Synthesis and active manipulation of magnetic liquid beads.

We report the fabrication and characterisation of magnetic liquid beads with a solid magnetic shell and liquid core using microfluidic techniques. The liquid beads consist of a fluorinated oil core and a polymer shell with magnetite particles. The beads are generated in a flow-focusing polydimethylsiloxane (PDMS) device and cured by photo polymerisation. We investigated the response of the liquid beads to an external magnetic field by characterising their motion towards a permanent magnet. Magnetic sorting of liquid beads in a channel was achieved with 90% efficiency. The results show that the liquid beads can be controlled magnetically and have potential applications in digital microfluidics including nucleic acid amplification, drug delivery, cell culture, sensing, and tissue engineering. The present paper also discusses the magnetophoretic behaviour of the liquid bead by varying its mass and magnetite concentration in the shell. We also demonstrated the two-dimensional self-assembly of magnetic liquid beads for potential use in digital polymerase chain reaction and digital loop mediated isothermal amplification.

Exploring potential anti-inflammatory effects of medicinal cannabis.

PURPOSE: Inflammation is thought to play a key role in malignant disease and may play a significant part in the expression of cancer-related symptoms. Cannabidiol (CBD) is a bioactive compound in cannabis and is reported to have significant anti-inflammatory properties. METHOD: Serial C-reactive protein (CRP) levels were measured in all participants recruited to a randomised controlled trial of CBD versus placebo in patients with symptoms related to advanced cancer. A panel of inflammatory cytokines was measured over time in a subset of these patients. RESULTS: There was no difference between the two arms in the trajectory of CRP or cytokine levels from baseline to day 28. CONCLUSION: We were unable to demonstrate an anti-inflammatory effect of CBD in cancer patients. TRIAL REGISTRATION: ANZCTR 26180001220257, registered 20/07/2018.

Stromal TRIM28-associated signaling pathway modulation within the colorectal cancer microenvironment.

BACKGROUND: Stromal gene expression patterns predict patient outcomes in colorectal cancer. TRIM28 is a transcriptional co-repressor that regulates an abundance of genes through the KRAB domain family of transcription factors. We have previously shown that stromal expression of TRIM28 is a marker of disease relapse and poor survival in colorectal cancer. Here, we perform differential epithelium-stroma proteomic network analyses to characterize signaling pathways associated with TRIM28 within the tumor microenvironment. METHODS: Reverse phase protein arrays were generated from laser capture micro-dissected carcinoma and stromal cells from fresh frozen colorectal cancer tissues. Phosphorylation and total protein levels were measured for 30 cancer-related signaling pathway endpoints. Strength and direction of associations between signaling endpoints were identified using Spearman's rank-order correlation analysis and compared to TRIM28 levels. Expression status of TRIM28 in tumor epithelium and stromal fibroblasts was assessed using IHC in formalin fixed tissue and the epithelium to stroma protein expression ratio method. RESULTS: We found distinct proteomic networks in the epithelial and stromal compartments which were linked to expression levels of TRIM28. Low levels of TRIM28 in tumor stroma (high epithelium: stroma ratio) were found in 10 out of 19 cases. Upon proteomic network analyses, these stromal high ratio cases revealed moderate signaling pathway similarity exemplified by 76 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). Furthermore, low levels of stromal TRIM28 correlated with elevated MDM2 levels in tumor epithelium (p = 0.01) and COX-2 levels in tumor stroma (p = 0.002). Low TRIM28 epithelium to stroma ratios were associated with elevated levels of caspases 3 and 7 in stroma (p = 0.041 and p = 0.036) and an increased signaling pathway similarity in stromal cells with 81 significant Spearman correlations (ρ ≥ 0.75, p ≤ 0.01). CONCLUSIONS: By dissecting TRIM28-associated pathways in stromal fibroblasts and epithelial tumor cells, we performed comprehensive proteomic analyses of molecular networks within the tumor microenvironment. We found modulation of several signaling pathways associated with TRIM28, which may be attributed to the pleiotropic properties of TRIM28 through its translational suppression of the family of KRAB domain transcription factors in tumor stromal compartments.

