Jerry Zhou

Cell Surface Markers in Colorectal Cancer Prognosis

The classification of colorectal cancers (CRC) is currently based largely on histologically determined tumour characteristics, such as differentiation status and tumour stage, i.e., depth of tumour invasion, involvement of regional lymph nodes and the occurrence of metastatic spread to other organs. These are the conventional prognostic factors for patient survival and often determine the requirement for adjuvant therapy after surgical resection of the primary tumour. However, patients with the same CRC stage can have very different disease-related outcomes. For some, surgical removal of early-stage tumours leads to full recovery, while for others, disease recurrence and metastasis may occur regardless of adjuvant therapy. It is therefore important to understand the molecular processes that lead to disease progression and metastasis and to find more reliable prognostic markers and novel targets for therapy. This review focuses on cell surface proteins that correlate with tumour progression, metastasis and patient outcome, and discusses some of the challenges in finding prognostic protein markers in CRC.

Surface antigen profiling of colorectal cancer using antibody microarrays with fluorescence multiplexing

A procedure is described for the disaggregation of colorectal cancers (CRC) and normal intestinal mucosal tissues to produce suspensions of viable single cells, which are then captured on customized antibody microarrays recognising 122 different surface antigens (DotScan CRC microarray). Cell binding patterns recorded by optical scanning of microarrays provide a surface profile of antigens on the cells. Sub-populations of cells bound on the microarray can be profiled by fluorescence multiplexing using monoclonal antibodies tagged with Quantum Dots or other fluorescent dyes. Surface profiles are presented for 6 CRC cell lines (T84, LIM1215, SW480, HT29, CaCo and SW620) and surgical samples from 40 CRC patients. Statistical analysis revealed significant differences between profiles for CRC samples and mucosal controls. Hierarchical clustering of CRC data identified several disease clusters that showed some correlation with clinico-pathological stage as determined by conventional histopathological analysis. Fluorescence multiplexing using Phycoerythrin- or Alexa Fluor 647-conjugated antibodies was more effective than multiplexing with antibodies labelled with Quantum Dots. This relatively simple method yields a large amount of information for each patient sample and, with further application, should provide disease signatures and enable the identification of patients with good or poor prognosis.

Comprehensive glycomics comparison between colon cancer cell cultures and tumours: Implications for biomarker studies

UNLABELLED Altered glycosylation is commonly observed in colorectal cancer. In vitro models are frequently used to study this cancer but little is known about the differences that may exist between these model cell systems and tumour tissue. We have compared the membrane protein glycosylation of five colorectal cancer cell lines (SW1116, SW480, SW620, SW837, LS174T) with epithelial cells from colorectal tumours using liquid chromatography tandem mass spectrometry. Remarkably, there were five abundant O-glycans in the tumour cells that were undetected in the low-mucin producing cell lines, although two were found in the mucinous LS174T cells. The O-glycans included the well-known glycan cancer marker, sialyl-Tn, which has been associated with mucins. Using qRT-PCR, sialyl-Tn expression was found to be associated with an increase in α2,6-sialyltransferase gene (ST6GALNAC1) and a decrease in core 1 synthase gene (C1GALT1) in LS174T cells. The expression of a subset of mucins (MUC2, MUC6, MUC5B) was also correlated with sialyl-Tn expression in LS174T cells. Overall, the membrane protein glycosylation of the model cell lines was found to differ from each other and from the epithelial cells of tumour tissue. These findings should be noted in the design of biomarker discovery experiments particularly when cell surface targets are being investigated. BIOLOGICAL SIGNIFICANCE The extent of protein glycosylation differences between in vitro cell lines and ex vivo tumours in colorectal cancer research is unknown. Our study expands current knowledge by characterising the membrane protein glycosylation profiles of five different colorectal cancer cell lines and of epithelial cells derived from resected colorectal cancer tumour tissue, using liquid chromatography tandem mass spectrometry. The detailed structural differences found in both N- and O-linked glycan structures on the membrane glycoproteins were determined and correlated with the mRNA expression of the relevant proteins in the cell lines. The glycosylation differences found between cultured cancer cell lines and epithelial cells from tumour tissue have important implications for glycan biomarker discovery.

