Quantum dot-based quantification revealed differences in subcellular localizationof EGFR and E-cadherin between EGFR-TKI sensitive and insensitive cancercells

Abstract

Nanoparticle quantum dots (QDs) provide sharper and more photostable fluorescent signalsthan organic dyes, allowing quantification of multiple biomarkers simultaneously.In this study, we quantified the expression of epidermal growth factor receptor(EGFR) and E-cadherin (E-cad) in the same cells simultaneously by using secondaryantibody-conjugated QDs with two different emission wavelengths (QD605 and QD565)and compared the cellular distribution of EGFR and E-cad between EGFR-tyrosine kinaseinhibitor (TKI)-insensitive and -sensitive lung and head and neck cancer cell lines.Relocalization of EGFR and E-cad upon treatment with the EGFR-TKI erlotinib in thepresence of EGF was visualized and analyzed quantitatively. Our results showed thatQD-immunocytochemistry (ICC)-based technology can not only quantify basal levels ofmultiple biomarkers but also track the localization of the biomarkers upon biostimulation.With this new technology we found that in EGFR-TKI-insensitive cells, EGFRand E-cad were located mainly in the cytoplasm; while in sensitive cells, theywere found mainly on the cell membrane. After induction with EGF, both EGFRand E-cad internalized to the cytoplasm, but the internalization capability insensitive cells was greater than that in insensitive cells. Quantification also showedthat inhibition of EGF-induced EGFR and E-cad internalization by erlotinib inthe sensitive cells was stronger than that in the insensitive cells. These studiesdemonstrate substantial differences between EGFR-TKI-insensitive and -sensitivecancer cells in EGFR and E-cad expression and localization both at the basallevel and in response to EGF and erlotinib. QD-based analysis facilitates theunderstanding of the features of EGFR-TKI-insensitive versus -sensitive cancercells and may be used in the prediction of patient response to EGFR-targetedtherapy.