Rapid and cost-efficient enumeration of rare cancer cells from whole blood by low-loss centrifugo-magnetophoretic purification under stopped-flow conditions.

We present a substantially improved design and functionality of a centrifugo-magnetophoretic platform which integrates direct immunoseparation and cost-efficient, bright-field detection of cancer cells in whole blood. All liquid handling takes place in a disposable cartridge with geometry akin to a conventional compact disc (CD). The instrumentation required to process such a "lab-on-a-disc" cartridge can be as simple and cost-efficient as the rotor on a common optical disc drive. In a first step, target cells in a blood sample are specifically bound to paramagnetic microbeads. The sample is then placed into the disc cartridge and spun. In the second step, magnetically tagged target cells are separated by a co-rotating, essentially lateral magnetic field from the background population of abundant blood cells, and also from unbound magnetic beads. A stream of target cells centrifugally sediments through a stagnant liquid phase into a designated detection chamber. The continuous, multiforce immunoseparation proceeds very gently, i.e. the mechanical and hydrodynamic stress to the target cells is minimized to mitigate the risk of cell loss by collective entrapment in the background cells or vigorous snapping against a wall. We successfully demonstrate the extraction of MCF7 cancer cells at concentrations as low as 1 target cell per µl from a background of whole blood, with capture efficiencies of up to 88%. Its short time-to-answer is a notable characteristic of this system, with 10% of target cells collected in the first minute after their loading to the system and the remainder captured within the following 10 min. All the above-mentioned factors synergetically combine to leverage the development of a prospective point-of-care device for CTC detection.

Relationship between epithelial and stromal TRIM28 expression predicts survival in colorectal cancer patients.

BACKGROUND AND AIM: TRIM28 is a multi-domain nuclear protein with pleotropic effects in both normal and tumor cells. In this study, TRIM28 expression in epithelial and stromal tumor microenvironment and its prognostic role in colorectal cancer were investigated. METHODS: Immunohistological staining of TRIM28 was evaluated in tissue microarrays constructed from 137 colorectal cancer patients. The correlations of TRIM28 expression with clinicopathological features and p53 expression were studied. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess overall survival (OS) and recurrence-free survival (RFS). RESULTS: Strong epithelial TRIM28 expression was found in 42% of colorectal cancer tissues. TRIM28 expression correlated significantly with p53 expression in matched cases (P=0.0168, Spearman rank test). A high epithelial to stromal TRIM28 expression ratio was associated with shorter OS (P=0.033; log-rank test) and RFS (P=0.043; log-rank test). Multivariate analysis showed that the epithelial to stromal TRIM28 expression ratio was an independent predictor of OS (hazard ratio=2.136; 95% confidence interval 1.015-4.498, P=0.046) and RFS (hazard ratio=2.100; confidence interval 1.052-4.191, P=0.035). CONCLUSION: A high TRIM28 expression ratio between stromal and epithelial compartments in colorectal cancer tissue is an independent predictor of poor prognosis. The pathophysiological role of TRIM28 in carcinogenesis may be dependent on expression levels and cell type within the tumor microenvironment.

Protein array processing software for automated semiquantitative analysis of serum antibody repertoires.

Effective immunotherapies activate natural antitumor immune responses in patients undergoing treatment. The ability to monitor immune activation in response to immunotherapy is critical in measuring treatment efficacy over time and across patient cohorts. Protein arrays are systematically arranged, large collections of annotated proteins on planar surfaces, which can be used for the characterization of disease-specific and treatment-induced antibody repertoires in individuals undergoing immunotherapy. However, the absence of appropriate image analysis and data processing software presents a substantial hurdle, limiting the uptake of this approach in immunotherapy research. We developed a first, automated semiquantitative open-source software package for the analysis of widely used protein macroarrays. The software allows accurate single array and inter-array comparative studies through the tackling of intra-array inconsistencies arising from experimental disparities. The innovative and automated image analysis process includes adaptive positioning, background identification and subtraction, removal of null signals, robust statistical analysis, and protein pair validation. The normalized values allow a convenient semiquantitative data analysis of different samples or timepoints. Enabling accurate characterization of sample series to identify disease-specific immune profiles or their relative changes in response to treatment may serve as a diagnostic or predictive tool of disease.