Surface profiles of live colorectal cancer cells and tumor infiltrating lymphocytes from surgical samples correspond to prognostic categories

Extensive surface profiles of colorectal cancer (CRC) cells and tumor infiltrating lymphocytes (TIL) have been obtained from 45 surgical resection samples. Live cells were captured on an antibody microarray and stained with fluorescently-labeled antibodies. Minimal panels of 11 CRC antigens (CD13, CD24, CD26, CD49d, CD138, CD166, CA-125, CA19-9, EGFR, Galectin-4 and HLA-DR) and 11 T-cell antigens (CD10, CD11b, CD11c, CD25, CD31, CD95, CD151, CD181, Galectin-4, CA19-9, TSP-1) provide signatures for relapse and survival. Hierarchical clustering of profiles from CRC cells and TIL identified groups of patients for survival, systemic relapse and death. The groups from CRC and TIL profiles for systemic relapse showed 79.2% concordance, enabling prediction of relapse after surgery. The results demonstrate communication between CRC cells and TIL.

The Potential for Gut Organoid Derived Interstitial Cells of Cajal in Replacement Therapy

Effective digestion requires propagation of food along the entire length of the gastrointestinal tract. This process involves coordinated waves of peristalsis produced by enteric neural cell types, including different categories of interstitial cells of Cajal (ICC). Impaired food transport along the gastrointestinal tract, either too fast or too slow, causes a range of gut motility disorders that affect millions of people worldwide. Notably, loss of ICC has been shown to affect gut motility. Patients that suffer from gut motility disorders regularly experience diarrhoea and/or constipation, insomnia, anxiety, attention lapses, irritability, dizziness, and headaches that greatly affect both physical and mental health. Limited treatment options are available for these patients, due to the scarcity of human gut tissue for research and transplantation. Recent advances in stem cell technology suggest that large amounts of rudimentary, yet functional, human gut tissue can be generated in vitro for research applications. Intriguingly, these stem cell-derived gut organoids appear to contain functional ICC, although their frequency and functional properties are yet to be fully characterised. By reviewing methods of gut organoid generation, together with what is known of the molecular and functional characteristics of ICC, this article highlights short- and long-term goals that need to be overcome in order to develop ICC-based therapies for gut motility disorders.

Antibody Microarrays and Multiplexing

This chapter presents a range of statistical methods for antibody microarray normalization and data analysis. Commonly used techniques for cluster generation, differential analysis, and classification are covered. The focus is on the implementation of each technique to the technology and its suitability in relation to sample types and experiment design.

Association between Drug Usage and Constipation in the Elderly Population of Greater Western Sydney Australia

The low socioeconomic region of Greater Western Sydney (GWS) has higher than average rates of gastrointestinal symptoms. The relationship between prescription drug usage and constipation has not been explored. The aim of this study was to investigate the impact of drug use on constipation in the elderly population of GWS (NSW, Australia). A random selection of elderly residents completed a postal questionnaire for constipation and drug use (response 30.7%). Bivariate associations between constipation and number of drug use and number of drug use with constipation adverse effect were compared. For multivariate analysis multiple logistic regression was performed for constipation with the number of drugs, use of drugs with known constipation side effects, and each drug class (Anatomical Therapeutic Chemical Classification System (ATC) level 4) as independent variables. The prevalence of constipation was 33.9%. There was a dose–response relationship between constipation and the number of drugs used (odds ratio 1.24, p < 0.001) and the usage of drugs with known constipation adverse effects (odds ratio 2.21, p = 0.009). These findings suggest that constipation is associated with the number of drugs used, particularly those with constipation adverse-effects, in the elderly of GWS.

