The effect of HYPE knock-out on the AMPylome of human OSU-CLL leukemia cells.

In humans, AMPylation of cellular proteins is carried out by Huntingtin yeast-interacting protein E (HYPE), activated under conditions of endoplasmic reticulum stress, such as in cancer cells. Extracts of the human chronic lymphocytic leukemia cell line, OSU-CLL, were fractionated using immuno-precipitation with antibodies against adenosine-phosphate and then AMP-Tyr. The proteins isolated were modified with AMP, the 'AMPylome.' AMP-labelled peptides isolated from HYPE wild-type (WT) and HYPE knock-out (KO) cells were identified using tandem mass spectrometry. A total of 213 proteins were identified from WT cell extracts, while only 23 of these were pulled down from KO cells, consistent with the presence of another AMPylator, besides HYPE. The KO cells were more sensitive to fludarabine nucleoside (2-FaraA) than WT cells. Ingenuity Pathway Analysis of the AMPylated proteins identified in WT cells clustered actin binding proteins of the cytoskeleton, and proteins of the RHO GTPase pathway that would jointly stimulate cell proliferation.

Adenylated proteins in mouse B16-F10 melanoma cells cluster in functional categories: a new paradigm for cellular regulation?

In mammals, AMPylation of cellular proteins is carried out by Huntingtin yeast-interacting protein E, and pseudokinase SelO. Lysates from mouse B16-F10 melanoma cells have been fractionated by immuno-precipitation using magnetic Dynabeads coated with antibodies against both adenosine 5'-monophosphate in phosphate ester linkage to tyrosine, and adenosine-phosphate. Proteins pulled down with both these antibodies were subject to post-translational modification, most likely AMPylation. Using tandem mass spectrometry, analysis of these protein fractions identified 333 proteins that could be pulled down by both antibodies. Many of these proteins clustered in 13 functional Ingenuity Pathway Analysis categories of 4 or more adenylated proteins including some from the cytoskeleton, and some involved with initiating the unfolded protein response.Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1995608 .

Fludarabine nucleoside induces major changes in the p53 interactome in human B-lymphoid cancer cell lines.

Triple combination FCR (fludarabine, cyclophosphamide and rituximab) is often used as front-line treatment for chronic lymphocytic leukemia (CLL) and non-Hodgkin's lymphoma. Results from our laboratory indicate that 2-FaraAMP (fludarabine) has multiple mechanisms of cytotoxicity that include accumulation of isoforms and phosphorylated derivatives of p53, and induction of the unfolded protein response (UPR). Using protein pull-downs with Dynabeads coated with p53 antibody, we have found that 2-FaraA (fludarabine nucleoside) induces major changes in the p53 interactome in human Raji lymphoma and IM9 multiple myeloma cells. These changes are likely driven by DNA strand breaks induced by 2-FaraA that activate protein kinases such as ATM, ATR and Chk1.

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. DotScan's 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.

Extensive surface protein profiles of extracellular vesicles from cancer cells may provide diagnostic signatures from blood samples.

Extracellular vesicles (EV) are membranous particles (30-1,000 nm in diameter) secreted by cells. Important biological functions have been attributed to 2 subsets of EV, the exosomes (bud from endosomal membranes) and the microvesicles (MV; bud from plasma membranes). Since both types of particles contain surface proteins derived from their cell of origin, their detection in blood may enable diagnosis and prognosis of disease. We have used an antibody microarray (DotScan) to compare the surface protein profiles of live cancer cells with those of their EV, based on their binding patterns to immobilized antibodies. Initially, EV derived from the cancer cell lines, LIM1215 (colorectal cancer) and MEC1 (B-cell chronic lymphocytic leukaemia; CLL), were used for assay optimization. Biotinylated antibodies specific for EpCAM (CD326) and CD19, respectively, were used to detect captured particles by enhanced chemiluminescence. Subsequently, this approach was used to profile CD19(+) EV from the plasma of CLL patients. These EV expressed a subset (~40%) of the proteins detected on CLL cells from the same patients: moderate or high levels of CD5, CD19, CD31, CD44, CD55, CD62L, CD82, HLA-A,B,C, HLA-DR; low levels of CD21, CD49c, CD63. None of these proteins was detected on EV from the plasma of age- and gender-matched healthy individuals.

