Abstract 6135: Dynamics of ligand-induced epidermal growth factor receptor internalization in cancer cell lines

Abstract

Antibody-drug conjugates (ADCs), comprised of cancer cell-specific antibodies linked to cytotoxic drugs, represent an innovative and promising class of anticancer agents. Due to its amplified expression in various tumor types, the epidermal growth factor receptor (EGFR) presents an attractive target for ADC therapy, and three anti-EGFR ADCs are currently studied in clinical trials. As ADCs depend on receptor internalization for intracellular release of drugs in the targeted cancer cells, studying the dynamics of receptor internalization can aid in their development and optimization. We studied the internalization of EGFR induced by its ligand EGF, as a mimic of ADCs, in cell lines with different surface receptor densities. BxPC-3 (pancreas) and RT-112 (bladder) cancer cells were seeded in 6-well plates and allowed to adhere for 24 hours. Cells were serum-starved overnight, followed by stimulation with 150 ng/mL EGF for 2, 4, 6, 24 and 48 hours. Internalization was studied by quantification of the number of surface-expressed receptors using flow cytometry with Quantibrite PE beads and EGFR-PE antibody. Moreover, incorporation of the ligand-receptor complex into acidic endo-lysosomes was studied using EGF conjugated to the fluorescent pH sensor pHrodo™ Green. Total EGFR and EGFR phosphorylation levels were assessed by western blot analysis. RNA was isolated and converted into cDNA to perform TaqMan qPCR. Unstimulated BxPC-3 cells showed higher numbers of EGFR per cell compared to unstimulated RT-112 cells. Since high receptor expression is key for some ADCs to induce effective endocytosis, this may indicate BxPC-3 cells as a more attractive model to study EGFR internalization. Stimulation with EGF induced a strong decrease in surface receptor density for both BxPC-3 and RT-112 cells during the first two hours, suggesting receptor internalization. Accordingly, treatment of BxPC-3 cells with pHrodo™ Green EGF demonstrated the incorporation of ligand-receptor complexes into endo-lysosomes. During following hours, the cell surface expression of EGFR remained stable, whereas it partially recovered after 24 to 48 hours. In contrast, the lysosomal incorporation of EGF-EGFR complexes was not decreased at 24 or 48 hours, suggesting that the increase in surface EGFR may be due to de novo protein synthesis rather than recycling. No increase of EGFR mRNA was, however, detected. Finally, increased phosphorylation of EGFR was observed after two to six hours of stimulation, whereas total EGFR levels were highest in unstimulated and 24- or 48-hour-stimulated cells, which is in concordance with the flow cytometry data. In conclusion, both BxPC-3 and RT-112 cells show dynamic changes in EGFR internalization upon EGF stimulation, despite differences in EGFR expression levels. These cell lines can therefore successfully be used for internalization studies, which may contribute to future ADC development. Citation Format: Mandy W. Smeets, Eef F. Smits, Janneke J. Melis, Dimitri Pappaioannou, Guido J. Zaman. Dynamics of ligand-induced epidermal growth factor receptor internalization in cancer cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6135.