Surface Profiling of Extracellular Vesicles from Plasma or Ascites Fluid Using DotScan Antibody Microarrays

DotScan antibody microarrays were initially developed for the extensive surface profiling of live leukemia and lymphoma cells. DotScans diagnostic capability was validated with an extensive clinical trial using mononuclear cells from the blood or bone marrow of leukemia or lymphoma patients. DotScan has also been used for the profiling of surface proteins on peripheral blood mononuclear cells (PBMC) from patients with HIV, liver disease, and stable and progressive B-cell chronic lymphocytic leukemia (CLL). Fluorescence multiplexing allowed the simultaneous profiling of cancer cells and leukocytes from disaggregated colorectal and melanoma tumor biopsies after capture on DotScan. In this chapter, we have used DotScan for the surface profiling of extracellular vesicles (EV) recovered from conditioned growth medium of cancer cell lines and the blood of patients with CLL. The detection of captured EV was performed by enhanced chemiluminescence (ECL) using biotinylated antibodies that recognized antigens expressed on the surface of the EV subset of interest. DotScan was also used to profile EV from the blood of healthy individuals and the ascites fluid of ovarian cancer patients. DotScan binding patterns of EV from human plasma and other body fluids may yield diagnostic or prognostic signatures for monitoring the incidence, treatment, and progression of cancers.

Is there a role for human pluripotent stem cells in modelling interstitial cells of Cajal and gut motility disorders

Gastrointestinal motility disorders affect millions of people worldwide, resulting in significant morbidity and mortality. Current treatments for these disorders are inadequate and often provide little to no relief for patients. As a result, gastrointestinal motility disorders produce substantial long-term social and economic burdens in both developed and developing countries. These limited treatment options arise largely from our relatively poor understanding of the molecular etiology for the majority of gastrointestinal motility disorders. In turn, this is due to our limited access to normal or diseased human gut tissue for use in research. In particular while the interstitial cells of Cajal (ICC) are known to be important for gastrointestinal motility, little is known of how these cells function or how they are involved in disease initiation and progression. The advent of human pluripotent stem cell technology offers an opportunity to generate large amounts of human tissue for both research and clinical applications. The application of this technology to gastrointestinal motility disorders is currently only in its infancy and as yet no studies have described ICC production from human pluripotent cells. By considering the present understanding of the anatomical, cellular and molecular basis of gut motility with particular emphasis on ICC, this review provides a clear framework for the application of human pluripotent stem cell technology to answer fundamental questions of ICC involvement in gut motility.

Semi-Solid and Solid Bolus Swallows in High-Resolution Oesophageal Manometry for the Detection of Motility Disorders

Background/Aims: High-resolution oesophageal manometry utilises water swallows to evaluate oesophageal function. However, small volumes of water are not representative of normal eating and as a result often produce normal manometry studies in patients with dysphagia. This study sets out to establish optimal diagnostic thresholds for semi-solid solid swallows and evaluate their ability to uncover motility abnormalities in patients with motility disorders. Method: Manometry was performed using ten 5-mL single water swallows followed by two semi-solid and two solid swallows in the upright position. Normative values for the adjunctive tests were obtained from patient controls while patients with major motility disorders were used to establish the optimal diagnostic thresholds. Diagnostic thresholds identified were prospectively tested in patients with normal water swallows but oesophagus related symptoms and in those with minor and major motility disorders. Results: Normal values for semi-solid and solid were determined in patient controls (n = 100). Development of diagnostic thresholds included 120 patients with major motility disorders. Optimal diagnostic thresholds identified for oesophagogastric junction dysfunction in semi-solid and solid swallows (IRP > 15.5 mmHg). Hypercontractilty and spasm used existing thresholds (>8000 mmHg-s-cm and < 4.5 s, respectively) but modified frequency of ≥50% of adjunctive swallows. Diagnostic thresholds were applied to symptomatic patients with normal water swallows (n = 70) identifying 12/70 (17%) to have abnormal adjunctive swallows. One of 30 patients (3%) with ineffective motility had abnormal adjunctive swallow and 12 patients with oesophageal spasm, oesophagogastric junction obstruction, and hypercontractility had abnormal adjunctive swallows that moved them up the motility disorder hierarchy. Conclusions: Semi-solid and solid challenge increase diagnostic yield of motility disorders.