Recent microfluidic advances in submicron to nanoparticle manipulation and separation.

Manipulation and separation of submicron and nanoparticles are indispensable in many chemical, biological, medical, and environmental applications. Conventional technologies such as ultracentrifugation, ultrafiltration, size exclusion chromatography, precipitation and immunoaffinity capture are limited by high cost, low resolution, low purity or the risk of damage to biological particles. Microfluidics can accurately control fluid flow in channels with dimensions of tens of micrometres. Rapid microfluidics advancement has enabled precise sorting and isolating of nanoparticles with better resolution and efficiency than conventional technologies. This paper comprehensively studies the latest progress in microfluidic technology for submicron and nanoparticle manipulation. We first summarise the principles of the traditional techniques for manipulating nanoparticles. Following the classification of microfluidic techniques as active, passive, and hybrid approaches, we elaborate on the physics, device design, working mechanism and applications of each technique. We also compare the merits and demerits of different microfluidic techniques and benchmark them with conventional technologies. Concurrently, we summarise seven standard post-separation detection techniques for nanoparticles. Finally, we discuss current challenges and future perspectives on microfluidic technology for nanoparticle manipulation and separation.

Isotypic analysis of anti-p53 serum autoantibodies and p53 protein tissue phenotypes in colorectal cancer.

The presence of IgA- and IgM-specific autoantibody (AAb) isotypes and their relationship to p53 tissue expression patterns are not well understood. This study aims to investigate the clinical utility of the anti-p53 AAb isotypes and tissue positivity in colorectal cancer (CRC). We analyzed anti-p53 IgG, IgM, and IgA AAbs in sera of 99 CRC patients and 99 non-cancer control subjects. Corresponding tissue expression of the p53 protein was evaluated by immunohistochemistry (IHC). Anti-p53 AAbs of the IgG isotype were present in the sera of 21 out of 99 patients (21%), whereas IgM AAbs were observed in 9 (9%) and IgA in 2 (2%) CRC patients. Anti-p53 AAbs of all 3 isotypes were generally associated with IHC staining indicative of mutated TP53. Seropositive anti-p53 IgM cases in the absence of anti-p53 IgG were linked to wild-type p53. Anti-p53 IgA in the absence of IgG AAbs was detected in 2 non-cancer controls indicating a potential p53 epitope mimicry. Although seropositivity was not associated with patient survival (P = .650), mutant-pattern p53 tissue expression was associated with reduced 5-year overall survival (P = .032); however, it was not an independent prognostic marker (multivariate Cox regression, P = .193). In conclusion, immunoglobulin isotyping revealed that anti-p53 IgM and IgA AAbs were predominantly concurrent with anti-p53 serum IgG and the mutant-pattern p53 tissue phenotype. IgM and IgA seropositive cases in absence of anti-p53 IgG were linked to wild-type p53 tissue phenotype indicating early anti-p53 immune responses preceding isotype class-switch (IgM) or p53 antigen mimicry (IgA).

IgM and IgA augmented autoantibody signatures improve early-stage detection of colorectal cancer prior to nodal and distant spread.

