Reverse-phase Microarray Research Articles

Functional proteomics based on protein microarray technology for biomedical research.

This chapter traces a route through Proteomics from its origins to the present day. The different proteomics applications are discussed with a focus on microarray technology. Analytical microarrays, functional microarrays and reverse phase microarrays and their different applications are discussed. Several studies are mentioned where the great versatility of this approach is shown. Finally, the advantages and future challenges of microarray technology are outlined.

Development of a Sol-gel-assisted Reverse-phase Microarray Platform for Simple and Rapid Detection of Prostate-specific Antigen from Serum

A sol-gel-based reverse-phase microarray was developed with improved sensitivity for prostatespecific antigen (PSA) from serum. The pore-sizecontrolled 3D sol-gel matrix was created with a large surface area to capture target molecules densely. Using the optically active sol-gel nanocomposites, human female serum was spiked with PSA and assessed using the reverse-phase protein microarray. The reverse-phase assay exhibited a limit of detection of 1 pg/mL for PSA and a dynamic range of 103 orders of magnitude. Notably, the platform matched the demand of the immunoassay in a simple and feasible manner. Moreover, the platform shortened the total assay time with an increased accuracy of diagnosis.

Proteomic Analysis Reveals Autophagy as Pro-Survival Pathway Elicited by Long-Term Exposure with 5-Azacitidine in High-Risk Myelodysplasia.

Azacytidine (5-AZA) is the standard first-choice treatment for high-risk myelodysplasia (MDS) patients. However, the clinical outcome for those patients who interrupt treatment or whose disease failed to respond is very poor. In order to identify the cellular pathways that are modified by long-term exposure to 5-AZA, we evaluated key proteins associated with the autophagy pathway by reverse-phase microarray (RPPA). Comparing bone marrow mononucleated cells (BMMCs) obtained from 20 newly-diagnosed patients and after four 5-AZA cycles we found an increased autophagy signaling. We then evaluated ex-vivo the effect of the combination of 5-AZA with autophagy inhibitors chloroquine (CQ) and leupeptin. Since 5-AZA and CQ showed synergism due to an increase of basal autophagy after 5-AZA exposure, we adopted a sequential treatment treating BMMCs with 5 μM 5-AZA for 72 h followed by 10 μM CQ for 24 h and found increased apoptosis, associated to a reduction of G2M phase and increase in G0-G1 phase. Long-term exposure to 5-AZA induced the reduction of the autophagic marker SQSTM1/p62, reversible by CQ or leupeptin exposure. In conclusion, we identified autophagy as a compensatory pathway occurring in MDS-BM after long-term exposure to 5-AZA and we provided evidences that a sequential treatment of 5-AZA followed by CQ could improve 5-AZA efficacy, providing novel insight for tailored therapy in MDS patients progressing after 5-AZA therapy.

Expression and Production of SH2 Domain Proteins.

The Src Homology 2 (SH2) domain lies at the heart of phosphotyrosine signaling, coordinating signaling events downstream of receptor tyrosine kinases (RTKs), adaptors, and scaffolds. Over a hundred SH2 domains are present in mammals, each having a unique specificity which determines its interactions with multiple binding partners. One of the essential tools necessary for studying and determining the role of SH2 domains in phosphotyrosine signaling is a set of soluble recombinant SH2 proteins. Here we describe methods, based on a broad experience with purification of all SH2 domains, for the production of SH2 domain proteins needed for proteomic and biochemical-based studies such as peptide arrays, mass-spectrometry, protein microarrays, reverse-phase microarrays, and high-throughput fluorescence polarization (HTP-FP). We describe stepwise protocols for expression and purification of SH2 domains using GST or poly His-tags, two widely adopted affinity tags. In addition, we address alternative approaches, challenges, and validation studies for assessing protein quality and provide general characteristics of purified human SH2 domains.

Dual Action of miR-125b As a Tumor Suppressor and OncomiR-22 Promotes Prostate Cancer Tumorigenesis.

