AtlasGrabber: a software facilitating the high throughput analysis of the human protein atlas online database.

BACKGROUND: The human protein atlas (HPA) is an online database containing large sets of protein expression data in normal and cancerous tissues in image form from immunohistochemically (IHC) stained tissue microarrays. In these, the tissue architecture is preserved and thus provides information on the spatial distribution and localization of protein expression at the cellular and extracellular levels. The database is freely available online through the HPA website but currently without support for large-scale screening and analysis of the images in the database. Features like spatial information are typically lacking in gene expression datasets from homogenized tissues or single-cell analysis. To enable high throughput analysis of the HPA database, we developed the AtlasGrabber software. It is available freely under an open-source license. Based on a predefined gene list, the software fetches the images from the database and displays them for the user. Several filters for specific antibodies or images enable the user to customize her/his image analysis. Up to four images can be displayed simultaneously, which allows for the comparison of protein expression between different tissues and between normal and cancerous tissues. An additional feature is the XML parser that allows the extraction of a list of available antibodies, images, and genes for specific tissues or cancer types from the HPA's database file. RESULTS: Compared to existing software designed for a similar purpose, ours provide more functionality and is easier to use. To demonstrate the software's usability, we identified six new markers of basal cells of the prostate. A comparison to prostate cancer showed that five of them are absent in prostate cancer. CONCLUSIONS: The HPA is a uniquely valuable database. By facilitating its usefulness with the AtlasGrabber, we enable researchers to exploit its full capacity. The loss of basal cell markers is diagnostic for prostate cancer and can help refine the histopathological diagnosis of prostate cancer. As proof of concept, with the AtlasGrabber we identified five new potential biomarkers specific for prostate basal cells which are lost in prostate cancer and thus can be used for prostate cancer diagnostics.

Inhibition of tumor cell proliferation and motility by fibroblasts is both contact and soluble factor dependent.

Normal human and murine fibroblasts can inhibit proliferation of tumor cells when cocultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. We showed previously that effective inhibition requires formation of a morphologically intact fibroblast monolayer before seeding of the tumor cells. Here we show that inhibition is extended to motility of tumor cells and we dissect the factors responsible for these inhibitory functions. We find that inhibition is due to two different sets of molecules: (i) the extracellular matrix (ECM) and other surface proteins of the fibroblasts, which are responsible for contact-dependent inhibition of tumor cell proliferation; and (ii) soluble factors secreted by fibroblasts when confronted with tumor cells (confronted conditioned media, CCM) contribute to inhibition of tumor cell proliferation and motility. However, conditioned media (CM) obtained from fibroblasts alone (nonconfronted conditioned media, NCM) did not inhibit tumor cell proliferation and motility. In addition, quantitative PCR (Q-PCR) data show up-regulation of proinflammatory genes. Moreover, comparison of CCM and NCM with an antibody array for 507 different soluble human proteins revealed differential expression of growth differentiation factor 15, dickkopf-related protein 1, endothelial-monocyte-activating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3.

Embedded Skyrmion Bags in Thin Films of Chiral Magnets.

Magnetic skyrmions are topologically nontrivial spin configurations that possess particle-like properties. Earlier research has mainly focused on a specific type of skyrmion with topological charge Q = -1. However, theoretical analyses of 2D chiral magnets have predicted the existence of skyrmion bags-solitons with arbitrary positive or negative topological charge. Although such spin textures are metastable states, recent experimental observations have confirmed the stability of isolated skyrmion bags in a limited range of applied magnetic fields. Here, by utilizing Lorentz transmission electron microscopy, the extraordinary stability of skyrmion bags in thin plates of B20-type FeGe is shown. In particular, it is shown that skyrmion bags embedded within a skyrmion lattice remain stable even in zero or inverted external magnetic fields. A robust protocol for nucleating such embedded skyrmion bags is provided. The results agree perfectly with micromagnetic simulations and establish thin plates of cubic chiral magnets as a powerful platform for exploring a broad spectrum of topological magnetic solitons.

Novel signatures of cancer-associated fibroblasts.

