ABSTRACT
Colorectal cancer (CRC) is one of the most common primary malignancies. Early stages of the disease are asymptomatic in the majority of cases, leading to late detection and high mortality. Available noninvasive diagnostic techniques are limited in sensitivity and specificity, and designing new ones is still a pressing problem. Exosomes are membrane-derived microvesicles secreted into human biological fluids and provide a novel way to assess the course of an oncology disease. The review describes the repertoire of exosomal surface biomarkers found in the blood of CRC patients and the prospects of employing multiplexed tests for exosomal markers in early noninvasive diagnosis of cancer.
Subject(s)
Biomarkers, Tumor/metabolism , Cell-Derived Microparticles/metabolism , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/metabolism , Exosomes/metabolism , Animals , Cell-Derived Microparticles/pathology , Colorectal Neoplasms/pathology , Exosomes/pathology , HumansABSTRACT
Exosomes are cell-derived vesicles that are secreted by both normal and cancer cells. Over the last decade, a few studies have revealed that exosomes cross talk and/or influence major tumor-related pathways such as angiogenesis and metastasis involving many cell types within the tumor microenvironment. The protein composition of the membrane of an exosome reflects that of the membrane of the cell of origin. Because of this, tumor-derived exosomes differ from exosomes that are derived from normal cells. The detection of tumor exosomes and analysis of their molecular composition hold promise for diagnosis and prognosis of cancer. Here, we present hydrogel microarrays (biochips), which contain a panel of immobilized antibodies that recognize tetraspanins (CD9, CD63, CD81) and prognostic markers for colorectal cancer (A33, CD147). These biochips make it possible to analyze the surface proteins of either isolated exosomes or exosomes that are present in the serum samples without isolation. These biochips were successfully used to analyze the surface proteins of exosomes from serum that was collected from a colorectal cancer patient and healthy donor. Biochip-guided immunofluorescent analysis of the exosomes has made it possible for us to detect the A33 antigen and CD147 in the serum sample of the colorectal cancer patient with normal levels of CEA and CA19-9.
Subject(s)
Antigens, CD/blood , Biomarkers, Tumor/blood , Colorectal Neoplasms/blood , Exosomes/metabolism , Hydrogels/chemistry , Lab-On-A-Chip Devices , Microchip Analytical Procedures/methods , Neoplasm Proteins/blood , Adult , Female , Humans , Male , Middle AgedABSTRACT
The objective of this work was to obtain preparations of recombinant squamous-cell carcinoma antigens (serpins B3 and B4) and to investigate their interactions with different monoclonal antibodies using hydrogel-based microarrays (biochips). Two genetic constructs encoding full-length serpin B3 and serpin B4 molecules were created to produce recombinant SPB3 and SPB4 proteins carrying a N-terminal His6-tag. Monoclonal antibodies against serpin B3 (H3, C5, H5, H81, and G9) were also obtained. An experimental gel-based biological microchip was designed to contain gel elements that carry immobilized antibodies against SPB3, immobilized commercial monoclonal SCC107 and SCC140 antibodies against squamous-cell carcinoma antigen (SCCA), and gel elements with immobilized SPB3 or SPB4. Judging by the specificity of recombinant SPB3 and SPB4, which bind to monoclonal antibodies against SCCA and, according to the manufacturer's data, can recognize conformational epitopes of both SPB3 and SPB4, it was concluded that the obtained recombinant serpins had the correct tertiary structure. A biochip-based direct immunoassay showed that SPB4 could bind effectively only to SCC107 and SCC140 antibodies, while SPB3 interacted specifically not only with these antibodies, but also with H3 and C5 monoclonal antibodies. Using biochip-based sandwich immunoassay, a pair of monoclonal antibodies SCC107/C5 that interacted specifically with serpin B3 but did not interact with serpin B4 was identified. Thus, it has been demonstrated that serpin B3 can be selectively determined in the presence of highly homologous serpin B4 using a biochip-based assay.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigens, Neoplasm/chemistry , Epitopes/chemistry , Hydrogels/chemistry , Serpins/chemistry , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Carcinoma, Squamous Cell/chemistry , Cloning, Molecular , Epitopes/genetics , Epitopes/immunology , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Lab-On-A-Chip Devices , Mice , Mice, Inbred BALB C , Microarray Analysis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Serpins/genetics , Serpins/immunologyABSTRACT
The biochip was constructed for simultaneous assay of total and free prostate-specific antigen, alpha-fetoprotein, cancer embryonic antigen, human chorionic gonadotropin, and neuron-specific enolase. These biochips represent an array of gel elements with covalently immobilized proteins. The major analytic characteristics of the developed method were obtained. It was shown that the results of simultaneous assay of six tumor markers in blood serum well correlated with routine measurement of each marker using enzyme immunoassay kits. This approach allowed us to reveal the hook effect of high concentrations during biochip assay, which prevents distortion of the diagnostic picture at high concentration of the analyte in the sample.