MicroRNAs (miRs) are a novel class of small RNA molecules, the dysregulation of which can contribute to cancer. A combinatorial approach was used to identify miRs that promote prostate cancer progression in a unique set of prostate cancer cell lines, which originate from the parental p69 cell line and extend to a highly tumorigenic/metastatic M12 subline. Together, these cell lines are thought to mimic prostate cancer progression in vivo. Previous network analysis and miR arrays suggested that the loss of hsa-miR-125b together with the overexpression of hsa-miR-22 could contribute to prostate tumorigenesis. The dysregulation of these two miRs was confirmed in human prostate tumor samples as compared to adjacent benign glandular epithelium collected through laser capture microdissection from radical prostatectomies. In fact, alterations in hsa-miR-125b expression appeared to be an early event in tumorigenesis. Reverse phase microarray proteomic analysis revealed ErbB2/3 and downstream members of the PI3K/AKT and MAPK/ERK pathways as well as PTEN to be protein targets differentially expressed in the M12 tumor cell compared to its parental p69 cell. Relevant luciferase+3’-UTR expression studies confirmed a direct interaction between hsa-miR-125b and ErbB2 and between hsa-miR-22 and PTEN. Restoration of hsa-miR-125b or inhibition of hsa-miR-22 expression via an antagomiR resulted in an alteration of M12 tumor cell behavior in vitro. Thus, the dual action of hsa-miR-125b as a tumor suppressor and hsa-miR-22 as an oncomiR contributed to prostate tumorigenesis by modulations in PI3K/AKT and MAPK/ERK signaling pathways, key pathways known to influence prostate cancer progression.

Reverse-Phase Microarray Analysis Reveals Novel Targets in Lymph Nodes of Bacillus anthracis Spore-Challenged Mice.

Anthrax is a frequently fatal infection of many animal species and men. The causative agent Bacillus anthracis propagates through the lymphatic system of the infected host; however, the specific interactions of the host and microbe within the lymphatics are incompletely understood. We report the first description of the phosphoprotein signaling in the lymph nodes of DBA/2 mice using a novel technique combining the reverse-phase microarray with the laser capture microdissesction. Mice were challenged into foot pads with spores of toxinogenic, unencapsulated Sterne strain. The spores quickly migrated to the regional popliteal lymph nodes and spread to the bloodstream as early as 3 h post challenge. All mice died before 72 h post challenge from the systemic disease accompanied by a widespread LN tissue damage by bacteria, including the hemorrhagic necrotizing lymphadenitis, infiltration of CD11b+ and CD3+ cells, and massive proliferation of bacteria in lymph nodes. A macrophage scavenger receptor CD68/macrosialin was upregulated and found in association with vegetative bacteria likely as a marker of their prior interaction with macrophages. The major signaling findings among the 65 tested proteins included the reduced MAPK signaling, upregulation of STAT transcriptional factors, and altered abundance of a number of pro- and anti-apoptotic proteins with signaling properties opposing each other. Downregulation of ERK1/2 was associated with the response of CD11b+ macrophages/dendritic cells, while upregulation of the pro-apoptotic Puma indicated a targeting of CD3+ T-cells. A robust upregulation of the anti-apoptotic survivin was unexpected because generally it is not observed in adult tissues. Taken together with the activation of STATs it may reflect a new pathogenic mechanism aimed to delay the onset of apoptosis. Our data emphasize a notion that the net biological outcome of disease is determined by a cumulative impact of factors representing the microbial insult and the protective capacity of the host.

