The Role of Immunoglobulin Superfamily Cell Adhesion Molecules in Cancer Metastasis

Metastasis is a major clinical problem and results in a poor prognosis for most cancers. The metastatic pathway describes the process by which cancer cells give rise to a metastatic lesion in a new tissue or organ. It consists of interconnecting steps all of which must be successfully completed to result in a metastasis. Cell-cell adhesion is a key aspect of many of these steps. Adhesion molecules belonging to the immunoglobulin superfamily (Ig-SF) commonly play a central role in cell-cell adhesion, and a number of these molecules have been associated with cancer progression and a metastatic phenotype. Surprisingly, the contribution of Ig-SF members to metastasis has not received the attention afforded other cell adhesion molecules (CAMs) such as the integrins. Here we examine the steps in the metastatic pathway focusing on how the Ig-SF members, melanoma cell adhesion molecule (MCAM), L1CAM, neural CAM (NCAM), leukocyte CAM (ALCAM), intercellular CAM-1 (ICAM-1) and platelet endothelial CAM-1 (PECAM-1) could play a role. Although much remains to be understood, this review aims to raise the profile of Ig-SF members in metastasis formation and prompt further research that could lead to useful clinical outcomes

A structural analysis of heparin-like glycosaminoglycans using MALDI-TOF mass spectrometry

Abstract. Mass spectrometry (MS) techniques have spear-headed the field of proteomics. Recently, MS has been used to structurally analyse carbohydrates. The heparin/heparan sulfate-like glycosaminoglycans (HLGAGs) present a special set of difficulties for structural analysis because they are highly sulfated and heterogeneous. We have used a matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-MS) technique in which heparin fragments are non-covalently bound to basic peptides of a known mass, so as to limit in-source desulfation and hence afford an accurate mass. We examined a range of different sized fragments with varying degrees of sulfation. The potential of combining the MALDI-MS technique with enzymatic digestion to obtain saccharide sequence information on heparin fragments was explored. A disaccharide analysis greatly assists in determining a sequence from MALDI-MS data. Enzymatic digestion followed by MALDI-MS allows structural data on heparin fragments too large for direct MALDI-MS to be obtained. We demonstrate that synthetic sulfated oligosaccharides can also be analysed by MALDI-MS. There are advantages and limitations with this methodology, but until superior MS techniques become readily accessible to biomedical scientists the MALDI-MS method provides a means to structurally analyse HLGAG fragments that have therapeutic potential because of their ability to bind to and functionally regulate a host of clinically important proteins

In vitro expansion of keratinocytes on human dermal fibroblast-derived matrix retains their stem-like characteristics

The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell's fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use

AI is a viable alternative to high throughput screening: a 318-target study

: High throughput screening (HTS) is routinely used to identify bioactive small molecules. This requires physical compounds, which limits coverage of accessible chemical space. Computational approaches combined with vast on-demand chemical libraries can access far greater chemical space, provided that the predictive accuracy is sufficient to identify useful molecules. Through the largest and most diverse virtual HTS campaign reported to date, comprising 318 individual projects, we demonstrate that our AtomNet® convolutional neural network successfully finds novel hits across every major therapeutic area and protein class. We address historical limitations of computational screening by demonstrating success for target proteins without known binders, high-quality X-ray crystal structures, or manual cherry-picking of compounds. We show that the molecules selected by the AtomNet® model are novel drug-like scaffolds rather than minor modifications to known bioactive compounds. Our empirical results suggest that computational methods can substantially replace HTS as the first step of small-molecule drug discovery

Fabrication and Evaluation of Electrospun Silk Fibroin/Halloysite Nanotube Biomaterials for Soft Tissue Regeneration

