Mass Spectrometry (Imaging) for Detection and Identification of Cyclic AMPs: Focus on Human Neutrophil Peptides (HNPs)

Antimicrobial peptides (AMPs) are known best for their role in innate immunity against bacteria, viruses, parasites and fungi. However, not only are they showing increasing promise as potential antimicrobial drug candidates, recently, it has been reported that certain AMPs also show a cytotoxic effect against cancer cells. Their possible antitumor effect could make AMPs interesting candidate cancer biomarkers and a possible lead for new anticancer therapy. Due to their cyclic structure, detection and identification of AMPs is challenging, however, mass spectrometry (imaging; MSI) has been shown as a powerful tool for visualization and identification of (unknown) cyclic AMPs. In this chapter, we will discuss how mass spectrometry (imaging), combined with the use of electron-transfer dissociation (ETD) as fragmentation technique, can be used as a reliable method to identify AMPs in their native cyclic state. Using this approach, we have previously detected and identified human neutrophil peptides (HNPs) as important AMPs in cancer, of which a detailed bacterial, viral and cancer-related overview will be presented

International breeder inquiry into the reproduction of the English bulldog

Dystocia may occur in all dog breeds, but the English bulldog is predisposed because of its conformation and specific problems such as anasarca pups. In this study, the frequency of abnormal pups and breeding problems in English bulldogs was investigated by a questionnaire containing information on 39 bitches in total. In 74.4% of these bitches, artificial insemination was performed. At the end of gestation, 25.8% of the bitches suffered from respiratory problems and partial anorexia. The average duration of gestation was 58.7 days counted from the first day of mating. Caesarean section was performed in 94.8% of the cases, with natural delivery in only 5.2% of the bitches. In this study, the average litter size was six pups. Thirteen percent of the pups were stillborn, 8.2% of the pups alive were deformed, with palatoschisis (38.8%) and anasarca (27.7%) being the most commonly observed abnormalities. Ten percent of the pups died before the age of weaning

The role of disease characteristics in the ethical debate on personal genome testing

Background: Companies are currently marketing personal genome tests directly-to-consumer that provide genetic susceptibility testing for a range of multifactorial diseases simultaneously. As these tests comprise multiple risk analyses for multiple diseases, they may be difficult to evaluate. Insight into morally relevant differences between diseases will assist researchers, healthcare professionals, policy-makers and other stakeholders in the ethical evaluation of personal genome tests. Discussion. In this paper, we identify and discuss four disease characteristics - severity, actionability, age of onset, and the somatic/psychiatric nature of disease - and show how these lead to specific ethical issues. By way of illustration, we apply this framework to genetic susceptibility testing for three diseases: type 2 diabetes, age-related macular degeneration and clinical depression. For these three diseases, we point out the ethical issues that are relevant to the question whether it is morally justifiable to offer genetic susceptibility testing to adults or to children or minors, and on what conditions. Summary. We conclude that the ethical evaluation of personal genome tests is challenging, for the ethical issues differ with the diseases tested for. An understanding of the ethical significance of disease characteristics will improve the ethical, legal and societal debate on personal genome testing

Comparative Oncogenomic Analysis of Copy Number Alterations in Human and Zebrafish Tumors Enables Cancer Driver Discovery

The identification of cancer drivers is a major goal of current cancer research. Finding driver genes within large chromosomal events is especially challenging because such alterations encompass many genes. Previously, we demonstrated that zebrafish malignant peripheral nerve sheath tumors (MPNSTs) are highly aneuploid, much like human tumors. In this study, we examined 147 zebrafish MPNSTs by massively parallel sequencing and identified both large and focal copy number alterations (CNAs). Given the low degree of conserved synteny between fish and mammals, we reasoned that comparative analyses of CNAs from fish versus human MPNSTs would enable elimination of a large proportion of passenger mutations, especially on large CNAs. We established a list of orthologous genes between human and zebrafish, which includes approximately two-thirds of human protein-coding genes. For the subset of these genes found in human MPNST CNAs, only one quarter of their orthologues were co-gained or co-lost in zebrafish, dramatically narrowing the list of candidate cancer drivers for both focal and large CNAs. We conclude that zebrafish-human comparative analysis represents a powerful, and broadly applicable, tool to enrich for evolutionarily conserved cancer drivers.Kathy and Curt Marble Cancer Research FundArthur C. MerrillNational Institutes of Health (U.S.) (Grant CA106416)National Institutes of Health (U.S.) (Grant ROI RR020833)National Institutes of Health (U.S.) (Grant 1F32GM095213-01

Mass Spectrometry Imaging Reveals Neutrophil Defensins as Additional Biomarkers for Anti-PD-(L)1 Immunotherapy Response in NSCLC Patients