Twist on Protein Microarrays: Layering Wax-Patterned Nitrocellulose to Create Customizable and Separable Arrays of Multiplexed Affinity Columns

We developed the simple and inexpensive FoRe microarray to simultaneously test several 1 μL samples for multiple proteins. By combining forward and reverse phase microarrays into an innovative three-dimensional format, the FoRe array exploits the advantages and eliminates several drawbacks of the traditional approaches (i.e., large sample volumes, protein loss, and cross-reactivity between detection antibodies). Samples are pipetted into an array of separable, multiplexed affinity columns. Several nitrocellulose membranes, each functionalized with a different capture antibody, are stacked to create a customizable affinity column. The nitrocellulose is patterned with wax to form 25 isolated microspots on each layer, allowing us to analyze multiple samples in parallel. After running the immunoassay, the stacks are quickly disassembled, revealing 2D microarrays of different fractions from multiple samples. By combining the stack-and-separate technique with wax patterning, we keep the arrays low cost and easily tailored to a variety of applications. We successfully performed 3D multiplexing using a model system with mouse and rabbit IgG. Binding proved to be independent of the position in the stack, and the limit of detection for a mouse IgG sandwich assay was 7.3 pM in BSA and 15 pM in human plasma. The FoRe microarray makes it possible to identify protein expression patterns across several minute volume samples; for example, it could be used to analyze cell lysate in drug response studies or pricks of blood from small animal studies.

Effect of distinct EGFR downstream pathways on resistance to dual EGFR/ErbB2 combined targeted therapy of lapatinib (LPT) and cetuximab (CTX).

91 Background: Resistance to EGFR therapy is poorly understood. We performed a phase I study in which pre- and post-treatment biopsies were profiled to assess the impact of CTX and LPT on downstream pathways that drive cancer progression. Methods: Between 10/2010 and 5/2012, 16 patients (pts) were enrolled in a 3+3 dose escalation trial, including: colon (7), lung (4), head and neck (3), and anal cancers (2). Prior EGFR therapy was allowed. CTX was 400mg/m2 on D1, then 250 mg/m2 weekly. LPT dose levels were 750 mg, 1000 mg, and 1250 mg daily. Cycles were 21 days in length; pts were assessed for response every 2 cycles. Pts underwent pre- and post-cycle 1 tumor biopsies. Biopsies were evaluated with a Reverse Phase MicroArray (RPMA) platform. Protein lysates were robotically immobilized in defined spots on nitrocellulose-coated slides; each printed array was then interrogated with a highly specific antibody. A total of 36 different proteins were analyzed, including total and phosphorylated EGFR, ErbB2, ErbB3, MET, IGF, MAPK, PI3K, Jak-Stat, and other subpathways. Relative baseline expression levels were determined, as were percent change in matched samples. Results: Of 12 pts evaluable for response, 2 had a partial response (PR), 5 had stable disease (SD), and 5 had disease progression (DP). Baseline tumor measurements of total ERK, p90RSK, and p38MAPK correlated with response (p<0.05), with a trend towards significance between response and expression of pEGFR, pMEK, pNFKB, and p4EBP1 (p<0.10). Nine patients had paired pre- and post-treatment biopsies. In tumors from pts with PR and SD, consistent down regulation of the EGFR pathway was detected. In the four patients with DP, up-regulation of distinct pathway components was detected, including of IGF (1 pt), Mek1/Erk (2 pts), mTOR (2 pts), and STAT3 (3 pts). Conclusions: A tumor profile was defined that correlated with clinical benefit to LPT+CTX. Resistance to this combination was due to upregulation of EGFR pathway components, components that are druggable. This analysis highlights the potential of personalizing therapy with combined EGFR therapies, and for adding additional targeted therapies to patients with resistant tumors.

Abstract 1542: CD38 is physically associated with CD49d and enhances CD49d-mediated adhesion of B-Cell chronic lymphocytic leukemia cells