The production of nanofibrous materials for soft tissue repair that resemble extracellular matrices (ECMs) is challenging. Electrospinning uniquely produces scaffolds resembling the ultrastructure of natural ECMs. Herein, electrospinning was used to fabricate Bombyx mori silk fibroin (SF) and SF/halloysite nanotube (HNT) composite scaffolds. Different HNT loadings were examined, but 1 wt% HNTs enhanced scaffold hydrophilicity and water uptake capacity without loss of mechanical strength. The inclusion of 1 wt% HNTs in SF scaffolds also increased the scaffold’s thermal stability without altering the molecular structure of the SF, as revealed by thermogravimetric analyses and Fourier transform infrared spectroscopy (FTIR), respectively. SF/HNT 1 wt% composite scaffolds better supported the viability and spreading of 3T3 fibroblasts and the differentiation of C2C12 myoblasts into aligned myotubes. These scaffolds coated with decellularised ECM from 3T3 cells or primary human dermal fibroblasts (HDFs) supported the growth of primary human keratinocytes. However, SF/HNT 1 wt% composite scaffolds with HDF-derived ECM provided the best microenvironment, as on these, keratinocytes formed intact monolayers with an undifferentiated, basal cell phenotype. Our data indicate the merits of SF/HNT 1 wt% composite scaffolds for applications in soft tissue repair and the expansion of primary human keratinocytes for skin regeneration

Structural integrity of decellularized muscle tissue.

<p>Scanning electron micrographs of 0.5 mm thick rat muscle sections (a, b) and 60 μm thick murine muscle sections (c). Images are representative of five samples. Scale bars are 10 μm (a–b) and 20 μm (c).</p

Immunofluorescent staining of mature C2C12 myotubes.

<p>C2C12 myoblasts were cultured for 4 and 6 days in differentiation medium on collagen I (a–c), fibronectin (d–f) and muscle matrix (g–i), and visualized using the mouse anti-MyHCB mAb (clone NOQ7.5.4D) and goat anti-mouse AF488. Arrows indicate striations (c, f, g and i). Nuclei are stained with DAPI (blue). Scale bars are 50 μm for a–b, d–e, g–h and g insert; and 25 μm for c, f and i. Myotube width was measured using ImageJ software (j), values are the mean and SD of 50 myotubes.</p

ECM proteins support C2C12 myoblast proliferation in serum free cultures.

<p>(A) Phase contrast images show C2C12 myoblasts cultured on collagen I (a, d), fibronectin (b, e) and solubilized muscle matrix (c, f) after 1 and 4 days in serum free culture. Scale bars are 150 μm. A section of “f” is shown at a higher magnification (Scale bar = 200 μm). Proliferation of C2C12 cells on protein substrates in serum free medium was assessed by a direct cell count (g). Data are mean ± SD of 4 independent experiments.</p

Differential preservation of collagen IV and fibronectin in sections treated with trypsin, SDS and PLA₂.

<p>Quadriceps muscle sections treated with trypsin (b, h), SDS (d, j) and PLA_<b>2</b> (f, l), and untreated controls (a, c, e, g, i, k), were fixed with 4% paraformaldehyde and stained with rabbit polyclonal antibodies against collagen IV and fibronectin followed by a goat anti-rabbit FITC conjugated secondary antibody. Nuclei are stained with DAPI (blue). Arrows indicate nuclear material remaining after decellularisation in SDS treated samples. Scale bar—100 μm.</p

SDS-PAGE and Western blotting indicates preservation of matrix proteins.

<p>(A) After PLA_<b>2</b> decellularisation ECM proteins were solubilised, resolved on a 7.5% SDS-PAGE and visualized by Coomassie Blue staining: PLA_<b>2</b> decellularised matrix (lane 3) and collagen 1 (lane 2). (B) Muscle extracts (untreated and PLA_<b>2</b> treated) were resolved on 4–15% gradient gels and membranes were probed with antibodies against collagen I, collagen VI, fibronectin, and perlecan (all rabbit polyclonals) and laminin α2 (rat, clone 4H8-2). Secondary antibodies were anti-rabbit or anti-rat conjugated to HRP, and blots were visualized using ECL. C: untreated muscle extract, PLA_<b>2</b>: decellularise muscle extract.</p