Protein profiles distinguish stable and progressive chronic lymphocytic leukemia.

Patients with a stable chronic lymphocytic leukemia (CLL) double their blood lymphocyte count in >5 years, but may develop progressive disease with lymphocytes doubling in <12 months. To identify a protein signature for progressive CLL, whole cell extracts of peripheral blood mononuclear cells from patients with CLL (n=27) were screened using iTRAQ (isobaric tags for relative and absolute quantification) analysis. A total of 84 differentially abundant proteins were identified from patients with stable and progressive CLL. Subsequently, 32 of these proteins were quantified by SRM (selected reaction monitoring) using extracts of purified CD19+ CLL cells from patients (n=50). Hierarchical clustering of these protein profiles showed two clusters of patients that correlated with progressive and stable CLL, providing signatures that should be useful for triaging patients. Some of the proteins in the progressive cluster have not been linked with CLL, for example, glutamate dehydrogenase 1 and transcription intermediary factor 1-beta.

Analysis of Post-Liver Transplant Hepatitis C Virus Recurrence Using Serial Cluster of Differentiation Antibody Microarrays.

BACKGROUND: Hepatitis C virus (HCV) reinfection of the liver allograft after transplantation is universal, with some individuals suffering severe disease recurrence. Predictive markers of recurrent disease severity are urgently needed. In this study, we used a cluster of differentiation (CD) microarray to predict the severity of HCV recurrence after transplantation. METHODS: The CD antibody microarray assays of live leukocytes were performed on peripheral blood taken in the first year after transplantation. The results were grouped into phases defined as; Pre-transplant (day 0), Early (day 3 to week 2), Mid (week 4 to week 10), and Late (week 12 to week 26). Hepatitis C virus severity was based on fibrosis stages in the first 2 years (F0-1 mild and F2-4 severe). RESULTS: Serial blood samples from 16 patients were taken before and after liver transplantation. A total of 98 assays were performed. Follow-up was 3 years or longer. Comparing recurrence severity, significantly greater numbers of CD antigens were differentially expressed on the pretransplant samples compared to any posttransplant timepoints. Five differentially expressed CD antigens before transplantation (CD27 PH, CD182, CD260, CD41, and CD34) were significantly expressed comparing severe to mild recurrence, whereas expression of only CD152 was significant in the late phase after transplantation. No relationship was observed between the donor or recipient interleukin-28B genotypes and HCV recurrence severity. CONCLUSIONS: This study shows that circulating leukocyte CD antigen expression has utility in assessing recurrent HCV disease severity after liver transplantation and serves as a proof of principle. Importantly, pretransplant CD antigen expression is most predictive of disease outcome.

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.

Cell surface phenotype profiles distinguish stable and progressive chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is clinically heterogeneous. While some patients have indolent disease for many years, 20-30% will progress and ultimately die of their disease. CLL may be classified by the Rai or Binet staging system, mutational status of the immunoglobulin variable heavy-chain gene (IGVH), ZAP-70 overexpression, cytogenetic abnormalities (13q-, + 12, 11q-, 17p-) and expression of several cell surface antigens (CD38, CD49d) that correlate with risk of disease progression. However, none of these markers identify all cases of CLL at risk. In a recent review, we summarized those CD antigens known to correlate with the prognosis of CLL. The present study has identified surface profiles of CD antigens that distinguish clinically progressive CLL from slow-progressive and stable CLL. Using an extended DotScan(™) CLL antibody microarray (Version 3; 182 CD antibodies), and with refined analysis of purified CD19 + B-cells, the following 27 CD antigens were differentially abundant for progressive CLL: CD11a, CD11b, CD11c, CD18, CD19, CD20 (two epitopes), CD21, CD22, CD23, CD24, CD25, CD38, CD40, CD43, CD45, CD45RA, CD52, CD69, CD81, CD84, CD98, CD102, CD148, CD180, CD196 and CD270. The extensive surface profiles obtained provide disease signatures with an accuracy of 79.2%, a sensitivity of 83.9% and a specificity of 72.5% that could provide the basis for a rapid test to triage patients with CLL according to probability of clinical progression and potential earlier requirement for treatment.