Abstract CD49d, a recently identified negative prognosticator in chronic lymphocytic leukemia (CLL), is an adhesion molecule mediating microenvironmental interactions of CLL cells in bone marrow and lymph nodes. Through CD49d-dependent adhesion to VCAM-1 and fibronectin (FN), CLL cells receive survival support that renders them less susceptible to conventional therapies. The closely associated expression of CD49d with CD38, another negative prognosticator in CLL, and the propensity of CD38 to associate and functionally cooperate with other molecules, prompted us to investigate whether: i) CD38/CD49d physical interactions may also occur on CLL cell membranes; ii) CD38 may potentiate the CD49d-mediated adhesion in CLL. Confocal microscopy and biochemical approaches were used to study the membrane organization of CD49d and CD38 in CLL cells. The CLL-derived CD49d+CD38- cell line Mec-1, and a subclone in which CD38 expression was induced by viral transduction (Mec-1/CD38) were utilized for adhesion experiments. Co-capping experiments in CD49d+/CD38+ CLL cells demonstrated a membrane relationship between CD38 and CD49d. Anti-CD49d monoclonal antibodies (mAbs) induced capping in approximately 75% of CLL cells, with a 80% redistribution of CD38 in the context of the capping area. The CD38/CD49d lateral association was confirmed by immunoprecipitation experiments with anti-CD49d mAbs and subsequent painting of immunoprecipitates using anti-CD38 mAbs. CD38/CD49d association was also maintained after engagement of CD49d with its natural ligands, as witnessed by co-localization of CD49d and CD38 in uropods of CLL cells adhered and spread onto VCAM-1 and FN. To investigate whether the CD38/CD49d association had also a functional meaning, adhesion assays on VCAM-1-coated plates were performed with the Mec-1 cell model. Mec-1/CD38 showed a marked increase in VCAM-1 adhesion compared to Mec-1 (mean values of adhered cells relative to control=5.4 vs. 2.3 and 5.4 vs. 1.9 after 15 and 30 minutes, respectively; p=0.01). Moreover, phase-contrast and immunofluorescence microscopy highlighted clear differences in the morphology of adhered cells, with Mec-1/CD38 cells characterized by a more complex pattern of uropods than Mec-1, and a clear co-localization of CD38 and CD49d in adhesion sites. Notably adhesion of Mec-1/CD38 cells to VCAM-1 resulted in higher levels of phosphorylation of the guanidine nucleotide exchange factor for Rho proteins Vav-1 and, subsequently, of higher F-actin polymerization, as compared to non transfected Mec-1 cells. CD49d and CD38 are physically associated on CLL cells membranes. CD38 directly contributes in enhancing CD49d-dependent adhesion to VCAM-1. Post-translationally modified proteins involved in CD49d-mediated cell adhesion will be quantified on CD49d-expressing CLL cells with different CD38 levels using Reverse Phase Microarray technology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1542. doi:10.1158/1538-7445.AM2011-1542

Reverse-phase protein microarrays (RPPA) as a diagnostic and therapeutic guide in multidrug resistant leukemia

Reverse-phase microarray assays using phospho-specific antibodies (RPPA) can directly measure levels of phosphorylated protein isoforms. In the current study, lysates from parental and multidrug resistant (MDR) CEM leukemia cells were spotted onto reverse-phase protein microarrays and probed with a panel of phospho-antibodies to ERK, PCK and Akt pathways. In particular, the Akt pathway is considered to play significant roles in leukemia and Akt inhibitor therapy has been proposed as a potential tool in the treatment of this disease. The RPPA data prompted us to investigate deeper this pathway. Here, we found that whereas total Akt1 protein level is higher in parental CEM cells, the activated isoform content, p-Akt1, increases in doxorubicin-selected CEM cells (MDR-CEM). This was backed up by Western blot analysis, confirming that Akt1 activity/phosphorylation may be up-regulated in MDR-CEM cells. Further exploration of inhibitory therapy in this system was evaluated. The TNF-related apoptosis-inducing ligand, TRAIL, has been shown to selectively kill tumor cells. Herein, we describe that in MDR-CEM cells TRAIL responsiveness correlates with a reduced expression of endogenous Akt1, suggesting that the MDR phenotype associated to P-gp sensitizes cells to TRAIL therapy.

