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High-throughput assessment of the antibody profile in ovarian cancer ascitic fluids.

ABSTRACT: The identification of effective biomarkers for early diagnosis, prognosis, and response to treatments remains a challenge in ovarian cancer (OC) research. Here, we present an unbiased high-throughput approach to profile ascitic fluid autoantibodies in order to obtain a tumor-specific antigen signature in OC. We first reported the reactivity of immunoglobulins (Igs) purified from OC patient ascites towards two different OC cell lines. Using a discovery set of Igs, we selected tumor-specific antigens from a phage display cDNA library. After biopanning, 700 proteins were expressed as fusion protein and used in protein array to enable large-scale immunoscreening with independent sets of cancer and noncancerous control. Finally, the selected antigens were validated by ELISA. The initial screening identified eight antigenic clones: CREB3, MRPL46, EXOSC10, BCOR, HMGN2, HIP1R, OLFM4, and KIAA1755. These antigens were all validated by ELISA in a study involving ascitic Igs from 153 patients (69 with OC, 34 with other cancers and 50 without cancer), with CREB3 showing the highest sensitivity (86.95%) and specificity (98%). Notably, we were able to identify an association between the tumor-associated (TA) antibody response and the response to a first-line tumor treatment (platinum-based chemotherapy). A stronger association was found by combining three antigens (BCOR, CREB3, and MRLP46) as a single antibody signature. Measurement of an ascitic fluid antibody response to multiple TA antigens may aid in the identification of new prognostic signatures in OC patients and shift attention to new potentially relevant targets.

SUBMITTER: Antony F

PROVIDER: S-EPMC6685609 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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High-throughput assessment of the antibody profile in ovarian cancer ascitic fluids.

Antony Frank F Deantonio Cecilia C Cotella Diego D Soluri Maria Felicia MF Tarasiuk Olga O Raspagliesi Francesco F Adorni Fulvio F Piazza Silvano S Ciani Yari Y Santoro Claudio C Macor Paolo P Mezzanzanica Delia D Sblattero Daniele D

Oncoimmunology 20190604 9

The identification of effective biomarkers for early diagnosis, prognosis, and response to treatments remains a challenge in ovarian cancer (OC) research. Here, we present an unbiased high-throughput approach to profile ascitic fluid autoantibodies in order to obtain a tumor-specific antigen signature in OC. We first reported the reactivity of immunoglobulins (Igs) purified from OC patient ascites towards two different OC cell lines. Using a discovery set of Igs, we selected tumor-specific antig ...[more]

PMID: 31428516

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Project description:Ovarian cancer is the most lethal gynecologic disease due to delayed diagnosis, and ascites production is a characteristic of patients in advanced stages. The aim of this study was to perform the proteomic analysis of ascitic fluids of Mexican patients with ovarian carcinoma, in order to detect proteins with a differential expression pattern in the continuing search to identify biomarkers for this disease.Samples were collected from 50 patients from the Instituto Nacional de Cancerología of México under informed consent and with approval of the bioethics and scientific committees. After elimination of abundant proteins (Albumin/IgGs) samples were processed for 2D electrophoresis and further protein identification by Mass Spectrometry (MALDI-TOF). Molecules of interest were followed by western blot and lectin binding assays, and their tissue location by histo-immunofluorescence and confocal analysis.An area with a differential expression pattern among samples was located in the 2D gels. Identified proteins were 6 alpha 1 isoforms and 1 alpha 2 isoform of Haptoglobin, and 2 isoforms of Transthyretin. While Transthyretin isoforms were constitutively expressed in all samples, clear differences in the expression pattern of Haptoglobin alpha isoforms were found. Moreover, increased levels of fucosylation of Haptoglobin alpha isoforms analyzed in 40 samples by Aleuria aurantia lectin binding by 1D overlay assay showed a positive correlation with advanced stages of the disease. Tissue detection of Haptoglobin and its fucosylated form, by histo-immunofluorescence in biopsies of ovarian cancer, also showed a correlation with ovarian cancer progression. Moreover, results show that fucosylated Haptoglobin is produced by tumor cells.Increased numbers of highly fucosylated Haptoglobin alpha isoforms in ascitic fluids and the presence of fucosylated Haptoglobin in tumor tissues of ovarian cancer Mexican patients associated with advanced stages of the disease, reinforce the potential of fucosylated Haptoglobin alpha isoforms to be characterized as biomarkers for disease progression.

Project description:Resistance to anticancer drugs is a critical issue in cancer treatment. Alterations in gene expression and DNA methylation profiles that accompany the acquisition of drug resistance are associated with resistance mechanisms. To analyze chemotherapy-associated alterations in gene expression and DNA methylation in gastric cancer cells obtained from ascites, ascitic fluids were collected from a patient with gastric cancer before chemotherapy with capecitabine and oxaliplatin (CapeOX), and after the disease had progressed. The fluids were cultured for 10 days, passaged into new flasks, and cultured for an additional 2 weeks. Normal cells, including white blood cells and mesothelial cells, were removed. The expression and DNA methylation profiles of 18,185 genes were analyzed using microarray, and compared between cells in ascitic fluids collected before and after the chemotherapy with CapeOX. In addition, fluorouracil- and oxaliplatin-resistant AGS cells were established and analyzed. Pathways having genes with expression profiles altered by CapeOX included those associated with 'signaling by G-protein-coupled receptor' and the 'immune system'. Genes that were commonly expressed at higher levels in CapeOX-resistant ascitic cells, fluorouracil-resistant AGS cells. and oxaliplatin-resistant AGS cells compared with those in untreated cells included telomerase reverse transcriptase (TERT), apolipoprotein C1 (APOC1) and serine/threonine/tyrosine kinase 1 (STYK1), whereas genes commonly expressed at lower levels in the three drug-resistant cell types compared with the untreated cells included defensin β4A (DEFB4A). A comparatively large number of genes exhibited altered methylation levels in drug-resistant AGS cells compared with the CapeOX-resistant cells. In addition, among the genes expressed at higher levels in decitabine-treated AGS cells, the majority were expressed at higher levels in fluorouracil-resistant AGS cells, and exhibited lower methylation levels. Taken together, the present study has demonstrated that comparing the expression profiles of gastric cancer cells obtained from ascitic fluids before and after chemotherapy with the expression profiles of drug-resistant cultured cells is a useful method for analyzing the molecular mechanisms underlying chemotherapy resistance.

Dataset Information

High-throughput assessment of the antibody profile in ovarian cancer ascitic fluids.

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High-throughput assessment of the antibody profile in ovarian cancer ascitic fluids.

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