Abstract
Epithelial to mesenchymal transition (EMT) is believed to be crucial for primary tumors to escape their original residence and invade and metastasize. To properly define EMT, there is a need for ligands that can identify this phenomenon in tumor tissue and invivo. A phage-display selection screening was performed to select novel binding phage peptides for identification of EMT in breast cancer. Epithelial breast cancer cell line, MCF-7 was transformed to mesenchymal phenotype by TGF-β treatment and was used for selection. Breast fibroblasts were used for subtractive depletion and breast cancer metastatic cell lines MDA-MB-231, T47D-shNMI were used for specificity assay. The binding peptides were identified, and their binding capacities were confirmed by phage capture assay, phage-based ELISA, immunofluorescence microscopy. The phage peptide bearing the 7-amino acid sequence, LGLRGSL, demonstrated selective binding to EMT phenotypic cells (MCF-7/TGF-β and MDA-MB-231) as compared to epithelial subtype, MCF-7, T47D and breast fibroblasts (Hs578T). The selected phage was also able to identify metastatic breast cancer tumor in breast cancer tissue microarray (TMA). These studies suggest that the selected phage peptide LGLRGSL identified by phage-display library, showed significant ability to bind to mesenchymal-like breast cancer cells/ tissues and can serve as a novel probe/ligand for metastatic breast cancer diagnostic and imaging.
Highlights
Breast cancer is the most common cancer in women and the second leading causes of death due to cancer [1]
MCF-7 breast cancer cells were transformed into mesenchymal phenotype by long-term treatment with transforming growth factor-β (TGFβ) (1 ng/mL for 16 days)
Extensive depletion of the phage library against plastic, breast fibroblasts before enrichment of phage that interact with MCF-7-TGFβ breast cancer cells was employed for a robust selection of phage clones specific for cancer cells
Summary
Breast cancer is the most common cancer in women and the second leading causes of death due to cancer [1]. The cause of death in breast cancer is often due metastasis to distant sites, resulting in organ failure accounting for a 5-year survival rate of 23%. Studies have shown that dissemination of cancer cells and metastasis into distant organs is often preceded by an epithelial to mesenchymal transition. TGFβ treatment changes epithelial cells from cubodial shape to more elongated ones with concomitant loss of epithelial markers and increased expression of mesenchymal markers vimentin, fibronectin and α-smooth muscle actin [11]. These EMT markers are present in activated cancer-associated fibroblasts (CAF’s), which contributes to the pathogenesis of tumor progression and invasiveness [12]. There is a lack of specific ligands that can recognize mesenchymal-like cancer cells and define EMT in tumor and in cancer-associated fibroblasts
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