Profiles of surface mosaics on chronic lymphocytic leukemias distinguish stable and progressive subtypes.

PURPOSE: Chronic lymphocytic leukemia (CLL) is a heterogeneous disease, some patients may survive for many years, while 20-30% of patients progress and may die within several years. Currently, there is not a single procedure that enables accurate prognosis and triaging of those patients who need immediate and aggressive treatment. All CLL cells are characterised by the expression of the B-cell antigens CD19, CD20, CD21, CD22 and CD23, with aberrant expression of the T-cell antigen CD5. METHODS: We have developed a CD antibody microarray (DotScan) containing 182 immobilised CD antibodies that has been used to obtain extensive surface profiles of CLL cells obtained from 96 patients. RESULTS: Of these 182 antigens, 27 were significantly differentially expressed between stable, stable-progressive and progressive CLL. Some of these antigens are not expressed on normal B-cells and may be targets for therapeutic antibodies against CLL. Unsupervised hierarchical clustering of the surface profiles from 96 patients showed that those with progressive CLL could be distinguished based solely upon this 'disease signature'. The sensitivity (proportion of actual positives correctly identified) was 67.9%, the specificity (proportion of negatives correctly identified) was 77.5%, and the accuracy was 71.9%. CONCLUSIONS: Considerable effort by a number of research groups has resulted in identification of individual markers for progressive CLL, but their collective use is yet to provide a test that identifies CLL patients at risk. Data presented here provide a basis for development of a simple test using an antibody microarray.

Analysis of human liver disease using a cluster of differentiation (CD) antibody microarray.

BACKGROUND: A CD antibody microarray has been previously developed allowing semi-quantitative identification of greater than 80 CD antigens on circulating leucocytes from peripheral blood samples. This assay, which uses a live cell-capture technique, enables an extensive leucocyte immunophenotype determination in a single analysis and to date this has been used successfully to characterise diseases including human leukaemias and HIV infection. AIMS: To determine CD antigen expression profiles for patients with various liver diseases and to look for preserved disease-specific signatures. METHODS: Three liver disease groups including hepatitis C (HCV) (n = 35), non-alcoholic steatohepatitis (NASH) (n = 21) and alcohol-related liver disease (n = 14) were compared with a normal group (n = 23). Hierarchal Clustering (HCL) and Principal Component Analysis (PCA) of the data revealed distinct binding patterns for patients with and without cirrhosis. RESULTS: Patients with cirrhosis and portal hypertension compared with those without cirrhosis had significantly reduced expression of several markers of T-cell function including CD45, CD8, CD28 and TCR α/ß. Disease prediction algorithms based on the expression data were able to discriminate cirrhotics from non-cirrhotics with 71% overall success, which improved to 77% when only patients with HCV were considered. CONCLUSIONS: These results demonstrate disease-specific consensus patterns of expression of CD antigens for patients with chronic liver disease, suggesting that the CD antibody array is a promising tool in the analysis of human liver disease, and with further refinement may have future research and clinical utility.

Surface profiles for subclassification of chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) has a variable clinical course. Some patients have stable disease while others progress and require treatment. Levels of several cluster of differentiation (CD) antigens are known to correlate with prognosis and may be used to stratify patients according to risk. In this review, we summarize current information on surface CD antigens found on CLL, their pathological significance and their detection using CD antibody microarrays. The use of extensive immunophenotypic patterns or surface profiles as disease signatures for CLL subclassification, prognosis and patient management is discussed with a focus on triaging patients with CLL with progressive disease.

