Nuclear Delivery of Radiosensitizing EGFR-Targeted Nanoparticles Is Abrogated in Isogenic Cervical Cancer Cell Lines Following Mutagenesis of the Ligand Binding Domain

Abstract

The development of targeted treatment regimens for cervical cancer remains a critically unexplored area of research. One such possible target is epidermal growth factor receptor (EGFR). Our laboratory has previously synthesized radiosensitizing nanoparticles (NPs) that specifically target cells that express EGFR. Here, we test the hypothesis that nuclear localization following treatment of EGFR-targeted NPs is preferentially achieved in cervical cancer cell lines with high levels of EGFR expression and is dependent upon an intact ligand binding domain. A panel of cervical cancer cell lines (HeLa, SiHa, CaSki, ME180, and C33A) with varying levels of endogenous and genetically manipulated EGFR expression were cultured and treated with Fe3O4@TiO2 NPs conjugated to EGF “mimic” (B-loop) peptide (EGFR-targeted NPs) or its scrambled version (scrambled non-targeted NPs). Transient overexpression and knockdown of EGFR was achieved with transfection of EGFR DNA or small interfering RNA (siRNA). Isogenic cell lines were expanded from single cell clones of the ME-180 cervical cancer cell line with heterozygous or homozygous mutations, introduced by CRISPR-Cas9 technology, of the critical amino acids Leucine-14 (Leu-14) and Tyrosine-45 (Tyr-45) located within the ligand binding domain of the EGFR protein. Immunocytochemistry with confocal microscopy and flow cytometry was utilized to assess EGF-binding ability of isogenic cell lines as well ascellular delivery, subcellular distribution, and receptor binding ability of EGFR-targeted NPs and scrambled non-targeted NPs in this panel of cell lines. All tested cervical cancer cell lines endogenously produced both full-length EGFR mRNA variant 1 (v1), as well as shorter mRNA variants 3 and 4 which encode soluble isoforms of EGFR, while expression of full-length EGFR (v1) varied dramatically across cell lines. Cell lines with high levels of endogenous or genetically upregulated EGFR expression experienced increased nuclear uptake of EGFR-targeted NPs compared to cell lines with low levels of endogenous EGFR expression or following siRNA-mediated knockdown, while uptake of scrambled non-targeted NPs was similar across all cell lines. Isogenic clones of the ME-180 cervical cancer cell line demonstrated impaired EGF-binding as well as nuclear localization of EGFR-targeted NPs but scrambled non-targeted NPs. Nuclear accumulation of EGFR-targeted radiosensitizing NPs, but not scrambled non-targeted NPs, occurred preferentially in cervical cancer cell lines with high expression of EGFR and was abrogated following mutation of the ligand binding domain. Further studies are required to demonstrate in vivo radiosensitization of cervical cancer following treatment with EGFR-targeted NPs.