterning techniques often used are limited to creating 2D functional surfaces on glass and silicon. In addition, in general, these procedures are not easy to implement in conventional biological laboratories. Here, we show the formation of a living poly­(ethylene glycol) (PEG) layer that can be patterned with visible light on plastic surfaces. This new and simple method can be expanded to pattern <i>multiple</i> types of biomolecule on either a previously formed PEG layer or a plastic substrate. Using common plastic wares (i.e., polyethylene films and polystyrene cell culture Petri-dishes), we demonstrate that these PEG-modified surfaces have a high resistance to protein adsorption and cell adhesion, while at the same time, being capable of undergoing further molecular grafting with bioactive motifs. With a photomask and a fluid delivery system, we illustrate a flexible way to immobilize biological functions with a high degree of 2D and 3D spatial control. We anticipate that our method can be easily implemented in a typical life science laboratory (without the need for specialized lithography equipment) offering the prospect of imparting desirable properties to plastic products, for example, the creation of functional microenvironments in biological studies or reducing biological adhesion to surfaces