Colorectal cancer cell surface protein profiling using an antibody microarray and fluorescence multiplexing.

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority of CRC cases, cell dysfunction is the result of numerous mutations that modify protein expression and post-translational modification(1). A number of cell surface antigens, including cluster of differentiation (CD) antigens, have been identified as potential prognostic or metastatic biomarkers in CRC. These antigens make ideal biomarkers as their expression often changes with tumour progression or interactions with other cell types, such as tumour-infiltrating lymphocytes (TILs) and tumour-associated macrophages (TAMs). The use of immunohistochemistry (IHC) for cancer sub-classification and prognostication is well established for some tumour types(2,3). However, no single 'marker' has shown prognostic significance greater than clinico-pathological staging or gained wide acceptance for use in routine pathology reporting of all CRC cases. A more recent approach to prognostic stratification of disease phenotypes relies on surface protein profiles using multiple 'markers'. While expression profiling of tumours using proteomic techniques such as iTRAQ is a powerful tool for the discovery of biomarkers4, it is not optimal for routine use in diagnostic laboratories and cannot distinguish different cell types in a mixed population. In addition, large amounts of tumour tissue are required for the profiling of purified plasma membrane glycoproteins by these methods. In this video we described a simple method for surface proteome profiling of viable cells from disaggregated CRC samples using a DotScan CRC antibody microarray. The 122-antibody microarray consists of a standard 82-antibody region recognizing a range of lineage-specific leukocyte markers, adhesion molecules, receptors and markers of inflammation and immune response(5), together with a satellite region for detection of 40 potentially prognostic markers for CRC. Cells are captured only on antibodies for which they express the corresponding antigen. The cell density per dot, determined by optical scanning, reflects the proportion of cells expressing that antigen, the level of expression of the antigen and affinity of the antibody(6). For CRC tissue or normal intestinal mucosa, optical scans reflect the immunophenotype of mixed populations of cells. Fluorescence multiplexing can then be used to profile selected sub-populations of cells of interest captured on the array. For example, Alexa 647-anti-epithelial cell adhesion molecule (EpCAM; CD326), is a pan-epithelial differentiation antigen that was used to detect CRC cells and also epithelial cells of normal intestinal mucosa, while Phycoerythrin-anti-CD3, was used to detect infiltrating T-cells(7). The DotScan CRC microarray should be the prototype for a diagnostic alternative to the anatomically-based CRC staging system.

An extended antibody microarray for surface profiling metastatic melanoma.

An antibody microarray was developed for profiling the surface proteome of melanoma cells, which may facilitate melanoma sub-classification and provide important prognostic information useful in predicting the clinical behavior of the melanoma (e.g., likely sites of metastatic spread), patient outcome and treatment response. Forty-eight antibodies were selected based on their correlation with melanoma development, progression and/or prognosis and printed on nitrocellulose slides. The immobilised antibodies capture live cells expressing corresponding antigens to produce a cell binding dot pattern representing the surface antigen profile (immunophenotype) of the melanoma. Surface antigen signatures were determined for a normal melanocyte and 6 melanoma cell lines and cell suspensions prepared from 10 surgically excised melanoma lymph node metastases. A procedure for obtaining separate surface antigen profiles for melanoma cells and leukocytes from clinical lymph node samples was also developed using anti-CD45 magnetic beads. The capture of live, bead-bound leukocytes on these antibody microarrays provides a significant enhancement of this microarray technology. The antibody microarray will be used to profile panels of surgically excised melanoma lymph node metastases (melanoma and leukocyte fractions) to determine whether the immunophenotypes correlate with clinicopathological characteristics, disease progression and clinical outcome.

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.

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.

Cladribine and Fludarabine Nucleoside Change the Levels of CD Antigens on B-Lymphoproliferative Disorders.

