GlycoFibroTyper: A Novel Method for the Glycan Analysis of IgG and the Development of a Biomarker Signature of Liver Fibrosis.

Our group has recently developed the GlycoTyper assay which is a streamlined antibody capture slide array approach to directly profile N-glycans of captured serum glycoproteins including immunoglobulin G (IgG). This method needs only a few microliters of serum and utilizes a simplified processing protocol that requires no purification or sugar modifications prior to analysis. In this method, antibody captured glycoproteins are treated with peptide N-glycosidase F (PNGase F) to release N-glycans for detection by MALDI imaging mass spectrometry (IMS). As alterations in N-linked glycans have been reported for IgG from large patient cohorts with fibrosis and cirrhosis, we utilized this novel method to examine the glycosylation of total IgG, as well as IgG1, IgG2, IgG3 and IgG4, which have never been examined before, in a cohort of 106 patients with biopsy confirmed liver fibrosis. Patients were classified as either having no evidence of fibrosis (41 patients with no liver disease or stage 0 fibrosis), early stage fibrosis (10 METAVIR stage 1 and 18 METAVIR stage 2) or late stage fibrosis (6 patients with METAVIR stage 3 fibrosis and 37 patients with METAVIR stage 4 fibrosis (cirrhosis)). Several major alterations in glycosylation were observed that classify patients as having no fibrosis (sensitivity of 92% and a specificity of 90%), early fibrosis (sensitivity of 84% with 90% specificity) or significant fibrosis (sensitivity of 94% with 90% specificity).

Defining the Tumor Microenvironment by Integration of Immunohistochemistry and Extracellular Matrix Targeted Imaging Mass Spectrometry.

Breast stroma plays a significant role in breast cancer risk and progression yet remains poorly understood. In breast stroma, collagen is the most abundantly expressed protein and its increased deposition and alignment contributes to progression and poor prognosis. Collagen post-translation modifications such as hydroxylated-proline (HYP) control deposition and stromal organization. The clinical relevance of collagen HYP site modifications in cancer processes remains undefined due to technical issues accessing collagen from formalin-fixed, paraffin-embedded (FFPE) tissues. We previously developed a targeted approach for investigating collagen and other extracellular matrix proteins from FFPE tissue. Here, we hypothesized that immunohistochemistry staining for fibroblastic markers would not interfere with targeted detection of collagen stroma peptides and could reveal peptide regulation influenced by specific cell types. Our initial work demonstrated that stromal peptide peak intensities when using MALD-IMS following IHC staining (αSMA, FAP, P4HA3 and PTEN) were comparable to serial sections of nonstained tissue. Analysis of histology-directed IMS using PTEN on breast tissues and TMAs revealed heterogeneous PTEN staining patterns and suggestive roles in stromal protein regulation. This study sets the foundation for investigations of target cell types and their unique contribution to collagen regulation within extracellular matrix niches.

Mass Spectrometry Imaging of Fibroblasts: Promise and Challenge.

INTRODUCTION: Fibroblasts maintain tissue and organ homeostasis through output of extracellular matrix that affects nearby cell signaling within the stroma. Altered fibroblast signaling contributes to many disease states and extracellular matrix secreted by fibroblasts has been used to stratify patient by outcome, recurrence, and therapeutic resistance. Recent advances in imaging mass spectrometry allow access to single cell fibroblasts and their ECM niche within clinically relevant tissue samples. AREAS COVERED: We review biological and technical challenges as well as new solutions to proteomic access of fibroblast expression within the complex tissue microenvironment. Review topics cover conventional proteomic methods for single fibroblast analysis and current approaches to accessing single fibroblast proteomes by imaging mass spectrometry approaches. Strategies to target and evaluate the single cell stroma proteome on the basis of cell signaling are presented. EXPERT OPINION: The promise of defining proteomic signatures from fibroblasts and their extracellular matrix niches is the discovery of new disease markers and the ability to refine therapeutic treatments. Several imaging mass spectrometry approaches exist to define the fibroblast in the setting of pathological changes from clinically acquired samples. Continued technology advances are needed to access and understand the stromal proteome and apply testing to the clinic.

CD8+ T-cells negatively regulate inflammation post-myocardial infarction.

The adaptive immune response is key for cardiac wound healing post-myocardial infarction (MI) despite low T-cell numbers. We hypothesized that CD8+ T-cells regulate the inflammatory response, leading to decreased survival and cardiac function post-MI. We performed permanent occlusion of the left anterior descending coronary artery on C57BL/6J and CD8atm1mak mice (deficient in functional CD8+ T-cells). CD8atm1mak mice had increased survival at 7 days post-MI compared with that of the wild-type (WT) and improved cardiac physiology at day 7 post-MI. Despite having less mortality, 100% of the CD8atm1mak group died because of cardiac rupture compared with only 33% of the WT. Picrosirius red staining and collagen immunoblotting indicated an acceleration of fibrosis in the infarct area as well as remote area in the CD8atm1mak mice; however, this increase was due to elevated soluble collagen implicating poor scar formation. Plasma and tissue inflammation were exacerbated as indicated by higher levels of Cxcl1, Ccl11, matrix metalloproteinase (MMP)-2, and MMP-9. Immunohistochemistry and flow cytometry indicated that the CD8atm1mak group had augmented numbers of neutrophils and macrophages at post-MI day 3 and increased mast cell markers at post-MI day 7. Cleavage of tyrosine-protein kinase MER was increased in the CD8atm1mak mice, resulting in delayed removal of necrotic tissue. In conclusion, despite having improved cardiac physiology and overall survival, CD8atm1mak mice had increased innate inflammation and poor scar formation, leading to higher incidence of cardiac rupture. Our data suggest that the role of CD8+ T-cells in post-MI recovery may be both beneficial and detrimental to cardiac remodeling and is mediated via a cell-specific mechanism.NEW & NOTEWORTHY We identified new mechanisms implicating CD8+ T-cells as regulators of the post-myocardial infarction (MI) wound healing process. Mice without functional CD8+ T-cells had improved cardiac physiology and less mortality 7 days post MI compared with wild-type animals. Despite having better overall survival, animals lacking functional CD8+ T-cells had delayed removal of necrotic tissue, leading to poor scar formation and increased cardiac rupture, suggesting that CD8+ T-cells play a dual role in the cardiac remodeling process.