(1) Background: Therapeutic blocking of the interaction between programmed death-1 (PD-1) with its ligand PD-L1, an immune checkpoint, is a promising approach to restore the antitumor immune response. Improved clinical outcomes have been shown in different human cancers, including non-small cell lung cancer (NSCLC). Unfortunately, still a high number of NSCLC patients are treated with immunotherapy without obtaining any clinical benefit, due to the limitations of PD-L1 protein expression as the currently sole predictive biomarker for clinical use; (2) Methods: In this study, we applied mass spectrometry imaging (MSI) to discover new protein biomarkers, and to assess the possible correlation between candidate biomarkers and a positive immunotherapy response by matrix-assisted laser desorption/ionization (MALDI) MSI in 25 formalin-fixed paraffin-embedded (FFPE) pretreatment tumor biopsies (Biobank@UZA); (3) Results: Using MALDI MSI, we revealed that the addition of neutrophil defensin 1, 2 and 3 as pretreatment biomarkers may more accurately predict the outcome of immunotherapy treatment in NSCLC. These results were verified and confirmed with immunohistochemical analyses. In addition, we provide in-vitro evidence of the immune stimulatory effect of neutrophil defensins towards cancer cells; and (4) Conclusions: With proteomic approaches, we have discovered neutrophil defensins as additional prospective biomarkers for an anti-PD-(L)1 immunotherapy response. Thereby, we also demonstrated that the neutrophil defensins contribute in the activation of the immune response towards cancer cells, which could provide a new lead towards an anticancer therapy

MALDI Mass Spectrometry Imaging Linked with Top-Down Proteomics as a Tool to Study the Non-Small-Cell Lung Cancer Tumor Microenvironment

Advanced non-small-cell lung cancer (NSCLC) is generally linked with a poor prognosis and is one of the leading causes of cancer-related deaths worldwide. Since only a minority of the patients respond well to chemotherapy and/or targeted therapies, immunotherapy might be a valid alternative in the lung cancer treatment field, as immunotherapy attempts to strengthen the body’s own immune response to recognize and eliminate malignant tumor cells. However, positive response patterns to immunotherapy remain unclear. In this study, we demonstrate how immune-related factors could be visualized from single NSCLC tissue sections (Biobank@UZA) while retaining their spatial information by using matrix assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI), in order to unravel the molecular profile of NSCLC patients. In this way, different regions in lung cancerous tissues could be discriminated based on the molecular composition. In addition, we linked visualization (MALDI MSI) and identification (based on liquid chromatography higher resolution mass spectrometry) of the molecules of interest for the correct biological interpretation of the observed molecular differences within the area in which these molecules are detected. This is of major importance to fully understand the underlying molecular profile of the NSCLC tumor microenvironment

Implementation of MALDI Mass Spectrometry Imaging in Cancer Proteomics Research: Applications and Challenges

Studying the proteome–the entire set of proteins in cells, tissues, organs and body fluids—is of great relevance in cancer research, as differential forms of proteins are expressed in response to specific intrinsic and extrinsic signals. Discovering protein signatures/pathways responsible for cancer transformation may lead to a better understanding of tumor biology and to a more effective diagnosis, prognosis, recurrence and response to therapy. Moreover, proteins can act as a biomarker or potential drug targets. Hence, it is of major importance to implement proteomic, particularly mass spectrometric, approaches in cancer research, to provide new crucial insights into tumor biology. Recently, mass spectrometry imaging (MSI) approaches were implemented in cancer research, to provide individual molecular characteristics of each individual tumor while retaining molecular spatial distribution, essential in the context of personalized disease management and medicine

A proteomic approach to better understand the role of human neutrophil peptides in the NSCLC microenvironment

Aim: Although treatment of lung cancer, one of the deadliest diseases worldwide, with immune checkpoint inhibitors (ICIs) has shown promising outcomes, these survival outcomes are only observed in a relatively small subset of lung cancer patients. In a previous study, we elucidated that the presence of human neutrophil peptide (HNP) 1, 2 and 3 in non-small cell lung cancerous (NSCLC) biopsies is associated with a clinical response towards treatment with PD-1/PD-L1 immune checkpoint inhibitors. Furthermore, HNP1 has shown in vitro an immune-activated function towards lung cancer cells, but the specific role of HNP1 in (lung) cancer is still unknown. The aim of this study was to provide a better understanding of HNP1 in an NSCLC microenvironment.Methods: To gain better insights into the role of HNP1 on cancer growth and to unravel immune responses, in vitro (SILAC-labelled) A549/PBMC (from three healthy donors) cocultures were set up and treated/not treated with HNP1. After 5 days, both secretome and cellular analysis using mass spectrometry were performed on these cocultures. After protein identification in all different tested conditions, pathway analyses (MetaCoreTM) were performed to investigate the biological significance of HNP1 stimulation on the A549/PBMC cocultures. The proteomic outcomes were confirmed by multiplex ELISA for a proinflammatory cytokine panel (TNF-α, IL-1β, IL-2, IL-4, IL-6, IL-12p70 and IL-18). Results: A number of biological pathways and process networks were observed to be upregulated after treatment of the coculture with HNP1. HNP1 stimulation leads to an increase in pathways and proteins stimulating chemotaxis (including plasmin signaling, leucocyte recruitment, CCL2 and CXCL8 expression), proinflammatory cytokine secretion (including IL-1β, IL-6 and TNF-α), dendritic cell (DC) maturation, phagocytosis and antigen presentation, leading to a more efficient adaptive anti-tumoral immunity.Conclusion: These results enhance our understanding of the role of HNP1 in the tumor microenvironment and suggest that HNP1 may be able to induce tumor necrosis by inducing prostimulatory immune responses