OBJECTIVES: Tumor-associated autoantibodies (AAbs) in individuals with cancer can precede clinical diagnosis by several months to years. The objective of this study was to determine whether the primary immune response in form of IgM and gut mucosa-associated IgA can aid IgG AAbs in the detection of early-stage colorectal cancer (CRC). METHODS: We developed a novel protein array comprising 492 antigens seropositive in CRC. The array was used to profile IgG, IgM and IgA antibody signatures in 99 CRC patients and 99 sex- and age-matched non-cancer controls. A receiver operating curve (ROC), Kaplan-Meier survival analysis and univariate and multivariate Cox regression analyses were conducted. RESULTS: We identified a panel of 16 multi-isotype AAbs with a cumulative sensitivity of 91% and specificity of 74% (AUC 0.90, 95% CI: 0.850-0.940) across all CRC stages. IgM and IgG isotypes were conversely associated with disease stage with IgM contributing significantly to improved stage I and II sensitivity of 96% at 78% specificity (AUC 0.928, 95% CI: 0.884-0.973). A single identified IgA AAb reached an overall sensitivity of 5% at 99% specificity (AUC 0.520, 95% CI: 0.440-0.601) balanced across all CRC stages. Kaplan-Meier analysis revealed that se33-1 (ZNF638) IgG AAbs were associated with reduced 5-year overall survival (log-rank test, P = 0.012), whereas cumulative IgM isotype signatures were associated with improved 5-year overall survival (log-rank test, P = 0.024). CONCLUSION: IgM AAbs are associated with early-stage colorectal cancer. Combining IgG, IgM and IgA AAbs is a novel strategy to improve early diagnosis of cancers.

A Nucleotide Analog Prevents Colitis-Associated Cancer via Beta-Catenin Independently of Inflammation and Autophagy.

BACKGROUND & AIMS: Chronic bowel inflammation increases the risk of colon cancer; colitis-associated cancer (CAC). Thiopurine treatments are associated with a reduction in dysplasia and CAC in inflammatory bowel disease (IBD). Abnormal Wnt/ß-catenin signalling is characteristic of >90% of colorectal cancers. Immunosuppression by thiopurines is via Rac1 GTPase, which also affects Wnt/ß-catenin signalling. Autophagy is implicated in colonic tumors, and topical delivery of the thiopurine thioguanine (TG) is known to alleviate colitis and augment autophagy. This study investigated the effects of TG in a murine model of CAC and potential mechanisms. METHODS: Colonic dysplasia was induced by exposure to azoxymethane (AOM) and dextran sodium sulfate (DSS) in wild-type (WT) mice and mice harboring intestinal epithelial cell-specific deletion of autophagy related 7 gene (Atg7ΔIEC). TG or vehicle was administered intrarectally, and the effect on tumor burden and ß-catenin activity was assessed. The mechanisms of action of TG were investigated in vitro and in vivo. RESULTS: TG ameliorated DSS colitis in wild-type but not Atg7ΔIEC mice, demonstrating that anti-inflammatory effects of locally delivered TG are autophagy-dependent. However, TG inhibited CAC in both wild-type and Atg7ΔIEC mice. This was associated with decreased ß-catenin activation/nuclear translocation demonstrating that TG's inhibition of tumorigenesis occurred independently of anti-inflammatory and pro-autophagic actions. These results were confirmed in cell lines, and the dependency on Rac1 GTPase was demonstrated by siRNA knockdown and overexpression of constitutively active Rac1. CONCLUSIONS: Our findings provide evidence for a new mechanism that could be exploited to improve CAC chemoprophylactic approaches.

Rapid characterization of binding specificity and cross-reactivity of antibodies using recombinant human protein arrays.

Antibodies are routinely used as research tools, in diagnostic assays and increasingly as therapeutics. Ideally, these applications require antibodies with high sensitivity and specificity; however, many commercially available antibodies are limited in their use as they cross-react with non-related proteins. Here we describe a novel method to characterize antibody specificity. Six commercially available monoclonal and polyclonal antibodies were screened on high-density protein arrays comprising of ~10,000 recombinant human proteins (Imagenes). Two of the six antibodies examined; anti-pICln and anti-GAPDH, bound exclusively to their target antigen and showed no cross-reactivity with non-related proteins. However, four of the antibodies, anti-HSP90, anti-HSA, anti-bFGF and anti-Ro52, showed strong cross-reactivity with other proteins on the array. Antibody-antigen interactions were readily confirmed using Western immunoblotting. In addition, the redundant nature of the protein array used, enabled us to define the epitopic region within HSP90 of the anti-HSP90 antibody, and identify possible shared epitopes in cross-reacting proteins. In conclusion, high-density protein array technology is a fast and effective means for determining the specificity of antibodies and can be used to further improve the accuracy of antibody applications.