Increasing evidence indicates the importance of the tumor microenvironment, in particular cancer-associated fibroblasts, in cancer development and progression. In our study, we developed a novel, visually based method to identify new immunohistochemical signatures of these fibroblasts. The method employed a protein list based on 759 protein products of genes identified by RNA profiling from our previous study, comparing fibroblasts with differential growth-modulating effect on human cancers cells, and their first neighbors in the human protein interactome. These 2,654 proteins were analyzed in the Human Protein Atlas online database by comparing their immunohistochemical expression patterns in normal versus tumor-associated fibroblasts. Twelve new proteins differentially expressed in cancer-associated fibroblasts were identified (DLG1, BHLHE40, ROCK2, RAB31, AZI2, PKM2, ARHGAP31, ARHGAP26, ITCH, EGLN1, RNF19A and PLOD2), four of them can be connected to the Rho kinase signaling pathway. They were further analyzed in several additional tumor stromata and revealed that the majority showed congruence among the different tumors. Many of them were also positive in normal myofibroblast-like cells. The new signatures can be useful in immunohistochemical analysis of different tumor stromata and may also give us an insight into the pathways activated in them in their true in vivo context. The method itself could be used for other similar analysis to identify proteins expressed in other cell types in tumors and their surrounding microenvironment.

Rho GTPases link cellular contractile force to the density and distribution of nanoscale adhesions.

The ability of cells to adhere and to exert contractile forces governs their capacity to move within an organism. The cytoskeletal regulators of the Rho GTPase proteins are involved in control of the contractile forces of cells. To elucidate the basis of cell migration, we analyzed contractile forces and nanoscale adhesion-related particles in single cells expressing constitutively active variants of Rho GTPases by using traction-force microscopy and ultra-high-resolution stimulated emission depletion microscopy, respectively. RhoAV14 induced large increases in the contractile forces of single cells, with Rac1L61 and RhoDV26 having more moderate effects. The RhoAV14- and RhoDV26-induced forces showed similar spatial distributions and were accompanied by reduced or unaltered cell spreading. In contrast, the Rac1L61-induced force had different, scattered, force distributions that were linked to increased cell spreading. All three of these Rho GTPase activities caused a loss of thick stress fibers and focal adhesions and a more homogenous distribution of nanoscale adhesion-related particles over the ventral surface of the cells. Interestingly, only RhoAV14 increased the density of these particles. Our data suggest a Rac1-specific mode for cells to generate contractile forces. Importantly, increased density and a more homogenous distribution of these small adhesion-related particles promote cellular contractile forces.

The architecture of fibroblast monolayers of different origin differentially influences tumor cell growth.

Normal human and murine fibroblasts can inhibit proliferation of tumor cells when co-cultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. It requires direct cell-to-cell contact and is not transferable with supernatant. Here, we show that effective inhibition also requires the formation of a morphologically intact fibroblast monolayer before the seeding of the tumor cells. Interference with the formation of the monolayer impairs the inhibition. Subclones of TERT-immortalized fibroblasts were selected on the basis of differences in the growth pattern and related inhibitory activity. Whereas the well-organized "whirly" (WH) growth pattern was associated with strong inhibition, the disorganized "crossy" (CR) growth pattern was linked to reduced inhibition. Time lapse imaging of tumor-fibroblast co-cultures using extended field live cell microscopy revealed that fibroblast monolayers with growth inhibitory capacity also reduced the motility of the tumor cells whereas noninhibitory monolayers had no effect on tumor cell motility. Gene expression pattern of two isogenic pairs of fibroblasts, WH and CR subclones of the TERT immortalized line (inhibitory, and less inhibitory subsequently) and freshly explanted skin (inhibitory) and hernia (noninhibitory) fibroblasts derived from the same patient, identified a set of genes that co-segregated with the inhibitory phenotype. This suggests that our model system may reveal molecular mechanisms involved in contact-mediated microenvironmental surveillance that may protect the organism from the outgrowth of disseminated tumor cells.

Decreased decorin expression in the tumor microenvironment.

Decorin is a small leucine-rich proteoglycan, synthesized and deposited by fibroblasts in the stroma where it binds to collagen I. It sequesters several growth factors and antagonizes numerous members of the receptor tyrosine kinase family. In experimental murine systems, it acted as a potent tumor suppressor. Examining the Human Protein Atlas online database of immunostained tissue samples we have surveyed decorin expression in silico in several different tumor types, comparing them with corresponding normal tissues. We found that decorin is abundantly secreted and deposited in normal connective tissue but its expression is consistently decreased in the tumor microenvironment. We developed a software to quantitate the difference in expression. The presence of two closely related proteoglycans in the newly formed tumor stroma indicated that the decreased decorin expression was not caused by the delay in proteoglycan deposition in the newly formed connective tissue surrounding the tumor.