Identificação e caracterização de biomarcadores teciduais e sorológicos no câncer de mama por Phage Display

Abstract

CHAPTER II: Breast cancer is one of the main causes of death among women, as there is no primary prevention. Early detection is the main objective aiming decrease mortality and increase survival. We used phage display technology to isolate ligand peptides to breast cancer tissues in order to select potential biomarkers for the improvement of diagnosis and treatment. Two random peptide libraries with seven and twelve residues expressed in fusion with the pIII protein of the M13 bacteriophage, Ph.D.-7 and Ph.D.-12, respectively, were firstly submitted to a pre clearing by placing them in contact with proteins of normal breast tissues, in oder to eliminate peptides that may recognize healthy tissues. Remaining phages in the supernatant was further selected through three rounds of positive selection. DNA of selected phage were sequenced and translated. Peptide sequences were then submitted to bioinformatic analyzes, ELISA, Dot-blotting, western-blotting and immunohistochemistry assays to confirm the selection strategy and to provide further evidences of the putative biological targets. The immunohistochemistry analysis was performed with six selected phage (BC04, BC05, BC07, BC11, BC12, BC17) in breast cancer, and a mixture of three phage (BC11, BC12, BC17) has been tested in breast and ovary cancers, non-Hodgkin lymphoma, and melanoma. All phages presented high information content with significant ELISA indexes. The Dot-blotting and western-blotting assays have validated the selection by demonstrating high specificity to tumor proteins. The immunohistochemistry analyzes have shown specific binding only to tumor cells. The mixture of phages has recognized all types of tumor tissues, except melanoma. Three selected phage, BC04, BC05 and BC07 have specifically marked the vascular walls of tumor cells. The selected phage may be used in the future as tissue biomarkers for diagnosis and therapeutic procedures in breast cancer. The high information content and the significant data presented by all immunological assays have validated the Phage Display subtractive selection strategy. CHAPTER III: Breast cancer is the most frequent malignant pathology in the Brazilian women population, which is aggravated by diagnosis in later stages of the disease. The aim of this investigation was to select and identify ligand peptides to serum proteins of women with breast cancer through phage display in order to use them as serological biomarkers. Selection of phages was performed in three steps using a random peptide library of 12 residues (Ph.D.-12). The first two selection steps consisted of a pre clearing using the phage libraries against sera proteins of healthy individuals, followed by a second selection against sera proteins of patients with benign breast diseases. After subtraction, the library was submitted to a positive selection against sera of patients with breast cancer. Phage ligands were selected, amplified, and the DNA was sequenced and translated. Peptide sequences were subimtted bioinformatic analyses and to ELISA and dot-blotting assays to confirm selection success. All clones presented significant ELISA indexes and a high information content. The sequence IETYESTHQSPP was the most frequent and presented a high reactivity against IgG from breast cancer patients´ in the dot-blotting analysis. Clones T03, T18 and T09 presented similarity with the ubiquitin and clones T06, T19, T04, T10, T11, T14, T17 with zinc finger motifs. The fact of different clones to present similarity for the same type of protein suggests that different motifs of one same protein can be epitopes recognized by auto-antibodies or that these motifs are interacting with proteins associated to breast cancer, since biopanning was made with total serum.