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Summary Protein misfolding is a common theme in aging and several agerelated diseases such as Alzheimer’s and Parkinson’s disease. The processes involved in the development of these diseases are many and complex. Here, we show that components of the basement membrane (BM), particularly laminin, affect protein integrity of the muscle cells they support. We knocked down gene expression of epi-1, a laminin a-chain, and found that this resulted in increased proteotoxicity in different Caenorhabditis elegans transgenic models, expressing aggregating proteins in the body wall muscle. The effect could partially be rescued by decreased insulin-like signaling, known to slow the aging process and the onset of various age-related diseases. Our data points to an underlying molecular mechanism involving proteasomal degradation and HSP-16 chaperone activity. Furthermore, epi-1depleted animals had altered synaptic function and displayed hypersensitivity to both levamisole and aldicarb, an acetylcholine receptor agonist and an acetylcholinesterase inhibitor, respectively. Our results implicate the BM as an extracellular modulator of protein homeostasis in the adjacent muscle cells. This is in agreement with previous research showing that imbalance in neuromuscular signaling disturbs protein homeostasis in the postsynaptic cell. In our study, proteotoxicity may indeed be mediated by the neuromuscular junction which is part of the BM, where laminins are present in high concentration, ensuring the proper microenvironment for neuromuscular signaling. Laminins are evolutionarily conserved, and thus the BM may play a much more causal role in protein misfolding diseases than currently recognized.

With the advent of modern technologies enabling single cell analysis, it has become clear that small sub-populations of cells or even single cells can drive the phenotypic appearance of tissue, both diseased and normal. Nucleic acid based technologies allowing single cell analysis has been faster to mature, while technologies aimed at analysing the proteome at a single cell level is still lacking behind, especially technologies which allow single cell analysis in tissue. Introducing methods, that allows such analysis, will pave the way for discovering new biomarkers with more clinical relevance, as these may be unique for microenvironments only present in tissue and will avoid artifacts introduced by in vitro studies. Here, we introduce a technology enabling biomarker identification on small sub-populations of cells within a tissue section. Phage antibody libraries are applied to the tissue sections, followed by washing to remove non-bound phage particles. To eliminate phage antibodies binding to antigens ubiquitously expressed and retrieve phage antibodies binding specifically to antigens expressed by the sub-population of cells, the area of interest is protected by a ‘shadow stick’. The phage antibodies on the remaining areas on the slide are exposed to UV light, which introduces cross-links in the phage genome, thus rendering them non-replicable. In this work we applied the technology, guided by CD31 expressing endothelial cells, to isolate recombinant antibodies specifically binding biomarkers expressed either by the cell or in the microenvironment surrounding the endothelial cell.

Breast cancer tumors are composed of heterogeneous cell populations. These populations display a high variance in morphology, growth and metastatic propensity. They respond differently to therapeutic interventions, and some may be more prone to cause recurrence. Studying individual subpopulations of breast cancer may provide crucial knowledge for the development of individualized therapy. However, this process is challenged by the availability of biomarkers able to identify subpopulations specifically. Here, we demonstrate an approach for phage display selection of recombinant antibody fragments on cryostat sections of human breast cancer tissue. This method allows for selection of recombinant antibodies binding to antigens specifically expressed in a small part of the tissue section. In this case, a CD271+ subpopulation of breast cancer cells was targeted, and these may be potential breast cancer stem cells. We isolated an antibody fragment LH 7, which in immunohistochemistry experiments demonstrates specific binding to breast cancer subpopulations. The selection of antibody fragments directly on small defined areas within a larger section of malignant tissue is a novel approach by which it is possible to better target cellular heterogeneity in proteomic studies. The identification of novel biomarkers is relevant for our understanding and intervention in human diseases. The selection of the breast cancer-specific antibody fragment LH 7 may reveal novel subpopulation-specific biomarkers, which has the potential to provide new insight and treatment strategies for breast cancer. Electronic supplementary material The online version of this article (doi:10.1007/s12026-015-8657-x) contains supplementary material, which is available to authorized users.

Background Various by-products of the cellular metabolism, such as reactive carbonyl species (RCS) are potentially harmful to cells and tissues, and play a role in many physiological and pathological processes. Among various RCS is the highly reactive dicarbonyl glyoxal (GO), which is a natural physiological metabolite produced by the auto-oxidation of glucose, and can form covalent adducts known as advanced glycation endproducts (AGE). We have previously reported that GO accelerates ageing and causes premature senescence in normal human skin fibroblasts. Results Using a bone marrow-derived telomerase-immortalised mesenchymal stem cell line hMSC-TERT we have observed that an exposure of cells to 0.75 mM and 1 mM GO induces irreversible cellular senescence within 3 days. Induction of senescence in hMSC-TERT was demonstrated by a variety of markers, including characteristic cell morphology and enlargement, vacuolisation, multinucleation, induction of senescence associated β-galactosidase, cell cycle arrest, and increased levels of a cell cycle inhibitor p16. These changes were accompanied by increased extent of DNA breaks as measured by the comet assay, and increased levels of the AGE product, carboxymethyl-lysine (CML). Furthermore, the in vitro differentiation potential of hMSC-TERT to become functional osteoblasts was highly reduced in GO-treated stem cells, as determined by alkaline phosphatase (ALP) activity and mineralized matrix (MM) formation. Conclusions The results of our study imply that an imbalanced glucose metabolism can reduce the functioning ability of stem cells in vivo both during ageing and during stem cell-based therapeutic interventions.

BACKGROUND/AIM: Primary tumors display a great level of intra-tumor heterogeneity in breast cancer. The current lack of prognostic and predictive biomarkers limits accurate stratification and the ability to predict response to therapy. The aim of the present study was to select recombinant antibody fragments specific against breast cancer subpopulations, aiding the discovery of novel biomarkers.MATERIALS AND METHODS: Recombinant antibody fragments were selected by phage display. A novel shadowstick technology enabled the direct selection using tissue sections of antibody fragments specific against small subpopulations of breast cancer cells. Selections were performed against a subpopulation of breast cancer cells expressing CD271(+), as these previously have been indicated to be potential breast cancer stem cells. The selected antibody fragments were screened by phage ELISA on both breast cancer and myoepithelial cells. The antibody fragments were validated and evaluated by immunohistochemistry experiments.RESULTS: Our study revealed an antibody fragment, LH8, specific for breast cancer cells. Immunohistochemistry results indicate that this particular antibody fragment binds an antigen that exhibits differential expression in different breast cancer subpopulations.CONCLUSION: Further studies characterizing this antibody fragment, the subpopulation it binds and the cognate antigen may unearth novel biomarkers of clinical relevance.