The purine analogs, fludarabine nucleoside (FdA), and cladribine (CdA) (1 µM, 24 hours), significantly changed the levels of some surface antigens on the human B-cell lines MEC2 and Raji. Changes in the surface proteins were identified using a Cluster of Differentiation (CD) antibody microarray that captures live cells and confirmed by flow cytometry. For Raji cells, CdA up-regulated CD10, CD54, CD80, and CD86, with repression of CD22, while FdA up-regulated CD20, CD54, CD80, CD86 and CD95. For MEC2 cells, CdA up-regulated CD11a, CD20, CD43, CD45, CD52, CD54, CD62L, CD80, CD86, and CD95, but FdA had no effect. Up-regulation of particular CD antigens induced on a B-cell lymphoproliferative disorder by a purine analog could provide targets for therapeutic antibodies with synergistic cell killing.

Profiling CD antigens on leukaemias with an antibody microarray.

Cluster of differentiation (CD) antigens are defined when a surface molecule found on some members of a standard panel of human cells reacts with at least one novel antibody, and there is good accompanying molecular data. Monoclonal antibodies to surface CD antigens on leukocytes have been used for flow cytometry, and more recently to construct microarrays that capture live cells. These DotScan microarrays enable the rapid and highly parallel characterization of repertoires of CD antigens whose expression patterns may be correlated with discrete leukaemia subtypes, or used to define biomarker 'signatures' for non-hematological diseases. DotScan with fluorescence multiplexing enables profiling of CD antigens for minor subsets of cells, such as colorectal cancer cells and tumour-infiltrating lymphocytes from a surgical sample.

The applicability of a cluster of differentiation monoclonal antibody microarray to the diagnosis of human disease.

Recent advances in antibody microarray technology have facilitated the development of multiplexed diagnostic platforms. Highly parallel antigen expression data obtained from these arrays allow disease states to be characterized using protein patterns rather than individual protein markers. The development of an antibody microarray platform of general applicability requires careful consideration of the array content. The human cluster of differentiation (CD) antigens constitute a promising candidate set, being united by their common expression at the leukocyte cell surface and the fact that the majority perform critical functions in the human immune response. The diagnostic potential of a microarray, containing 82 cluster of differentiation monoclonal antibodies (DotScan microarrays) has been demonstrated for a variety of infectious and neoplastic disease states, including HIV, many acute and chronic leukemias, and colorectal cancer. It is likely that these microarrays will have more general utility that extends to other pathological categories, including autoimmune, metabolic, and degenerative diseases.

Longitudinal microarray analysis of cell surface antigens on peripheral blood mononuclear cells from HIV+ individuals on highly active antiretroviral therapy.

BACKGROUND: The efficacy of highly active antiretroviral therapy (HAART) determined by simultaneous monitoring over 100 cell-surface antigens overtime has not been attempted. We used an antibody microarray to analyze changes in the expression of 135 different cell-surface antigens overtime on PBMC from HIV+ patients on HAART. Two groups were chosen, one (n = 6) achieved sustainable response by maintaining below detectable plasma viremia and the other (n = 6) responded intermittently. Blood samples were collected over an average of 3 years and 5-8 time points were selected for microarray assay and statistical analysis. RESULTS: Significant trends over time were observed for the expression of 7 cell surface antigens (CD2, CD3epsilon, CD5, CD95, CD36, CD27 and CD28) for combined patient groups. Between groups, expression levels of 10 cell surface antigens (CD11a, CD29, CD38, CD45RO, CD52, CD56, CD57, CD62E, CD64 and CD33) were found to be differential. Expression levels of CD9, CD11a, CD27, CD28 and CD52, CD44, CD49d, CD49e, CD11c strongly correlated with CD4+ and CD8+ T cell counts, respectively. CONCLUSION: Our findings not only detected markers that may have potential prognostic/diagnostic values in evaluating HAART efficacy, but also showed how density of cell surface antigens could be efficiently exploited in an array-like manner in relation to HAART and HIV-infection. The antigens identified in this study should be further investigated by other methods such as flow cytometry for confirmation as biological analysis of these antigens may help further clarify their role during HAART and HIV infection.