Defining the molecular target of an antibody derived from nuclear extract of Jurkat cells using protein arrays.

Many diagnostic antibodies are generated by immunization with whole cells or cell extracts and are shown by screening on tissue sections to label specific cell populations. However, their target molecule then needs to be identified, and this can be technically demanding. Here we describe the use of protein arrays to define the targets of new or uncharacterized monoclonal antibodies. The technique involves screening protein arrays containing thousands of recombinant human proteins. An initial experiment showed that a well-characterized monoclonal antibody against nucleophosmin identified 22 clones on the array encoding this protein. Next, the antibody JJ166, for which the antigen had not yet been identified, was screened. This antibody was generated by immunizing with a nuclear extract of Jurkat cells and was detected in immunohistochemistry due to its distinctive nuclear staining of lymphoid tissue cells. However, its molecular target had remained unidentified using traditional approaches. A protein array screen rapidly identified the mitotic spindle-associated molecule NUMA1 (nuclear mitotic apparatus protein 1). To confirm this putative specificity, JJ166 was shown to react with COS-1 cells transiently transfected with the complementary DNA for NUMA1. Furthermore, a commercially available antibody against NUMA1 showed nearly identical staining in immunohistochemistry on human tissue and cells. Overall, this method represents an effective and quick strategy for defining the protein targets of new or uncharacterized monoclonal antibodies identified as having diagnostic or other potential value on the basis of their immunostaining patterns.

Microfluidic Array Chip for Parallel Detection of Waterborne Bacteria.

The polymerase chain reaction (PCR) is a robust technique used to make multiple copies of a segment of DNA. However, the available PCR platforms require elaborate and time-consuming operations or costly instruments, hindering their application. Herein, we introduce a sandwiched glass-polydimethylsiloxane (PDMS)-glass microchip containing an array of reactors for the real-time PCR-based detection of multiple waterborne bacteria. The PCR solution was loaded into the array of reactors in a single step utilising capillary filling, eliminating the need for pumps, valves, and liquid handling instruments. Issues of generating and trapping bubbles during the loading chip step were addressed by creating smooth internal reactor surfaces. Triton X-100 was used to enhance PCR compatibility in the chip by minimising the nonspecific adsorption of enzymes. A custom-made real-time PCR instrument was also fabricated to provide thermal cycling to the array chip. The microfluidic device was successfully demonstrated for microbial faecal source tracking (MST) in water.

Measurement of the IgM and IgG Autoantibody Immune Responses in Human Serum has High Predictive Value for the Presence of Colorectal Cancer.

INTRODUCTION: Colorectal cancer is a major public health issue, with incidences continuing to rise owing to the growing and aging world population. Current screening strategies for colorectal cancer diagnosis suffer from various limitations, including invasiveness and poor uptake. Consequently, there is an unmet clinical need for a minimally invasive, sensitive, and specific method for detecting the presence of colorectal cancer and pre-malignant lesions. PATIENTS AND METHODS: An indirect enzyme-linked immunosorbent assay was used to measure the primary (IgM) and secondary (IgG) adaptive humoral immune responses to a panel of previously identified cancer antigens in the sera of normal and adenoma samples, and sera from patients with colorectal cancer. RESULTS: An optimal panel of 7 biomarkers capable of identifying patients with colorectal cancer as distinct from both normal and adenoma samples is identified. The cumulative sensitivity and specificity of the assay are 70.8% and 86.5%, respectively. The positive and negative predictive values of the cohort are 77.3% and 82.1%. This assay was not able to accurately discriminate between normal and adenoma samples. Patients whose serum was positive for the presence of anti-ICLN IgM autoantibodies had a significantly poorer 5-year survival than patients whose serum was negative (P = .004). CONCLUSION: This study describes a novel minimally invasive enzyme-linked immunosorbent assay-based method, capable of identifying patients with colorectal cancer as distinct from both normal and adenoma samples. Patients are likely to be far more amenable to a blood-based test such as the one described herein, rather than a fecal-based test, likely leading to increased patient uptake.