Activated VEGF Receptor Shed Into the Vitreous in Eyes With Wet AMD

To examine whether phosphorylated vascular endothelial growth factor (VEGF) receptors shed into the vitreous reflect the ongoing retinal and choroidal signal pathway activity in wet age-related macular degeneration (AMD). Vitreous samples obtained immediately prior to anti-VEGF injection from 11 patients with choroidal neovascularization were analyzed using reverse-phase microarrays. Two patients had samples collected at the time of injection and 1 month later. Samples from 5 patients were collected prior to vitrectomy for macular hole, epiretinal membrane, or retinal detachment. Phosphorylated forms of VEGF receptor (VEGFR Y996 and Y1175), platelet-derived growth factor receptor beta (PDGFRbeta Y716 and Y751), and c-KIT (Y703) were present in the vitreous. A significant difference in PDGFRbeta Y751 (P < .002), VEGFR Y996 (P < .04), and VEGFR Y1175 (P < .006), but not c-KIT Y703 (P < .05) or PDGFRbeta Y716 (P < .96), was noted for the responders to treatment (n = 5) compared with nonresponders (n = 6) and controls (n = 5). Vitreous levels of activated receptors constitute a new class of biomarkers. Activated forms of VEGF and PDGF receptors, previously not known to exist in the vitreous, correlate with response to anti-VEGF therapy. These findings could provide the basis for the development of individualized treatment and discovery of new therapeutic targets.

Quantitative cell signalling analysis reveals down‐regulation of MAPK pathway activation in colorectal cancer

Mitogen-activated protein kinases (MAPK) are considered to play significant roles in colonic carcinogenesis and kinase inhibitor therapy has been proposed as a potential tool in the treatment of this disease. Reverse-phase microarray assays using phospho-specific antibodies can directly measure levels of phosphorylated protein isoforms. In the current study, samples from 35 cases of untreated colorectal cancer colectomies were laser capture-microdissected to isolate epithelium and stroma from cancer as well as normal (i.e. uninvolved) mucosa. Lysates generated from these four tissue types were spotted onto reverse-phase protein microarrays and probed with a panel of antibodies to ERK, p-ERK, p38, p-p38, p-JNK, MEK and p-MEK. Whereas total protein levels were unchanged, or slightly elevated (p38, p = 0.0025) in cancers, activated isoforms, including p-ERK, p-p38 and p-JNK, were decreased two- to four-fold in cancers compared with uninvolved mucosa (p < 0.0023 in all cases except for p-JNK in epithelium, where decrement was non-significant). This was backed up by western blotting. Dukes' stage B and C cancers displayed lower p-ERK and p-p38 expression than Dukes' stage A cancers, although this was not statistically significant. It is concluded that MAPK activity may be down-regulated in colorectal cancer and that further exploration of inhibitory therapy in this system should be carefully evaluated if this finding is confirmed in larger series.

Pegylated, Steptavidin-Conjugated Quantum Dots Are Effective Detection Elements for Reverse-Phase Protein Microarrays

Protein microarray technologies provide a means of investigating the proteomic content of clinical biopsy specimens in order to determine the relative activity of key nodes within cellular signaling pathways. A particular kind of protein microarray, the reverse-phase microarray, is being evaluated in clinical trials because of its potential to utilize limited amounts of cellular material obtained through biopsy. Using this approach, cellular lysates are arrayed in dilution curves on nitrocellulose substrates for subsequent probing with antibodies. To improve the sensitivity and utility of reverse-phase microarrays, we tested whether a new reporter technology as well as a new detection instrument could enhance microarray performance. We describe the use of an inorganic fluorescent nanoparticle conjugated to streptavidin, Qdot 655 Sav, in a reverse-phase protein microarray format for signal pathway profiling. Moreover, a pegylated form of this bioconjugate, Qdot 655 Sav, is found to have superior detection characteristics in assays performed on cellular protein extracts over the nonpegylated form of the bioconjugate. Hyperspectral imaging of the quantum dot microarray enabled unamplified detection of signaling proteins within defined cellular lysates, which indicates that this approach may be amenable to multiplexed, high-throughput reverse-phase protein microarrays in which numerous analytes are measured in parallel within a single spot.