MUC13 promotes the development of colitis-associated colorectal tumors via ß-catenin activity.

Many adenocarcinomas, including colorectal cancer (CRC), overexpress the MUC13 cell surface mucin, but the functional significance and mechanisms are unknown. Here, we report the roles of MUC13 in colonic tumorigenesis and tumor progression. High-MUC13 expression is associated with poor survival in two independent patient cohorts. In a comprehensive series of in vivo experiments, we identified a critical role for MUC13 in the development of this malignancy, by promoting survival and proliferation of tumor-initiating cells and driving an immunosuppressive environment that protects tumors from checkpoint inhibitor immunotherapy. In Muc13-deficient mice, fewer tumors are generated after exposure to carcinogens and inflammation, they have markedly reduced ß-catenin signaling, have more tumor-infiltrating CD103+ dendritic cells and CD8+ T lymphocytes, fewer myeloid-derived suppressor cells, and are rendered sensitive to checkpoint inhibitor immunotherapy (anti-PD-L1). Mechanistically, we show that MUC13 protects ß-catenin from degradation, by interacting with GSK-3ß, which increases ß-catenin nuclear translocation and promotes its signaling, thereby driving cancer initiation, progression, invasion, and immune suppression. Therefore, MUC13 is a potential marker of poor prognosis in colorectal cancer, and inhibiting MUC13 may be useful in the treatment of colitis-associated cancer and sensitizing tumors to immunotherapy.

Referencing cross-reactivity of detection antibodies for protein array experiments.

Protein arrays are frequently used to profile antibody repertoires in humans and animals. High-throughput protein array characterisation of complex antibody repertoires necessitates the use of extensively validated secondary detection antibodies. This article details the validation of an affinity-isolated anti-chicken IgY antibody produced in rabbit and a goat anti-rabbit IgG antibody conjugated with alkaline phosphatase using protein arrays consisting of 7,390 distinct human proteins. Probing protein arrays with secondary antibodies in absence of chicken serum revealed non-specific binding to 61 distinct human proteins. Despite the identified non-specific binding, the tested antibodies are well suited for use in protein array experiments as the cross-reactive binding partners can be readily excluded from further analysis. The evident cross-reactivity of the tested secondary detection antibodies points towards the necessity of platform-specific antibody characterisation studies for all secondary immunoreagents. Furthermore, secondary antibody characterisation using protein arrays enables the generation of reference lists of cross-reactive proteins, which can be then marked as potential false positives in follow-up experiments. Providing such cross-reactivity reference lists accessible to the wider research community may help to interpret data generated with the same antibodies in applications not only related to protein arrays such as immunoprecipitation, Western blots or other immunoassays.

Real-time monitoring of cell migration, phagocytosis and cell surface receptor dynamics using a novel, live-cell opto-microfluidic technique.

We report an opto-microfluidic method for continuous and non-interfering monitoring of cell movement and dynamic molecular processes in living cells enabled by the microfluidic "Lab-in-a-Trench" (LiaT) platform. To demonstrate real-time monitoring of heterogeneous cell-cell interactions, cell tracking and agent-induced cell activation dynamics, we observe phagocytosis of Escherichia coli by murine macrophages, migration of active macrophages and LPS-induced CD86 expression in macrophages. The visualization of phagocytosis is facilitated through the loading of green fluorescent protein (GFP) expressing E. coli to the array of cell capture modules before the introduction of macrophages. Simple migration tracking of active macrophages is enabled by a spatio-temporal control of the environment conditions within the LiaT platform. Furthermore, we report an interference-free monitoring of non-modified, endogenous changes in protein expression on the surface of living cells using traditional, antibody immuno-reagents. Throughout the experiment, murine macrophages were captured in the LiaT device and exposed to sub-background levels of fluorescently labeled anti-CD86 antibody. Upon lipopolysaccharide (LPS) stimulation, CD86 changes were visualized in real-time by time-lapse microscopy. This novel opto-microfluidic effect is controlled by the equilibrium of convective-diffusive replenishment of fluorescently labeled antibodies and antibody affinity. Overall, our non-interfering analysis method allows the studying of active cellular processes and endogenous protein dynamics in live cells in a simple and cost-efficient manner.

Diagnostic potential of zinc finger protein-specific autoantibodies and associated linear B-cell epitopes in colorectal cancer.

Colorectal cancer is one of the most common cancers worldwide with almost 700,000 deaths every year. Detection of colorectal cancer at an early stage significantly improves patient survival. Cancer-specific autoantibodies found in sera of cancer patients can be used for pre-symptomatic detection of the disease. In this study we assess the zinc finger proteins ZNF346, ZNF638, ZNF700 and ZNF768 as capture antigens for the detection of autoantibodies in colorectal cancer. Sera from 96 patients with colorectal cancer and 35 control patients with no evidence of cancer on colonoscopy were analysed for the presence of ZNF-specific autoantibodies using an indirect ELISA. Autoantibodies to individual ZNF proteins were detected in 10-20% of colorectal cancer patients and in 0-5.7% of controls. A panel of all four ZNF proteins resulted in an assay specificity of 91.4% and sensitivity of 41.7% for the detection of cancer patients in a cohort of non-cancer controls and colorectal cancer patients. Clinicopathological and survival analysis revealed that ZNF autoantibodies were independent of disease stage and did not correlate with disease outcome. Since ZNF autoantibodies were shared between patients and corresponding ZNF proteins showed similarities in their zinc finger motifs, we performed an in silico epitope sequence analysis. Zinc finger proteins ZNF700 and ZNF768 showed the highest sequence similarity with a bl2seq score of 262 (E-value 1E-81) and their classical C2H2 ZNF motifs were identified as potential epitopes contributing to their elevated immunogenic potential. Our findings show an enhanced and specific immunogenicity to zinc finger proteins, thereby providing a multiplexed autoantibody assay for minimally invasive detection of colorectal cancer.

High CerS5 expression levels associate with reduced patient survival and transition from apoptotic to autophagy signalling pathways in colorectal cancer.

Ceramide synthase 5 is involved in the de novo synthesis of ceramide, a sphingolipid involved in cell death and proliferation. In this study, we investigated the role of ceramide synthase 5 in colorectal cancer by examining ceramide synthase 5 expression, clinico-pathological parameters and association with survival/death signalling pathways in cancer. Immunohistochemical analysis of CerS5 was performed on 102 colorectal cancer samples using tissue microarrays constructed from formalin-fixed and paraffin-embedded tissues. We found strong membranous ceramide synthase 5 staining in 57 of 102 (56%) colorectal cancers. A multivariate Cox regression analysis of ceramide synthase 5 expression adjusted for disease stage, differentiation and lymphovascular invasion revealed reduced 5-year overall survival (p = 0.001) and 5-year recurrence-free survival (p = 0.002), with hazard ratios of 4.712 and 4.322, respectively. The effect of ceramide synthase 5 expression on tumourigenic processes was further characterised by reverse phase protein array analysis. Reverse phase protein arrays were generated from laser capture microdissection-enriched carcinoma cells from 19 fresh-frozen colorectal cancer tissues. Measurements of phosphorylation and total levels of signalling proteins involved in apoptosis, autophagy and other cancer-related pathways revealed two distinct signalling networks; weak membranous ceramide synthase 5 intensity was associated with a proteomic network dominated by signalling proteins linked to apoptosis, whereas strong ceramide synthase 5 intensity was associated with a proteomic sub-network mostly composed of proteins linked to autophagy. In conclusion, high ceramide synthase 5 expression was found in colorectal cancer tissue and was associated with poorer patient outcomes. Our findings suggest that this may be mediated by a transition from apoptotic to autophagy signalling pathways in ceramide synthase 5 High expressing tumours, thus implicating ceramide synthase 5 in the progression of colorectal cancer.